Towards the identification of a genetic basis for Landau-Kleffner syndrome

Towards the identification of a genetic basis for

Landau-Kleffner syndrome*†Judith Conroy, †‡Paul A. McGettigan, §DaraMcCreary, ¶Naisha Shah, **Kevin Collins,

**Bronwyn Parry-Fielder, §**MargaretMoran, ††DonnchaHanrahan, ‡‡ThierryW.Deonna,

§§ChristianM. Korff, ¶¶DavidWebb, †***Sean Ennis, *†***Sally A. Lynch, and †§MaryD. King

Epilepsia, **(*):1–8, 2014doi: 10.1111/epi.12645

Dr. Judith Conroyis a postdoctoralresearcher at theChildren’sUniversity Hospitaland UniversityCollege Dublin,Ireland.

SUMMARY

Objective: To establish the genetic basis of Landau-Kleffner syndrome (LKS) in a

cohort of two discordantmonozygotic (MZ) twin pairs and 11 isolated cases.

Methods: We used a multifaceted approach to identify genetic risk factors for LKS.

Array comparative genomic hybridization (CGH) was performed using the Agilent

180K array. Whole genome methylation profiling was undertaken in the two discor-

dant twin pairs, three isolated LKS cases, and 12 control samples using the Illumina

27K array. Exome sequencing was undertaken in 13 patients with LKS including two

sets of discordant MZ twins. Data were analyzed with respect to novel and rare vari-

ants, overlapping genes, variants in reported epilepsy genes, and pathway enrichment.

Results: A variant (cG1553A) was found in a single patient in theGRIN2A gene, causing

an arginine to histidine change at site 518, a predicted glutamate binding site. Follow-

ing copy number variation (CNV),methylation, and exome sequencing analysis, no sin-

gle candidate gene was identified to cause LKS in the remaining cohort. However, a

number of interesting additional candidate variants were identified including variants

in RELN, BSN, EPHB2, andNID2.

Significance: A single mutation was identified in the GRIN2A gene. This study has iden-

tified a number of additional candidate genes including RELN, BSN, EPHB2, andNID2.

KEYWORDS: Rolandic epilepsy, Verbal auditory agnosia, Copy number variation, Ex-

ome sequencing, Discordantmonozygotic twins, Candidate genes.

Landau-Kleffner syndrome (LKS, OMIM 245570) is arare childhood epileptic encephalopathy also known asacquired epileptic aphasia, and it is considered part of the

spectrum of idiopathic focal, particularly rolandic, epilep-sies. Age of onset is between 3 and 9 years, with a male-to-female ratio of 2:1. Typically there is abrupt or gradualonset of language regression due to verbal auditory agnosia,often with seizures. Sleep electroencephalography mayshow continuous spikes and waves in slow-wave sleep butmay fluctuate and be normal in the early phases of the dis-ease.1 Recovery is complete in some patients, whereas oth-ers are left with severe permanent aphasia and/orbehavioral/learning disabilities.

The presentation of this disorder in siblings indicates apossible genetic/epigenetic cause, although environmentalinfluences cannot be excluded as either the sole cause or asa modifier of a genetic cause of LKS. Discordant monozy-gotic (MZ) twins have also been reported, indicating thepossibility of a heterogeneous etiologic basis including thefollowing: (1) postzygotic de novo mutations, (2) postzygot-ic de novo copy number variations (CNVs), or (3) differen-tial methylation as a cause for the discordance, in addition

AcceptedMarch 28, 2014.*Department of Genetics, Children’s University Hospital, Dublin, Ire-

land; †Academic Centre on Rare Diseases, School of Medicine andMedicalScience, University College Dublin, Dublin, Ireland; ‡School of Agricul-ture and Food Science, University College Dublin, Dublin, Ireland;§Department of Neurology, Children’s University Hospital, Dublin, Ire-land; ¶School of Medicine and Medical Science, University College Dub-lin, Dublin, Ireland; **Royal Children’s Hospital, Melbourne, VIC,Australia; ††Royal Belfast Hospital for Sick Children, Belfast, UnitedKingdom; ‡‡University Hospital of Lausanne, Lausanne, Switzerland;§§University Hospitals, Geneva, Switzerland; ¶¶Department of Neurology,Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland; and ***TheNational Centre for Medical Genetics, Our Lady’s Children’s HospitalCrumlin, Dublin, Ireland

Address correspondence to Judith Conroy, School of Medicine andMedical Sciences, University College Dublin, Room C328, Belfield, Dub-lin 4, Ireland. E-mail: [email protected]

Wiley Periodicals, Inc.© 2014 International League Against Epilepsy

1

FULL-LENGTHORIGINALRESEARCH

to a number of possible acquired and environmental factors(see Table 1 for further information). A number of recentstudies have highlighted the role of the GRIN2A mutationsand CNVs in familial and sporadic cases of LKS.2–7 Muta-tions in this gene are estimated to affect 5–20% of familial/sporadic cases with LKS, continuous spike and waves dur-ing slow-wave sleep or atypical rolandic epilepsy. However,the etiology of the disorder in the remaining patientsremains unclear. A recent study has highlighted the likeli-hood that diseases of the epilepsy–aphasia spectrum(including LKS) have an inheritance pattern that suggests acomplex genetic disorder.8 It is also possible that there areother genes/variants of major effect but of low frequency inthe LKS population. This study aims to identify potentialgenetic causes and candidate genes for LKS through the useof CNV, methylation, and exome sequencing analysis in acohort of patients that includes two sets of discordant MZtwins, including theMZ pair previously reported by Feekeryet al. (1993).18

Material and MethodsPatients

Patients with LKS were identified in Ireland (n = 8),Switzerland (n = 3), Northern Ireland (n = 1), and Austra-lia (n = 1), and they were diagnosed with LKS on the basisof clinical presentation, electroencephalography (EEG)findings, and natural history. The diagnoses were in accor-dance with the guidelines proposed by Scheltens-de Boer(2009).19 Clinical information for each patient is outlinedin Table 2. The presence of epilepsy, language impair-ment, and/or intellectual disability in the related familymembers is also noted for each individual. Monozygosityfor the twins was confirmed through single nucleotidepolymorphism (SNP) genotyping of over one millionmarkers using the Human 1M genotyping array (IlluminaInc., San Diego, CA, U.S.A.). Ethical approval for thestudy was granted by the Children’s University Hospital,Temple Street, Dublin, Ireland. Informed consent wasobtained for each participating individual.

CNV analysisCNVs were identified using the Agilent array comparative

genomic hybridization (CGH) platform and the SurePrint G3Human 180K microarray kit (Agilent Technologies, SantaClara, CA, U.S.A.). CNVs were filtered using an aberrationdetection method 2 (ADM-2) threshold of 5.0, window of0.2 Mb, a cutoff of 0.25, and at least three contiguousprobes. CNVs <500 kb with no RefSeq genes, events locatedentirely within segmental duplications and CNVs with >50%overlap with control CNVs in the Database of GenomicVariants were excluded. CNV validation was undertakenusing the HumanCytoSNP-12 genotyping array (IlluminaInc.). Samples were processed according to the manufac-turer’s standard protocol, and CNV calls were generatedusing the PennCNV algorithm (Wang et al., 2007).20

CNVs were classified into four different subgroupsaccording to the classification system used by Bartnik et al.(2012).21 Group A CNVs are of clinical relevance (occur-ring in epilepsy CNV hotspots, containing known epilepsygenes or are discordant deletions in the affected MZ twin).Group B type are of likely pathogenic effect (CNVs >1 Mbin length and/or discordant duplications in the MZ twinpairs). A group of CNVs of possible pathogenic effect(Group C) are CNVs > 500 kb containing no RefSeq genes,or CNVs > 300 kb with at least one RefSeq gene. Group Dclassification comprises all remaining CNVs. CNVs thatoverlapped by greater than 50% in each twin were classifiedas concordant CNVs.

Methylation analysisGenome-wide methylation analysis was undertaken using

the Illumina Infinium Human Methylation27 Beadchip(Illumina Inc.). The samples were processed using themanufacturers recommended protocol, and scanned on theIllumina BeadArray Reader using default settings. Dataanalysis was performed using the methylation module ofIllumina BeadStudio platform. These included the discor-dant MZ twins, three isolated LKS samples (patients 3, 4,and 5) and 14 controls. Quality control criteria for inclusionin the analysis included a 95% Cytosine-phosphate-guanine(CpG) site call rate, background signal <1,000 units andclear separation in the nonpolymorphic controls dashboard.Two control samples were excluded from the study. Individ-ual CpG sites were examined using the detection p-valuemetric provided by Illumina. A threshold of p < 0.05 wasused as a cut-off (1,422 probes excluded). Differences inaverage b of at least |0.3| were deemed to indicate differentialmethylation status.

Exome sequencing and data analysisWhole exome sequencing was performed at Atlas Biolabs

(http://www.atlas-biolabs.de). The exomes of individualswere enriched using three different enrichment kits (seeTable S1 for more information) and sequenced on an Illu-mina HiSeq platform. All chromosome locations are based

Table 1. Summary of the possible factors associated with

the etiopathogenesis of Landau-Kleffner syndrome (LKS)

Possible factors associated with LKS

Genetic Copy number variations2–4

Single mutations5–7

Somatic mosaicism

Complex genetic inheritance8

Environmental Immune response9,10

Metabolic changes11,12

Lead poisoning13

Structural abnormalities Astrocytoma14

Perisylvian polymicrogyria15

Focal cortical microdysgenesis16

Neurocysticercosis17


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on the March 2009 (NCBI37/hg19) assembly. Candidatevariants and genes were prioritized according to predictedprotein effects, protein length, brain expression patterns,and potential links with epilepsy. Further information relat-ing to exome sequencing, bioinformatic analysis, and geneprioritization can be found in Supplementary Methods. Asubset of these variants has been validated by Sangersequencing, and inheritance patterns were tested wherepossible (See Tables S4, S6, S8, and S10).

Finally, to test for enriched functional terms or pathways,all variants with novel or low frequency variants wereassessed using the Ingenuity Pathway Analysis (IPA) toolset(Ingenuity Systems, www.ingenuity.com).

ResultsCNV results

No group A or group B CNVs were identified in thispatient cohort. A single group C CNV was called

(chr12:50854891–51489850). This 634 kb deletion of12q13.13 in patient 8 contains 10 RefSeq genes; LARP4,DIP2B, ATF1, TMPRSS12, METTL7A, HIGD1C, SLC11A2,LETMD1, CSRNP2, and TFCP2. Fourteen group D CNVs,containing 16 genes, were identified (Table S2).

Methylation resultsCpG methylation was assessed in >27,000 CpG loci

across the genome. For twin set 1, there were 83 loci withincreased and 72 loci with decreased methylation in theaffected twin. This is comparable to the 162 increased and177 decreased methylated loci in twin set 2. As methylationdifferences between MZ twin pairs increase with age, thishigher number of differentially methylated probes in twinset2 is not unexpected.22 The correlation coefficient (0.91) ofthis twin set is lower than the younger twinset (0.95). Four-teen loci (13 genes) were found to be significantly differen-tially methylated in both twin sets. Five showed a signal inthe same direction (PEG10, CASP2, FGD2, GPR37L1, and

Table 2. Summary of clinical details of patients with Landau-Kleffner syndrome (LKS)

Patient ID number Gender

Family history of epilepsy, learning difficulties or

language impairment Current age Age at onset EEG abnormality ESES Epilepsy

1-a F No family history. Unaffected MZ twin remains

unaffected with normal EEG

8.5 years 3 years Yes Yes Rare

2-a M No family history. Unaffected MZ twin remains

unaffected with normal EEG

33 years 5 years Yes NA Yes

3 F No family history 20 years 5.5 years Yes Yes Yes

4 F No family history 29 years 3 years Yes Yes Yes

5 F No family history 15 years 4 years Yes Yes Yes

6 M Stammer present in father, paternal grandfather

and paternal uncle. Father also

has subtle language difficulties

17 years 4.5 years Yes Yes Yes

7 M No family history 10 years 6 years Yes Yes No

8 F Twomaternal aunts with speech delay 10 years 6.5 years Yes Yes Yes

9 F No family history 25 years 2.5 years Yes Yes Rare

10 M No family history 7 years 4 years Yes Yes Rare

11 M No family history 24 years 6.5 years Yes Yes Yes

12 M Cousin (paternal side) with autism and frequent

slow waves during sleep

12 years 4 years Yes Yes Rare

13 M No family history 6 years 5 years Yes Yes No

Patient ID number Treatment Response to Treatment Outcome - speech Outcome - IQ Outcome - EEG

1-a AED, Steroids Poor No speech Normal Normal

2-a AED Good Mild language disorder Mild LD Abnormal

3 AED, Steroids, Surgery Poor No speech Severe LD Abnormal

4 AED, Steroids Poor No speech Severe LD Abnormal

5 Steroids Good Good Normal None

6 AED, Steroids Good Good Mild LD Normal

7 Steroids Good, steroid responsive

relapses

Poor speech Borderline +Behavior disorder

Abnormal

8 Steroids Poor Poor speech Normal Abnormal

9 AED, Steroids Poor No speech Normal Normal

10 AED, Steroids Good Good Normal Normal

11 AED, Steroids Excellent Excellent Normal Normal

12 AED, Steroids Good Good Normal Normal

13 Steroids Good Good Normal Normal

M, Male; F, Female; ESES, electrical status epilepticus in sleep; IQ, intelligence quotient; AED, antiepileptic drugs; LD, Learning difficulties; NA, data not available.


3

LKS: A Comprehensive Genetic Study

GUCY2C, Table S3). When the affected individuals (n = 5)were compared against unaffected individuals (n = 12), nosignificantly different loci with a |Db| > 0.3 were identified.Twelve loci were identified at the |Db| > 0.25 level. Allmethylation results are presented in Table S3.

Exome sequencing resultsExome sequencing resulted in high quality data with

median targeted exome coverage of 94% for a minimum of10-fold coverage. Additional data quality details can befound in Table S1. The median number of novel variants perpatient was 44. Ranking of these variants and candidategenes using the criteria listed in Supplementary Methodsproduced normalized scores ranging from 94 in theSLC7A6OS gene to 0 in the EEPK1 gene (Table 3 andTable S4). It is important to note that low normalized scoresdo not eliminate a gene as a risk gene; it is merely beingused as a method to prioritize “more likely” candidategenes. Information on splice-site variants can be found inTable S5.

Rare variants with a minor allele frequency (MAF) of<1% were also identified in each patient (Table S6). A med-ian of 140 rare variants was identified per patient (n = 13).These variants were combined with the novel variants andassessed for gene overlap and variants within knownepilepsy genes (mucin and olfactory receptor genes wereexcluded). The maximum overlap of patients with novel orrare variants encompassed two genes (HSPG2 and NID2)present in five patients. The remaining number of overlap-ping genes in which rare or novel variants were identified isoutlined as follows: 9 genes in 4 patients, 45 genes in 3patients, and 241 genes in 2 patients. When analysis isrestricted to novel variants only, the overlap was reduced to28 genes in 2 patients and no overlapping genes in 3 or more

patients. Information on these variants can be found inTable S4 and Table S7. Mucins and olfactory receptors areexcluded based on a long protein length and high rates ofmutation. Novel variants in these genes are common andhighly unlikely to be related to the development of LKS.Further information on the NID2 and HSPG2 genes withrare variants and the patients with the variants can be foundin Table S8. Untranslated region (UTR) UTR variants werealso identified in five patients (5, 10, 11, 12, and 13) (seeTable S9). Interpretation of this data requires caution giventhe smaller number of control sequences with high readdepth. As a result this data set is more likely to contain non–disease-causing variants.

Forty-six novel and rare variants were identified in previ-ously reported epilepsy genes (n = 18 and 28, respectively)(Table 4 and Table S10). Five genes occurred more thanonce. These genes are BSN, COL18A1, GALR1, L2HGDH,and RELN. However, not all variants are expected to beprotein damaging (Table S10). A single variant was identi-fied in the GRIN2A gene. Additional information for thevariants located within epilepsy genes, CNVs, and com-pound heterozygotes can be found in Tables S4, S6, S7, S9,and S10.

Pathway analysis was conducted using all of the genesidentified with rare and novel variants. Although a numberof interesting pathways and functional terms had low p-values,none passed the significance threshold. A selection of thesepathways/terms, the genes they include, and the associatedp-values can be found in Table S11.

DiscussionThis study assessed the genetic contribution to the devel-

opment of LKS in a cohort of 13 patients with LKS from

Table 3. Top prioritized candidate novel variants per patient

Patient chromLoc Gene cDNA change Protein change Normalized final score Ref Variant Candidate variant

1 7:142693820–142693820 TMEM139 C427T R143X 83 C T

2 X:54065436–54065436 PHF8 C274T R92W 80 G A

3 8:109297869–109297869 EIF3E A899G Y300C 83 T C

4 7:138680968–138680968 C7orf55 G320A G107E 80 G A

5 1:22983809–22983809 EPHB2 G464A R155H 87 G A

6 3:81622288–81622288 GBE1 G2069C R690T 75 C G

7 16:9842103–9842103 GRIN2A G1553A R518H 80 C T

7 1:151500113–151500113 LOR G64T G22C 80 G T

8 19:14450329–14450329 GIPC1 G901A A301T 87 C T

9 1:22983809–22983809 EPHB2 G464A R155H 87 G A

10 16:66895521–66895521 SLC7A6OS A587G Y196C 94 T C

11 8:72918628–72918628 MSC G340A A114T 83 C T

12 20:47044443–47044443 ARFGEF2 G3022C G1008R 83 G C

12 2:9465724–9465724 ITGB1BP1 A350G Y117C 83 T C

13 11:33695548–33695548 CD59 G113A C38Y 87 C T

The top prioritized gene(s) are reported here for each patient. Prioritization is based on the likely protein effect (scored using Mutation Taster, SIFT, and Poly-phen2), protein length, brain expression (Human Protein atlas and UCSC), and links to epilepsy (using biograph.com). Details on scoring each category and the individ-ual ranking scores can be found in the Supplementary Methods and Table S4. Final scores have been normalized to 100. If more than one variant has an equal topscore, both variants are presented. All chromosome locations are based on the February 2009 (GRCh37/hg19) assembly. NA, data not available or variant not scored.


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four countries, including 11 singletons and two sets ofdiscordant MZ twins. CNVs, methylation, and exomesequencing analysis were all undertaken in an effort togenerate multifaceted analyses. A variant in the GRIN2Agene was identified in one patient (patient 7). The low num-ber of common genes highlights the complex genetic natureof this rare disorder, where each susceptibility variant mayaccount for the development of the disorder in only a smallproportion of the affected individuals. This is not uncom-mon in epilepsy research. Despite this, a number of interest-ing candidate genes have been cataloged. Additional studieswith independent samples and/or functional work may helpestablish rare causative mutations/genes.

A major strength of this study was the presence of twosets of discordant MZ twins. In rare diseases such asLKS, the number of such twins is extremely limited.Based on the observed incidence of LKS of one in978,000, the chance of MZ twin births, and assuming thatat least 50% of MZ twins are concordant for LKS, theoverall number of such a discordant twin set is estimatedto be one in 652 million.23,24 Several biologic processeshave been proposed and documented to explain diseasediscordance in MZ twin sets. These include discordantCNVs, discordant methylation, and somatic mosaicism.Although some studies report differences in CNV profilesbetween MZ twins, these numbers tend to be low. Theidentification of no discordant CNVs between two MZtwin-pairs is therefore not unexpected and has beenreported previously.25 With regard to the lack of variant

differences detected by exome sequencing in the discor-dant twins, again the low rate of de novo mutations withinthe exome region of the genome is in agreement with thedata published by other groups.25,26 Methylation studiesin both sets of discordant twins and in a subset of the iso-lated cases also failed to reveal large common statisticallysignificant changes between these twins, similar to previ-ous discordant twin studies.25,27 Furthermore a random Xchromosome inactivation pattern was found in theaffected twin (twin-pair 1, data not shown).

Somatic mosaicism can be identified experimentallyusing next-generation sequencing but has not yet beenassessed on our twin sets. Difficulties can arise if a variant isat a low frequency in the tissue undertaken for sequencing,for example, blood. In such cases, establishing true mosaicvariants while ruling out multiple false-positive results canbe particularly challenging. A recent study by Weber-Lehmann et al. (2013) was undertaken in an effort to determinethe usefulness of next-generation sequencing for differentiatingbetween MZ twins in a forensic setting.26 With ultra-deepwhole-genome sequencing they identified 12 potentialsomatic SNP candidates that could be used to distinguishbetween MZ twins, of which 7 were excluded based on mul-tiple alignment hits in the genome. Of interest, only one ofthe remaining five mutations were identified in the bloodDNA compared to the buccal and sperm DNA, highlightingthe need for multiple tissue sources to ensure the number offalse negatives is minimized. This illustrates the difficultiesthat can arise in somatic mosaic variant identification.

Table 4. Top epilepsy candidate genes

Patient chromLoc Gene Ref Variant Novel or rare variant MT final score SIFT score

7 16:9842103–9842103 GRIN2A C T Novel Disease causing Damaging

7 9:13178971–13178971 MPDZ T G Novel Disease causing Damaging

9 16:29732523–29732523 PRRT2 C T Rare Disease causing Damaging

8 3:49666434–49666434 BSN T A Rare Disease causing NS

11 3:49666438–49666438 BSN C T Rare Disease causing NS

12 9:13130070–13130070 MPDZ C T Rare Polymorphism Tolerated

3 7:102951126–102951126 RELN C T Rare Disease causing Tolerated

9 7:102989563–102989563 RELN C T Rare Disease causing Tolerated

5 2:166768840–166768840 SCN9A A G Rare Disease causing Tolerated

3 2:27333199–27333199 SLC30A3 C T Novel Polymorphism Damaging

Patient Polyphen 2 score cDNA change Protein change

7 Probably damaging c.G1553A p.R518H

7 Probably damaging c.A2176C p.T726P

9 Probably damaging c.C647T p.P216L

8 Probably damaging c.T4441A p.S1481T

11 Probably damaging c.C4445T p.P1482L

12 Possibly damaging c.G3919A p.E1307K

3 Benign c.G7438A p.G2480S

9 Benign c.G5284A p.V1762I

5 Benign c.T4612C p.W1538R

3 Possibly damaging c.G844A p.A282T

Epilepsy genes were identified in Carpedb.ua.eu, epiGad.org, and orpha.net. Rare and novel variants within these genes were noted, and their predicted proteineffect was estimated through the use of Mutation Taster, SIFT, and Polyphen2. All chromosome locations are based on the February 2009 (GRCh37/hg19) assem-bly. NA, data not available or variant not scored.


5


The presenting LKS phenotype may occur due to geneticvariants and/or environmental influences. Stochastic bio-logic processes in Caenorhabditis elegans have also beenobserved to mask the effect of some inherited mutations.28

There may be many paths to LKS, and not every individualwith a susceptible genotype may actually develop thesyndrome. This may also explain the discordant phenotypein the two sets of MZ twins. Recent studies suggest that the“missing heritability” in complex traits in yeast arise frommany loci, each of small effect.29 Large studies are requiredto map these loci and to identify potential environmentaltriggers, and thus pose a challenge in studies of LKS andother rare diseases.

Clearly there are limitations to exomic sequencing datain that the disease-causing variants may not be exomic orin an untranslated region (UTR). In an effort to avoid miss-ing disease-causing variants in the UTR regions of the gen-ome, the final four samples were processed using theAgilent All Exon version 5’+UTRs kit. Multiple geneswere found with overlapping UTR variants, including oneUTR variant (rs71528908) identified in a gene that wasalso found to have differential methylation in both discor-dant LKS twin pairs (see Table S9 for more information onUTR variants). However these results must be viewed withcaution. As the number of full genome sequencesincreases, many more rare and non–disease-causing vari-ants will be identified. Another limitation relates to regionsof low read depth across the exome. However, the effectsof this limitation are reduced. First, although regions oflow read depth do occur and it is also possible that exonshave not been captured by the array, these regions varyfrom patient to patient, and the degree of overlap is low.Second, the location of mutations can span multiple generegions (e.g., the GRIN2A gene).5–7 Third, the read depthin this study was high (median coverage with at least 10reads is 94%). Combining these three observations reduces,but does not eliminate, the possibility that a single disease-causing gene has not been detected in this analysis.

The overlap of genes with rare or novel variants wassmall. The maximum overlap was for two genes, each withvariants in five patients. These genes (HSPG2 and NID2)transcribe basement membrane proteins that interactdirectly with each other. Studies investigating the role of theother protein in the nidogen family (NID1) have alreadybeen reported as leading to deficits in neuronal excitabilityand synaptic plasticity.30 Nid1 knockout mice develop anepilepsy phenotype in the absence of obvious structuralabnormalities.31 The locations of the variants in thesepatients vary throughout the gene, but four of five variantsare located in domains that are highly conserved betweenNID1 and NID2. Two patients (7 and 12) share an identicalNID2 variant (C718T, pP240S). Additional variants in base-ment membrane proteins have also been reported to causeepilepsy including RELN, laminins, and collagens.32–34

Patients in this cohort have novel or rare variants in LAMA5,

LAMA1, LAMA4, NCAN, RELN, and ITGB5. However,because of the length of the HSPG2 and NID2 proteins(4,391 and 1,375 amino acids, respectively), it is possiblethat such overlaps are due to chance alone. In addition, thepredicted effects of these variants are highly variable (TableS8), and in the absence of functional work, the role of thesegenes in the development of LKS is speculative.

Although each gene that is identified is worthy of furtherconsideration and cannot be excluded as a candidate gene,those genes previously associated with epilepsy should bereviewed with particular care. Overall in this study 18genes associated with epilepsy contained novel variants,and 24 genes contained rare variants (n = 28). One variantof particular interest is a variant in the GRIN2A gene thatresults in an arginine to histidine change at site 518, a pre-dicted glutamate-binding site (patient 7). The GRIN2Agene has already been reported to be a candidate gene forrolandic-type epilepsy, including LKS, in a number ofstudies. Reutlinger et al. (2010) identified deletions inthree separate patients with rolandic epilepsy/electricalstatus epilepticus of sleep (ESES), with the critical regioncontaining just the GRIN2A gene.2 In a study of 61 LKSand continuous spike and waves during slow-wave sleepsyndrome (CSWSS) patients, a de novo deletion partiallyspanning the gene was also identified in one child withLKS without ESES.3 Three recent publications by Carvillet al.,5 Lemke et al.,7 and Lesca et al.6 identified GRIN2Amutations in a subset of patients with LKS.5–7 Althoughthe occurrence of just one variant in the GRIN2A gene mayappear low (7.6%), this occurrence is similar to that of theprevious studies, where the GRIN2A mutations were identi-fied in 5–20% of familial/sporadic cases with LKS. Fur-thermore, the finding of the Arg518His mutation in thisstudy, previously reported with functional studies by Lescaet al.6, supports a role for this variant in the developmentof LKS. Rare variants in two N-methyl-D-aspartate(NMDA) receptors, GRIN3A and GRIN2C, were also iden-tified in two other patients with LKS (two and four). TheseGRIN genes have not yet been linked with LKS and theirinheritance from unaffected parents reduces the likelihoodof their involvement in this form of epilepsy.

Another epilepsy gene that should be considered as acandidate gene for LKS is the BSN gene. This study hasidentified three variants, two rare and one novel, in twopatients. A mouse BSN mutant model exists in which micewith deletions of exons 4 and 5 develop myoclonic and/orgeneralized clonic epileptic seizures.35 One of these vari-ants lies within exon 5 and is a highly conserved amino acid(P1482L, phyloP score = 5.53, PhastCon score = 1). Addi-tional epilepsy associated genes of interest include SCN9Aand SLC30A3. A de novo variant in the RELN gene alsomay highlight a candidate gene of interest. The variant inpatient 3 (c.G7438A, p.G2480S) is predicted to result in theloss of the epidermal growth factor like (EGF-like) domainof the protein. This variant (rs150236371) has been reported


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previously in a small number of control individuals(MAF = 0.002). The gene has been linked to temporal lobeepilepsy and lissencephaly, in addition to a number of otherdevelopmental and psychiatric disorders (see Folsom andFatemi,36 for a recent review). Brain magnetic resonanceimaging (MRI) revealed no abnormalities in this patient.The predicted effect of this variant on protein function andstructure remains inconclusive. The Mutation Taster pro-gram concludes it has a “disease causing effect” (http://www.mutationtaster.org) whereas the SIFT (http://sift.jcvi.org) and Polyphen2 (http://genetics.bwh.harvard.edu/pph2/)programs suggest it is a “tolerated/benign” change. Many ofthe remaining candidate epilepsy genes, can be excludedbased on their involvement in genetic syndromes with fea-tures not seen in patients with LKS, for example, Mowat-Wilson syndrome (ZEB2), Warbug micro-syndrome(RAB3GAP1), and mitochondrial DNA depletion syndrome(POLG).

It is also important to assess nonepilepsy genes for theirpotential to cause LKS. The ephrin family of receptors hasbeen shown to be a major player in many developmentalprocesses, including the establishment of correct tonotopicconnections in the auditory system.37,38 In addition, ephrinproteins (including receptors) have been linked with epi-lepsy through the interactions with RELN (EFNB1/2/3),hippocampal synaptic plasticity, glutamate transporterabundance, and a reduction in seizure frequency incaMEK1-induced epilepsy.39,40 The EPHB2 protein has theability to promote the clustering of NMDA receptors andthe sensitization of the neurons to the effect of glutamate.Two patients with LKS (patients 5 and 9) have the samenovel variant in EPHB2 (G464A, R155H), which is pre-dicted to have detrimental effects on the protein. Two addi-tional patients had variants in EPHA5 (patient 10 =G2558A, R853Q; patient 12 = C717A, H239Q), althoughthe latter variant is predicted to have little effect.

In conclusion, this study identified a GRIN2Amutation ina patient with LKS and it has highlighted a number of inter-esting candidate genes. Exome sequencing of a larger cohortof patients with LKS and functional studies will be neces-sary to identify patient overlaps and to clarify the role ofthese genes in the development of this rare epilepsy syn-drome. If there are multiple genes in which mutations leadto the development of LKS, then the sample size may be toosmall to identify common overlaps. This is a commonoccurrence with rare and ultra-rare disorders, where samplecollections are limited primarily by the prevalence of thesedisorders in the general population. In such cases the over-lap of many smaller studies will lead to identification ofadditional candidate genes.

AcknowledgmentsThe authors of this paper would like to thank the patients and family

members who participated in the research. We would also like to thank the

Children’s Fund for Health, Children’s University Hospital, Temple Street,for funding this project.

DisclosureNone of the authors has any conflict of interest to disclose. We confirm

that we have read the Journal’s position on issues involved in ethical publi-cation and affirm that this report is consistent with those guidelines.

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Supporting InformationAdditional Supporting Information may be found in the

online version of this article:Table S1. Sequence output and quality information.Table S2. CNVs of unlikely clinical consequence.Table S3.Methylation status in discordant MZ twin pairs

and case-controls.Table S4.Novel variants and their prioritization scores.Table S5. Rare splice-site variants identified in LKS

cohort.Table S6. Rare variants identified in the LKS cohort.Table S7.Genes with novel variants in two patients.Table S8. Evaluation of predicted protein effects in

HSPG2 and NID2 genes.Table S9.UTR variants identified in LKS cohort.Table S10. Epilepsy candidate genes.Table S11. Ingenuity pathway analysis results.Data S1. Supplementary information.


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J. Conroy et al.

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Towards the identification of a genetic basis for Landau-Kleffner syndrome