University of Groningen Genotyping and phenotyping epilepsies of childhood Vlaskamp, Danique

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Genotyping and phenotyping epilepsies of childhood

Vlaskamp, Danique

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Chapter 2

Copy number variation in

a hospital-based cohort of

children with epilepsy

Published as: DRM Vlaskamp, PMC Callenbach, P Rump, et al. Copy number variation in a hospital-based cohort of children with epilepsy. Epilepsia Open, 2017; 2: 244–254.

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Danique RM Vlaskamp1,2_{, Petra MC Callenbach}1*_{, Patrick Rump}2*_{, Lucia AA Giannini}2_{, Trijnie}

Dijkhuizen2_{, Oebele F Brouwer}1_{, Conny MA van Ravenswaaij-Arts}2

1_{University of Groningen, University Medical Center Groningen, Department of Neurology,} Groningen the Netherlands. 2_{University of Groningen, University Medical Center Groningen,} Department of Genetics, Groningen, the Netherlands. * These authors contributed equally to this work

Acknowledgements. P.M.C. Callenbach received an unrestricted research grant from UCB Pharma BV, the Netherlands. UCB Pharma BV had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. We thank K. McIntyre for editing the manuscript and J. Anderson for helping identifying CNVs for epilepsy reported in the literature.

Disclosures. None of the authors has any conflicts of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

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ABSTRACT

Objective. To evaluate the diagnostic yield of microarray analysis in a hospital-based cohort of children with seizures and to identify novel candidate genes and susceptibility loci for epilepsy. Methods. Of all children who presented with their ﬁrst seizure in the University Medical Center Groningen (January 2000 through May 2013) (n = 1,368), we included 226 (17%) children who underwent microarray analysis before June 2014. All 226 children had a deﬁnite diagnosis of epilepsy. All their CNVs on chromosomes 1-22 and X that contain protein-coding genes and have a prevalence of <1% in healthy controls were evaluated for their pathogenicity.

Results. Children selected for microarray analysis more often had developmental problems (82% vs 25%, p < 0.001), facial dysmorphisms (49% vs 8%, p < 0.001) or behavioral problems (41% vs 13%, p < 0.001) than children who were not selected. We found known clinically relevant CNVs for epilepsy in 24 of the 226 children (11%). Seventeen of these 24 children had been diagnosed with symptomatic focal epilepsy not otherwise specified (71%) and five with West syndrome (21%). Of these 24 children, many had developmental problems (100%), behavioral problems (54%) or facial dysmorphisms (46%). We further identified five novel CNVs comprising four potential candidate genes for epilepsy: MYT1L, UNC5D, SCN4B and NRXN3.

Significance. The 11% yield in our hospital-based cohort underscores the importance of microarray analysis in diagnostic evaluation of children with epilepsy.

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INTRODUCTION

Genetic factors play an important role in the etiology of epilepsy,1_{as demonstrated by the large}

number of genes and regions that cause or predispose to epilepsy newly identiﬁed by various genome-wide technologies.2_{Chromosomal microarray analysis, in particular, enables the}

identiﬁcation of chromosomal deletions (losses) or duplications (gains), called Copy Number Variants (CNVs).3_{CNVs may contribute to epilepsy in two ways. First, CNVs that comprise epilepsy-related}

genes could lead to epilepsy following a Mendelian inheritance. For example, both KCNQ2 sequence variants and whole gene deletions can cause benign familial neonatal seizures.4,5_{Second, CNVs}

that occur more frequently in patients compared to healthy controls may increase an individual’s susceptibility to developing epilepsy, with the responsible haploinsuﬃcient gene(s) often being unknown. Large cohort studies have identiﬁed such susceptibility CNVs in several chromosomal regions, including well-known CNVs located at 15q11.2 (BP1-BP2), 15q13.3 and 16p13.11.6-8

Studies using microarray analysis have most often been performed in research cohorts of children who were selected based on their epilepsy diagnosis, e.g. idiopathic generalized epilepsies, focal epilepsies and/or fever-associated epilepsies.6-8_{Only a few studies have addressed the yield of}

microarray analysis in clinical cohorts of all children presenting with any type of seizures in a clinical setting.9-11_{We therefore aimed to evaluate the diagnostic yield of microarray analysis in a}

hospital-based cohort of children with epilepsy for whom detailed phenotypic information was available, with the further goal of identifying novel candidate genes or susceptibility loci for epilepsy.

MATERIAL AND METHODS

Study cohort

The study cohort was derived from the childhood seizure database of the University Medical Center Groningen (UMCG), a regional referral center for children with epilepsy. In this database, we retrospectively included all children who presented with their ﬁrst febrile or afebrile seizure before the age of 18 years between January 2000 and June 2013, and who were seen and/or treated by a child neurologist of the UMCG (n = 1,368). Epilepsy was diagnosed in 91% of these children using the current International League Against Epilepsy (ILAE) practical clinical deﬁnition of epilepsy.12_{Of the remaining children (9%), 7% had febrile seizures only and 2% had only one}

afebrile seizure. The UMCG database contains phenotype information and was independently completed by two researchers (DRMV and PMCC). Phenotypic inconsistencies and epilepsy classification were discussed until agreement was reached using the information in the database as well as in the original medical records (PMCC and OFB). Epilepsy syndromes and seizure types were classified according to the 2006 ILAE classification.13_{Children were included in this study if}

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Formal independent review board evaluation was waived by the Institutional Medical Ethical Committee of the UMCG because of the retrospective and observational character of this study.

Chromosomal microarray analysis and data interpretation

Microarray analyses were performed using an oligonucleotide array (Agilent 105K or 180K custom HD-DGH microarray; Agilent Technologies, Santa Clara, USA) or a single nucleotide polymorphism (SNP) array (Illumina Omni Express 12-V1.0; Illumina, San Diego, USA). Cartagenia Bench Lab CNV software was used for storage, analysis and reporting of the structural genomic data (Cartagenia, Leuven, Belgium; part of Agilent Technologies, Santa Clara, USA). The chromosomal coordinates of CNVs were reported relative to the Genome Reference Consortium Human Reference genome version 37 (GRCh37/hg19).

CNVs on chromosome 1-22 or X identiﬁed by at least three (SNP microarray) or four (oligonucleotide microarray) consecutive probes were evaluated for their pathogenicity (Figure 1). CNVs were excluded from further analysis when they did not contain (protein-coding) genes or had ≥90% overlap with CNVs seen in ≥1% of healthy controls. The prevalence of CNVs in healthy controls was calculated using the International Database of Genomic Variance (n = 14,316, last updated February 2013),14_{the Low Lands Consortium database of oligonucleotides (n}

= 2,402, last updated December 2012) and SNP microarray results of healthy parents of children who underwent microarray analysis in ﬁve Dutch genetic centers (n = 749, last updated October 2014). Remaining CNVs were categorized into two groups: (1) CNVs with <90% overlap with CNVs observed in healthy controls and (2) CNVs with ≥90% overlap with CNVs observed in <1% of the healthy controls (Figure 1). CNVs in both groups were marked as potentially clinically relevant if they had overlap with genetic regions previously associated with epilepsy. These regions were identiﬁed by performing a literature search using PubMed, complemented with information from the Decipher database and Cartagenia Bench Lab CNV software. The remaining CNVs in both groups were evaluated for novel candidate genes or susceptibility loci for epilepsy. CNVs with <90% overlap with CNVs of healthy controls were of interest if they contained a gene with an expression or function in the brain or a gene associated with an autosomal dominant or X-linked neuropsychiatric disease, and if they occurred in at least one (for deletions) or two (for duplications) unrelated children in our cohort. In the group of CNVs with ≥90% overlap with CNVs observed in <1% of the healthy controls, overlapping regions between CNVs in at least two unrelated children were of interest if these regions contained protein-coding genes and were 10 times more prevalent in our cohort compared to healthy controls.

Statistical analyses

SPSS Statistics Version 22.0 (IBM Corporation, NY, USA) was used to perform descriptive and comparative statistics. Diﬀerences in categorical and ordinal phenotypic data between children were analyzed using Fisher’s exact and Mann-Whitney U tests, respectively.

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RESULTS

Characteristics of the study cohort

In 226 (17%) children, microarray analysis was performed in the context of their diagnostic work-up. Their phenotypic characteristics are summarized in Table 1. All children had a deﬁnite diagnosis of epilepsy, except for one who had a single febrile status epilepticus.

Children with epilepsy who underwent microarray analysis had significantly more developmental problems (82% vs 25%, p < 0.001), facial dysmorphisms (49% vs 8%, p < 0.001) or behavioral problems (41% vs 13%, p < 0.001) when compared to children in our database who did not undergo microarray analysis. To reduce bias, our comparisons were limited to children with epilepsy onset after December 31, 2005, when microarray analysis was introduced in our center (n = 158 for children with array; n = 271 for children without array or another identified genetic cause). The presence of a positive family history for epilepsy, known in 141/158 children who did and in 206/271 children who did not undergo microarray, did not differ significantly (33% vs 30%, p = 0.56) between the two groups.

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Figure 1: Flowchart for evaluating Copy Number Variants (CNVs) in our hospital-based cohort of

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Table 1: Characteristics of the study cohort (n = 226) Characteristics

Male (%) 132 (58.4)

Deceased (%) 17 (7.5)

Median age at evaluation (range) 8y 10mo (1y 8mo – 23y 3mo)

Median age at epilepsy onset (range) 1y 1mo (0 days – 15y 11mo)

Seizure types (%) GTCS 10 (4.4) Absences 5 (2.2) Myoclonic seizures 18 (8.0) Epileptic spasms 36 (15.9) Atonic seizures 12 (5.3) Focal seizures 176 (77.9)

Secondarily generalized seizures 123 (54.4)

Neonatal seizures 33 (14.6)

Unclassiﬁed 1 (0.4)

Status epilepticus (%) 74 (32.7)

Epilepsy syndrome (%)

Benign (familial) neonatal seizures 7 (3.1)

Neonatal seizures (not benign) 1 (0.4)

Ohtahara syndrome 1 (0.4)

Benign familial infantile seizures 4 (1.8)

West syndrome 36 (15.9)

Myoclonic epilepsy in infancy 3 (1.3)

Myoclonic encephalopathy in nonprogressive disorders 1 (0.4)

Benign epilepsy with centrotemporal spikes 5 (2.2)

Childhood absence epilepsy 1 (0.4)

Epilepsy with myoclonic absences 1 (0.4)

CSWS / Landau-Kleﬀner syndrome 9 (4.0)

Lennox-Gastaut syndrome 5 (2.2)

Juvenile absence epilepsy 1 (0.4)

Symptomatic focal epilepsies n.o.s. 148 (65.5)

Localization-related cryptogenic epilepsy 24 (10.6)

Other symptomatic generalized epilepsy 2 (0.9)

Epilepsy with both generalized and focal seizures 3 (1.3)

Febrile seizures plus 5 (2.2)

Febrile infection related epilepsy syndrome 1 (0.4)

One febrile status epilepticus 1 (0.4)

One seizure likely to re-occur 2 (0.9)

Epilepsies of unknown cause 1 (0.4)

Seizure free* (%) 89/192 (46.4)

Family history of epilepsy** (%) 68/200 (34.0)

Developmental problems in speech, language, motor skills

and/or cognition (%) 195 (86.3) Behavioral/psychiatric problems (%) 104 (46.0) Microcephaly (≤ -2SD) (%) 40 (17.7) Macrocephaly (≥2 SD) %) 13 (5.8) Short stature (≤ -2SD) (%) 39 (17.3) Tall stature (≥2 SD) %) 13 (5.8) Facial dysmorphisms (%) 109 (48.2) Congenital anomalies (%) 72 (31.9) MRI abnormalities (%) 118/218 (54.1)

Epilepsy syndromes and seizure types were classified according to the International League Against Epilepsy (ILAE) classification of 2006.13_{* Seizure freedom was defined as present if a patient had no clinical seizures for at least one year at the time of}

evaluation. ** Family history of epilepsy was positive if a ﬁrst or second degree relative has epilepsy. Abbreviations: CSWS = continuous spike during slow-wave sleep, GTCS = generalized seizures with tonic and/or clonic manifestations, n.o.s. = not otherwise speciﬁed, SD = standard deviation.

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Diagnostic yield of microarray analysis

Microarray analysis revealed 1,982 CNVs in 226 children (Figure 1). After excluding CNVs that contained no (protein-coding) genes and/or were identiﬁed as most likely benign polymorphisms (≥90% overlap in ≥1% of controls), 408 CNVs in 181 children remained to be evaluated for their pathogenicity. These 408 CNVs included 233 (57.1%) duplications with a median size of 198.8 kb (range 17.9 kb - 21.0 Mb) and 175 (42.9%) deletions with a median size of 168.4 kb (range 22.4 kb - 21.0Mb). Inheritance could be analyzed for 102 (25.0%) CNVs, with 23 (22.5%) occurring de novo and 79 (77.5%) being inherited.

Known clinically relevant CNVs for epilepsy were identified in 24 of the 226 (11%) children with epilepsy (Figure 1, Tables 2 and 3). Their epilepsy was most often classified as symptomatic focal epilepsy not otherwise specified (71%) or West syndrome (21%). All children had developmental problems (100%), and many had behavioral problems (54%) and facial dysmorphisms (46%). Overall, no significant differences were found between children with and without clinically relevant CNVs for epilepsy syndrome diagnosis or the presence of other phenotypic characteristics (data not shown). In 14 (7%) children, 15 known clinically relevant CNVs were found that do not occur in healthy controls (Table 2). Ten of these CNVs occurred de novo. For the remaining CNVs, inheritance was unknown. The phenotypes of these 14 children were compatible with previously reported phenotypes associated with these CNVs and included the well-established diagnoses: chromosome 1p36 deletion syndrome (MIM 607872), chromosome 2q23.1 deletion syndrome (MIM 156200), chromosome 18q deletion syndrome (MIM 601808), Angelman syndrome (MIM 105830), chromosome 15q11q13 duplication syndrome (MIM 608636), chromosome 16p11.2 deletion syndrome (MIM 611913), lissencephaly type 1 (MIM 607432), Phelan-McDermid syndrome (MIM 606232), and Juberg-Hellman syndrome (MIM 300088; epilepsy, female restricted, with mental retardation) (Table 2). One patient had a 2.6 Mb 8q22 deletion, comparable deletions have been published in six other cases.16,17 _{In 10 (4%) children, known clinically relevant CNVs}

were found that also occur in healthy controls, albeit in less than 1% (Table 3). Six of these CNVs (60%) were inherited from an affected (n = 2) or non-affected (n = 4) parent while two (20%) CNVs occurred de novo (the deletions including NRXN1 and CHRNA7). In three (30%) children, another cause for epilepsy - one not associated with the CNVs - was identified. These were developmental anomalies of cerebral structure (patient 822), a GLUT1-deficiency (patient 225) and polymicrogyria (patient 761) (Table 3).

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T able 2: Clinically r elevant CNV s with <90% ov er lap with CNV s obser ved in health y contr ols (n = 14) Pati en t Se x, a ge (y ) Micro arr ay r esu lts CNV size in k ilobases Inheritanc

e tal phenot aren (p

ype) Rele vant genes Epilepsy syndrome Age a t e pil ep sy on se t Age a t la st s eiz ure Dev elopmen tal problems Behavior al problems Mic ro ce ph aly ( ≤ -2 SD ) Ma cro ce ph aly ( ≥2 S D) Sh ort s ta tu re ( ≤ -2 SD ) Tal l sta tu re ( ≥2 S D) Facial dysmorphisms Conge nital anomalies MR I abnormalit ies 1 04 0 F, 3 .5 † Ar r 1 p 34 .1 p 33 (4 6, 08 9,4 75 -4 6, 73 8, 33 3)x 3 a 64 9 De n ov o Un kn ow n Fo ca l k 1y 3. 5y † + -+-+-+ +-10 32 M , 8 ar r 1 p 36 .3 3p 36 .3 1( 74 6, 419 -5 ,6 96 ,74 5) x1 b 4, 950 De n ov o GA BR D KL HL 17 W S 3m o 21 m o + - ++ -59 0 F, 1 0 ar r 1 p 36 .3 3p 36 .2 3( 56 4, 22 4-8, 10 4, 81 2) x1 b 7, 54 1 U n kn o w n GA BR D KLHL 17 KCNAB 2 WS, _foca l k 1m o N A + ---+ + + 1 83 M , 1 1 ar r 2 q 23 .1 (1 48 ,7 75 ,3 16 -1 49 ,0 02 ,6 34 )x 1 c 22 7 De n ov o M BD 5 Fo ca l k 2y _3mo 2y _3mo + + ---+ 1 10 5 F, 2 ar r 2 q 22. 3q 23 .3 (1 46, 50 6, 57 9 -1 51 ,3 55 ,7 90 )x 1 c 4, 84 9 U n kn o w n MBD 5 Fo ca l k 2m o 9m o + --+-+ + + 5 75 F, 6 ar r 8 q 22 .3 (1 01 ,7 95 ,0 20 -1 04 ,4 06 ,4 06 )x 1 2, 61 1 De n ov o Un kn ow n Un d et . l 1y 4. 5y + + ----+ + + 10 37 M , 4 † ar r 1 3q 31 .3 q 34 (9 4, 01 7, 65 5-11 5, 10 5,9 59) x3 , 18 q2 1. 32 q2 3 ( 56 ,9 21 ,0 91 -7 8, 01 0, 172 )x 1 d 21 ,0 88 21 ,0 89 Un kn ow n Un kn ow n Un kn ow n Un kn ow n WS, _foca l k 2y 4y † + -+-+-+ + U 1 079 M , 4 ar r 1 5q 11 .2q 13 .1 (2 2, 28 5, 09 1-28 ,9 40 ,2 39) x1 e 6, 65 5 De n ov o GA BR B3 UB E3 A Fo ca l k 7m o N A + ---+ U

2

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C ontinuation table 2 Pati en t Se x, a ge (y ) Micro arr ay r esu lts CNV size in k ilobases Inheritanc e (p aren tal phenot ype) Rele vant genes Epilepsy syndrome Age a t e pil ep sy on se t Age a t la st s eiz ure Dev elopmen tal problems Behavior al problems Mic ro ce ph aly ( ≤ -2 SD ) Ma cro ce ph aly ( ≥2 S D) Sh ort s ta tu re ( ≤ -2 SD ) Tal l sta tu re ( ≥2 S D) Facial dysmorphisms Conge nital anomalies MR I abnormalit ies 3 56 M , 1 6 ar r 1 5q 11 .2 q1 3. 1( 22 ,6 68, 85 2-29 ,0 45 ,4 87 )x 3 f 6, 37 7 De n ov o GA BR B3 UB E3 A Fo ca l k 7y 14 y + + - + 1 06 2 M , 5 ar r 1 5q 11 .2 q1 3. 1( 22 ,6 68, 85 2-29 ,0 60 ,6 34 )x 3 f 6, 39 2 De n ov o GA BR B3 UB E3 A Fo ca l k 8m o N A + + 3 19 F, 1 3 ar r 1 6p 11 .2 (29 ,6 20, 489 -3 0, 19 9, 50 7) x1 g 579 De n ov o PR RT 2 BF IS 4m o 4y + + 1 08 1 M , 4 ar r 1 7p 13. 3( 2, 35 5, 35 3-3, 32 2,77 9) x1 h 96 7 D e n ov o PA FA H1B1 WS, fo cal k 3m o N A + -+ ---+ 2 01 M , 1 6 ar r 22 q1 3. 3( 51 ,1 25 ,3 51 -5 1, 21 9, 15 0) x1 i 94 U n kn o w n SH A N K3 Un d et . l 9y N A + + ----+ + 8 31 F, 1 1 ar r X q 22 .1 (9 9, 58 2, 92 1-99 ,6 71 ,0 28 )x 1 j 88 D e nov o PC DH 19 Fo ca l k 2y N A + ---+ + -Th e chro m o so mal c o o rdinat es are rep o rt ed relati ve t o th e G en o m e Ref er enc e C on sor ti u m H u m an Ref er enc e ge nome v ers ion 3 7 ( G RC h 37 /hg 19 ). a P re vi o u sl y p u b lis h ed b y s o m e o f u s. 15 b C h ro m o so m e 1 p 36 d el et io n s yn d ro m e ( M IM 6 07 87 2) . c C h ro m o so m e 2 q 23 .1 d el et io n s yn d ro m e ( M IM 1 56 20 0) . d C h ro m o so m e 1 8q d el et io n s yn d ro m e ( M IM 6 01 80 8) . e Angel m an s ynd rome (M IM 1 05 83 0) . f Chro m o so m e 1 5q 11 -1 5q 13 dup lic ati o n s yn d ro m e ( M IM 6 08 636 ). g Chr o m o so m e 1 6p 11 .2 d el etio n s yn d ro m e ( M IM 6 11 91 3) . h L iss en ce p h al y t yp e 1 ( M IM 6 07 43 2) . i Ph el an -M cD er m id sy n d ro m e ( M IM 6 0 62 32 ). j J u b er g -H el lm an s yn d ro m e ( M IM 3 0 0 08 8; e p ile p sy , f em al e r es tr ic te d w it h m en ta l r et ar d at io n ( EF M R )). k Sy mpt o mati c fo ca l ep ileps y n o t oth er w is e sp eci ﬁe d . l Ot h er un d et er m in ed ep ileps y w ith b o th g en er ali ze d an d fo ca l s ei zure s. + P h en ot yp e is p re sen t in th e chil d . – P h en ot yp e is abs en t in t h e chil d . A b b rev iati o n s: B FI S = b eni gn f amilial inf an til e s ei zure s, F = femal e, M = mal e, m o = m o n ths , NA = n o t ap p lic ab le (n ot s ei zure -f re e) , U = unk n ow n, W S = W es t s yn d ro m e, y = year s, † = d ec eas ed .

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2

T able 3: Clinically r elevant CNV s with ≥90% ov er lap with CNV s obser ved in <1% of health y contr ols (n = 10) Pati en t Se x, a ge (y ) Micro arr ay r esu lts CNV size in k ilobases Inheritanc

e tal phenot aren (p

ype) Rele vant genes Epilepsy syndrome Age a t e pil ep sy on se t Age a t la st s eiz ure Dev elopmen tal problems Behavior al problems Mic ro ce ph aly ( ≤ -2 SD ) Ma cro ce ph aly ( ≥2 S D) Sh ort s ta tu re ( ≤ -2 SD ) Tal l sta tu re ( ≥2 S D) Facial dysmorphisms Conge nital anomalies MR I abnormalit ies 8 22 F, 6 ar r 1 q 21 .1 (1 45 ,3 95 ,1 97 -1 46 ,0 89 ,2 61 )x 3 69 4 Pa t ( n on e) U nk n ow n W S, fo cal a 6m o N A + + ++ --+ 3 37 F, 1 3 ar r 2 p1 6. 3( 50 ,9 68, 25 2-51 ,5 79 ,8 62 )x 1 b 612 De n ov o N RX N 1 Fo ca l a 3. 5y N A +-+-+-+ + + 1000 M, 1 6 arr 1 5q 11 .2 (2 0, 27 9,34 3-23 ,3 00 ,4 38 )x 1 3,0 21 Pa t ( n one ) CY FI P1 , NIP A1 , NIP A 2 Fo ca l a 11 y 13 y + + ---+ 225 M , 1 1 ar r 1 5q 11 .2 (22 ,6 98, 322 -2 3, 21 7, 65 5) x1 c 51 9 Pa t ( n o n e) CY FI P1 , NIP A1 , NIP A 2 Fo ca l a 6y N A + + ---10 99 F, 8 ar r 15 q13 .3 (3 0, 92 1, 71 7-32 ,5 15 ,12 1) x1 1, 59 3 De n ov o C H RN A 7 Fo ca l a 4y N A + 3 72 M , 9 ar r 1 5q 13 .3 (3 0, 83 3,54 6 -3 2, 86 1, 76 7)x 3 2, 02 8 Ma t ( n on e) CH RN A 7 Fo ca l a 11 m o 6y + + ---+ 7 61 F, 1 3 ar r 1 6p 11 .2 (2 9, 62 0,4 88 -3 0, 19 8, 75 2) x3 57 8 U nk now n PR RT 2 C SW S 4 y N A + + ---+ 7 30 M , 1 2 ar r 1 6p 11 .2 (2 9,5 92 ,5 82 -3 0, 19 8, 75 2) x3 60 6 U nk now n PR RT 2 Fo ca l a 1m o 8y + + ---+ 2 70 M , 1 4 ar r 1 6p 13 .11 (1 4, 94 4, 35 9 -1 6,5 25 ,4 88 )x 1 1,5 81 Pa t ( FS) NDE 1 O n e F S 2. 5y 2. 5y + + -+ -U 1 04 5 M , 6 ar r 1 6p 13. 11 (1 4, 94 4, 36 0 -1 6, 56 1, 29 2) x1 1, 61 7 M at (F S) NDE 1 M A E 1. 5y N A + ---+ --Th e chro m o so mal c o o rdinat es are rep o rt ed relati ve t o th e G en o m e R eferen ce C o ns o rtium Human R eferen ce g en o m e ver si o n 3 7 ( G R C h3 7/ hg 19 ). a S ympt o ma tic fo ca l epileps y n o t o th er w is e sp ec iﬁ ed . b T h is p ati en t al so c ar ri es a chro m o so m e 1 4 q 31 .1 d el eti o n in clu d in g th e NR XN 3 ge ne . c T his p ati en t al so c ar ri es a likel y p ath o g eni c s equ en ce v ar ian t in th e SL C 2A 1 g en e ass o ciat ed w ith G LU T-1 d eﬁ ci en cy . + P h en ot yp e is p re sen t in th e chil d . – P h en ot yp e is abs en t in th e chil d . A b b rev iati o n s: B EC TS = b eni gn ep ile ps y w ith c en trot emp o ral sp ike s, C AE = chil dh o o d abs en ce ep ileps y, C SW S = c o n tinu o us sp ike w ave s dur in g sl ow -w ave sl ee p s yn d ro m e, F = femal e, F S = feb ril e s ei zure s, JM E = ju venil e m yo cl o ni c ep ileps y, M = mal e, mat . = mat er n al, M A E = ep ileps y w ith m yo cl o ni c abs en ce s, m o = m o n ths , NA = n o t ap p lic ab le ( n ot s ei zure -f re e) , U = unk n ow n, p at = p at er n al, W S = W es t s yn d ro m e, y = year s.

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Novel CNVs of interest for epilepsy

In five (2%) children, we identified novel CNVs of interest that were not found in healthy controls (Table 4). These CNVs comprised four potential candidate genes for epilepsy: MYT1L, UNC5D, SCN4B and NRXN3 (see Supplemental Table 1 for more information on these genes). In 16 (7%) children, eight different overlapping deletions (n = 4) and duplications (n = 4) occurred at a 10 times higher frequency in our cohort than in healthy controls (Supplemental Table 2). However, another cause for epilepsy was identified in seven (44%) of these children, with CNVs involving six of the eight regions. The two remaining regions did not contain genes that seem of particular interest for epilepsy (Supplemental Table 2).

DISCUSSION

Our study was performed in a university hospital-based cohort of children with epilepsy, who were selected to undergo microarray analysis as part of their diagnostic work-up based on their doctors’ preference. We found that microarray analysis yielded known clinically relevant CNVs for epilepsy in 11% of the children. We further identiﬁed ﬁve novel CNVs of interest for epilepsy in 2% of the children.

Diagnostic yield of microarray analysis

The 11% yield of microarray analysis in our cohort is comparable with the 9% yield found in another clinical cohort of American individuals with epilepsy.9_{Higher yields of 36-40% have been}

reported in smaller cohorts of Saudi individuals with epilepsy.10,11_{Diﬀerences in yield between}

studies is probably due to the selection of children for microarray analysis, which is often based on the presence of additional features other than epilepsy. For example, a higher yield of microarray analysis is found in individuals with epilepsy when the epilepsy is accompanied by global developmental delay or cognitive dysfunction.18_{In our database, children who}

underwent microarray analysis more often had developmental problems, facial dysmorphisms and behavioral problems when compared to those who did not, i.e. the presence of such comorbidities prompted the treating physicians to request a microarray analysis. Probably due to this selection, we found no diﬀerences in epilepsy syndrome diagnosis or the presence of other phenotypic characteristics between children with and without clinically relevant CNVs. We found a 2.6 Mb 8q22 deletion in one child. She (patient 575) is the seventh individual reported with such a deletion so far and shares the combination of absence seizures and focal seizures with two of the previously reported children.16,17_{An eighth individual, listed in the}

DECIPHER database (case 2846), also has a 8q22 deletion and absence seizures. Thus, both focal and generalized (especially absences) seizures may occur in patients with 8q22 deletions. The smallest region of overlap harbors two candidate genes for epilepsy, NCALD (MIM 606722) and

(14)

T able 4: Nov el CNV s in our cohor t of childr en with epilepsy (n = 5) Pati en t (se x, a ge i n ye ars ) Micro arr ay r esu lts, inheritance CNV size in k ilobases Rele vant genes Seizure t ypes (e stimate d number of seizures) Age a t a cti ve ep ile psy Epilep tiform activit

y on) ati (localiz EG on E

An ti-e pileptic dr ugs (eff ect iv en ess) Epilepsy syndrome Add iti on al f ea tu re s MR I abnormalit ies 98 1 (M , 1 6) ar r 2 p 25 .3 ( 1, 71 1, 39 9 -2, 07 8, 55 7) x1 , de nov o 376 MY T1 L Fr on ta l abs enc es (3 /d ay ), s ec . gen . sz . (4) 3y - 1 2y G en . 3 H z SW C, fo cal spik es an d SW C (f ro nto -temp., L> R) ET X (-) LE V ( + ) Fo ca l b De ve lo p m en ta l pr ob lems, auti sm , pu b er ta s prae co x, upslan ting p alp ebr al fis su re s N o rmal 626 (F, 9 ) ar r 8 p12 (3 5, 12 0, 62 1-35 ,3 58 ,3 15 )x 1, pa te rn al (f at h er has migr ain e) 23 8 UN C 5D Fo cal S E ( 1) , fo cal sz . ( 10 ) 3y – ongoi n g G en . SW C ( m ax . bif ron t.) , f o ca l sh ar p w aves an d SW C ( L t emp. ) VP A (s id e-effe ct s) C BZ ( + ) Fo ca l b D evelopm en ta l an d b eh aviora l pr oble m s MT S an d p o ssib le lef t c o rt ical dys pl as ia 31 (M , 23 ) ar r 1 1q 23 .3 ( 11 7, 95 1, 62 9 -11 8, 02 2, 70 0) x1 , u n kn o w n 71 SCN4 B Fo ca l s z. (u nk now n) , s ec . gen . s z. ( 1) 14 y – unk n ow n Norm al VP A (s id e-effe ct s) TP M ( + ) LT G ( u nk now n) Fo ca l b Pe rin ata l a sph yx ia , developm en ta l an d b eh aviora l pr oble m s, dys ki ne si a Diff u se whit e ma tt er abnorm al it y 33 7 (F , 1 3) ar r 1 4q 31 .1 (7 9, 33 5,4 93-79 ,6 54 ,2 45 )x 1 a, de nov o 31 9 NR XN 3 Fo ca l s z. (u n kn o w n) 3. 5y – ongoi n g Fo ca l s har p w aves ( o cc .) None F o ca l b De ve lo p m en ta l pr ob lems, h earing loss, micr o cep hal y, thick eyebr o ws, de ep ly s et eyes, en tr opion, thin

lips, high nas

al br idge , abnor mal p o si tion of e ar s, s hor t s ta tur e, pec tu s ex ca va tu m M ega ci st er na ma gna 96 9 (F , 8 ) ar r 1 4q 24 .3q 31 .1 (7 6, 62 1, 11 6 -7 9, 82 8, 26 9)x 1, de nov o 3, 20 7 NR XN 3 Fo cal S E ( 1) , fo cal sz . ( 2-4/ m o n th ) 2y - 4 y N o rmal V PA ( + ) Fo cal b PD D -NO S, epic an thal fo ld, fif th fi nger cl ino d ac ty ly N o rmal Th e chro m o so mal c o o rdinat es are rep o rt ed relati ve t o th e G en o m e R eferen ce C o ns o rtium Human R eferen ce g en o m e ver si o n 3 7 ( G R C h3 7/ hg 19 ). a Th is p ati en t al so c ar ri es a chro m o so m e 2 p1 6. 3 deletion including th e NR XN 1 ge ne . b S ym p to ma tic f o ca l epi leps y no t o ther w is e s p ec ifie d . + > 50 % se izu re f re q ue nc y r ed u ct ion. - < 50 % se izu re f re q ue nc y r ed u ct ion. Ab br evi ation s: bif ro n t. = b if ro n ta l, CB Z = c ar b ama zep in e, E T X = eth o su xi mi d e, F = femal e, f ro n to -t emp. = f ro n to -t emp o ral, g en . = g en er ali ze d, H z = H er tz , L = l ef t, LE V = l evetir ac et am, L TG = lam o tr ig in e, M = mal e, ma x. = ma xi mum, m o = m o n ths , M TS = m esi ot emp o ral s cl erosis , o cc . = o ccip it al, Pat = p at er n al, P D D -N O S = p er vasi ve d evel o p m en ta l dis o rd er n o t oth er w is e sp ec ifi c, R = r igh t, S E = s tat us ep ilepti cus , se c. g en . = s ec o n d ar ily g en er ali ze d, S W C = sp ike -w ave c o mp le xe s, s z. = s ei zure s, t emp. = t emp o ral, T PM = t o p ir amat e, V PA = v alp ro ic a ci d , y = year s.

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A large proportion of identiﬁed CNVs are also observed in <1% of the healthy controls. Among these CNVs were chromosome 15q11.2 and 15q13.3 deletions in three children with symptomatic focal epilepsy, while similar deletions are known to predispose to idiopathic generalized epilepsies (Table 3).6-8,19,20_{We also found a chromosome 15q13.3 duplication in a child with focal seizures. An}

association between 15q13.3 duplications and epilepsy has only been reported in a few cases so far.9,21

The observations in our cohort suggest that chromosome 15q11.2 deletions and 15q13.3 deletions and duplications might predispose to both generalized and focal epilepsies. Although these CNVs are regarded as susceptibility CNVs for epilepsy, one should always consider that other causes of epilepsy may also be present, as seen in 30% of the children with susceptibility CNVs in our cohort (Table 3).

Novel CNVs of interest for epilepsy

In ﬁve children, we identiﬁed novel CNVs that comprised four candidate genes for epilepsy (MYT1L, UNC5D, SCN4B and NRXN3) with either expression or function in the brain or a previous association with neurodevelopmental disorders (Supplemental Table 1).

We found a 376 kb deletion involving the MYT1L gene (MIM 613084) in a child with focal epilepsy and intellectual disability. MYT1L codes for a transcription factor that has an important role in the diﬀerentiation of cells to functional neurons.22_{It has been identiﬁed as a candidate gene}

for intellectual disability in patients with 2p25.3 deletions,23,24_{and seizures have been reported}

in 8/21 patients with such a deletion.7,23,25_{The DECIPHER database includes another individual}

(case 259324) with absence seizures, intellectual disability, autism and a large chromosome 2p25 deletion including MYT1L. Thus, based on our and previous observations, MYTL1 deletions are not only associated with intellectual disability but also with epilepsy.

We found a deletion of UNC5D in a child with focal epilepsy, mesiotemporal sclerosis and developmental and behavioral problems, as well as in his father who had migraine. UNC5D (MIM 616466) on chromosome 8p12 has been shown to be involved in cortical development and p53-dependent apoptosis in neuroblastoma cells.26,27_{UNC5D was considered as a candidate gene for}

neurodevelopmental phenotypes in a family with a t(6;8) balanced translocation that disrupted this gene in two aﬀected siblings with developmental delay (one with schizencephaly) and their asymptomatic mother.28_{Further conﬁrmation that deletion of the UNC5D gene may cause or}

predispose to epilepsy and developmental problems is, however, needed.

In one child from our cohort with focal epilepsy and developmental problems, we found a deletion of the sodium channel voltage gated type IV beta subunit gene, SCN4B (MIM 608256). SCN4B is expressed in rat brain and spinal cords, and its protein has been shown to inﬂuence SCN2A by altering channel properties and shifting the voltage dependence of activation in the hyperpolarizing direction.29_{Variants in the SCN2A gene are a well-known cause of benign familial}

neonatal and infantile seizures5_{and early infantile epileptic encephalopathy.}30_{Haploinsuﬃciency}

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Lastly, the NRXN3 gene (MIM 600567) was deleted in two unrelated children in our cohort. We had described one of them in a previous study: she has a severe developmental delay and a concomitant deletion of NRXN1 with no second NRXN1 sequence variant on the other allele (patient 337).31_{NRXN3 encodes a polymorphic cell surface protein, neurexin III, that is expressed in neurons}

and necessary for neurotransmission. Deletions and variants in the neurexin I gene, NRXN1, have been associated with moderate to severe intellectual disability, language delay, autism spectrum disorder and seizures.31_{The severe phenotype of our patient was not in line with the milder}

phenotypes previously reported in children with heterozygous NRXN1 deletions so far, and we speculated that her severe phenotype might be explained by the additional deletion of NRXN3.31_In

the current study, we found a second NRXN3 deletion in another unrelated child with symptomatic focal epilepsy and a pervasive developmental disorder not otherwise speciﬁed (Table 2). NRXN3 has been associated with bipolar disorder32_{and autism spectrum disorder.}33_{Recently, NRXN3}

deletions were reported in four individuals of three diﬀerent families with epilepsy (unclassiﬁed in three, progressive myoclonic epilepsy in one), behavioral problems and developmental delay, or intellectual disability.10_{The observation of NRXN3 deletions in two children in our cohort supports}

the idea that NRXN3 haploinsuﬃciency can be associated with epilepsy.

For all four candidate genes, MYT1L, UNC5D, SCN4B and NRXN3, additional patients with compatible genotypes and phenotypes, and/or supporting evidence from functional studies, are needed to conﬁrm their role in the pathophysiology of epilepsy.

In eight diﬀerent regions, we found CNVs that occurred 10 times more often in our study cohort than in healthy controls. The pathogenicity of these CNVs in epilepsy is doubtful because these children either had other identiﬁed epilepsy causes and/or the CNVs lacked genes of interest for epilepsy.

CONCLUSION

Our study demonstrates the importance of microarray analysis in the diagnostic work-up of epilepsy in childhood. We identified known clinically relevant CNVs for epilepsy in 11% of the children investigated. This yield was obviously influenced by the clinical selection of children, which was largely based on the presence of additional developmental or behavioral problems and/or facial dysmorphisms. Furthermore, we identified novel CNVs that include four new candidate genes for epilepsy: MYT1L, UNC5D, SCN4B and NRXN3. Analysis of these genes in larger study cohorts is warranted to further confirm their role in the etiology of epilepsy.

LINKING TO DATABASES

UCSC Genome Bioinformatics

DatabaseE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECIPHER) Online Mendelian Inheritance in Man (OMIM)

Database of Genomic Variants (DGV)

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REFERENCES

1. Thomas RH, Berkovic SF. The hidden genetics of epilepsy-a clinically important new paradigm. Nat Rev Neurol 2014; 10: 283-292

2. Myers CT, Meﬀord HC. Advancing epilepsy genetics in the genomic era. Genome Med 2015; 7: 91

3. Schaaf CP, Wiszniewska J, Beaudet AL. Copy number and SNP arrays in clinical diagnostics. Annu Rev Genomics Hum Genet 2011; 12: 25-51.

4. Heron SE, Cox K, Grinton BE, et al. Deletions or duplications in KCNQ2 can cause benign familial neonatal seizures. J Med Genet 2007; 44: 791-796.

5. Zara F, Specchio N, Striano P, et al. Genetic testing in benign familial epilepsies of the first year of life: Clinical and diagnostic significance. Epilepsia 2013; 54: 425-436. 6. Mefford HC, Muhle H, Ostertag P, et al. Genome-wide

copy number variation in epilepsy: Novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS Genet 2010; 6: e1000962.

7. de Kovel CG, Trucks H, Helbig I, et al. Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain 2010; 133: 23-32. 8. Jahn JA, von Spiczak S, Muhle H, et al. Iterative

phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers in pediatric epilepsies. Epilepsy Res 2014; 108: 109-116.

9. Olson H, Shen Y, Avallone J, et al. Copy number variation plays an important role in clinical epilepsy. Ann Neurol 2014; 75: 943-958.

10. Faheem M, Naseer MI, Chaudhary AG, et al. Array-comparative genomic hybridization analysis of a cohort of saudi patients with epilepsy. CNS Neurol Disord Drug Targets 2015; 14: 468-475.

11. Naseer MI, Faheem M, Chaudhary AG, et al. Genome wide analysis of novel copy number variations duplications/ deletions of diﬀerent epileptic patients in saudi arabia. BMC Genomics 2015; 16: S10.

12. Fisher RS, Acevedo C, Arzimanoglou A, et al. ILAE oﬃcial report: A practical clinical deﬁnition of epilepsy. Epilepsia 2014; 55: 475-482.

13. Engel J,Jr. Report of the ILAE classiﬁcation core group. Epilepsia 2006; 47: 1558-1568.

14. MacDonald JR, Ziman R, Yuen RK, et al. The database of genomic variants: A curated collection of structural variation in the human genome. Nucleic Acids Res 2014; 42: D986-992.

15. Hanemaaijer N, Dijkhuizen T, Haadsma M, et al. A 649 kb microduplication in 1p34.1, including POMGNT1, in a patient with microcephaly, coloboma and laryngomalacia; and a review of the literature. Eur J Med Genet 2009; 52: 116-119.

16. Kuechler A, Buysse K, Clayton-Smith J, et al. Five patients with novel overlapping interstitial deletions in 8q22.2q22.3. Am J Med Genet A 2011; 155A: 1857-1864. 17. Kuroda Y, Ohashi I, Saito T, et al. Reﬁnement of the

deletion in 8q22.2-q22.3: The minimum deletion size at 8q22.3 related to intellectual disability and epilepsy. Am J Med Genet A 2014; 164A: 2104-2108.

18. Mercimek-Mahmutoglu S, Patel J, Cordeiro D, et al. Diagnostic yield of genetic testing in epileptic encephalopathy in childhood. Epilepsia 2015; 56: 707-716. 19. Shinawi M, Schaaf CP, Bhatt SS, et al. A small recurrent

deletion within 15q13.3 is associated with a range of neurodevelop-mental phenotypes. Nat Genet 2009; 41: 1269-1271.

20. Helbig I, Meﬀord HC, Sharp AJ, et al. 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy. Nat Genet 2009; 41: 160-162.

21. Moreira DP, Griesi-Oliveira K, Bossolani-Martins AL, et al. Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in autism spectrum disorder brazilian individuals with and without epilepsy. PLoS One 2014; 9: e107705.

22. Pang ZP, Yang N, Vierbuchen T, et al. Induction of human neuronal cells by deﬁned transcription factors. Nature 2011; 476: 220-223.

23. Stevens SJ, van Ravenswaaij-Arts CM, Janssen JW, et al. MYT1L is a candidate gene for intellectual disability in patients with 2p25.3 (2pter) deletions. Am J Med Genet A 2011; 155A: 2739-2745.

24. De Rocker N, Vergult S, Koolen D, et al. Reﬁnement of the critical 2p25.3 deletion region: The role of MYT1L in intellectual disability and obesity. Genet Med 2015; 17: 460-466.

25. Mayo S, Rosello M, Monfort S, et al. Haploinsuﬃciency of the MYT1L gene causes intellectual disability frequently associated with behavioral disorder. Genet Med 2015; 17: 683-684.

26. Sasaki S, Tabata H, Tachikawa K, et al. The cortical subventricular zone-speciﬁc molecule Svet1 is part of the nuclear RNA coded by the putative netrin receptor gene Unc5d and is expressed in multipolar migrating cells. Mol Cell Neurosci 2008; 38: 474-483.

27. Wang H, Wu Q, Li S, et al. Unc5D regulates p53-dependent apoptosis in neuroblastoma cells. Mol Med Rep 2014; 9: 2411-2416.

28. Utami KH, Hillmer AM, Aksoy I, et al. Detection of chromosomal breakpoints in patients with developmental delay and speech disorders. PLoS One 2014; 9: e90852.

29. Yu FH, Westenbroek RE, Silos-Santiago I, et al. Sodium channel beta4, a new disulﬁde-linked auxiliary subunit with similarity to beta2. J Neurosci 2003; 23: 7577-7585. 30. Ogiwara I, Ito K, Sawaishi Y, et al. De novo mutations of

voltage-gated sodium channel alphaII gene SCN2A in intractable epilepsies. Neurology 2009; 73: 1046-1053. 31. Bena F, Bruno DL, Eriksson M, et al. Molecular and clinical

characterization of 25 individuals with exonic deletions of NRXN1 and comprehensive review of the literature. Am J Med Genet B 2013; 162B: 388-403.

32. Noor A, Lionel AC, Cohen-Woods S, et al. Copy number variant study of bipolar disorder in canadian and UK populations implicates synaptic genes. Am J Med Genet B 2014; 165B: 303-313.

33. Vaags AK, Lionel AC, Sato D, et al. Rare deletions at the neurexin 3 locus in autism spectrum disorder. Am J Hum Genet 2012; 90: 133-141.

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Supplemen tal F igur e 1: O ver view of chr omosome 8q22 deletions .

SUPPLEMENTAL DATA

Th e 8 q 22 d el eti o n s o b se rv ed in o n e p ati en t o f o u r c o h o rt , ﬁ ve in di vi dual s rep o rt ed by K u echl er et al, 1 o n e i n d iv id u al r ep o rt ed b y K u ro d a e t a l 2 an d o n e in di vi dual in th e D ECIP H ER dat ab as e ( h tt ps: // d ecip h er .s an g er .a c.uk /) . T h e chro m o so mal c o o rdinat es are rep o rt ed relati ve t o th e G en o m e R eferen ce C o ns o rtium Human R eferen ce g en o m e ver si o n 3 7 ( G R C h3 7/ h g1 9) . T h e small es t re gi o n o f over lap is in di ca te d by th e hi ghli gh te d zo n e. O n ly p rot ein -c o din g g en es are sh ow n . Re fe re nc es 1. Ku e ch le r A , B u ys se K , Clay to n -Sm it h J , e t a l. Fi ve p at ie n ts w it h n o ve l ove rla p p in g int e rs ti ti al d e le ti o ns in 8 q 22. 2 q22. 3. A m J M e d G e n e t A 20 11 ; 1 55 A : 1 8 5 7-18 6 4. 2. Ku ro d a Y , O h ashi I, S ai to T , e t a l. R e ﬁn e m e n t o f t h e d e le ti o n in 8 q 22. 2 -q 22. 3 : T h e m inimu m d e le ti o n si ze at 8 q 22. 3 re lat e d to int e lle c tu al d is ab ili ty a n d e p ile p sy . A m J M e d G e n e t 20 14 ; 1 6 4A : 20 14 -2 10 8 .

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Supplemental Table 1: Information on novel candidate genes

Gene (protein) MIM Information on expression, function and/or disease association

MYT1L

(myelin transcription factor 1-like)

613084 - Expression in embryonic rat brains from day 15, with maximal

expression at parturition and subsequent decline to low but

detectable levels in adult rat brains 1

- Functions as transcription factor to convert non-neuronal human

somatic cells or pluripotent stem cells into functional neurons 2

- Identiﬁed as candidate gene for intellectual disability in patients with

2p25.3 deletion syndrome 3,4

UNC5D

(unc-5 homolog D)

616466 - Expression in multipolar cells of the subventricular zone throughout

cortical development 5_{. The cortical subventricular zone-speciﬁc}

molecule Svet1 is part of the nuclear RNA coded by the putative netrin receptor gene Unc5d and is expressed in multipolar migrating cells. - Functions as transcriptional target of pro-apoptotic p53 and might

induce p53-dependent apoptosis by ser-15 phosphorlation in

neuroblastoma cells 6

- Disruption of UNC5D is reported in a patient with schizencephaly and

developmental delay due to a t(6;8) balanced translocation 7

SCN4B

(Sodium channel,

voltage-gated, type IV, beta subunit)

608256 - Expression in neurons in rat brain and spinal cord and some sensory

neurons 8

- Whole-cell recording in co-transfected embryonic kidney cells showed that SCN4B alters the channel properties of SCN2A. There is a shift in voltage dependent activation in the hyperpolarizing direction, but no

shift in the voltage dependent inactivation 8

NRXN3

(Neurexin III)

600567 - Polymorphic cell surface protein that is expressed in neurons 9

- Alpha-neurexin I, II and/or III knockout mice showed impaired function of synaptic calcium channels suggesting that alpha-neurexins organize

presynaptic terminals 10

- A clinical study including 4 families with at least one member with autism suggested that deletions comprising NRNX3 may predispose to

developing autism spectrum disorder 11

- NRXN3 deletions have been found in four individuals of three diﬀerent families with unclassiﬁed epilepsy (n = 3) or progressive myoclonic

epilepsy (n = 1) 12

Abbreviations: MIM = Mendelian Inheritance in Men database number (https://omim.org)

References

1. Kim JG, Armstrong RC, Agoston D, et al. Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc ﬁnger, is expressed in developing neurons in the mammalian central nervous system. J Neurosci Res 1997; 50: 272-290. 2. Pang ZP, Yang N, Vierbuchen T, Induction of human neuronal cells by deﬁned transcription factors. Nature 2011; 476: 220-223.

3. Stevens SJ, van Ravenswaaij-Arts CM, Janssen JW, et al. MYT1L is a candidate gene for intellectual disability in patients with 2p25.3 (2pter) deletions. Am J Med Genet A 2011; 155A: 2739-2745

4. De Rocker N, Vergult S, Koolen D, et al. Reﬁnement of the critical 2p25.3 deletion region: the role of MYT1L in intellectual disability and obesity. Genet.Med 2015; 17: 460-66

5. Sasaki S, Tabata H, Tachikawa K, et al. The cortical subventricular zone-speciﬁc molecule Svet1 is part of the nuclear RNA coded by the putative netrin receptor gene Unc5d and is expressed in multipolar migrating cells. Mol Cell Neurosci 2008; 38: 474-483.

6. Wang H, Wu Q, Li S, et al. Unc5D regulates p53-dependent apoptosis in neuroblastoma cells. Mol Med Rep 2014; 9: 2411-2416.

7. Utami KH, Hillmer AM, Aksoy I, et al. Detection of chromosomal breakpoints in patients with developmental delay and speech disorders. PLoS One 2014; 9: e90852.

8. Yu FH, Westenbroek RE, Silos-Santiago I, et al. Sodium channel beta4, a new disulﬁde-linked auxiliary subunit with similarity to beta2. J Neurosci 2003; 23: 7577-85. 9. Ushkaryov YA, Petrenko AG, Geppert M, et al. Neurexins:

synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin. Science 1992; 257: 50-56. 10. Hishimoto A, Liu QR, Drgon T, et al. Neurexin 3

polymorphisms are associated with alcohol dependence and altered expression of speciﬁc isoforms. Hum Mol Genet 2007; 16: 2880-91.

11. Vaags AK, Lionel AC, Sato D, et al. Rare deletions at the neurexin 3 locus in autism spectrum disorder. Am J Hum Genet 2012; 90: 133-141.

12. Faheem M, Naseer MI, Chaudhary AG, et al. Array-comparative genomic hybridization analysis of a cohort of Saudi patients with epilepsy. CNS Neurol Disord Drug Targets 2015; 14: 468-75.

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Supplemen tal T able 2: CNV s with a t en times higher fr equenc

y in the study cohor

t compar ed t o contr ols Region C o ordinates (s iz e i n k b ) Fr e quenc y in c o hor t (n =226 ) (% ) Fr e quenc y in D G V (n =1 4 316 ) (% ) Fr e quenc y in LL C ( n =24 0 2 ) (% ) Gen e s P h enot ype first pat ien t P h enot ype sec ond p a ti e n t Reason for in terpret ing th is r e g io n le ss li ke ly pat hogenic D e le ti o n s ( n = 4 ) 1q 21. 1-q2 1. 2 14 9, 29 8, 62 2 - 49 ,7 63 ,11 8 (4 65 ) 2 (0 .8 8) 0 (-) 2 (0 .0 8) FC GR 1A PPI A L4 C H IS T2H2B F FA M 23 1D FA M 72 C Pa tien t 5 74 ( F, 1 6y ): fo ca l epileps y a, dev elopme n ta l pr ob lems, epic an thal fo ld an d uptur n ed nas al tip Pa tien t 98 1 ( M , 1 6y ): fo ca l epileps y a , developm en ta l pr ob lems, auti sm , p u b er tas pr ae co x an d up slan ting p alp ebr al ﬁ ss ur es Ano th er m o re lik el y epileps y c au se : MY TL 1 deletion (p at ien t 98 1) N o gen es of in ter es t 7q 34 14 2, 39 3, 82 3 - 14 2, 48 6, 37 3 (93) 2 (0 .8 8) 3 (0 .0 2) 1 (0 .0 4) PR SS 1 Pa tien t 8 41 ( F, 6 y) : We st Sy n d ro m e an d developm en ta l pr oble m s Pa tien t 1 07 0 ( M , 6 y) : C SWS, de ve lopme n ta l pr oble m s, tr em or , a ta

xia, high for

eh ea d , de ep ly s et eyes an d thin upp er lip N o gen es of in ter es t 11 p1 2 40 ,3 39 ,4 14 - 40 ,4 84 ,7 07 (1 45 ) 2 (0 .8 8) 0 (-) 2 (0 .0 8) LR RC4C b Pa tien t 9 9 ( M , 9) : lo ca liz ation cr yp to genic epileps y, dev elopme n ta l pr oble m s, ma cr o cep hal y, auti sm an d t al l s ta tur e Pa tien t 7 19 ( M , 8 y) : fo cal epileps y a, developm en ta l an d b eh aviora l pr oble m s, br ain h yp o p las ia on MR I an d in ver te d nip p le s O th er lik el y epileps y ca u se :ca rb oh yd ra te de ﬁc ie n t g ly copr o te in sy n d ro m e t yp e IC (p at ien t 71 9) 22 q1 1. 21 20 ,3 45 ,6 68 - 20,7 09 ,0 90 (3 63 ) 2 (0 .8 8) 3 (0 .0 2) 2 (0 .0 8) USP 41 GG TL C3 FA M 23 0A RIMBP 3 Pa tien t 3 56 ( M , 1 6y ): fo ca l epileps y a, dev elopme n ta l pr oble m s, h yp o to n ia, auti sm , highl y ar ch ed eyebr o ws, h yp er telor is m, f u ll lips an d widely s p ac ed te eth Pa tien t 1 04 4 ( M , 6 y) : fo ca l epileps y a, developm en ta l an d b eh aviora l pr oble m s, h yp o to nia an d in ver te d nip p le s O th er lik el y epileps y c au se : a 1 5q 11 .2 -q 13 dup lic ation (pa tie n t 3 56 ) N o gen es of in ter es t D u plicat ions (n = 4 ) 7p 14 .3 32 ,2 26 ,3 19 - 32 ,3 83 ,34 5 (1 57 ) 2 (0 .8 8) 2 (0 .0 1) 2 (0 .0 8) PD E1 C Pa tien t 1 06 2 ( M , 5 y) : fo ca l epileps y a an d developm en ta l an d b eh aviora l pr oble m s. Pa tien t 1 11 6 ( F, 2 y) : fo ca l epileps y a, developm en ta l pr oble m s, abnorm al ities on MRI c o rr esp o nding t o p er ina tal asp h yx ia an d micr o cep hal y O th er lik el y epileps y c au se : a 1 5q 11 .2 -q 13 dup lic ation (p at ien t 1 06 2) N o gen es of in ter es t

2

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C ontinuation of Supplemental Table 2 Region C o ordinates (s iz e i n k b ) Fr e quenc y in c o hor t (n =226 ) (% ) Fr e quenc y in D G V (n =1 4 316 ) (% ) Fr e quenc y in LL C (n = 2 4 0 2) (% ) G e n e s Ph e n o ty p e fi rs t p a ti e n t P h enot ype sec ond p a ti e n t Reason for in terpret ing th is r e g io n le ss li ke ly pat hogenic 9q 21 .11 71 ,6 13 ,3 95 - 71 ,7 27 ,2 77 (1 14 ) 2 (0 .8 8) 0 (-) 1 (0 .0 4) PI P 5 K 1B c PR K A C G d FXN e Pa tien t 1 08 0 ( M , 4 y) : WS an d fo ca l epileps y a, developm en ta l pr oble m s, bra in a tr oph y a n d hy p o p la si a o n M RI , hy p o to n ia , b allism, t all s tat ure an d epic an thal fo ld Pa tien t 1 05 5 ( F, 5 y) : fo ca l epileps y a, G o m ez-Lo p ez- He rn ande z s ynd rome , de ve lopme n ta l pr oble m s, abnor malities an d h ydr o cep hal y on MR I, micr o cep hal y, h yp er tonia of th e le g s an d un sp ec ifie d fa cial d ysm o rp h isms O th er lik el y epileps y ca u ses : a m ito -c hond ria l re sp ira to ry chain d efe ct (p at ien t 1 08 0) an d G o me z-Lo p ez-Hernande z sy n d ro m e ( p at ie nt 1 05 5) 17 q1 2 34 ,5 39 ,666 -34 ,8 17 ,6 22 (2 78 ) 2 (0 .8 8) 6 (0 .0 4) 1 (0 .0 4) TBC 1D 3G CC L4 L2 CC L4 L1 CC L3 L1 TBC 1D 3C TBC 1D 3H Pa tien t 1 07 2 ( M , 1 7y ): fo ca l epileps y a, developm en ta l pr oble m s a n d P DD -NO S. Pa tien t 1 07 5 ( M ,4 y) : fo cal epileps y a, developm en ta l pr oble m s, h yp o to n ia, h yp o den se ar ea s in m es enc ep halon an d p o n s on MR I, high p ala te , t en te d m o uth, s mal l chin, s mal l ea rs , up lif te d e ar lob es, si ngle tr an sv er se p almar cr ea se a n d c lin o d ac ty ly digiti I V O th er lik el y epileps y c au se : a mit o chon dr ial dep letion sy n d ro m e (p at ien t 1 07 5) N o gen es of in ter es t Xq 28 15 5, 18 0, 28 8 - 15 5, 22 9, 11 4 (49 ) 2 (0 .8 8) 0 (-) 2 (0 .0 8) VA M P7 IL 9R Pa tien t 1 04 8 ( M , 5 y) : fo ca l epileps y a, developm en ta l pr oble m s, ac ro ce ph al y, co rp u s c al los um a g en es is an d as ym m etr ic ven tr icles on MR I, un der d evelop ed ala e nas i, h yp er telor is m, e xtr a nipp le , br onchomalacia and t al l s ta tur e Pa tien t 1 09 0 ( F, 4 y) : BF IS N o gen es of in ter es t Th e chro m o so mal c o o rdinat es are rep o rt ed relati ve t o th e G en o m e R eferen ce C o ns o rtium Human R eferen ce g en o m e ver si o n 3 7 ( G R C h3 7/ h g1 9) . G en es in b o ld are e xp re ss ed in th e b rain o r ass o ciat ed w ith a n eur o p sy chiat ri c di se as e. a Sy mpt o mati c fo ca l ep ileps y n o t oth er w is e sp eci fie d . b LRR 4C 4 ( M IM 6 08 81 7) is a m em b er o f t h e n et ri n f am ily o f a xo n g u id an ce m o le cu le s a n d is ex p re ss ed in a dul t an d fet al b rain . c PI PI 5K 1B ( M IM 6 02 74 5) en co d es a f u n cti o n al p h osp h ati d yl in osi to l p h osp h at e k inas e an d has b een is o lat ed f ro m th e re gi o n o f th e g en o m e as so ci at ed wit h Fr ie drie ch A ta xi a. d Va ri at io n s i n PR K A CG ( M IM 1 76 89 3) h av e b ee n a ss o ci at ed w it h s h o rt -t er m e p is o d ic m em o ry p er fo rm an ce . e C om p ou nd het er o zy gou s r ep ea t e xpa n sion of FX N (M IM 6 0 68 29 ) ha ve b een ass o ciat ed w ith F ri edrei ch ’s A ta xia . A b b rev iati o n s: B FI S = b eni gn f amilial inf an til e s ei zure s, C SW S = c o n tinu o us sp ik e w ave s dur in g sl ow -w ave sl ee p s yn d ro m e, D G V = dat ab as e o f g en o mi c v ar ian ts , LL C = Low L an d s C o ns o rtium dat ab as e, M R I = ma gn eti c re so nan ce ima g in g, P D D -N O S = p er vasi ve d evel o p m en ta l dis o rd er n o t oth er w is e sp ec ifi ed .

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