13
Polyhydramnios, megalencephaly and symptomatic epilepsy caused by a homozygous 7-kilobase deletion in LYK5 Erik G. Puffenberger, 1, * Kevin A. Strauss, 1, * Keri E. Ramsey, 2 David W. Craig, 2 Dietrich A. Stephan, 2 Donna L. Robinson, 1 Christine L. Hendrickson, 1 Steven Gottlieb, 3 David A. Ramsay, 4 Victoria M. Siu, 5 Gregory G. Heuer, 6 Peter B. Crino 7 and D. Holmes Morton 1 1 Clinic for Special Children, Strasburg, PA, USA, 2 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA, 3 Pediatric Neurology, Lancaster General Hospital, Lancaster, PA, USA, 4 Department of Pathology, London Health Sciences Centre, London, Ontario, Canada, 5 Medical Genetics Program, London Health Sciences Centre, London, Ontario, Canada, 6 Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA and 7 Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA *These authors contributed equally to this work. Correspondence to: Kevin A. Strauss, MD, Clinic for Special Children, 535 Bunker Hill Road, Strasburg, PA 17579, USA E-mail: [email protected] We used single nucleotide polymorphism (SNP) microarrays to investigate the cause of a symptomatic epilepsy syndrome in a group of seven distantly related Old Order Mennonite children. Autozygosity mapping was incon- clusive, but closer inspection of the data followed by formal SNP copy number analyses showed that all affected patients had homozygous deletions of a single SNP (rs721575) and their parents were hemizygous for this marker. The deleted SNP marked a larger deletion encompassing exons 9^13 of LYK5 , which encodes STE20 - related adaptor protein, a pseudokinase necessary for proper localization and function of serine/threonine kinase 11 (a.k.a. LKB1). Homozygous LYK5 deletions were associated with polyhydramnios, preterm labour and distinctive craniofacial features. Affected children had large heads, infantile-onset intractable multifocal sei- zures and severe psychomotor retardation. We designated this condition PMSE syndrome ( polyhydramnios, megalencephaly and symptomatic epilepsy). Thirty-eight percent (N ¼16) of affected children died during child- hood (ages 7 months to 6 years) from medical complications of the disorder, which included status epilepticus, congestive heart failure due to atrial septal defect and hypernatremic dehydration due to diabetes insipidus. A single post-mortem neuropathological study revealed megalencephaly, ventriculomegaly, cytomegaly and extensive vacuolization and astrocytosis of white matter. There was abundant anti-phospho-ribosomal S6 label- ling of large cells within the frontal cortex, basal ganglia, hippocampus and spinal cord, consistent with consti- tutive activation of the mammalian target of rapamycin (mTOR) signalling pathway in brain. Keywords: symptomatic epilepsy; mammalian target of rapamycin; Mennonite; single nucleotide polymorphism; syndromic developmental delay; tuberous sclerosis complex Abbreviations: GFAP ¼ glial acidic fibrillary protein; LFB-CV ¼ luxol fast blue-cresyl violet; mTOR ¼ mammalian target of rapamycin; P-S6 ¼ phospho-ribosomal S6 protein; SNP ¼ single nucleotide polymorphism; STK11 ¼serine/threonine kinase 11 (a.k.a. LKB1); STRAD ¼ STE20 -related adaptor protein; TSC ¼ tuberous sclerosis complex Received September 18, 2006. Revised January 28, 2007 . Accepted April 4, 2007 . Advance Access publication May 23, 2007 Introduction Developmental delay (Battaglia and Carey, 2003; Shevell et al., 2003) is the presenting problem for 35% of the approximately 125 Amish and Mennonite patients evalu- ated each year at the Clinic for Special Children. Population-based genetic knowledge allows us to reach an aetiological diagnosis for some of these children (Morton et al., 2003; Strauss et al., 2005, 2006), but many do not have a recognizable clinical syndrome and remain without a diagnosis after exhaustive biochemical and cytogenetic testing (Hunter, 2000; Battaglia and Carey, 2003). Although some consider brain MRI informative in doi:10.1093/brain/awm100 Brain (2007), 130, 1929 ^1941 ß The Author (2007). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]

Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

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Page 1: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

Polyhydramnios megalencephaly and symptomaticepilepsy caused by a homozygous 7-kilobase deletionin LYK5Erik G Puffenberger1 Kevin A Strauss1 Keri E Ramsey2 David WCraig2 Dietrich A Stephan2

Donna L Robinson1 Christine L Hendrickson1 Steven Gottlieb3 David A Ramsay4 Victoria M Siu5

Gregory G Heuer6 Peter B Crino7 and D Holmes Morton1

1Clinic for Special Children Strasburg PA USA 2Neurogenomics Division Translational Genomics Research InstitutePhoenix AZ USA 3Pediatric Neurology Lancaster General Hospital Lancaster PA USA 4Department of PathologyLondon Health Sciences Centre London Ontario Canada 5Medical Genetics Program London Health Sciences CentreLondon Ontario Canada 6Department of Neurosurgery University of Pennsylvania Philadelphia PA USA and7Department of Neurology University of Pennsylvania Philadelphia PA USA

These authors contributed equally to this work

Correspondence to Kevin A Strauss MD Clinic for Special Children 535 Bunker Hill Road Strasburg PA17579 USAE-mail kstraussclinicforspecialchildrenorg

We used single nucleotide polymorphism (SNP) microarrays to investigate the cause of a symptomatic epilepsysyndrome in a group of seven distantly related Old Order Mennonite children Autozygositymapping was incon-clusive but closer inspection of the data followed by formal SNP copy number analyses showed that all affectedpatients had homozygous deletions of a single SNP (rs721575) and their parents were hemizygous for thismarker The deleted SNP marked a larger deletion encompassing exons 9^13 of LYK5 which encodes STE20 -related adaptor protein a pseudokinase necessary for proper localization and function of serinethreoninekinase 11 (aka LKB1)Homozygous LYK5 deletions were associated with polyhydramnios preterm labour anddistinctive craniofacial features Affected children had large heads infantile-onset intractable multifocal sei-zures and severe psychomotor retardation We designated this condition PMSE syndrome (polyhydramniosmegalencephaly and symptomatic epilepsy)Thirty-eight percent (Nfrac1416) of affected children died during child-hood (ages 7 months to 6 years) from medical complications of the disorder which included status epilepticuscongestive heart failure due to atrial septal defect and hypernatremic dehydration due to diabetes insipidusA single post-mortem neuropathological study revealed megalencephaly ventriculomegaly cytomegaly andextensive vacuolization and astrocytosis of white matterThere was abundant anti-phospho-ribosomal S6 label-ling of large cells within the frontal cortex basal ganglia hippocampus and spinal cord consistent with consti-tutive activation of the mammalian target of rapamycin (mTOR) signalling pathway in brain

Keywords symptomatic epilepsy mammalian target of rapamycin Mennonite single nucleotide polymorphism syndromicdevelopmental delay tuberous sclerosis complex

Abbreviations GFAPfrac14glial acidic fibrillary protein LFB-CVfrac14 luxol fast blue-cresyl violet mTORfrac14mammalian target ofrapamycin P-S6frac14 phospho-ribosomal S6 protein SNPfrac14 single nucleotide polymorphism STK11frac14serinethreonine kinase 11(aka LKB1) STRADfrac14 STE20-related adaptor protein TSCfrac14 tuberous sclerosis complex

Received September 18 2006 Revised January 28 2007 Accepted April 4 2007 Advance Access publication May 23 2007

IntroductionDevelopmental delay (Battaglia and Carey 2003 Shevellet al 2003) is the presenting problem for 35 of theapproximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special ChildrenPopulation-based genetic knowledge allows us to reach an

aetiological diagnosis for some of these children

(Morton et al 2003 Strauss et al 2005 2006) but

many do not have a recognizable clinical syndrome and

remain without a diagnosis after exhaustive biochemical

and cytogenetic testing (Hunter 2000 Battaglia and Carey

2003) Although some consider brain MRI informative in

doi101093brainawm100 Brain (2007) 130 1929^1941

The Author (2007) Published by Oxford University Press on behalf of the Guarantors of Brain All rights reserved For Permissions please email journalspermissionsoxfordjournalsorg

such cases (Shevell et al 2003 Srour et al 2006) we findthat abnormal imaging results rarely imply a specificdiagnosisIn the present work we describe the focused application

of DNA microarrays to investigate a small cluster ofMennonite patients with syndromic epilepsy and develop-mental delay In 1998 we evaluated a Mennonite boy bornwith a large head and distinctive facial features He beganto have intractable multifocal seizures at 4 months of ageand his subsequent developmental progress was stagnant Abrain MRI showed ventriculomegaly focal subependymaldysplasia and multiple areas of high diffusion withinsubcortical white matter (Fig 2) There was no specificgenetic diagnosis after 6 years of detailed investigationsIn autumn of 2004 the boyrsquos mother identified several

other Mennonite children with similar physical featuresearly onset focal seizures and severe psychomotor retarda-tion We collected DNA from seven individuals with acommon phenotype and using a 10 000 single nucleotidepolymorphism (SNP) mapping strategy (Puffenberger et al2004 Strauss et al 2005 2006) found a homozygousdeletion of a single SNP in all affected individuals Thedeleted SNP marked a large deletion of LYK5 whichencodes the STE20-related adaptor protein (STRAD)important for normal in vitro localization and catalyticactivity of serinethreonine kinase 11 (STK11 aka LKB1)(Baas et al 2003 Boudeau et al 2003) This is the firstdescription of a human disease associated with a mutationin LYK5 The neurological phenotype implies a specific rolefor STRAD in human brain growth and development and amore general role for the mTOR signalling pathway incertain cortical malformation syndromes (Baybis et al2004 Shaw et al 2004 Kwiatkowski and Manning 2005)

Patients and MethodsThis study was approved by the Institutional Review Board ofLancaster General Hospital Parents consented in writing tomolecular testing and reproduction of photographs Seven affectedindividuals and their 14 parents were used for SNP genome-wideautozygosity mapping (Strauss et al 2005) After mapping thedisease gene to chromosome 17 nine additional Mennonitechildren with suggestive clinical features were tested for the LYK5deletion by direct molecular genetic analysis Phenotype informa-tion was based on clinical data from 16 patients ages 7 months to28 years

GenotypingTotal genomic DNA from whole blood was isolated using thePUREGENE DNA Isolation Kit (Gentra Systems MinneapolisMN USA) according to the manufacturerrsquos protocol SNPgenotyping was done using the GeneChip Mapping 10K AssayKit (Affymetrix Santa Clara CA USA) as previously described(Puffenberger et al 2004) Briefly 250 ng of double-strandedgenomic DNA was digested with XbaI the restriction fragmentswere ligated to adapters and the ligated products were amplifiedin quadruplicate using a generic primer PCR products werepurified fragmented with DNase labelled with terminal

deoxytransferase and finally hybridized to a GeneChip HumanMapping 10K Xba142 20 Array Microarrays were then washedwith a fluidics station incubated and scanned using the GeneChipScanner 3000 enabled by GeneChip GCOS software (Affymetrix)

Genome-wide autozygosity mappingData were analysed in Microsoft Excel spreadsheets (MicrosoftCorporation Redmond WA USA) that were custom-formatted atthe Clinic for Special Children SNP positions came fromAffymetrix genome annotation files and genotype data camefrom the Affymetrix GeneChip Human Mapping 10K Xba 142 20Arrays Excel spreadsheets were designed to identify genomicregions that were identical-by-descent between all affectedindividuals This analysis assumes mutation and locus homo-geneity Two-point LOD scores were calculated for each genotypedSNP using an approach similar to Broman and Weber (1999a b)Cumulative two-point LOD scores for a block of homozygousSNPs were considered the location score for a region providing arelative measure of likelihood that the region harbours the diseasegene Genotype data from this study and others (Puffenbergeret al 2004 Strauss et al 2005 2006) were used to estimatepopulation-specific SNP allele frequencies

DNA sequencingThe target sequence was amplified by using specific oligonucleo-tide primers and 30ndash50 ng of genomic DNA from affected andunaffected family members Primer sequences for PCR amplifica-tion and sequencing were designed using Primer3 (httpfrodowimiteducgi-binprimer3primer3_wwwcgi) PCR pro-ducts were purified using QiaQuick columns (Qiagen ValenciaCA USA) as per manufacturerrsquos instructions and then sequencedusing the BigDye Terminator cycle sequencing protocol (AppliedBiosystems Foster City CA USA) Extension products weresubsequently size-fractionated on an ABI PRISM 310 GeneticAnalyzer (Applied Biosystems) and data were compared to thehuman genome reference sequence and dbSNP in order to identifysequence variants Population-based control samples were studiedin an identical fashion

Genome-wide copy number analysisThe SNP copy number generated by the copy number analysistool (CNAT) of the Affymetrix Genotyping software wasstandardized for analyses For each SNP on the array we firstgenerated a mean copy number and standard deviation from areference population of 100 healthy (control) individuals from thePlain community Copy number data for each patient were thenexported to Excel files and SNP copy number z-scores (standardscores) were calculated according to the formula zfrac14 (patientvalue control mean)control standard deviation The resultingsingle-point copy number z-scores and log2 ratios were plottedusing chart and trendline functions in Excel Within a normalpopulation 998 of z-scores fall between 3 and thorn3 scoresoutside of these limits were investigated in more detail

Histology and immunohistochemistryAn autopsy was performed on one female patient who died of anundetermined cause at 7 months of age Paraffin-embeddedsections from liver kidney heart lung and brain were stainedwith haematoxylinndasheosin Additional brain specimens were stained

1930 Brain (2007) 130 1929^1941 EG Puffenberger et al

with luxol fast blue-cresyl violet (LFB-CV) and routine immuno-peroxidase methods using antibodies against glial fibrillary acidicprotein (GFAP) Sections of frontal cortex striatum hippocampusand spinal cord were probed with anti-phospho-ribosomal S6(P-S6) antibodies (Ser235236 1 50 dilution Cell Signaling NewEngland Biolabs Beverly MA USA) (Baybis et al 2004) whichwere visualized with biotinylated anti-rabbit IgG (1 1000dilution Vector Laboratories Burlingame CA USA) afteravidinndashbiotin conjugation (Vectastain ABC Elite VectorLaboratories) and treatment with 330-diaminobenzadine

ResultsClinical phenotypeClinical data from 16 affected individuals (ages 7 months to28 years) are summarized in Table 1 All affectedpregnancies were complicated by polyhydramniosSpontaneous onset of labour occurred between 25 and36 weeks gestation in 12 pregnancies (75) All but oneaffected child had macrocephaly (head circumference 2SD for age) (Fig 1) Linear growth was normal and bodymass index was consistently low (13 kgm2) due to lowmuscle mass Several young children had persistent restlesslimb movements a sign we interpreted as mild chorea Thiswas not a finding in older patients

Each affected child had a long face large foreheadpeaked eyebrows broad nasal bridge hypertelorism and alarge mouth with thick lips Craniofacial structure changedconsiderably with age (Fig 2A) During infancy and earlychildhood the skull was shaped like an inverted pyramidreflecting prominence of the cranial vault relative to thelower face As children grew older their appearancechanged due to overgrowth of the mandible enlargementof the mouth and thickening of the lips (Fig 2B)Radiological skeletal surveys available for only two infantswere normal

Cranial magnetic resonance imaging (MRI) studies at 15Tesla were available from only four patients ages 4 monthsto 5 years These images were acquired 5 to 10 years agoand thus the quality was poor relative to current imagingstandards The MRI from the two youngest children whosehead circumferences were greater than four standarddeviations above normal showed only mild ventriculome-galy that could not fully account for increased head sizeStudies from the two older children showed ventriculome-galy evidence of subependymal dysplasia and multiple areasof high water diffusion within the subcortical white matter(Fig 3)

Seizures started between 3 and 7 months of age in allpatients These were most commonly complex partialseizures that would occasionally spread to involve one orboth cerebral hemispheres Electroencephalographic record-ings typically showed high-voltage spike and slow wavedischarges arising from bilateral independent electroence-phalographic foci over the frontal temporal parietal andoccipital regions These spike-slow wave discharges wouldsometimes change into a long run of spikes or into arhythmic slow wave focus (25 to 3 cycles per second)

Table 1 Clinical features of 16 patients with homozygousLYK5 deletions

Clinical variable Percent of patients affected(nfrac1416)

PrenatalPolyhydramnios 100Preterm laboura 75

PostnatalMacrocephalyb 100Infantile-onset partial epilepsy 100Hypotonia 100Craniofacial dysmorphism 100Skeletal muscle hypoplasia 100Strabismus 56Atrial septal defect 25Nephrocalcinosisc 13Diabetes insipidusd 13Supraventricular tachycardia 6Leucaemia 6

Developmental domaine Maximum developmentalage (range in months)

Gross motor 6^14Fine motor-adaptive 4^10Language^ communication 2^6Personal^ social 2^6

aMean onset of labour at 31weeks gestation range 25^37 weeksbDue to a combination of megalencephaly and hydrocephaluscRenal ultrasounds were only performed in four patients two ofwhom (imaged at ages 6 and 28 years) had nephrocalcinosisdOne patient with DI had bilateral nephrocalcinosis on CTscanThe cause of DI was mixed with both central and nephrogeniccomponentseDevelopmental outcomes based on Denver DevelopmentalScreeningTest II

Fig 1 Occipitofrontal head circumference (in cm) of 16 childrenwith PMSE syndrome Males are depicted as circles females asinverted triangles Although mild enlargement of the ventricularsystem was seen on the four available neuroimaging studies weattributed increased head circumference primarily tomegalencephaly

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1931

Fig 2 Dysmorphic features (A) Young persons with homozygous LYK5 deletions had a long face large forehead peaked eyebrows broadnose and wide-set eyes As patients got older (from upper left to lower right) the mouth became enlarged and the lips thickened (B)Individual craniofacial structure changed considerably with age Serial pictures of one affected individual show macrocephaly frontal bos-sing hypertelorism and broad nasal bridge shortly after birth (left panel) lengthening of the face during early childhood (middle panel) andovergrowth of the mandible enlargement of the mouth and thickening of the lips during adolescence (right panel)

1932 Brain (2007) 130 1929^1941 EG Puffenberger et al

lasting 10 to 15min Two young patients were diagnosedwith infantile spasms based on clinical and electroencepha-lographic criteria One child had right-sided epilespiapartialis continua during a 24-h period of outpatientmonitoring In all patients seizures were frequent andmedically intractable during infancy and early childhoodClusters of brief partial seizures secondary seizure general-ization and status epilepticus were common causes ofhospitalization Two young children died of seizure-relatedcomplicationsAll patients had severe psychomotor retardation

Neurodevelopmental outcomes were uniformly poorProjected adult mental ages were between 6 and12 months (Table 1) Most of the affected individualswere confined to wheelchairs Four patients learned to walkwith assistive devices but subsequently regressed Allsubjects were mute and fully dependent on others forfeeding and self-careFour patients (25) had atrial septal defects and one

of these children developed congestive heart failure at3 months of age The atrial defects could not be classified(ie secundum primum or sinus venosus) based onultrasound reports Diabetes insipidus was present in twopatients that were formally tested and two additional patientshad a clinical history suggestive of an osmoregulatory defectDiabetes insipidus appeared to be of both central andnephrogenic origin One 5-year-old subject had chronicpolydipsia and polyuria and presented with recurrentepisodes of hypernatremic dehydration During one suchepisode (serum sodium 151mEqml serum osmolarity327mOsml) plasma argininendashvasopressin was inappropri-ately low (91 pgml) but urine was dilute (557 mOsml)relative to the argininendashvasopressin level This patient andone other had bilateral nephrocalcinosis despite essentiallynormal urinary calcium levels (calciumcreatinine ratio019ndash047mgmg upper limit of normal for age 042)

Among 16 children with PMSE syndrome six (38) diedbetween ages 7 months and 6 years Causes of death werestatus epilepticus (nfrac14 2) hypovolemic shock (secondary todiabetes insipidus) and leucaemia Two young patients diedsuddenly and unexpectedly of undetermined causes

Gene mappingAll seven children used for autozygosity mapping were OldOrder Mennonite four were from Lancaster CountyPennsylvania two were from New York and one wasfrom Iowa They shared the same clinical phenotype andwere related to one another albeit distantly (Fig 4A)Assuming mutation homogeneity we searched SNP geno-type profiles for homozygous regions shared among affectedindividuals Surprisingly this analysis did not show anylarge homozygous blocks that were common to all sevenpatients (Fig 4B) Analysis of the data with the GeneSpringGT software package (Agilent Technologies) yielded similarnegative results

In an effort to understand the inconclusive mappingresults we tested several alternative hypotheses for the

Fig 3 MRI findings associated with homozygous LYK5 deletionsCoronal and axial fluid-attenuated inversion recovery images of a4-year-old child with PMSE syndrome show subependymal dyspla-sia (black arrows) and large ventricles with irregular contours(asterisk) (left and middle panels) Mild ventriculomegaly in this childcannot account for the large occipitofrontal head circumference(44 standard deviations above normal for age) suggesting thatmegalencephaly was the primary cause An axial apparent diffusioncoefficient map shows areas of high diffusion (white arrows) withinsubcortical white matter corresponding to areas of vacuolizationand gliosis seen microscopically (right panel) (see eg Fig 7)

Fig 4 Autozygosity mapping of syndromic cortical dysplasia usingDNA samples from seven Old Order Mennonite patients with asimilar distinctive phenotype (A) Genealogical analyses confirmedthat all patients shared distant common ancestry although nopatients were closely related to one another Patients were widelydistributed geographically living in settlements in PennsylvaniaNewYork and Iowa (B) Autozygosity mapping using Affymetrix10KGeneChip Mapping Arrays failed to unequivocally localize thedisease geneThe SNP genotype data is aligned in physical order bychromosome on the X-axis The graphs depict location scoresassociated with homozygous regions identified in all patients Thetop graph (purple) displays the location scores for PMSE syndromeNo regions of the autosomal genome provided strong evidence ofsharing The lower graph (red) depicts a successful mapping studyusing seven Mennonite patients affected with posterior columnataxia and retinitis pigmentosa (AXPC1)The gene for this disorderwas previously mapped to chromosome 1 (Higgins et al 1999)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1933

apparent lack of autozygosity including mutation hetero-geneity locus heterogeneity and digenic inheritance All ofthese tests were uninformative We then concentrated onchromosomal regions with insufficient SNP coverageWhile examining one such region on chromosome 17 wenoticed a single SNP rs721575 which produced lsquono callsrsquofor all seven patients (Fig 5A) This SNP had a prior callrate greater than 985 (Nfrac14 144 samples) making theresults for this marker highly suspicious Indeed we foundthat all 14 parents of affected children were lsquohomozygousrsquofor their respective rs721575 allele This suggested that themarker was deleted in all patients and hemizygous inparentsTo further test this conclusion we performed two

separate analyses of the genotype data using the CNAT

module of the Affymetrix software The first analysisplotted log2 ratio data generated by the copy numbertool (Fig 5B) whereas the second calculated average copynumber z-scores for each SNP from the seven affectedpatients In the seven patients the average z-score forrs721575 was 416 only one other SNP (Nfrac14 10 033) felloutside the conservative cutoff range of 3 to 3 Resultsfrom both analyses were consistent with homozygousdeletion of rs721575 in all affected children (Fig 5)

We then proceeded to delineate the deletion boundariesThe Affymetrix protocol relies on genomic DNA digestionwith the restriction endonuclease XbaI followed byamplification of restriction fragments in the 250ndash1000 bpsize range Amplified patient DNA is then labelled andhybridized to GeneChip arrays Thus the failure of

Fig 5 Evidence for homozygous deletions of SNP rs721575 in children with PMSE syndrome (A) Genotype data are shown for sevenaffected individuals and their parents All seven patients produced lsquono callsrsquo for SNP rs721575 on chromosome 17 while all 14 parentsappeared homozygous for their respective allele This suggested homozygous deletion of rs721575 in patients and hemizygosity of parents(B) A plot of log2 ratios exported from the copy number tool of the Genotyper software (Affymetrix) shows a log2 ratio for SNP rs721575of less than 8 (arrow) consistent with homozygous deletion in patients Copy number z-scores produced similar results (rs721575 z-scorefrac14416)

1934 Brain (2007) 130 1929^1941 EG Puffenberger et al

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 2: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

such cases (Shevell et al 2003 Srour et al 2006) we findthat abnormal imaging results rarely imply a specificdiagnosisIn the present work we describe the focused application

of DNA microarrays to investigate a small cluster ofMennonite patients with syndromic epilepsy and develop-mental delay In 1998 we evaluated a Mennonite boy bornwith a large head and distinctive facial features He beganto have intractable multifocal seizures at 4 months of ageand his subsequent developmental progress was stagnant Abrain MRI showed ventriculomegaly focal subependymaldysplasia and multiple areas of high diffusion withinsubcortical white matter (Fig 2) There was no specificgenetic diagnosis after 6 years of detailed investigationsIn autumn of 2004 the boyrsquos mother identified several

other Mennonite children with similar physical featuresearly onset focal seizures and severe psychomotor retarda-tion We collected DNA from seven individuals with acommon phenotype and using a 10 000 single nucleotidepolymorphism (SNP) mapping strategy (Puffenberger et al2004 Strauss et al 2005 2006) found a homozygousdeletion of a single SNP in all affected individuals Thedeleted SNP marked a large deletion of LYK5 whichencodes the STE20-related adaptor protein (STRAD)important for normal in vitro localization and catalyticactivity of serinethreonine kinase 11 (STK11 aka LKB1)(Baas et al 2003 Boudeau et al 2003) This is the firstdescription of a human disease associated with a mutationin LYK5 The neurological phenotype implies a specific rolefor STRAD in human brain growth and development and amore general role for the mTOR signalling pathway incertain cortical malformation syndromes (Baybis et al2004 Shaw et al 2004 Kwiatkowski and Manning 2005)

Patients and MethodsThis study was approved by the Institutional Review Board ofLancaster General Hospital Parents consented in writing tomolecular testing and reproduction of photographs Seven affectedindividuals and their 14 parents were used for SNP genome-wideautozygosity mapping (Strauss et al 2005) After mapping thedisease gene to chromosome 17 nine additional Mennonitechildren with suggestive clinical features were tested for the LYK5deletion by direct molecular genetic analysis Phenotype informa-tion was based on clinical data from 16 patients ages 7 months to28 years

GenotypingTotal genomic DNA from whole blood was isolated using thePUREGENE DNA Isolation Kit (Gentra Systems MinneapolisMN USA) according to the manufacturerrsquos protocol SNPgenotyping was done using the GeneChip Mapping 10K AssayKit (Affymetrix Santa Clara CA USA) as previously described(Puffenberger et al 2004) Briefly 250 ng of double-strandedgenomic DNA was digested with XbaI the restriction fragmentswere ligated to adapters and the ligated products were amplifiedin quadruplicate using a generic primer PCR products werepurified fragmented with DNase labelled with terminal

deoxytransferase and finally hybridized to a GeneChip HumanMapping 10K Xba142 20 Array Microarrays were then washedwith a fluidics station incubated and scanned using the GeneChipScanner 3000 enabled by GeneChip GCOS software (Affymetrix)

Genome-wide autozygosity mappingData were analysed in Microsoft Excel spreadsheets (MicrosoftCorporation Redmond WA USA) that were custom-formatted atthe Clinic for Special Children SNP positions came fromAffymetrix genome annotation files and genotype data camefrom the Affymetrix GeneChip Human Mapping 10K Xba 142 20Arrays Excel spreadsheets were designed to identify genomicregions that were identical-by-descent between all affectedindividuals This analysis assumes mutation and locus homo-geneity Two-point LOD scores were calculated for each genotypedSNP using an approach similar to Broman and Weber (1999a b)Cumulative two-point LOD scores for a block of homozygousSNPs were considered the location score for a region providing arelative measure of likelihood that the region harbours the diseasegene Genotype data from this study and others (Puffenbergeret al 2004 Strauss et al 2005 2006) were used to estimatepopulation-specific SNP allele frequencies

DNA sequencingThe target sequence was amplified by using specific oligonucleo-tide primers and 30ndash50 ng of genomic DNA from affected andunaffected family members Primer sequences for PCR amplifica-tion and sequencing were designed using Primer3 (httpfrodowimiteducgi-binprimer3primer3_wwwcgi) PCR pro-ducts were purified using QiaQuick columns (Qiagen ValenciaCA USA) as per manufacturerrsquos instructions and then sequencedusing the BigDye Terminator cycle sequencing protocol (AppliedBiosystems Foster City CA USA) Extension products weresubsequently size-fractionated on an ABI PRISM 310 GeneticAnalyzer (Applied Biosystems) and data were compared to thehuman genome reference sequence and dbSNP in order to identifysequence variants Population-based control samples were studiedin an identical fashion

Genome-wide copy number analysisThe SNP copy number generated by the copy number analysistool (CNAT) of the Affymetrix Genotyping software wasstandardized for analyses For each SNP on the array we firstgenerated a mean copy number and standard deviation from areference population of 100 healthy (control) individuals from thePlain community Copy number data for each patient were thenexported to Excel files and SNP copy number z-scores (standardscores) were calculated according to the formula zfrac14 (patientvalue control mean)control standard deviation The resultingsingle-point copy number z-scores and log2 ratios were plottedusing chart and trendline functions in Excel Within a normalpopulation 998 of z-scores fall between 3 and thorn3 scoresoutside of these limits were investigated in more detail

Histology and immunohistochemistryAn autopsy was performed on one female patient who died of anundetermined cause at 7 months of age Paraffin-embeddedsections from liver kidney heart lung and brain were stainedwith haematoxylinndasheosin Additional brain specimens were stained

1930 Brain (2007) 130 1929^1941 EG Puffenberger et al

with luxol fast blue-cresyl violet (LFB-CV) and routine immuno-peroxidase methods using antibodies against glial fibrillary acidicprotein (GFAP) Sections of frontal cortex striatum hippocampusand spinal cord were probed with anti-phospho-ribosomal S6(P-S6) antibodies (Ser235236 1 50 dilution Cell Signaling NewEngland Biolabs Beverly MA USA) (Baybis et al 2004) whichwere visualized with biotinylated anti-rabbit IgG (1 1000dilution Vector Laboratories Burlingame CA USA) afteravidinndashbiotin conjugation (Vectastain ABC Elite VectorLaboratories) and treatment with 330-diaminobenzadine

ResultsClinical phenotypeClinical data from 16 affected individuals (ages 7 months to28 years) are summarized in Table 1 All affectedpregnancies were complicated by polyhydramniosSpontaneous onset of labour occurred between 25 and36 weeks gestation in 12 pregnancies (75) All but oneaffected child had macrocephaly (head circumference 2SD for age) (Fig 1) Linear growth was normal and bodymass index was consistently low (13 kgm2) due to lowmuscle mass Several young children had persistent restlesslimb movements a sign we interpreted as mild chorea Thiswas not a finding in older patients

Each affected child had a long face large foreheadpeaked eyebrows broad nasal bridge hypertelorism and alarge mouth with thick lips Craniofacial structure changedconsiderably with age (Fig 2A) During infancy and earlychildhood the skull was shaped like an inverted pyramidreflecting prominence of the cranial vault relative to thelower face As children grew older their appearancechanged due to overgrowth of the mandible enlargementof the mouth and thickening of the lips (Fig 2B)Radiological skeletal surveys available for only two infantswere normal

Cranial magnetic resonance imaging (MRI) studies at 15Tesla were available from only four patients ages 4 monthsto 5 years These images were acquired 5 to 10 years agoand thus the quality was poor relative to current imagingstandards The MRI from the two youngest children whosehead circumferences were greater than four standarddeviations above normal showed only mild ventriculome-galy that could not fully account for increased head sizeStudies from the two older children showed ventriculome-galy evidence of subependymal dysplasia and multiple areasof high water diffusion within the subcortical white matter(Fig 3)

Seizures started between 3 and 7 months of age in allpatients These were most commonly complex partialseizures that would occasionally spread to involve one orboth cerebral hemispheres Electroencephalographic record-ings typically showed high-voltage spike and slow wavedischarges arising from bilateral independent electroence-phalographic foci over the frontal temporal parietal andoccipital regions These spike-slow wave discharges wouldsometimes change into a long run of spikes or into arhythmic slow wave focus (25 to 3 cycles per second)

Table 1 Clinical features of 16 patients with homozygousLYK5 deletions

Clinical variable Percent of patients affected(nfrac1416)

PrenatalPolyhydramnios 100Preterm laboura 75

PostnatalMacrocephalyb 100Infantile-onset partial epilepsy 100Hypotonia 100Craniofacial dysmorphism 100Skeletal muscle hypoplasia 100Strabismus 56Atrial septal defect 25Nephrocalcinosisc 13Diabetes insipidusd 13Supraventricular tachycardia 6Leucaemia 6

Developmental domaine Maximum developmentalage (range in months)

Gross motor 6^14Fine motor-adaptive 4^10Language^ communication 2^6Personal^ social 2^6

aMean onset of labour at 31weeks gestation range 25^37 weeksbDue to a combination of megalencephaly and hydrocephaluscRenal ultrasounds were only performed in four patients two ofwhom (imaged at ages 6 and 28 years) had nephrocalcinosisdOne patient with DI had bilateral nephrocalcinosis on CTscanThe cause of DI was mixed with both central and nephrogeniccomponentseDevelopmental outcomes based on Denver DevelopmentalScreeningTest II

Fig 1 Occipitofrontal head circumference (in cm) of 16 childrenwith PMSE syndrome Males are depicted as circles females asinverted triangles Although mild enlargement of the ventricularsystem was seen on the four available neuroimaging studies weattributed increased head circumference primarily tomegalencephaly

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1931

Fig 2 Dysmorphic features (A) Young persons with homozygous LYK5 deletions had a long face large forehead peaked eyebrows broadnose and wide-set eyes As patients got older (from upper left to lower right) the mouth became enlarged and the lips thickened (B)Individual craniofacial structure changed considerably with age Serial pictures of one affected individual show macrocephaly frontal bos-sing hypertelorism and broad nasal bridge shortly after birth (left panel) lengthening of the face during early childhood (middle panel) andovergrowth of the mandible enlargement of the mouth and thickening of the lips during adolescence (right panel)

1932 Brain (2007) 130 1929^1941 EG Puffenberger et al

lasting 10 to 15min Two young patients were diagnosedwith infantile spasms based on clinical and electroencepha-lographic criteria One child had right-sided epilespiapartialis continua during a 24-h period of outpatientmonitoring In all patients seizures were frequent andmedically intractable during infancy and early childhoodClusters of brief partial seizures secondary seizure general-ization and status epilepticus were common causes ofhospitalization Two young children died of seizure-relatedcomplicationsAll patients had severe psychomotor retardation

Neurodevelopmental outcomes were uniformly poorProjected adult mental ages were between 6 and12 months (Table 1) Most of the affected individualswere confined to wheelchairs Four patients learned to walkwith assistive devices but subsequently regressed Allsubjects were mute and fully dependent on others forfeeding and self-careFour patients (25) had atrial septal defects and one

of these children developed congestive heart failure at3 months of age The atrial defects could not be classified(ie secundum primum or sinus venosus) based onultrasound reports Diabetes insipidus was present in twopatients that were formally tested and two additional patientshad a clinical history suggestive of an osmoregulatory defectDiabetes insipidus appeared to be of both central andnephrogenic origin One 5-year-old subject had chronicpolydipsia and polyuria and presented with recurrentepisodes of hypernatremic dehydration During one suchepisode (serum sodium 151mEqml serum osmolarity327mOsml) plasma argininendashvasopressin was inappropri-ately low (91 pgml) but urine was dilute (557 mOsml)relative to the argininendashvasopressin level This patient andone other had bilateral nephrocalcinosis despite essentiallynormal urinary calcium levels (calciumcreatinine ratio019ndash047mgmg upper limit of normal for age 042)

Among 16 children with PMSE syndrome six (38) diedbetween ages 7 months and 6 years Causes of death werestatus epilepticus (nfrac14 2) hypovolemic shock (secondary todiabetes insipidus) and leucaemia Two young patients diedsuddenly and unexpectedly of undetermined causes

Gene mappingAll seven children used for autozygosity mapping were OldOrder Mennonite four were from Lancaster CountyPennsylvania two were from New York and one wasfrom Iowa They shared the same clinical phenotype andwere related to one another albeit distantly (Fig 4A)Assuming mutation homogeneity we searched SNP geno-type profiles for homozygous regions shared among affectedindividuals Surprisingly this analysis did not show anylarge homozygous blocks that were common to all sevenpatients (Fig 4B) Analysis of the data with the GeneSpringGT software package (Agilent Technologies) yielded similarnegative results

In an effort to understand the inconclusive mappingresults we tested several alternative hypotheses for the

Fig 3 MRI findings associated with homozygous LYK5 deletionsCoronal and axial fluid-attenuated inversion recovery images of a4-year-old child with PMSE syndrome show subependymal dyspla-sia (black arrows) and large ventricles with irregular contours(asterisk) (left and middle panels) Mild ventriculomegaly in this childcannot account for the large occipitofrontal head circumference(44 standard deviations above normal for age) suggesting thatmegalencephaly was the primary cause An axial apparent diffusioncoefficient map shows areas of high diffusion (white arrows) withinsubcortical white matter corresponding to areas of vacuolizationand gliosis seen microscopically (right panel) (see eg Fig 7)

Fig 4 Autozygosity mapping of syndromic cortical dysplasia usingDNA samples from seven Old Order Mennonite patients with asimilar distinctive phenotype (A) Genealogical analyses confirmedthat all patients shared distant common ancestry although nopatients were closely related to one another Patients were widelydistributed geographically living in settlements in PennsylvaniaNewYork and Iowa (B) Autozygosity mapping using Affymetrix10KGeneChip Mapping Arrays failed to unequivocally localize thedisease geneThe SNP genotype data is aligned in physical order bychromosome on the X-axis The graphs depict location scoresassociated with homozygous regions identified in all patients Thetop graph (purple) displays the location scores for PMSE syndromeNo regions of the autosomal genome provided strong evidence ofsharing The lower graph (red) depicts a successful mapping studyusing seven Mennonite patients affected with posterior columnataxia and retinitis pigmentosa (AXPC1)The gene for this disorderwas previously mapped to chromosome 1 (Higgins et al 1999)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1933

apparent lack of autozygosity including mutation hetero-geneity locus heterogeneity and digenic inheritance All ofthese tests were uninformative We then concentrated onchromosomal regions with insufficient SNP coverageWhile examining one such region on chromosome 17 wenoticed a single SNP rs721575 which produced lsquono callsrsquofor all seven patients (Fig 5A) This SNP had a prior callrate greater than 985 (Nfrac14 144 samples) making theresults for this marker highly suspicious Indeed we foundthat all 14 parents of affected children were lsquohomozygousrsquofor their respective rs721575 allele This suggested that themarker was deleted in all patients and hemizygous inparentsTo further test this conclusion we performed two

separate analyses of the genotype data using the CNAT

module of the Affymetrix software The first analysisplotted log2 ratio data generated by the copy numbertool (Fig 5B) whereas the second calculated average copynumber z-scores for each SNP from the seven affectedpatients In the seven patients the average z-score forrs721575 was 416 only one other SNP (Nfrac14 10 033) felloutside the conservative cutoff range of 3 to 3 Resultsfrom both analyses were consistent with homozygousdeletion of rs721575 in all affected children (Fig 5)

We then proceeded to delineate the deletion boundariesThe Affymetrix protocol relies on genomic DNA digestionwith the restriction endonuclease XbaI followed byamplification of restriction fragments in the 250ndash1000 bpsize range Amplified patient DNA is then labelled andhybridized to GeneChip arrays Thus the failure of

Fig 5 Evidence for homozygous deletions of SNP rs721575 in children with PMSE syndrome (A) Genotype data are shown for sevenaffected individuals and their parents All seven patients produced lsquono callsrsquo for SNP rs721575 on chromosome 17 while all 14 parentsappeared homozygous for their respective allele This suggested homozygous deletion of rs721575 in patients and hemizygosity of parents(B) A plot of log2 ratios exported from the copy number tool of the Genotyper software (Affymetrix) shows a log2 ratio for SNP rs721575of less than 8 (arrow) consistent with homozygous deletion in patients Copy number z-scores produced similar results (rs721575 z-scorefrac14416)

1934 Brain (2007) 130 1929^1941 EG Puffenberger et al

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 3: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

with luxol fast blue-cresyl violet (LFB-CV) and routine immuno-peroxidase methods using antibodies against glial fibrillary acidicprotein (GFAP) Sections of frontal cortex striatum hippocampusand spinal cord were probed with anti-phospho-ribosomal S6(P-S6) antibodies (Ser235236 1 50 dilution Cell Signaling NewEngland Biolabs Beverly MA USA) (Baybis et al 2004) whichwere visualized with biotinylated anti-rabbit IgG (1 1000dilution Vector Laboratories Burlingame CA USA) afteravidinndashbiotin conjugation (Vectastain ABC Elite VectorLaboratories) and treatment with 330-diaminobenzadine

ResultsClinical phenotypeClinical data from 16 affected individuals (ages 7 months to28 years) are summarized in Table 1 All affectedpregnancies were complicated by polyhydramniosSpontaneous onset of labour occurred between 25 and36 weeks gestation in 12 pregnancies (75) All but oneaffected child had macrocephaly (head circumference 2SD for age) (Fig 1) Linear growth was normal and bodymass index was consistently low (13 kgm2) due to lowmuscle mass Several young children had persistent restlesslimb movements a sign we interpreted as mild chorea Thiswas not a finding in older patients

Each affected child had a long face large foreheadpeaked eyebrows broad nasal bridge hypertelorism and alarge mouth with thick lips Craniofacial structure changedconsiderably with age (Fig 2A) During infancy and earlychildhood the skull was shaped like an inverted pyramidreflecting prominence of the cranial vault relative to thelower face As children grew older their appearancechanged due to overgrowth of the mandible enlargementof the mouth and thickening of the lips (Fig 2B)Radiological skeletal surveys available for only two infantswere normal

Cranial magnetic resonance imaging (MRI) studies at 15Tesla were available from only four patients ages 4 monthsto 5 years These images were acquired 5 to 10 years agoand thus the quality was poor relative to current imagingstandards The MRI from the two youngest children whosehead circumferences were greater than four standarddeviations above normal showed only mild ventriculome-galy that could not fully account for increased head sizeStudies from the two older children showed ventriculome-galy evidence of subependymal dysplasia and multiple areasof high water diffusion within the subcortical white matter(Fig 3)

Seizures started between 3 and 7 months of age in allpatients These were most commonly complex partialseizures that would occasionally spread to involve one orboth cerebral hemispheres Electroencephalographic record-ings typically showed high-voltage spike and slow wavedischarges arising from bilateral independent electroence-phalographic foci over the frontal temporal parietal andoccipital regions These spike-slow wave discharges wouldsometimes change into a long run of spikes or into arhythmic slow wave focus (25 to 3 cycles per second)

Table 1 Clinical features of 16 patients with homozygousLYK5 deletions

Clinical variable Percent of patients affected(nfrac1416)

PrenatalPolyhydramnios 100Preterm laboura 75

PostnatalMacrocephalyb 100Infantile-onset partial epilepsy 100Hypotonia 100Craniofacial dysmorphism 100Skeletal muscle hypoplasia 100Strabismus 56Atrial septal defect 25Nephrocalcinosisc 13Diabetes insipidusd 13Supraventricular tachycardia 6Leucaemia 6

Developmental domaine Maximum developmentalage (range in months)

Gross motor 6^14Fine motor-adaptive 4^10Language^ communication 2^6Personal^ social 2^6

aMean onset of labour at 31weeks gestation range 25^37 weeksbDue to a combination of megalencephaly and hydrocephaluscRenal ultrasounds were only performed in four patients two ofwhom (imaged at ages 6 and 28 years) had nephrocalcinosisdOne patient with DI had bilateral nephrocalcinosis on CTscanThe cause of DI was mixed with both central and nephrogeniccomponentseDevelopmental outcomes based on Denver DevelopmentalScreeningTest II

Fig 1 Occipitofrontal head circumference (in cm) of 16 childrenwith PMSE syndrome Males are depicted as circles females asinverted triangles Although mild enlargement of the ventricularsystem was seen on the four available neuroimaging studies weattributed increased head circumference primarily tomegalencephaly

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1931

Fig 2 Dysmorphic features (A) Young persons with homozygous LYK5 deletions had a long face large forehead peaked eyebrows broadnose and wide-set eyes As patients got older (from upper left to lower right) the mouth became enlarged and the lips thickened (B)Individual craniofacial structure changed considerably with age Serial pictures of one affected individual show macrocephaly frontal bos-sing hypertelorism and broad nasal bridge shortly after birth (left panel) lengthening of the face during early childhood (middle panel) andovergrowth of the mandible enlargement of the mouth and thickening of the lips during adolescence (right panel)

1932 Brain (2007) 130 1929^1941 EG Puffenberger et al

lasting 10 to 15min Two young patients were diagnosedwith infantile spasms based on clinical and electroencepha-lographic criteria One child had right-sided epilespiapartialis continua during a 24-h period of outpatientmonitoring In all patients seizures were frequent andmedically intractable during infancy and early childhoodClusters of brief partial seizures secondary seizure general-ization and status epilepticus were common causes ofhospitalization Two young children died of seizure-relatedcomplicationsAll patients had severe psychomotor retardation

Neurodevelopmental outcomes were uniformly poorProjected adult mental ages were between 6 and12 months (Table 1) Most of the affected individualswere confined to wheelchairs Four patients learned to walkwith assistive devices but subsequently regressed Allsubjects were mute and fully dependent on others forfeeding and self-careFour patients (25) had atrial septal defects and one

of these children developed congestive heart failure at3 months of age The atrial defects could not be classified(ie secundum primum or sinus venosus) based onultrasound reports Diabetes insipidus was present in twopatients that were formally tested and two additional patientshad a clinical history suggestive of an osmoregulatory defectDiabetes insipidus appeared to be of both central andnephrogenic origin One 5-year-old subject had chronicpolydipsia and polyuria and presented with recurrentepisodes of hypernatremic dehydration During one suchepisode (serum sodium 151mEqml serum osmolarity327mOsml) plasma argininendashvasopressin was inappropri-ately low (91 pgml) but urine was dilute (557 mOsml)relative to the argininendashvasopressin level This patient andone other had bilateral nephrocalcinosis despite essentiallynormal urinary calcium levels (calciumcreatinine ratio019ndash047mgmg upper limit of normal for age 042)

Among 16 children with PMSE syndrome six (38) diedbetween ages 7 months and 6 years Causes of death werestatus epilepticus (nfrac14 2) hypovolemic shock (secondary todiabetes insipidus) and leucaemia Two young patients diedsuddenly and unexpectedly of undetermined causes

Gene mappingAll seven children used for autozygosity mapping were OldOrder Mennonite four were from Lancaster CountyPennsylvania two were from New York and one wasfrom Iowa They shared the same clinical phenotype andwere related to one another albeit distantly (Fig 4A)Assuming mutation homogeneity we searched SNP geno-type profiles for homozygous regions shared among affectedindividuals Surprisingly this analysis did not show anylarge homozygous blocks that were common to all sevenpatients (Fig 4B) Analysis of the data with the GeneSpringGT software package (Agilent Technologies) yielded similarnegative results

In an effort to understand the inconclusive mappingresults we tested several alternative hypotheses for the

Fig 3 MRI findings associated with homozygous LYK5 deletionsCoronal and axial fluid-attenuated inversion recovery images of a4-year-old child with PMSE syndrome show subependymal dyspla-sia (black arrows) and large ventricles with irregular contours(asterisk) (left and middle panels) Mild ventriculomegaly in this childcannot account for the large occipitofrontal head circumference(44 standard deviations above normal for age) suggesting thatmegalencephaly was the primary cause An axial apparent diffusioncoefficient map shows areas of high diffusion (white arrows) withinsubcortical white matter corresponding to areas of vacuolizationand gliosis seen microscopically (right panel) (see eg Fig 7)

Fig 4 Autozygosity mapping of syndromic cortical dysplasia usingDNA samples from seven Old Order Mennonite patients with asimilar distinctive phenotype (A) Genealogical analyses confirmedthat all patients shared distant common ancestry although nopatients were closely related to one another Patients were widelydistributed geographically living in settlements in PennsylvaniaNewYork and Iowa (B) Autozygosity mapping using Affymetrix10KGeneChip Mapping Arrays failed to unequivocally localize thedisease geneThe SNP genotype data is aligned in physical order bychromosome on the X-axis The graphs depict location scoresassociated with homozygous regions identified in all patients Thetop graph (purple) displays the location scores for PMSE syndromeNo regions of the autosomal genome provided strong evidence ofsharing The lower graph (red) depicts a successful mapping studyusing seven Mennonite patients affected with posterior columnataxia and retinitis pigmentosa (AXPC1)The gene for this disorderwas previously mapped to chromosome 1 (Higgins et al 1999)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1933

apparent lack of autozygosity including mutation hetero-geneity locus heterogeneity and digenic inheritance All ofthese tests were uninformative We then concentrated onchromosomal regions with insufficient SNP coverageWhile examining one such region on chromosome 17 wenoticed a single SNP rs721575 which produced lsquono callsrsquofor all seven patients (Fig 5A) This SNP had a prior callrate greater than 985 (Nfrac14 144 samples) making theresults for this marker highly suspicious Indeed we foundthat all 14 parents of affected children were lsquohomozygousrsquofor their respective rs721575 allele This suggested that themarker was deleted in all patients and hemizygous inparentsTo further test this conclusion we performed two

separate analyses of the genotype data using the CNAT

module of the Affymetrix software The first analysisplotted log2 ratio data generated by the copy numbertool (Fig 5B) whereas the second calculated average copynumber z-scores for each SNP from the seven affectedpatients In the seven patients the average z-score forrs721575 was 416 only one other SNP (Nfrac14 10 033) felloutside the conservative cutoff range of 3 to 3 Resultsfrom both analyses were consistent with homozygousdeletion of rs721575 in all affected children (Fig 5)

We then proceeded to delineate the deletion boundariesThe Affymetrix protocol relies on genomic DNA digestionwith the restriction endonuclease XbaI followed byamplification of restriction fragments in the 250ndash1000 bpsize range Amplified patient DNA is then labelled andhybridized to GeneChip arrays Thus the failure of

Fig 5 Evidence for homozygous deletions of SNP rs721575 in children with PMSE syndrome (A) Genotype data are shown for sevenaffected individuals and their parents All seven patients produced lsquono callsrsquo for SNP rs721575 on chromosome 17 while all 14 parentsappeared homozygous for their respective allele This suggested homozygous deletion of rs721575 in patients and hemizygosity of parents(B) A plot of log2 ratios exported from the copy number tool of the Genotyper software (Affymetrix) shows a log2 ratio for SNP rs721575of less than 8 (arrow) consistent with homozygous deletion in patients Copy number z-scores produced similar results (rs721575 z-scorefrac14416)

1934 Brain (2007) 130 1929^1941 EG Puffenberger et al

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 4: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

Fig 2 Dysmorphic features (A) Young persons with homozygous LYK5 deletions had a long face large forehead peaked eyebrows broadnose and wide-set eyes As patients got older (from upper left to lower right) the mouth became enlarged and the lips thickened (B)Individual craniofacial structure changed considerably with age Serial pictures of one affected individual show macrocephaly frontal bos-sing hypertelorism and broad nasal bridge shortly after birth (left panel) lengthening of the face during early childhood (middle panel) andovergrowth of the mandible enlargement of the mouth and thickening of the lips during adolescence (right panel)

1932 Brain (2007) 130 1929^1941 EG Puffenberger et al

lasting 10 to 15min Two young patients were diagnosedwith infantile spasms based on clinical and electroencepha-lographic criteria One child had right-sided epilespiapartialis continua during a 24-h period of outpatientmonitoring In all patients seizures were frequent andmedically intractable during infancy and early childhoodClusters of brief partial seizures secondary seizure general-ization and status epilepticus were common causes ofhospitalization Two young children died of seizure-relatedcomplicationsAll patients had severe psychomotor retardation

Neurodevelopmental outcomes were uniformly poorProjected adult mental ages were between 6 and12 months (Table 1) Most of the affected individualswere confined to wheelchairs Four patients learned to walkwith assistive devices but subsequently regressed Allsubjects were mute and fully dependent on others forfeeding and self-careFour patients (25) had atrial septal defects and one

of these children developed congestive heart failure at3 months of age The atrial defects could not be classified(ie secundum primum or sinus venosus) based onultrasound reports Diabetes insipidus was present in twopatients that were formally tested and two additional patientshad a clinical history suggestive of an osmoregulatory defectDiabetes insipidus appeared to be of both central andnephrogenic origin One 5-year-old subject had chronicpolydipsia and polyuria and presented with recurrentepisodes of hypernatremic dehydration During one suchepisode (serum sodium 151mEqml serum osmolarity327mOsml) plasma argininendashvasopressin was inappropri-ately low (91 pgml) but urine was dilute (557 mOsml)relative to the argininendashvasopressin level This patient andone other had bilateral nephrocalcinosis despite essentiallynormal urinary calcium levels (calciumcreatinine ratio019ndash047mgmg upper limit of normal for age 042)

Among 16 children with PMSE syndrome six (38) diedbetween ages 7 months and 6 years Causes of death werestatus epilepticus (nfrac14 2) hypovolemic shock (secondary todiabetes insipidus) and leucaemia Two young patients diedsuddenly and unexpectedly of undetermined causes

Gene mappingAll seven children used for autozygosity mapping were OldOrder Mennonite four were from Lancaster CountyPennsylvania two were from New York and one wasfrom Iowa They shared the same clinical phenotype andwere related to one another albeit distantly (Fig 4A)Assuming mutation homogeneity we searched SNP geno-type profiles for homozygous regions shared among affectedindividuals Surprisingly this analysis did not show anylarge homozygous blocks that were common to all sevenpatients (Fig 4B) Analysis of the data with the GeneSpringGT software package (Agilent Technologies) yielded similarnegative results

In an effort to understand the inconclusive mappingresults we tested several alternative hypotheses for the

Fig 3 MRI findings associated with homozygous LYK5 deletionsCoronal and axial fluid-attenuated inversion recovery images of a4-year-old child with PMSE syndrome show subependymal dyspla-sia (black arrows) and large ventricles with irregular contours(asterisk) (left and middle panels) Mild ventriculomegaly in this childcannot account for the large occipitofrontal head circumference(44 standard deviations above normal for age) suggesting thatmegalencephaly was the primary cause An axial apparent diffusioncoefficient map shows areas of high diffusion (white arrows) withinsubcortical white matter corresponding to areas of vacuolizationand gliosis seen microscopically (right panel) (see eg Fig 7)

Fig 4 Autozygosity mapping of syndromic cortical dysplasia usingDNA samples from seven Old Order Mennonite patients with asimilar distinctive phenotype (A) Genealogical analyses confirmedthat all patients shared distant common ancestry although nopatients were closely related to one another Patients were widelydistributed geographically living in settlements in PennsylvaniaNewYork and Iowa (B) Autozygosity mapping using Affymetrix10KGeneChip Mapping Arrays failed to unequivocally localize thedisease geneThe SNP genotype data is aligned in physical order bychromosome on the X-axis The graphs depict location scoresassociated with homozygous regions identified in all patients Thetop graph (purple) displays the location scores for PMSE syndromeNo regions of the autosomal genome provided strong evidence ofsharing The lower graph (red) depicts a successful mapping studyusing seven Mennonite patients affected with posterior columnataxia and retinitis pigmentosa (AXPC1)The gene for this disorderwas previously mapped to chromosome 1 (Higgins et al 1999)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1933

apparent lack of autozygosity including mutation hetero-geneity locus heterogeneity and digenic inheritance All ofthese tests were uninformative We then concentrated onchromosomal regions with insufficient SNP coverageWhile examining one such region on chromosome 17 wenoticed a single SNP rs721575 which produced lsquono callsrsquofor all seven patients (Fig 5A) This SNP had a prior callrate greater than 985 (Nfrac14 144 samples) making theresults for this marker highly suspicious Indeed we foundthat all 14 parents of affected children were lsquohomozygousrsquofor their respective rs721575 allele This suggested that themarker was deleted in all patients and hemizygous inparentsTo further test this conclusion we performed two

separate analyses of the genotype data using the CNAT

module of the Affymetrix software The first analysisplotted log2 ratio data generated by the copy numbertool (Fig 5B) whereas the second calculated average copynumber z-scores for each SNP from the seven affectedpatients In the seven patients the average z-score forrs721575 was 416 only one other SNP (Nfrac14 10 033) felloutside the conservative cutoff range of 3 to 3 Resultsfrom both analyses were consistent with homozygousdeletion of rs721575 in all affected children (Fig 5)

We then proceeded to delineate the deletion boundariesThe Affymetrix protocol relies on genomic DNA digestionwith the restriction endonuclease XbaI followed byamplification of restriction fragments in the 250ndash1000 bpsize range Amplified patient DNA is then labelled andhybridized to GeneChip arrays Thus the failure of

Fig 5 Evidence for homozygous deletions of SNP rs721575 in children with PMSE syndrome (A) Genotype data are shown for sevenaffected individuals and their parents All seven patients produced lsquono callsrsquo for SNP rs721575 on chromosome 17 while all 14 parentsappeared homozygous for their respective allele This suggested homozygous deletion of rs721575 in patients and hemizygosity of parents(B) A plot of log2 ratios exported from the copy number tool of the Genotyper software (Affymetrix) shows a log2 ratio for SNP rs721575of less than 8 (arrow) consistent with homozygous deletion in patients Copy number z-scores produced similar results (rs721575 z-scorefrac14416)

1934 Brain (2007) 130 1929^1941 EG Puffenberger et al

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 5: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

lasting 10 to 15min Two young patients were diagnosedwith infantile spasms based on clinical and electroencepha-lographic criteria One child had right-sided epilespiapartialis continua during a 24-h period of outpatientmonitoring In all patients seizures were frequent andmedically intractable during infancy and early childhoodClusters of brief partial seizures secondary seizure general-ization and status epilepticus were common causes ofhospitalization Two young children died of seizure-relatedcomplicationsAll patients had severe psychomotor retardation

Neurodevelopmental outcomes were uniformly poorProjected adult mental ages were between 6 and12 months (Table 1) Most of the affected individualswere confined to wheelchairs Four patients learned to walkwith assistive devices but subsequently regressed Allsubjects were mute and fully dependent on others forfeeding and self-careFour patients (25) had atrial septal defects and one

of these children developed congestive heart failure at3 months of age The atrial defects could not be classified(ie secundum primum or sinus venosus) based onultrasound reports Diabetes insipidus was present in twopatients that were formally tested and two additional patientshad a clinical history suggestive of an osmoregulatory defectDiabetes insipidus appeared to be of both central andnephrogenic origin One 5-year-old subject had chronicpolydipsia and polyuria and presented with recurrentepisodes of hypernatremic dehydration During one suchepisode (serum sodium 151mEqml serum osmolarity327mOsml) plasma argininendashvasopressin was inappropri-ately low (91 pgml) but urine was dilute (557 mOsml)relative to the argininendashvasopressin level This patient andone other had bilateral nephrocalcinosis despite essentiallynormal urinary calcium levels (calciumcreatinine ratio019ndash047mgmg upper limit of normal for age 042)

Among 16 children with PMSE syndrome six (38) diedbetween ages 7 months and 6 years Causes of death werestatus epilepticus (nfrac14 2) hypovolemic shock (secondary todiabetes insipidus) and leucaemia Two young patients diedsuddenly and unexpectedly of undetermined causes

Gene mappingAll seven children used for autozygosity mapping were OldOrder Mennonite four were from Lancaster CountyPennsylvania two were from New York and one wasfrom Iowa They shared the same clinical phenotype andwere related to one another albeit distantly (Fig 4A)Assuming mutation homogeneity we searched SNP geno-type profiles for homozygous regions shared among affectedindividuals Surprisingly this analysis did not show anylarge homozygous blocks that were common to all sevenpatients (Fig 4B) Analysis of the data with the GeneSpringGT software package (Agilent Technologies) yielded similarnegative results

In an effort to understand the inconclusive mappingresults we tested several alternative hypotheses for the

Fig 3 MRI findings associated with homozygous LYK5 deletionsCoronal and axial fluid-attenuated inversion recovery images of a4-year-old child with PMSE syndrome show subependymal dyspla-sia (black arrows) and large ventricles with irregular contours(asterisk) (left and middle panels) Mild ventriculomegaly in this childcannot account for the large occipitofrontal head circumference(44 standard deviations above normal for age) suggesting thatmegalencephaly was the primary cause An axial apparent diffusioncoefficient map shows areas of high diffusion (white arrows) withinsubcortical white matter corresponding to areas of vacuolizationand gliosis seen microscopically (right panel) (see eg Fig 7)

Fig 4 Autozygosity mapping of syndromic cortical dysplasia usingDNA samples from seven Old Order Mennonite patients with asimilar distinctive phenotype (A) Genealogical analyses confirmedthat all patients shared distant common ancestry although nopatients were closely related to one another Patients were widelydistributed geographically living in settlements in PennsylvaniaNewYork and Iowa (B) Autozygosity mapping using Affymetrix10KGeneChip Mapping Arrays failed to unequivocally localize thedisease geneThe SNP genotype data is aligned in physical order bychromosome on the X-axis The graphs depict location scoresassociated with homozygous regions identified in all patients Thetop graph (purple) displays the location scores for PMSE syndromeNo regions of the autosomal genome provided strong evidence ofsharing The lower graph (red) depicts a successful mapping studyusing seven Mennonite patients affected with posterior columnataxia and retinitis pigmentosa (AXPC1)The gene for this disorderwas previously mapped to chromosome 1 (Higgins et al 1999)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1933

apparent lack of autozygosity including mutation hetero-geneity locus heterogeneity and digenic inheritance All ofthese tests were uninformative We then concentrated onchromosomal regions with insufficient SNP coverageWhile examining one such region on chromosome 17 wenoticed a single SNP rs721575 which produced lsquono callsrsquofor all seven patients (Fig 5A) This SNP had a prior callrate greater than 985 (Nfrac14 144 samples) making theresults for this marker highly suspicious Indeed we foundthat all 14 parents of affected children were lsquohomozygousrsquofor their respective rs721575 allele This suggested that themarker was deleted in all patients and hemizygous inparentsTo further test this conclusion we performed two

separate analyses of the genotype data using the CNAT

module of the Affymetrix software The first analysisplotted log2 ratio data generated by the copy numbertool (Fig 5B) whereas the second calculated average copynumber z-scores for each SNP from the seven affectedpatients In the seven patients the average z-score forrs721575 was 416 only one other SNP (Nfrac14 10 033) felloutside the conservative cutoff range of 3 to 3 Resultsfrom both analyses were consistent with homozygousdeletion of rs721575 in all affected children (Fig 5)

We then proceeded to delineate the deletion boundariesThe Affymetrix protocol relies on genomic DNA digestionwith the restriction endonuclease XbaI followed byamplification of restriction fragments in the 250ndash1000 bpsize range Amplified patient DNA is then labelled andhybridized to GeneChip arrays Thus the failure of

Fig 5 Evidence for homozygous deletions of SNP rs721575 in children with PMSE syndrome (A) Genotype data are shown for sevenaffected individuals and their parents All seven patients produced lsquono callsrsquo for SNP rs721575 on chromosome 17 while all 14 parentsappeared homozygous for their respective allele This suggested homozygous deletion of rs721575 in patients and hemizygosity of parents(B) A plot of log2 ratios exported from the copy number tool of the Genotyper software (Affymetrix) shows a log2 ratio for SNP rs721575of less than 8 (arrow) consistent with homozygous deletion in patients Copy number z-scores produced similar results (rs721575 z-scorefrac14416)

1934 Brain (2007) 130 1929^1941 EG Puffenberger et al

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 6: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

apparent lack of autozygosity including mutation hetero-geneity locus heterogeneity and digenic inheritance All ofthese tests were uninformative We then concentrated onchromosomal regions with insufficient SNP coverageWhile examining one such region on chromosome 17 wenoticed a single SNP rs721575 which produced lsquono callsrsquofor all seven patients (Fig 5A) This SNP had a prior callrate greater than 985 (Nfrac14 144 samples) making theresults for this marker highly suspicious Indeed we foundthat all 14 parents of affected children were lsquohomozygousrsquofor their respective rs721575 allele This suggested that themarker was deleted in all patients and hemizygous inparentsTo further test this conclusion we performed two

separate analyses of the genotype data using the CNAT

module of the Affymetrix software The first analysisplotted log2 ratio data generated by the copy numbertool (Fig 5B) whereas the second calculated average copynumber z-scores for each SNP from the seven affectedpatients In the seven patients the average z-score forrs721575 was 416 only one other SNP (Nfrac14 10 033) felloutside the conservative cutoff range of 3 to 3 Resultsfrom both analyses were consistent with homozygousdeletion of rs721575 in all affected children (Fig 5)

We then proceeded to delineate the deletion boundariesThe Affymetrix protocol relies on genomic DNA digestionwith the restriction endonuclease XbaI followed byamplification of restriction fragments in the 250ndash1000 bpsize range Amplified patient DNA is then labelled andhybridized to GeneChip arrays Thus the failure of

Fig 5 Evidence for homozygous deletions of SNP rs721575 in children with PMSE syndrome (A) Genotype data are shown for sevenaffected individuals and their parents All seven patients produced lsquono callsrsquo for SNP rs721575 on chromosome 17 while all 14 parentsappeared homozygous for their respective allele This suggested homozygous deletion of rs721575 in patients and hemizygosity of parents(B) A plot of log2 ratios exported from the copy number tool of the Genotyper software (Affymetrix) shows a log2 ratio for SNP rs721575of less than 8 (arrow) consistent with homozygous deletion in patients Copy number z-scores produced similar results (rs721575 z-scorefrac14416)

1934 Brain (2007) 130 1929^1941 EG Puffenberger et al

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 7: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

rs721575 to function in patients could have been due eitherto polymorphism within the flanking XbaI restriction sitesor to polymorphic insertions or deletions between orinvolving the XbaI sites To investigate these possibilitieswe designed PCR primers to amplify rs721575 as well as theflanking XbaI sites We were unable to generate PCRproducts using patient DNA However parent and controlDNA produced appropriately sized products whichmatched the normal human genome reference sequenceand confirmed lsquohomozygosityrsquo for rs721575 in all parentsThese results indicated that patients were homozygous for adeletion encompassing both rs721575 and the flanking XbaIsitesThe SNP rs721575 lies within intron 10 of the LYK5

gene PCR primers were generated for all LYK5 exons(isoform 1) and used to amplify patient samples Wesuccessfully amplified LYK5 exons 1ndash8 in patients but notexons 9ndash13 All 13 exons could be amplified from controlDNA samples Exon 17 of MAP3K3 distal to LYK5 and inan opposite orientation was determined by PCR to bepresent in patient samples Using the forward primer forexon 8 of LYK5 additional reverse primers were fashionedevery 1 kb to delimit the size of the deletion One primerset produced a product in patients but not in controlsSequencing of this product revealed a homozygous 7304 bpdeletion that encompassed the terminal 5 exons of LYK5but spared adjacent genes LOC440455 MGC10986 andMAP3K3 (Fig 6)

Using primers designed to span the deletionbreakpoints we genotyped 100 healthy Old OrderMennonite controls We identified four 7 kb deletionheterozygotes yielding an estimated carrier frequency forthe mutant allele in Lancaster County Old OrderMennonites of approximately 4

Descriptive pathologyA 7-month-old child with PMSE syndrome died unexpect-edly following a prolonged cluster of seizures At the timeof death she was 7 months old 697 kg (11 SD) 70 cm inlength (thorn09 SD) and had a head circumference of 49 cm(thorn40 SD) Pathological examination did not reveal thecause of death The visceral anatomy was normal as wasmicroscopic histology of lungs myocardium liver ovariesskeletal muscle bone marrow kidneys thymus pancreasthyroid and parathyroid glands

The fresh brain was 1134 g (normal 95 CI for age750 184 g) The cerebral ventricles were enlarged and hadpatent outflow pathways Leptomeningeal glioneuronalheterotopias were present at the ventrolateral surface ofthe pons and adjacent to the lateral geniculate body butwere not found over the neocortical surface

Enlarged neurons with granular cytoplasm diminishedNissl substance areas of vacuolization and indistinct nucleiwere found in the cerebellar Pukinje cell layer hilus of thehippocampal dentate gyrus trochlear (IV) nerve nuclei

Fig 6 Delineation of a 7304bp deletion in LYK5Genotype and copy number analyses suggested that seven patients with syndromic cor-tical dysplasia harboured a homozygous deletion of rs721575 (in red top)This SNP maps to intron10 of the LYK5 geneThe gene contains13exons (isoform 1) Analysis of these coding regions indicated that exons 9^13 were deleted in patients (middle) Using an upstream primerknown to be outside the deletion additional reverse primers were fashioned to span deletion breakpoints A unique PCR product wasgenerated from patients and subsequently sequenced to precisely determine the deletion boundaries (bottom)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1935

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 8: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

substantia nigra anterior pituitary and anterior horn of thespinal cord (Fig 7A and B) Large dysmorphic cells withsimilar characteristics were also seen in the frontal cortexlentiform nuclei and pallidum (Fig 8) Although neuronswith these abnormal structural features were diffuselydistributed we did not find any definite abnormalities ofcortical lamination or focal areas of dysplasia in the singlebrain specimen examinedSmall optically clear vacuoles were widely distributed

throughout the white matter and were most prominent atthe neocortical and entorhinal gray matterndashwhite mattertransition zones (Fig 7C and D) These vacuoles wereorganized in linear arrays parallel to and often displacingintact nerve fibres with normal appearing myelin Therewas also marked vacuolization within the globus pallidushead of the caudate putaminal white matter bundlesexternal and extreme capsules pontine tegmentum ventralmedulla and the dentate nuclei of the cerebellum (Fig 7Eand F) In contrast corticospinal and pontocerebellar tractsappeared normalAstrocytes were abundant throughout the vacuolated

white matter regions of the brain (Fig 7C D and F)There was also prominent astrocytosis in the hippocampalformation particularly within the hilus and adjacent to thedentate granule-cell layer (Fig 7G and H) Astrocyteprocesses were often in physical contact with fluid vacuoles(Fig 7D inset) or neuronal cell bodies (Fig 8D) and someastrocytes were large and in the process of duplication(Fig 7G inset)

Labelling of mTOR cascade componentsThere was a low-level of P-S6 expression in control braintissue In the brain of a child with PMSE syndrome P-S6expression was increased throughout all six layers ofneocortex and was most prominent in pyramidal cells oflayer V Within the entorhinal cortex there was robust P-S6expression within islands of large pyramidal cells of layer IIIn the hippocampus large dysmorphic P-S6-labelledneurons were present in the hilus of the dentate gyrusand there was modest P-S6 expression in CA sectors Therewas robust P-S6 expression in large neurons of the anteriorhorn of the spinal cord cerebellar Purkinje cell layer andbasal ganglia (Fig 8C) and P-S6 was evident in manycranial motor nuclei (eg III VI and VII) of the brainstemAs only a limited amount of tissue from a single brain

was available for pathological inspection additional ana-lyses of gene and protein expression were not possible

DiscussionThe use ofmicroarrays for investigating novelclinical phenotypesSince 1998 we have identified molecular lesions for morethan 65 monogenic disorders segregating in Plain popula-tions (Morton et al 2003) Developmental delay is the

presenting problem for 29 (45) of these 10 of which areassociated with distinctive physical features Neverthelessevaluating a child with syndromic developmental delayremains a difficult diagnostic problem For such patientswe increasingly rely upon small-scale microarray studies toprovide focus and direction (Puffenberger et al 2004Strauss et al 2005 2006) Indeed SNP genotyping is oftenthe first step of the diagnostic process When coupled topopulation-based genetic knowledge (Morton et al 2003Puffenberger 2003) it is an efficient way to detect DNAcopy number abnormalities scan candidate regions forhomozygosity and determine shared haplotypes among asfew as two patients with a similar phenotype

Among the present group of patients there were no cluesfrom the clinical or MRI data to implicate LYK5 deficiencyand no analyte testing could have uncovered the diagnosisThus a small-scale mapping study was the only conceivableway to investigate the problem Because we work with ayoung genetically isolated population (Puffenberger 2003Puffenberger et al 2004 Strauss et al 2005 2006) weexpected autozygosity mapping would be straightforwardOur initial results (Fig 4B) highlight important pitfalls ofsmall mapping studies The lack of significant homozygousblocks could have resulted from mutation or locusheterogeneity Identity-by-descent analyses assume muta-tion and locus homogeneity however even in small isolatedpopulations such as the Amish and Mennonites hetero-geneity does exist (Puffenberger 2003) For this studyfurther statistical analyses were necessary to show thatmutation and locus heterogeneity were not present

A dearth of SNPs in the disease gene region was theprimary reason that autozygosity mapping failed to reveal alarge shared homozygous block Although most genomicregions are well represented on the Mapping 10K Arraycoverage is uneven and significant gaps exist LYK5 lies inone of those gaps The flanking SNPs rs1960286 andrs230572 are 3845Mb apart (Fig 5A) For comparisonour mapping study of SIDDT syndrome identified a 36Mbhomozygous region shared among four Amish patients(Puffenberger et al 2004) If the region surrounding LYK5had the average SNP coverage 3 SNPs per Mb (for theMapping 10K Array) then identifying the region byautozygosity mapping would have been simpler as wehave shown previously (Puffenberger et al 2004 Strausset al 2005 2006) For this study use of a higher densityarray would have likely solved this problem

Fortunately the single SNP included on the Mapping10K Array that localized to the 4 Mb region betweenrs1960286 and rs230572 happened to lie within the deletedregion of LYK5 Although not explored in our earlierstudies we show here that the homozygous deletion of asingle SNP is easily detected when appropriate analyses areemployed Average lsquono callrsquo rates for individual SNPs in areference population provided an important first clue toidentification of the molecular lesion in our patientsFormal assessment of copy number changes using the

1936 Brain (2007) 130 1929^1941 EG Puffenberger et al

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 9: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

Fig 7 Histological findings in the brain of a 7-month-old infant who had PMSE syndrome (A) In the anterior spinal cord motor neuronsare enlarged with rounded cell bodies granular cytoplasm and an indistinct nuclear membrane Neurons with similar morphology werefound in select areas of the brainstem cerebellum basal ganglia anterior pituitary and cortex (LFB-CV 600) For comparison panel Bshows normal appearing anterior horn cells from a control spinal cord specimen that was stained in parallel and viewed at similar magni-fication (C) Astrocytes are abundant within the vacuolated gray matter^white matter transition zone (dotted line) of the frontal cortex(GFAP 20) (D) A higher magnification view (100) shows optically clear vacuoles in linear arrays often in direct contact with astrocytes(arrow inset) (E and F) Vacuolization and astrocytosis in the dentate nucleus of the cerebellum (40 panel Efrac14LFB-CV panel Ffrac14GFAP)(G) There is astocytosis in the hippocampal formation particularly within the hilus and adjacent to the dentate granule-cell layer (dottedline) (GFAP 40) Some astrocytes are in the process of duplication (arrow inset) Panel H is a magnified view of hippocampal dentategranule cells with adjacent astrocytes (GFAP 400)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1937

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 10: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

Affymetrix Copy Number Analysis Tool (CNAT) as well asthe calculation of standard scores (z-scores) was indis-pensable for identification of the LYK5 deletion Given theincreasing density of SNP microarrays we anticipate thatcopy number analysis will become a routine part of diseasegene mapping The same set of SNP genotypes can be usednot only to identify autozygous genomic regions but alsoto detect loss of alleles even for a single SNP

Biological relevance of the PMSE syndromeThe LYK5 gene product STRAD is expressed in humanembryonic specimens and a wide variety of mature tissuesincluding kidney muscle peripheral nerve and brain(httpcgapncinihgov) It is part of a trimeric complexwith serinendashthreonine kinase 11 (STK11 aka LKB1) andMO25 (aka calcium binding protein 39 CAB39) (Fig 9)(Boudeau et al 2003 Baas et al 2004b) LYK5 is necessaryfor normal cytosolic localization and kinase activity ofSTK11 (Boudeau et al 2003)STK11-STRAD-MO25 functions primarily as a tumour

suppressor (Baas et al 2003 2004a b) Haploinsufficiency

of STK11 is associated with the PeutzndashJeghers syndromewhich is characterized by the development of multiplegastrointestinal hamartomatous polyps and a wide spec-trum of benign and malignant tumors (Hemminki 1999)While one affected child from our series died of leukaemia(Table 1) we cannot prove that it was causally related tothe LYK5 mutation and other tumours were not evidentamong patients Our cohort was young (median age 16years range 7 months to 28 years) however and apredisposition to tumours may turn out to be a latermanifestation of PMSE syndrome Moreover none of ourpatients had colonoscopies and polyposis would have goneunrecognized

The clinical manifestations of PMSE syndrome could notbe anticipated from prior in vitro studies of STRAD proteindistribution and function (Hemminki 1999 Milburn et al2004 de Leng et al 2005) The phenotype associated withLYK5 deletions in humans (Table 1) shows that STRAD hasa key role in shaping the nervous system and may alsoinfluence cerebrospinal fluid transport skeletal musclegrowth cardiac septation and renal physiology Ourfindings underscore the fact that data about protein

Fig 8 P-S6 immunohistochemistry and cytomegaly in the putamen of a child with PMSE sydrome (A) Numerous large cells (arrows) areseen with a LFB-CV at 100 (B) At higher magnification (LFB-CV 400) these cells are rounded have granular cytoplasm loss of Nisslsubstance and indistinct nuclei and nucleoli they are structurally similar to the anterior horn cells depicted in Fig 7A (C) Large cellsappear to have neuronal morphology and are strongly immunoreactive for P-S6 (inset) Similar anti-P-S6 reactivity was seen in large neu-rons of the caudate nucleus hippocampus and anterior horn of the spinal cord (D) GFAP immunostaining shows intense vacuolization andastrogliosis around these large neurons (asterisks) (400)

1938 Brain (2007) 130 1929^1941 EG Puffenberger et al

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 11: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

function derived from animal knockout and in vitro studiesmay be misleading when extrapolated to human biology(Strauss et al 2006)

LYK5 and mTOR activationIn one post-mortem case available for analysis we foundevidence of megalencephaly cytomegaly astrocytic gliosisand white matter vacuolization (Figs 7 and 8) A focalcortical dysplasia was not available to examine from thissingle brain specimen but limited MRI evidence shows thatsuch lesions may be associated with homozygous LYK5deletions in some patients (Fig 3) Thus although thehistological observations should be interpreted cautiouslythey suggest that this condition is a new syndromicmalformation of cortical development Further histopatho-logical studies of post-mortem or surgical tissue arenecessary to define the exact nature and extent of corticaldysplasia associated with LYK5 mutations Magneticresonance imaging at 15 Tesla does not have sufficientresolution for this purpose (Strauss et al 2006)Interestingly the widespread cytomegalic cells expressing

P-S6 in the brain of an infant with PMSE syndrome weresimilar to the giant cells of tuberous sclerosis complex andthe balloon cells of focal cortical dysplasia type IIB (Baybiset al 2004 Miyata et al 2004) This observation suggeststhat loss of LYK5 function during brain development maylead to downstream activation of the mTOR cascadeIndeed in normal cells LYK5 (as part of trimeric complexwith STK11 and MO25) participates in the phosphorylation

of AMPK which in turn phosphorylates the TSC2 proteinat Ser1345 thereby inhibiting mTOR (Fig 9)

We postulate that mTOR is constitutively activated as aresult of homozygous LYK5 mutations and that this leadsto inappropriate phosphorylation of p70 S6 kinase and P-S6(Fig 9) Thus the cellular mechanisms leading to abnormalbrain development from LYK5 deletions may be similar tothose in TSC Quantitative analysis of protein expression(ie reduced phospho-AMPK or enhanced phospho-mTOR) was not feasible because frozen brain tissue wasnot available additional studies of both human tissue andexperimental animals are necessary to fully explore thishypothesis Here we show that P-S6 expression wasincreased in large dysmorphic neurons throughout theneuraxis We speculate that high P-S6 expression in variousbrain regions may be associated with abnormal cellularfunctions that manifest as severe cognitive disabilities andepilepsy in all affected children and movement disorders insome younger patients

Widespread white matter vacuolization and astrocytosiswere the most prominent histological features of the singlebrain we studied (Fig 7CndashF) Increased numbers ofastrocytes are also found in cortical tubers (Scheithauerand Regan 1999) and abnormal white matter structure hasbeen described in TSC brains (Ridler et al 2001) In bothTSC and PMSE syndrome proliferation of astrocytes mayresult from constitutive activation of mTOR (Fig 9) eitherfocally (TSC) or diffusely (PMSE) Alternatively gliosis maysimply be a reactive process in response to abnormalneuronal structure and activity The mechanism of whitematter vacuolization is unknown

Fig 9 A simplified scheme showing the possible effect of STRAD mutations on the mTOR pathwayThe LYK5 gene product STRAD (red)is part of a trimeric complex with MO25 and STK11 (aka LKB1) Assembly of the complex is necessary for normal localization and cata-lytic activity of STK11 in vivo (Boudeau et al 2003 2004) STK11-STRAD-MO25 phosphorylates (thornp) and thereby activates (arrow) AMP-activated protein kinase (AMPK) which in turn activates theTSC1TSC2 complex and inhibits mTOR-GbL-Raptor through phosphorylation(Kwiatkowski and Manning 2005) Downstream phosphorylations along the mTOR pathway influence cell growth and survival throughproteins such as eIF4B and S6 (blue) (Shahbazian et al 2006) Phosphorylated S6 protein (P-S6) is overexpressed in cortical tubers (Baybiset al 2004) and was abundant throughout the brain and spinal cord of an infant with homozygous LYK5 deletions In principle compoundssuch as rapamycin that can cross the blood^brain barrier and inhibit mTOR might influence the course of brain disease caused by LYK5deficiency In the figure heavy arrows indicate constitutive overactivity dashed lines represent underactivity and thornp indicates phosphory-lation (httpwwwgenomejpkeggpathwayhsa)

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1939

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 12: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

Conclusions and future directionsWe used the LYK5 mutation information to develop arapid inexpensive molecular diagnostic test This test willspare children and families protracted costly and unin-formative diagnostic evaluations (Strauss et al 2005) Inpractice this will allow us to anticipate problems like heartfailure hypernatremic dehydration and status epilepticusThese are serious clinical conditions for which preventionnot symptomatic treatment is the goal (Morton et al2003)The identification of LYK5 deletions in affected patients

is a starting point for considerations about treatmentwhich may for example focus on mTOR or relatedsignalling pathways (Fig 9) (Kenerson et al 2005Kwiatkowski and Manning 2005 Kesari et al 2006Doherty et al 2006) The success of such therapies willdepend on diagnosing the condition early in life beforeintractable focal seizures injure the developing brain(Holmes and Ben-Ari 1998 2001)

Disclosure StatementThe authors declare that the Affymetrix GeneChip Scannerused for a portion of this study was a charitable donationto the non-profit Clinic for Special Children (EIN 23-2555373) from the Affymetrix Corporation

Supplementary materialSupplementary material is available at BRAIN online

AcknowledgementsThe authors dedicate this paper to all of the families whocooperated to make this work possible The authors extenda special thank you to Mark and Dorothy Weaver forinitially identifying the seven LYK5 deletion patients usedfor mapping and organizing the families to participate inthis study We are grateful to Christine Weir and thetechnical staff of the Lancaster MRI group as well asRebecca LeVier and her colleagues in the Department ofPathology Lancaster General Hospital This study wassupported in part by grant U24NS051872 from the NationalInstitutes of Health to Dietrich A Stephan

ReferencesBaas AF Boudeau J Sapkota GP Smit L Medema R Morrice NA et al

Activation of the tumour suppressor kinase LKB1 by the STE20-like

pseudokinase STRAD EMBO J 2003 22 3062ndash72

Baas AF Kuipers J van der Wel NN Batlle E Koerten HK Peters PJ et al

Complete polarization of single intestinal epithelial cells upon activation

of LKB1 by STRAD Cell 2004a 116 457ndash66

Baas AF Smit L Clevers H LKB1 tumor suppressor protein PARtaker in

cell polarity Trends Cell Biol 2004b 14 312ndash9

Battaglia A Carey JC Diagnostic evaluation of developmental delaymental

retardation an overview Am J Med Genet C Semin Med Genet 2003

117 3ndash14

Baybis M Yu J Lee A Golden JA Weiner H McKhann G 2nd et al

mTOR cascade activation distinguishes tubers from focal cortical

dysplasia Ann Neurol 2004 56 478ndash87

Boudeau J Baas AF Deak M Morrice NA Kieloch A Schutkowski M

et al MO25alphabeta interact with STRADalphabeta enhancing their

ability to bind activate and localize LKB1 in the cytoplasm EMBO J

2003 22 5102ndash14

Boudeau J Scott JW Resta N Deak M Kieloch A Komander D et al

Analysis of the LKB1-STRAD-MO25 complex J Cell Sci 2004 117

6365ndash75

Broman KW Weber JL Long homozygous chromosomal segments in

reference families from the centre drsquoEtude du polymorphisme humain

Am J Hum Genet 1999a 65 1493ndash500

Broman KW Weber JL Method for constructing confidently ordered

linkage maps Genet Epidemiol 1999b 16 337ndash43

de Leng WW Keller JJ Luiten S Musler AR Jansen M Baas AF et al

STRAD in Peutz-Jeghers syndrome and sporadic cancers J Clin Pathol

2005 58 1091ndash5

Doherty L Gigas DC Kesari S Drappatz J Kim R Zimmerman J et al

Pilot study of the combination of EGFR and mTOR inhibitors in

recurrent malignant gliomas Neurology 2006 67 156ndash8

Hemminki A The molecular basis and clinical aspects of Peutz-Jeghers

syndrome Cell Mol Life Sci 1999 55 735ndash50

Higgins JJ Morton DH Loveless JM Posterior column ataxia with

retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

Neurology 1999 52 146ndash50

Holmes GL Ben-Ari Y Seizures in the developing brain perhaps not so

benign after all Neuron 1998 21 1231ndash4

Holmes GL Ben-Ari Y The neurobiology and consequences of epilepsy in

the developing brain Pediatr Res 2001 49 320ndash5

Hunter AG Outcome of the routine assessment of patients with

mental retardation in a genetics clinic Am J Med Genet 2000 90

60ndash8

Kenerson H Dundon TA Yeung RS Effects of rapamycin in the

Eker rat model of tuberous sclerosis complex Pediatr Res 2005 57

67ndash75

Kesari S Ramakrishna N Sauvageot C Stiles CD Wen PY Targeted

molecular therapy of malignant gliomas Curr Oncol Rep 2006 8

58ndash70

Kwiatkowski DJ Manning BD Tuberous sclerosis a GAP at the crossroads

of multiple signaling pathways Hum Mol Genet 2005 14 Spec No 2

R251ndash8

Milburn CC Boudeau J Deak M Alessi DR van Aalten DM

Crystal structure of MO25 alpha in complex with the C terminus of

the pseudo kinase STE20-related adaptor Nat Struct Mol Biol 2004 11

193ndash200

Morton DH Morton CS Strauss KA Robinson DL Puffenberger EG

Hendrickson C et al Pediatric medicine and the genetic disorders of the

Amish and Mennonite people of Pennsylvania Am J Med Genet C

Semin Med Genet 2003 121 5ndash17

Puffenberger EG Genetic heritage of the Old Order Mennonites of

Southeastern Pennsylvania Am J Med Genet Part C (Semin Med Genet)

2003 121C 18ndash31

Puffenberger EG Hu-Lince D Parod JM Craig DW Dobrin SE

Conway AR et al Mapping of sudden infant death with dysgenesis

of the testes syndrome (SIDDT) by a SNP genome scan and

identification of TSPYL loss of function Proc Natl Acad Sci USA

2004 101 11689ndash94

Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J et al

The mTORPI3K and MAPK pathways converge on eIF4B to control its

phosphorylation and activity EMBO J 2006 25 2781ndash91

Shaw RJ Bardeesy N Manning BD Lopez L Kosmatka M DePinho RA

et al The LKB1 tumor suppressor negatively regulates mTOR signaling

Cancer Cell 2004 6 91ndash9

Shevell M Ashwal S Donley D Flint J Gingold M Hirtz D et al Practice

parameter evaluation of the child with global developmental delay

report of the Quality Standards Subcommittee of the American

1940 Brain (2007) 130 1929^1941 EG Puffenberger et al

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941

Page 13: Polyhydramnios, megalencephaly and symptomatic epilepsy ... · approximately 125 Amish and Mennonite patients evalu-ated each year at the Clinic for Special Children. Population-based

Academy of Neurology and The Practice Committee of the Child

Neurology Society Neurology 2003 60 367ndash80

Srour M Mazer B Shevell MI Analysis of clinical features predicting

etiologic yield in the assessment of global developmental delay

Pediatrics 2006 118 139ndash45

Strauss KA Puffenberger EG Craig DW Panganiban CB Lee AM

Hu-Lince D et al Genome-wide SNP arrays as a diagnostic tool clinical

description genetic mapping and molecular characterization of Salla

disease in an Old Order Mennonite population Am J Med Genet A

2005 138 262ndash7

Strauss KA Puffenberger EG Huentelman MJ Gottlieb S Dobrin SE

Parod JM et al Recessive symptomatic focal epilepsy and

mutant contactin-associated protein-like 2 N Engl J Med 2006 354

1370ndash7

LYK5 deletion syndrome Brain (2007) 130 1929^1941 1941