� 2008 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 9999:1–5 (2008)

Primary Microcephaly With ASPM Mutation ShowsSimplified Cortical Gyration With Antero-Posterior

Gradient Pre- and Post-Natally

Julie Desir,1,2* Marie Cassart,3 Philippe David,3 Patrick Van Bogaert,4 and Marc Abramowicz1,2

1DepartmentQ1 of Medical Genetics, Hopital Erasme, Brussels, Belgium2Laboratory of Medical Genetics, ULB, Free University of Brussels, Brussels, Belgium

3Department of Radiology, Hopital Erasme, Brussels, Belgium4Department of Pediatric Neurology, Hopital Erasme, Brussels, Belgium

Received 24 December 2007; Accepted 19 February 2008

Primary microcephaly is a disorder of brain developmentcharacterized by a congenitally small but normally formedbrain, and non-progressive mild-to-moderate mental retarda-tion. Most cases are inherited in an autosomal recessivepattern, with genetic heterogeneity, the ASPM locus beingmost common. Postnatal imaging data are scarce andprenatal imaging unreported. Microcephaly with simplifiedgyral pattern shares features with primary microcephaly,but it is not clear whether these disorders are part of aphenotypic continuum. We examined a consanguineousfamily with a daughter affected with primary microcephalyand an ongoing pregnancy. We performed prenatal andpostnatal brain magnetic resonance imaging and geneticanalyses in the course of genetic evaluation. The affecteddaughter and the fetus were homozygous for polymorphicmarkers linked to the ASPM locus, and we identified a novel,

truncating ASPM mutation by direct sequencing of the gene.Imaging at 30 and 35 gestational weeks showed micro-cephaly with simplified gyration, more severe anteriorly. Theantero-posterior gradient of gyration persisted 1 week afterbirth. Brain imaging in the affected sister also showed somedegree of a predominantly anterior simplification of gyra-tion. Our data suggest that one form of autosomal recessivemicrocephaly is allelic to at least a subset of microcephalywith simplified gyral pattern, and that the neuronal depletionassociated with the ASPM defect predominantly affects theanterior cortex. � 2008 Wiley-Liss, Inc.

Key words: brain development; primary microcephaly;prenatal diagnosis; genotype–phenotype correlation; con-sanguinity; asymmetric cell division

How to cite this article: Desir J, Cassart M, David P, Van Bogaert P, Abramowicz M. 2008. PrimaryMicrocephaly with ASPM mutation shows simplified cortical gyration with antero-posterior gradient

pre- and post-natally. Am J Med Genet Part A 9999:1–5.

INTRODUCTION

Primary microcephaly is a disorder of brain volumedevelopment with a congenitally small but normallyformed brain, and non-progressive mild-to-moderatemental retardation. It is usually transmitted as anautosomal recessive trait, and is referred to as MCPH[Woods et al., 2005]. Autosomal recessive micro-cephaly is genetically heterogeneous with at leastseven loci, and four genes identified so far [Cox et al.,2006]. Three of these genes encode centrosomalproteins, and the cellular knock-down of the beststudied of these genes, ASPM, hampers symmetriccell division of neural progenitors [Fish et al., 2006],consistent with a decreased production of neuronsduring fetal development [Kouprina et al., 2005].Mutations in ASPM mutation are prevalent amongpatients with MCPH [Woods et al., 2005]. Most of themutations are truncating, with no obvious clustering

of mutations and no apparent genotype–phenotypecorrelation [Bond et al., 2003].

Primary microcephaly with simplified gyral pattern(MSG; OMIM 603802) is defined by congenitalmicrocephaly, reduced number and shallow appea-rance of gyri, and normal to thin cortex [Peifferet al., 1999]. Both MCPH and MSG are classified ingroup I of malformations of cortical developmentand both diseases are believed to result from

AJMA-07-0941.R1(32312)

This article contains supplementary material, which may be viewedat the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/1552-4825/suppmat/index.html.

Grant sponsor: FRSMQ2; Grant number: 3.4567.02; Grant sponsor:Erasme Fund, Hopital Erasme, ULB.

*Correspondence to: Julie Desir, IRIBHM, 808 Route de Lennik,Brussels 1070, Belgium. E-mail: jdesir@ulb.ac.be

Published online 00 Month 2008 in Wiley InterScience(www.interscience.wiley.com)

DOI 10.1002/ajmg.a.32312

abnormal neuronal proliferation [Barkovich et al.,2005]. Primary microcephaly and MSG might hencerepresent a phenotypic continuum. Five types ofMSG have however been delineated on the basis ofMRI and neurodevelopmental findings [Barkovichet al., 1998], and the clinical phenotype may be moresevere than for MCPH. Mental retardation rangesfrom mild to moderate (MSG type 1) to severe (types2–5), and pyramidal signs may be observed. Someforms are associated with early-onset seizures anda poor prognosis. A novel, lethal form of MSGwas recently reported [Rajab et al., 2007]. Althoughautosomal recessive transmission of the latter andother types of MSG have been reported, no locus hasbeen identified to date. It is thus unclear whether theMSG phenotype relates to mutations in the samegenes as MCPH. Published reports of brain imagingdata are scarce in children and adults with MCPH,and to our knowledge virtually no data are reportedin affected human fetuses. We report on prenatal andpostnatal MRI findings in siblings with a novel ASPMmutation.

CLINICAL REPORT

The proband, a female first child born to healthyyoung consanguineous parents of Moroccan de-scent, presented with congenital microcephaly. She

was born at term after an unremarkable pregnancy,with an OFC of 30 cm (��3.5 SD), a weight of 3.3 kg(�50th centile) and a length of 47.5 cm (�10thcentile). Microcephaly was observed prenatally byultrasonography at 31 weeks of gestation. Motordevelopment was normal. Language was delayed.The child presented two episodes of seizures at age4 years, which were evaluated but eventually nottreated. A brain MRI was performed at age 5 years(Fig. 2)Q3. It showed a simplified pattern of gyration,more severe in the frontal lobes, with a decreasingseverity towards the parietal and temporal regions.She was evaluated again at the age of 6 years forhyperactivity—attention deficit disorder. Her IQ was50 (McCarthy scale, homogenous profile) and shewas referred to special schooling. At that time shehad an OFC of 42 cm (��7 SD) with no dysmorphicfeatures, an otherwise normal physical examination,normal motor milestones, and hyperactive behavior.

The parents consulted us for recurrence riskcounseling during their second pregnancy, whenthe proband was 5 years old. The pregnancy was inits 26th week. Fetal ultrasonography at 26 showed aBPD of 59 mm, an OFD of 80 mm and a calculatedOFC of 219 mm (�10th centile). At 30 weeks, BPDwas at 70 mm (�3rd centile). The parents were toldof the very likely autosomal recessive inheritanceof microcephaly in their family, and of an a priori

FIG. 1. Pre- and post-natal brain imaging in affected brother. A: Axial and (B) sagittal T2-weighted images of the affected fetus at 30 weeks of gestational age. Thefrontal lobes are short and hypoplastic, with an anterior to posterior (i.e., anterior more severe than posterior) gradient of simplification of the gyral pattern. C: Axial and(D) sagittal T2-weighted images of the same fetus at 35 weeks of gestational age. Gyration has progressed, but the anomalies noted at 30 weeks persist and maintain thegyral gradient pattern. E: Axial T2-weighted and (F) sagittal T1-weighted images exutero at 7 days of age, showing a global reduction in the sizeof the cerebral structuresassociated with microcephaly, a sloping forehead, and preserved midline structures.

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25% risk of recurrence in each pregnancy. Theywere told that recurrence was likely in the currentpregnancy considering the fetal head measurementson ultrasonography. Fetal brain MRI was performedat 30 weeks of gestation (Fig. 1A,B). It showeda reduced number of cortical gyri, more severeanteriorly, that is, an anterior to posterior gradient ofgyration. The frontal lobes of the fetal brain weresmall and squared-off. To evaluate the progressionof gyration, a second MRI of the fetal brain wasperformed at 35 weeks gestation (Fig. 1C,D). Itshowed short and hypoplastic frontal lobes, withdiffuse, albeit anterior more than posterior, simpli-fication of the gyral pattern. The results of theimaging studies and their interpretation were dis-cussed with the parents at 26, 30, 32, and 35 weeks ofgestation. Termination of the pregnancy was pre-sented as a possible option and was declined by theparents. A baby boy was born at term by standardvaginal vertex delivery. His weight, length andOFC were 3.250 kg (�25th centile), 48.5 cm (�10thcentile) and 31 cm (��3 SD), respectively. Bloodwas sampled from the umbilical cord with informedconsent for DNA analysis. Physical and neurologicalexaminations were normal except for congenitalmicrocephaly. Abrain MRIwas repeated 1 week afterbirth, and showed improved but persistent simplifi-cation of the gyral pattern with anterior-to-posteriordecreasing severity (Fig. 1E,F). The boy was fol-lowed until age 2 years. He walked at 18 months,persistently on tiptoes.He hadno spasticity. Hehad aspeechdelaywithonly 10 isolatedwords at the ageof2 years, and a hyperactive behavior with attentiondeficit disorder like his sister. His electroencephalo-gram was normal.

LINKAGE AND MUTATION ANALYSES

Peripheral blood was sampled from the probandand both parents and cord blood from the boywith informed consent for DNA analysis. DNAwas extracted by the standard phenol–chloroform

method [Sambrook et al., 1989]. Linkage analyses tothe six known MCPH loci and the RELN gene wascarried out with microsatellite markers (see Supple-mentary Data). Marker order was obtained fromelectronic databases (see Electronic Databases Sec-tion). DNA was amplified by PCR using 15 ng of eachindividual’s DNA in a 15 ml final volume, followed byPolyAcrylamide Gel Electrophoresis and silver stain-ing. No evidence of linkage was found to the RELNgene nor to MCPH1, 2, 3, 4 and 6. A 14 cM region ofhomozygosity, shared between the two affectedsiblings, was identified at the MCPH5 (ASPM) locusat chromosome 1q31.2–q32.1 between markersD1S518 and D1S1171 (Fig. 3).

The 28 exons and flanking intronic sequencesof the ASPM gene were direct sequenced fromgenomic DNA of both parents and affected sibs. PCRproducts were purified using ExoSAP-IT1 ForPCR Product Clean-Up (USB) and sequenced usingthe Big Dye Terminator cycle sequencing kit v2

FIG. 2. Brain MRI in proband at age 5 years. A: Axial and (B) sagittal T1-weighted images. The gyral pattern is simplified with an anterior to posteriorgradient. The thickness of the cortex is normal. The midline structures arepreserved. Sloping of the forehead reflects the severe decrease in the cranial-to-facial proportions.

FIG. 3. Linkage analysis. The parents are first cousins once removed, the twoaffected children are represented in black. The ASPM locus is closely flankedby markers GATA135F02 and D1S1660. Genetic distances are indicated incentimorgans (cM). The homozygous haplotype shared by the affected siblingsis boxed. No evidence of linkage was found to the RELN gene nor to the fiveother MCPH loci reported to date (data not shown).

GYRATION GRADIENT IN ASPM MICROCEPHALY 3

American Journal of Medical Genetics Part A

(Applied Biosystems, Foster City, CA), and analyzedon a 3130 Genetic Analyzer sequencing machine(Applied Biosystems). Sequences were inspected insilico for mutations using the SeqScape softwareversion 2.0 (Applied Biosystems).

We identified a previously unreported mutation,consisting of an insertion of one base pair in exon 18,c.4195_4196insA (GenBank reference NM_018136.2)that introduced a frameshift and a premature stopcodon (p.Thr1399AsnfsX20). The mutation washomozygous in the two affected siblings, and, asexpected from the haplotype analysis, heterozygous

in both parents (Fig. 4). This study was approved bythe Ethical Committee of ULB, Hopital Erasme.

DISCUSSION

We were consulted for genetic evaluation duringthe second pregnancy of a young consanguineouscouple whose first child presented with congenitalmicrocephaly, moderate mental retardation andattention-hyperactivity disorder. The clinical picturewas typical of primary microcephaly. In view ofparental consanguinity, MCPH was likely.

FIG. 4. Mutation c.4195_4196insA (p.Thr1399AsnfsX20) of the ASPM gene. The black arrow indicates nucleotide 4195, where an A was inserted, causing a frameshiftand an in-frame premature stop codon (TGA, underlined). The mutation was heterozygous in the unaffected mother and father, while homozygous in the proband andaffected brother, and absent in a control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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American Journal of Medical Genetics Part A

Serial MRIs in the fetus confirmed microcephaly.At the same time, genetic analysis of polymorphicmarkers was consistent with linkage of microcephalyin the proband and fetus to the ASPM locus.

Prenatal imaging of the brain has, to our knowl-edge, not been reported in primary microcephaly.Postnatal data are scarce [Bond et al., 2002]. The MSGphenotype is described as being distinct from that ofprimary microcephaly, although it shares featureswith the latter [Peiffer et al., 1999]. In both diseases,the cortex is of normal or reduced thickness andpresumably has a normal laminar architecture withsix layers of neurons, which contrasts with lissence-phaly syndromes. Classic (type I) lissencephaly ischaracterized by microcephaly with an abnormallythick cortex and only four, or two, layers of cells[Forman et al., 2005]. Within the type I lissencephalygroup, mutations of DCX or RELN result in an anteriormore severe than posterior gradient, while mutationsof LIS1 or ARX lead to a posterior more severethan anterior gradient [Kato and Dobyns, 2003;Guerrini and Marini, 2006]. The phenotype of type Ilissencephaly is usually more severe, and clearlydistinct, from that observed in the family reportedhere, and other MCPH patients.

Taken together, serial MRIs in the two patientsreported here showed microcephaly with an anteriormore severe than posterior gradient of gyralsimplification. Gyral simplification was more pro-nounced at earlier prenatal stages. It was notapparent in the occipital region at the age of 5 yearsin the proband. These observations suggest thatneuronal depletion due to an ASPM mutation[Fish et al., 2006] predominantly affects the anteriorcortex. It is presently unknown whether the patternof brain development observed in this family isfrequent in patients with ASPM mutations, andwhether it is also present in patients with mutationsin other MCPH genes, for example, Microcephalin,CENPJ, or CDK5RAP3.

ELECTRONIC DATABASES

Marshfield Comprehensive Human GeneticMaps: http://research.marshfieldclinic.org/genetics/geneticresearch/compmaps.asp.

UCSC Genome Browser: http://genome.ucsc.edu/cgi-bin/hggateway.

ACKNOWLEDGMENTS

JD is a fellow of the Belgian Kids’ Fund. Thisstudy was supported by the FRSM grant 3.4567.02

(F.N.R.S., Belgium) and the Transdisciplinary Grantof the Erasme Fund, Hopital Erasme, ULB. We thankthe patients for their collaboration, S. Strollo forexpert technical assistance, P. Vanderhaeghen foruseful discussions and G. Vassart for interest andsupport.

REFERENCES

Barkovich AJ, Ferriero DM, Barr RM, Gressens P, Dobyns WB,Truwit CL, Evrard P. 1998. Microlissencephaly: A heteroge-neousmalformation of cortical development. Neuropediatrics29:113–119.

Barkovich AJ, Kuzniecky RI, Jackson GD, Guerrini R, DobynsWB. 2005. A developmental and genetic classification formalformations of cortical development. Neurology 65:1873–1887.

Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, AskhamJM, Springell K, Mahadevan M, Crow YJ, Markham AF, WalshCA, Woods CG. 2002. ASPM is a major determinant of cerebralcortical size. Nat Genet 32:316–320.

Bond J, Scott S, Hampshire DJ, Springell K, Corry P, AbramowiczMJ, Mochida GH, Hennekam RC, Maher ER, Fryns JP, AlswaidA, Jafri H, Rashid Y, Mubaidin A, Walsh CA, Roberts E, WoodsCG. 2003. Protein-truncating mutations in ASPM causevariable reduction in brain size. Am J Hum Genet 73:1170–1177.

Cox J, Jackson AP, Bond J, Woods CG. 2006. What primarymicrocephaly can tell us about brain growth. Trends Mol Med12:358–366.

Fish JL, Kosodo Y, Enard W, Paabo S, Huttner WB. 2006. Aspmspecifically maintains symmetric proliferative divisions ofneuroepithelial cells. Proc Natl Acad Sci USA 103:10438–10443.

Forman MS, Squier W, Dobyns WB, Golden JA. 2005. Genotypi-cally defined lissencephalies show distinct pathologies.J Neuropathol Exp Neurol 64:847–857.

Guerrini R, Marini C. 2006. Genetic malformations of corticaldevelopment. Exp Brain Res 173:322–333.

Kato M, Dobyns WB. 2003. Lissencephaly and the molecularbasis of neuronal migration. Hum Mol Genet 12:R89–R96.

Kouprina N, Pavlicek A, Collins NK, Nakano M, Noskov VN,Ohzeki J, Mochida GH, Risinger JI, Goldsmith P, Gunsior M,Solomon G, Gersch W, Kim JH, Barrett JC, Walsh CA, Jurka J,Masumoto H, Larionov V. 2005. The microcephaly ASPM geneis expressed in proliferating tissues and encodes for a mitoticspindle protein. Hum Mol Genet 14:2155–2165.

Peiffer A, Singh N, Leppert M, Dobyns WB, Carey JC. 1999.Microcephaly with simplified gyral pattern in six relatedchildren. Am J Med Genet 84:137–144.

Rajab A, Manzini MC, Mochida GH, Walsh CA, Ross ME. 2007. Anovel form of lethal microcephaly with simplified gyralpattern and brain stem hypoplasia. Am J Med Genet Part A143A:2761–2767.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: Alaboratory manual, 2nd edition. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory.

Woods CG, Bond J, Enard W. 2005. Autosomal recessiveprimary microcephaly (MCPH): A review of clinical, molec-ular, and evolutionary findings. Am J Hum Genet 76:717–728.

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Softproofing for advanced Adobe Acrobat Users - NOTES toolNOTE: ACROBAT READER FROM THE INTERNET DOES NOT CONTAIN THE NOTES TOOL USED IN THIS PROCEDURE.

Acrobat annotation tools can be very useful for indicating changes to the PDF proof of your article.By using Acrobat annotation tools, a full digital pathway can be maintained for your page proofs.

The NOTES annotation tool can be used with either Adobe Acrobat 4.0, 5.0 or 6.0. Other annotation tools are also available in Acrobat 4.0, but this instruction sheet will concentrateon how to use the NOTES tool. Acrobat Reader, the free Internet download software from Adobe,DOES NOT contain the NOTES tool. In order to softproof using the NOTES tool you must havethe full software suite Adobe Acrobat 4.0, 5.0 or 6.0 installed on your computer.

Steps for Softproofing using Adobe Acrobat NOTES tool:

1. Open the PDF page proof of your article using either Adobe Acrobat 4.0, 5.0 or 6.0. Proofyour article on-screen or print a copy for markup of changes.

2. Go to File/Preferences/Annotations (in Acrobat 4.0) or Document/Add a Comment (in Acrobat6.0 and enter your name into the “default user” or “author” field. Also, set the font size at 9 or 10point.

3. When you have decided on the corrections to your article, select the NOTES tool from theAcrobat toolbox and click in the margin next to the text to be changed.

4. Enter your corrections into the NOTES text box window. Be sure to clearly indicate where thecorrection is to be placed and what text it will effect. If necessary to avoid confusion, you canuse your TEXT SELECTION tool to copy the text to be corrected and paste it into the NOTEStext box window. At this point, you can type the corrections directly into the NOTES textbox window. DO NOT correct the text by typing directly on the PDF page.

5. Go through your entire article using the NOTES tool as described in Step 4.

6. When you have completed the corrections to your article, go to File/Export/Annotations (inAcrobat 4.0) or Document/Add a Comment (in Acrobat 6.0).

7. When closing your article PDF be sure NOT to save changes to original file.

8. To make changes to a NOTES file you have exported, simply re-open the original PDFproof file, go to File/Import/Notes and import the NOTES file you saved. Make changes and re-export NOTES file keeping the same file name.

9. When complete, attach your NOTES file to a reply e-mail message. Be sure to include yourname, the date, and the title of the journal your article will be printed in.