The genetic landscape of infantile spasms.

HMG Advance Access published May 12, 2014 Human Molecular Genetics, 2014 doi:10.1093/hmg/ddu199

1–13

The genetic landscape of infantile spasms Jacques L. Michaud1,2,3, Mathieu Lachance3, Fadi F. Hamdan3, Lionel Carmant1,2,3, Anne Lortie1,2,3, Paola Diadori1,2,3, Philippe Major1,2,3, Inge A. Meijer3, Emmanuelle Lemyre1,3, Patrick Cossette2,4, Heather C. Mefford5, Guy A. Rouleau6 and Elsa Rossignol1,2,3,∗ 1

Department of Pediatrics and 2Department of Neurosciences, Universite´ de Montreál, Montreál, QC, Canada, CHU Ste-Justine Research Center, Montreál, QC, Canada, 4CHUM, Montreál, QC, Canada, 5Department of Pediatrics, University of Washington, Seattle, WA, USA and 6Department of Neurosciences, Montreal Neurological Institute, McGill University, Montreál, QC, Canada 3

Received January 3, 2014; Revised April 3, 2014; Accepted April 24, 2014

INTRODUCTION Infantile spasms (IS) is one of the classical epileptic encephalopathies occurring in 2.5 per 10 000 live births (1). IS are characterized by the developmental onset of flexion or extension limb and trunk spasms, accompanied by electrodecremential responses and a disorganized high-amplitude background, i.e. hypsarrhythmia, on electroencephalograms (EEG). IS frequently lead to long-term neurological impairment including developmental delay (DD)/ intellectual deficiency (ID) in 70% of cases, autism spectrum disorder in 45% of cases, and chronic refractory epilepsy in 70% of cases (1). The poor neurodevelopmental outcome of IS is thought to reflect the severity of the underlying pathology as well as the disruptive consequences of seizures and disorganized network activities on developing neuronal connectivity.

In a majority of cases (60 – 75%), an underlying etiology can be identified through metabolic, infectious and radiological investigations (2,3). These symptomatic IS cases typically result from diffuse brain lesions occurring in the context of neonatal infections, strokes, hypoxic ischemic or metabolic encephalopathies (4–8), chromosomal anomalies (6,9–15), neurocutaneous disorders (16) or cerebral malformations (17–20). However, in up to 40% of cases, no etiology is identified. Genetic studies conducted in children with unexplained IS have revealed mutations on the X chromosome in genes such as ARX (21) and CDKL5 (STK9) (22– 24) as well as de novo mutations in autosomal genes, including MAGI2 (25), STXBP1 (26), SCN1A (27) and SCN2A (28), together explaining a minority of cases. De novo mutations in other genes may thus underlie a large fraction of the remaining unexplained IS cases.

∗ To whom correspondence should be addressed at: Elsa Rossignol, CHU Ste-Justine Research Center, Lab 5994, 3175 Coˆte Ste-Catherine, Montreal, QC, Canada H3T 1C5. Tel: +1 5143454931 ext. 7241; Fax: +1 5143452372; Email: [email protected]

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Downloaded from http://hmg.oxfordjournals.org/ at Maastricht University on July 9, 2014

Infantile spasms (IS) is an early-onset epileptic encephalopathy of unknown etiology in ∼40% of patients. We hypothesized that unexplained IS cases represent a large collection of rare single-gene disorders.We investigated 44 children with unexplained IS using comparative genomic hybridisation arrays (aCGH) (n 5 44) followed by targeted sequencing of 35 known epilepsy genes (n 5 8) or whole-exome sequencing (WES) of familial trios (n 5 18) to search for rare inherited or de novo mutations. aCGH analysis revealed de novo variants in 7% of patients (n 5 3/44), including a distal 16p11.2 duplication, a 15q11.1q13.1 tetrasomy and a 2q21.3-q22.2 deletion. Furthermore, it identified a pathogenic maternally inherited Xp11.2 duplication. Targeted sequencing was informative for ARX (n 5 1/14) and STXBP1 (n 5 1/8). In contrast, sequencing of a panel of 35 known epileptic encephalopathy genes (n 5 8) did not identify further mutations. Finally, WES (n 5 18) was very informative, with an excess of de novo mutations identified in genes predicted to be involved in neurodevelopmental processes and/or known to be intolerant to functional variations. Several pathogenic mutations were identified, including de novo mutations in STXBP1, CASK and ALG13, as well as recessive mutations in PNPO and ADSL, together explaining 28% of cases (5/18). In addition, WES identified 1–3 de novo variants in 64% of remaining probands, pointing to several interesting candidate genes. Our results indicate that IS are genetically heterogeneous with a major contribution of de novo mutations and that WES is significantly superior to targeted re-sequencing in identifying detrimental genetic variants involved in IS.

2

Human Molecular Genetics, 2014

Collectively, these observations suggest that IS is characterized by great genetic heterogeneity. We postulate that a majority of unexplained IS cases are due to genetic anomalies that disrupt fundamental processes in neuronal maturation or function. Using a combination of array comparative genomic hybridization, targeted sequencing and whole-exome sequencing (WES) in a large cohort of children with IS, we have identified de novo variants expected to affect neuronal maturation and network development in a majority of unexplained IS cases. We also demonstrate that WES has a significantly higher diagnostic yield than targeted sequencing in this population.

RESULTS We recruited 44 sporadic cases of unexplained IS followed at the pediatric epilepsy clinic of the CHU Ste-Justine. Family history was unremarkable and none of the parents were related. Extensive metabolic investigation was performed in each proband and was unremarkable (Supplementary Material, Table S1). Brain magnetic resonance imaging (MRI) was normal in 11 patients (26%) or revealed non-specific changes including nonfocal cortical and subcortical atrophy (n ¼ 21, 47%), delayed myelination (n ¼ 15, 34%), thinning of the corpus callosum (n ¼ 8, 18%) and/or basal ganglia T2 hyperintensities (n ¼ 3, 7%) without evidence of lactic acidosis or metabolic disturbances on blood and urinary screens. IS occurred at a mean age of 5.5 months (range: 0 – 40 months), and the majority of patients required the addition of adrenocorticotropic hormone (ACTH) or topiramate to control the IS (n ¼ 30, 68%). Furthermore, most patients developed other seizure types in the course of their disease, including focal seizures, tonic seizures, generalized tonic-clonic seizures, atonic seizures and/or myoclonic seizures (n ¼ 35, 80%). The majority of patients had global DD (n ¼ 40, 91%), and of those that were evaluated by a psychologist after the age of 5 years, a majority had ID, ranging from moderate to severe (n ¼ 18/25, 72%). Clinical data for the entire cohort are summarized in Table 1. Novel copy number variants (CNVs) associated with IS All probands were investigated for pathogenic CNVs using comparative genomic hybridisation arrays (aCGH) (Fig. 1). We identified de novo variants in 7% of patients (n ¼ 3/44) and a rare hemizygous variant in 2% of patients (n ¼ 1/44). These variants are likely pathogenic as detailed below. In addition, we found rare inherited variants in 14% of patients (n ¼ 6/44, of which two patients had two inherited variants), which were considered benign. Additionally, we identified rare variants of uncertain inheritance in 4.5% of patients (n ¼ 2/44) when aCGH could not be obtained from both parents. Clinical data for patients with presumed pathogenic CNVs (de novo and hemizygous) are summarized in Table 2. Detailed patient descriptions are found in Supplementary Material. Variants identified in our cohort are summarized in Table 3. Of the de novo variants identified, one patient with moderateto-severe ID and refractory seizures carried a de novo 7.9 Mb deletion encompassing 2q21.3-q22.2 (Supplementary Material).

N (%) Total Gender Males Females Ethnicity Caucasian Arabic Asian American Indian Age onset (Mean, months) Additional seizure types Focal Tonic GTC Atonic Myoclonic # AED (Mean) Development Normal Global delay MRI Normal Atrophy/ISAS Myelination delay CC anomaly BG Hyperintensities

44 22 (50) 22 (50) 34 (77) 7 (16) 2 (5) 1 (2) 5.5 26 (59) 11 (25) 6 (14) 2 (5) 7 (16) 5 4 (9) 40 (91) 11 (26) 21 (50) 15 (34) 8 (18) 3 (7)

GTC, generalized tonic-clonic; CC, corpus callosum; BG, basal ganglia; ISAS, increased sub-arachnoid spaces.

Deletions in this region have been associated with intellectual disability, behavioral difficulties and autism spectrum disorder (ASD) (29– 31) but not with epilepsy to date. Among potential candidate genes included in this interval, CXCR4 and NXPH2 are expressed in neurons and are involved in fundamental neurodevelopmental processes. CXCR4 encodes the C-X-C chemokine receptor type 4, which is activated by its Cxcl12 chemokine ligand to regulate cortical GABAergic interneuron distribution and the development of cortical inhibitory tone (32 –35). Furthermore, Cxcr4-mediated signaling is involved in neuronal survival following injury (36), which might be required to reduce neuronal damage following repeated seizures. NXPH2 encodes neurexophilin-2, a member of the a-neurexinbinding neurexophilin family (37,38). Alpha-neurexins bind neuroligins to generate transynaptic complexes involved in synapse formation, stabilization and in the regulation of synaptic release (39,40). A second patient, who showed early-onset seizures and moderate – severe ID, carried a de novo 7.8 Mb tetrasomy of the proximal segment of chromosome 15q up to 15q13.1, encompassing the Angelman/Prader – Willi syndrome interval, including the GABRA5, GABRB3 and UBE3A genes (Supplementary Material). Maternal inheritance of duplications or triplications of this interval are associated with epilepsy, global DD and intellectual disabilities (41,42). A third patient with microcephaly, severe ID and wellcontrolled seizures carried a de novo 0.452 Mb duplication affecting the distal segment of the 16p11.2 region (Supplementary Material). 16p11.2 distal deletions have been associated


Clinical characteristics

Table 1. Clinical characteristics


3

Table 2. Clinical data for cases with deleterious genetic variants identified in this study Gender

Origin

Age DX (months)

Seizures

RX

EEG

Dev.

MRI

CGH (inheritance)

M M F M

C C/Ar C C/A

5 17 8 0.5

IS, F, T, M IS, AT, F IS, T, AT IS, F

5 8 2 8

H, M G, F, M M H, M

G, ID-M G, ID-S G, ID-S G, ID-M

ISS MD ISS, TCC ISS, TCC

M F M F F F

C C C C As C

2 0.2 0.25 4 36 0.02

IS IS, CPC IS, F IS, F IS IS, M

1 4 7 3 4 9

M M M H, M N M

G G, ID-M G G, ID-S G, ID-S G

N ISS, MD AT, BGH AT AT AT, ISS

M

C

7

IS, F

2

H, G

G, ID-M

N

Del 2q22.2q21.3 (d.n.) Tetrasomy 15q11.1q13.1(d.n.) Dup 16p11.2 (d.n.) Dup Xp11.23 (mat) Dup Xp11.23p11.22 (mat) N N N Del 9p24.2 (mat) N Dup 3q13.12q13.13 (mat) Del 8q23.2q23.3 (pat) N

Targeted sequencing

WES

ARX STXBP1 STXBP1 ALG13 CASK PNPO ADSL

Dx, diagnosis; Origin: C, Caucasian; Ar, Arabic; A, African; AI, Amerindian; As, Asian; Seizures: IS, infantile spasms; F, focal; T, tonic; AT, atonic; GTC, generalized tonic-clonic; M, myoclonic; EEG: H, hypsarrhythmia; G, generalized; F, focal; M, multifocal; Development: G, global developmental delay; ID, intellectual deficiency (M, moderate; S, severe). MRI: ISS, increased sub-arachnoid spaces; MD, myelination delay; TCC, thin corpus callosum; AT, atrophy; BGH, basal ganglia hyperintensities; CGH: inheritance: d.n., de novo; mat, maternal inheritance; pat, paternal inheritance.

with obesity, DD, autism or schizophrenia and seizures (43,44). Duplications of the distal 16p11.2 segment, as found in our patient, have been variably associated with developmental or

speech delay, ID, autism, attention deficit hyperactivity disorder, dimorphisms/overgrowth and epilepsy with reduced penetrance (43,44). The genomic interval duplicated in our patient includes


Figure 1. Summary of genetic investigations and findings. Schematic representation of the genetic investigations conducted in our cohort of 44 children with sporadic unexplained IS. Array comparative genomic hybridization studies (CGH) were performed in all patients and identified de novo or hemizygous CNVs, which were presumed pathogenic, in four patients (see Table 3). In addition, two patients carried CNVs for which inheritance could not be confirmed and were presumed pathogenic. Targeted sequencing of selected genes (ARX, STXBP1 and CDKL5, see text) identified pathogenic mutations in ARX and STXBP1 in one patient each. Targeted re-sequencing of 35 known epilepsy genes in 8 patients did not identify pathogenic mutations. However, WES conducted in 18 patients identified mutations that were predicted pathogenic (i.e. with SIFT score ,0.5 or PolyPhen v2 score .0.85, see Table 4) in known epilepsy genes in five patients. In 8 of the remaining 13 patients, de novo mutations predicted to be pathogenic were identified in 11 new putative epilepsy candidate genes.

4


Table 3. CNVs revealed by aCGH analysis

De novo CNVs Del 2q22.2q21.3

Position

Genes included

Prediction

(hg19: 135,134,483-143,066,371)

THSD7B, LRP1B, MIR5590, MIR128-1, MGAT5, CXCR4, R3HDM1, HNRNPKP2, NXPH2, ACMSD, UBXN4, UBBP1, CCNT2, MCM6, MAP3K19, DARS, RAB3GAP1, ZRANB3 GABRA5, GABRB3, MAGEL2, SNRPN, UBE3A SULT1a1, EIF3C, NPIPL1, ATXN21, TUFM, SH2B1, ATP2A1, RABEP2, CD19

P

OTUD5, KCND1, GRIPAP1, TFE3, PRAF2, WDR45, PLP2, PRICKLE3, SYP,CACNA1F, FOXP3, CCDC120, GPKOW, MAGIX, CCDC22, PPP1R3F PAGE4, CLCN5, LOC158572, USP27X, MIR532, MIR188, MIR500A, MIR362, MIR501, MIR500B, MIR660, MIR502

P

interrupts PTGER3 HHLA2, MYH15, KIAA1524, DZIP3 KCNV1, CSMD3, MIR2053 interrupts SLC1A1 interrupts CHRNA7 interrupts RGMA, CHD2

B B B B B B

interrupts FGD4, DNM1L DGCR2, DGCR14, TSSK2, GSC2, SLC25A1, CLTCL1

U B

Tetr 15q13.1 Dup 16p11.2 Hemizygous CNVs Dup Xp11.23

Identified on karyotype (hg 18: 28,518,059-28,970,054)

Dup Xp11.23p11.22

(hg 18: 49,463,797-49,721,060)

(hg 18: 48,696,007-49,031,414)

U

Dup, Duplication; Del, deletion; Predicted impact: P, pathogenic; U, uncertain; B, benign.

Table 4. Rare de novo variants revealed through WES ID

Chr.

Position

Ref.

Mut.

Variant

Gene

Detailed annotation

SIFTa

PolyPhen v2b

2 3 4

X 5 19 5 7 2 9 9 1 10 5 3 6 19 3 X 1 1 20

1 10 928 268 1 79 247 941 17 312 954 92 921 132 44 579 758 1 52 222 651 1 30 430 439 1 34 371 230 36 301 467 29 747 509 1 67 689 699 1 24 774 668 1 29 704 291 14 160 107 1 22 640 860 41 712 458 2 07 941 154 22 188 503 62 221 927

A C G C G T G G G C G G G CCCCT G G C G C

G T A A A C A A A T A T A C T A T A A

SNV SNV SNV SNV SNV SNV SNV SNV SNV SNV SNV SNV SNV fs_del. SNV stopgain SNV SNV SNV

ALG13 SQSTM1 MYO9B NR2F1 NPC1L1 TNFAIP6 STXBP1 PRRC2B EIF2C4 SVIL TENM2 HEG1 LAMA2 IL27RA SEMA5B CASK CD46 HSPG2 GMEB2

NM_001099922:exon3:c.A320G:p.N107S NM_003900:exon1:c.C5T:p.A2V NM_001130065:exon28:c.G4678A:p.V1560M NM_005654:exon1:c.C403A:p.R135S NM_013389:exon2:c.C238T:p.R80C NM_007115:exon3:c.T314C:p.F105S NM_003165:exon10:c.G875A:p.R292H NM_013318:exon31:c.G6659A:p.R2220Q NM_017629:exon13:c.G1597A:p.G533S NM_021738:exon37:c.G6412A:p.V2138I NM_001122679:exon29:c.G8182A:p.G2728R NM_020733:exon1:c.C67A:p.L23M NM_000426:exon35:c.G4984A:p.E1662K NM_004843:exon10:c.1384_1387del:p.462_463del NM_001256348:exon11:c.C1416A:p.S472R NM_001126054:exon2:c.C82T:p.R28X NM_172359:exon8:c.C932T:p.A311V NM_005529:exon38:c.C4846T:p.P1616S NM_012384:exon10:c.G1108T:p.A370S

0 0.01 0 0 0.01 0.18 0.00 0.57 0.001 0.07 0.00 0.00 0.05 n/a 0.003 n/a 0.4 0.012 0.55

0.538 0.289 1 1.0 0.957 0.953 1.0 0.026 0.972 0.778 0.999 0.999 0.618 n/a 0.94 n/a 0.0 0.998 0.567

5 7 9 10 11 12 14 15 16

ID, patient identifier; Chr., chromosome; Ref., reference allele; Mut., mutant allele; fs_del., frameshift deletion; n/a, not applicable. Predicted pathogenicity based on bioinformatic scores: a SIFT: ,0.05, damaging, .0.05, tolerated. b PolyPhen v2 score: .0.85, probably damaging, .0.15, possibly damaging, ≤0.15, benign.

several genes, of which SULT1A1, EIF3C, TUFM, SH2B1, RABEP2 are known to be expressed in the brain and might contribute to the phenotype observed. Finally, one male patient, with ID and intractable seizures, was found to carry a complex chromosomal rearrangement that includes the duplication of two adjacent regions on chromosome Xp (0.335 Mb Xp11.22-23 and 0.257 Mb Xp11.23) (Supplementary Material). These duplications were maternally inherited but were not present in the maternal grandparents of

the proband, suggesting that they occurred de novo in his mother. Larger duplications that include the segments affected in our patient have been reported to cause ID, ASD, language impairment and refractory epilepsy in girls with skewed X inactivation (45). The documentation of these CNVs in our patient thus leads to a better delineation of the critical region underlying this neurodevelopmental phenotype, with the possible contribution of genes affected exclusively by the larger duplications and of genes located in the regions of overlap between the larger and


Inherited autosomal CNVs Del 1p31.1 (hg 18: 71,145,303-71,230,018) Dup 3q13.12q13.13 (hg18: 109,468,131-109,925,023) Del 8q23.2q23.3 (hg 18: 110,830,845-114,121,392) Del 9p24.2 (hg 18: 4,398,900-4,548,776) Dup 15q13.3 (hg 18: 29,816,893-30,226,405) Dup 15q26.1 (hg18: 91,326,934-91,392,664) Unconfirmed inheritance Dup 12p11.21 (hg 18: 32,683,821-32,726,913) Dup 22q11.21 (hg 18: 17,429,152-17,575,845)

P P


De novo mutations in STXBP1 or ARX explain a significant proportion of IS cases Sanger sequencing with or without deletion/duplication analysis of at least one known IS gene was performed in most cases (Fig. 1). This revealed one mutation in ARX (1/14) and one mutation in STXBP1 (1/8). The de novo deletion in STXBP1 was identified in a patient with moderate ID and intractable seizures (n ¼ 1/8 patients tested, 13%) (Supplementary Material). This mutation was deemed pathogenic as it deletes exons 8 – 11, truncating the protein. STXBP1 (MUNC18-1), encoding syntaxin-binding protein 1, controls synaptic release by binding syntaxin-1 in an essential step leading to the pre-synaptic fusion of synaptic vesicles (54). Mutations in this gene have been extensively associated with severe early-onset epileptic encephalopathies including Ohtahara and other epileptic phenotypes (55– 59). Sequencing of the ARX gene revealed a maternally inherited poly-alanine expansion in the ARX gene (NM_139058.2) at c.441_464dup24, p.Ala148_Ala155dup in one patient. Overall, ARX sequencing was contributory in 7% of patients tested (n ¼ 1/14). Therefore, ARX mutations probably explain a small fraction of IS cases. Mutations in this gene have been associated

with X-linked ID, lissencephaly with abnormal genitalia, Ohtahara syndrome and isolated IS with DD (60– 64). Sequencing of the CDKL5 gene in 20 girls with IS was noncontributory. CDKL5 mutations were initially reported in girls with Rett-like disorder and/or severe early-onset seizures (22,24,65), but they have recently been reported to cause severe early-onset epileptic encephalopathy with brain atrophy in both genders (66–69) and sequencing of this gene should now be offered to patients of either gender with early-onset epilepsy. Finally, sequencing of a panel of 35 epileptic encephalopathy genes (Fig. 1 and Supplementary Material, Table S2) was performed in eight patients and failed to identify a molecular diagnosis in these patients (n ¼ 0/8). Identification of recessive mutations by exome sequencing in unexplained IS cases Exome sequencing was performed in 18 families (probands and both parents) when aCGH and targeted sequencing of one or two known IS genes failed to identify an etiology (Fig. 1). We first focused our attention on candidate recessive mutations. Homozygous rare variants were identified in 12 probands (n ¼ 12/18, 67%), with a mean of 1.4 homozygous rare variants per proband (Supplementary Material, Tables S3 and S4). One of these mutations, c.674G.A (p.R225H) in the PNPO gene (NM_018129.3), was found in a patient with early-onset intractable seizures (Supplementary Material). Mutations in the PNPO gene lead to pyridoxamine 5′ -phosphate oxidase (PNPO) deficiency and result in pyridoxal-phosphate responsive epileptic encephalopathy (70). The p.R225H mutation is extremely rare [not reported in 1000 Genomes or Exome Variant Server (EVS) datasets] and is predicted damaging with both SIFT (score ¼ 0.00) and POLYPHEN-2 (score ¼ 0.999). It lies in a highly conserved sequence and is adjacent to R229 whose recessive mutation was previously reported to cause epileptic encephalopathy (71). Furthermore, p.R225H was shown to reduce PNPO catalytic function in vitro (G. Mitchell, personal communication, CHU Ste-Justine, Montreal, Canada). No de novo mutations were found in this patient. We conclude that this mutation is pathogenic. Interestingly, extensive investigation including spinal fluid measures of pyridoxal-5′ -phosphate had failed to reveal this treatable disease in our patient illustrating potential pitfalls in the traditional biochemical investigations of such neurometabolic disorders and a potential significant impact of quick genetic testing in these patients. Compound heterozygous variants with bi-parental inheritance were identified in all probands, with a mean rate of 6.1 sets of variants per proband (Supplementary Material, Tables S3 and S4). One patient with moderate ID and wellcontrolled seizures carried two rare mutations in the ADSL gene (NM_000026) with bi-parental inheritance confirming a compound heterozygous state (Supplementary Material). Both mutations are extremely rare and were not reported in 1000 Genome or ESV databases. One mutation, c.1191+5G.C, is predicted to impair exon 11 splicing by abolishing the donor splice site (HSF Splice Finder: http://www.umd.be/HSF/ and Mutation Taster). The other mutation, c.T1342C (p.S448P) in exon 12, is predicted to be pathogenic by SIFT (score ¼ 0.00) but potentially benign according to POLYPHEN-2 (score ¼ 0.146). This patient had elevated succinyladenosine urinary


smaller duplications. Among the candidate genes located in these overlapping regions, the WDR45 gene is of special interest. In males, de novo mutations in WDR45 cause a neuroferritinopathy, which is characterized by static encephalopathy during childhood, evolving towards neurodegeneration in the adult through brain iron accumulation (SENDA) (46,47). WDR45 deletions occasionally present as an apparently isolated epileptic encephalopathy (48). Among the two patients carrying variants of unconfirmed inheritance, one patient carried a 22q11.21 microduplication, not inherited from his mother, but for which the paternal inheritance could not be assessed. This variant is a 147 kb duplication corresponding to a small segment of the proximal 22q11 Di George interval (49,50). The variant described here was found in 5 out of 11 000 cases studied in our center (frequency: 0.00045%) including unaffected individuals. This variant is likely a benign variant. The other variants detected in our cohort were considered benign as they were inherited from unaffected parents. However, we cannot exclude the possibility that these variants are associated with a reduced penetrance or that they modify the severity of the phenotype in some cases. Of particular interest, one patient carried an inherited short 0.065 Mb 15q26.1 duplication, which intersected two genes, RGMA and CHD2, without affecting any other genes. CHD2 deletions have recently been reported to cause epileptic encephalopathy in children (48). Another patient carried an inherited 0.41 Mb 15q13.3 duplication, intersecting the CHRNA7 gene, a neuronal nicotinic cholinergic receptor subunit associated with generalized epilepsy (51), schizophrenia (52) and, in one case, IS (53), but has also been reported in asymptomatic controls (local database). It is not clear whether these two duplications affect the expression of the intersected genes. In both cases, the variants were inherited from an asymptomatic parent, suggesting that they are not pathogenic or that they are associated with decreased penetrance.

5

6


excretion, confirming a dysfunction of adenylosuccinate lyase (ADSL). ADSL deficiency is an autosomal recessive defect in purine nucleotide metabolism leading to refractory seizures with variable degrees of hypotonia, global DD, autistic traits and progressive brain atrophy (72). The relatively mild clinical presentation of our patient might be attributable to residual enzymatic function. Identification of de novo mutations by exome sequencing in unexplained IS cases

DISCUSSION This study illustrates the diagnostic efficiency of complementary cytogenetic and genomic approaches in children with IS of unknown etiology. We were able to identify deleterious genetic variants in 11 out of 44 patients (25%) with IS, including cases with de novo (n ¼ 7) or inherited (n ¼ 4) mutations. aCGH was found to be informative as it revealed de novo CNVs in 7% of patients, and variants of uncertain inheritance in 4.5% of patients. Our data support previous reports of high rate of CNVs in children with severe epileptic encephalopathy (82–86), including IS (86) and supports the use of this test as a first-line screening tool in patients with unexplained IS. Together, targeted and exome sequencing identified mutations in genes previously associated with epilepsy or intellectual disability, including STXBP1, ALG13, PNPO, ADSL, CASK and ARX, in seven additional cases. Other known IS genes, such as CDKL5, MAGI2, SCN1A and SCN2A, did not contribute to the pathogenesis of IS in our cohort, and targeted re-sequencing of 35 known epilepsy gene was not informative. Finally, exome sequencing revealed several candidate de novo mutations in the remaining unexplained cases. Therefore, although IS appears to be a relatively welldefined and clinically homogeneous epileptic encephalopathy


We identified rare de novo non-synonymous variants (not inherited from the parents) in the majority of families (n ¼ 12/18, 67%), with 1 – 3 de novo variant per proband (mean of 1.1) (Table 4 and Supplementary Material, Table S3). These, de novo mutations occurred more frequently in genes related to brain development or neuronal processes than what we could have expected from random occurrence. Indeed, 21% of genes identified as carrying de novo mutations in our cohort are involved in nervous system development as compared with 6% of genes as annotated genome-wide in the Panther database (http://www.pantherdb.org) (73) (n ¼ 4/19 versus 1146/18 331, x2 with Yates correction ¼ 4.783, P ¼ 0.0287, two-tailed). Furthermore, 32% of the genes with de novo mutations are involved in neuronal processes compared with 10.4% of genes annotated genome-wide (n ¼ 6/19, versus 1917/18 331, x2 with Yates correction ¼ 6.915, P ¼ 0.0085, two-tailed). Review of the literature (Pubmed, OMIM), consultation of public genomic (NCBI) and proteomic (UniProt) databases and analysis of the variants impact on amino acid sequence or protein structure through SIFT and POLYPHEN-2 scores were used to assess putative pathogenicity of the de novo variants identified. Most variants were missense variants (n ¼ 17/19 variants, 89%), and were identified in 61% of patients (n ¼ 11/18). Most of these variants were expected to be damaging by SIFT (n ¼ 10/17, 59%) and POLYPHEN-2 (n ¼ 12/17, 71%). In addition, we identified two clearly detrimental mutations in 11% of our patients (n ¼ 2 variants/18 patients), one frameshift deletion (in IL27RA) and one stop gain mutation (in CASK). The de novo variants identified were enriched in genes predicted to be intolerant to functional variations as 50% of variants occurred in genes scoring below the 25th percentile for intolerance, according to the Residual Variance Intolerance Score (RVIS) (74), which differed significantly from expectations (n ¼ 9/18 versus n ¼ 4264/16 957 genes with IVS scores, x2 with Yates correction ¼ 4.651, P ¼ 0.031, two-tailed). Together, this data suggest that a large fraction of de novo variants identified will be pathogenic and relevant to IS. These de novo mutations preferentially involved genes reported in UNIPROT or Pubmed to be involved in various fundamental cellular and/or neurodevelopmental processes including gene regulation, protein modification, signal transduction, neurogenesis, cellular differentiation, axonal guidance, neuronal migration, cell adhesion, synaptogenesis and synaptic release (Fig. 2). Furthermore, a gene network analysis weighted for biological processes (geneMANIA v.3.1.2.6; www.genema nia.org) revealed considerable interactions between 14 of the genes identified in this study (STXBP1, SQSTM1, IL27RA, MYO9B, SVIL, EIF2C4, NR2F1, ALG13, HSPG2, WDR45, LAMA2, SEMA5B, CASK, NPC1L1) and other genes associated

with IS or other early-onset epileptic encephalopathies in at least two unrelated cases in the literature (Pubmed) (Fig. 3). Detrimental mutations in these 14 genes are therefore likely to contribute to the pathogenesis of IS. In three patients, de novo mutations were identified in known epilepsy or ID genes and were deemed pathogenic. One patient with moderate ID and intractable seizures carried a de novo missense variant, c.875G.A (p.R292H), in STXBP1 (NM_003165.3) (Supplementary Material). This variant modifies a highly conserved residue and is predicted to be damaging (SIFT score: 0; POLYPHEN-2 score: 1.0). In a second patient, a girl with severe ID and relative microcephaly, a de novo nonsense mutation, c.82C.T (p.R28X), was identified in the CASK gene (NM_001126054.2) on chromosome Xp11.4 (Supplementary Material). CASK loss-of-function mutations cause microcephaly with profound ID and cerebellar and/or pontine atrophy in girls (75,76). CASK mutations are not usually associated with epilepsy. However, CASK binds the neuronal actin cytoskeleton and the pre-synaptic release machinery and participates in synaptogenesis, synaptic release, synaptic plasticity and dendritic spine stabilization (77– 80) and could therefore lead to epilepsy and encephalopathy. In a third patient, a girl with severe ID, exome sequencing revealed a unique de novo mutation, c.320A.G (p.N107S) in the ALG13 gene (NM_001099922.2) on the X chromosome (Supplementary Material). This mutation replaces an asparagine by a serine, predicted to be damaging by SIFT (score ¼ 0), but was considered potentially benign by POLYPHEN-2 (score 0.54). This same mutation has been reported in three females with epileptic encephalopathy (48,81). In the remaining 13 patients, 15 de novo mutations were identified in genes never reported before to cause ID or epilepsy (Table 4). In these cases, further validation in larger cohorts of patients with epileptic encephalopathy and functional validation will be required to confirm pathogenicity.


7


Figure 2. UNIPROT functions for genes with de novo mutations. Upper panel: Relevant functions annotated in UNIPROT database for the 20 genes with de novo variants identified through exome sequencing or targeted sequencing. Some gene products have multiple functions. Bottom panel: Same genes clustered into broader functional groups with regards to known or presumed functions in UniProt.

syndrome, our data suggest that its genetic determinants are multiple, with very few cases explained by mutations in the same gene. Our findings suggest that a majority of unexplained sporadic IS cases involve de novo detrimental genetic variants (CNVs or missense/nonsense mutations) as only a few predicted pathogenic homozygous or compound heterozygous mutations were identified. Indeed, the fact that very few cases of familial

recurrent IS have been described to date would be consistent with the more frequent involvement of de novo mutations. However, in a few sporadic cases, exome sequencing was instrumental in identifying recessive disorders, such as pyridoxalphosphate responsive epileptic encephalopathy (PNPO gene) and ADSL deficiency (ADSL gene), with significant impact on genetic counseling.

8



Figure 3. Gene network analysis. Gene network analysis conducted on the genes with de novo, predicted pathogenic homozygous or compound heterozygous mutations identified in our cohort of children with sporadic IS (black circles with yellow highlight). Other genes associated with IS or other early-onset epileptic encephalopathies (black circles, no highlight) as well as other genes known to interact with the above (grey dots) were included in the analysis. Lines connecting the dots represent known genetic interactions (green), protein interactions reported in interProt (grey), known gene ontology biological pathways (blue) and known direct protein interactions (pink). Note that HSPG2 and LAMA2 are connected with other members of this network in Consolidated Pathways 2013 (lines not shown to simplify overall diagram) and are depicted close to the clusters. (Data analyzed with http://www.genemania.org/ on 5 March 2014).


fundamental neurodevelopmental processes converge to induce epilepsy and possibly cognitive impairment in IS. In a more circuit-specific manner, emerging data support the involvement of GABAergic inhibitory circuits in neurodevelopmental disorders such as epilepsy (reviewed in 107). In this respect, some of the genes discovered in this study have a clear biased effect on GABAergic neurons. For instance, ARX and CXCR4 are essential for proper migration and laminar positioning of cortical GABAergic interneurons (32– 35). Furthermore, CASK encodes a calcium/calmodulin-dependent serine protein kinase, which interacts with a-neurexin and the pre-synaptic release machinery to regulate synaptic vesicle exocytosis (79), with predominant impact on GABA release (80). Therefore, deleterious mutations in these genes might be expected to cause epileptic encephalopathy that might be amendable to cellbased therapies with GABAergic precursors (108). In summary, using a combination of genetic investigation approaches, we identified deleterious variants in a large proportion of patients with unexplained IS. Furthermore, exome sequencing enabled us to uncover de novo mutations in putative new epilepsy genes. The majority of these genes are known to affect fundamental cellular and neurodevelopmental processes such as gene regulation, cell signaling, neuronal differentiation and specification, axonal guidance and neuronal migration, cell adhesion, synaptogenesis and/or synaptic release. Our data suggest that unexplained IS are genetically heterogeneous and can result from abnormalities in most fundamental processes of brain development. Further validation in large multicentric international cohorts as well as functional validation in cellular and animal models will help clarify the impact of mutations in these genes in epilepsy and will shed light on some fundamental processes involved in epileptogenesis.

MATERIAL AND METHODS Patient recruitment We recruited 44 children with a history of unexplained IS followed in our pediatric epilepsy clinic at the CHU Ste-Justine in Montreál. All patients had received a diagnosis of IS based on clinical evaluation by a pediatric epileptologist (AL, LC, PD, PM), according to ILAE guidelines. Extensive radiological, metabolic, infectious investigations were obtained to exclude readily identifiable etiologies (Supplementary Material, Table S1). Patients were treated with vigabatrin as first-line treatment, with rapid dose increase in non-responders up to 150 mg/kg/day over 2–3 weeks, followed by ACTH treatment in refractory cases, with additional topiramate in non-responders. Other anticonvulsive treatments were added when required over time at the discretion of the treating neurologist. Patients were followed by their treating neurologists, who documented seizure types, frequency, EEG anomalies and developmental progresses. Genomic DNA was extracted from a blood aliquot obtained from each proband and their parents, after obtaining informed consent for genetic investigation in accordance with our Ethics Committee Board. Array comparative genomic hybridization (aCGH) aCGH analysis was performed in all probands, using a 135K-feature whole-genome microarray (SignatureChip


Another recent study based on the use of exome sequencing in familial trios also provided evidence for the involvement of de novo mutations in IS (48). In both this study and ours, the majority of cases remained unexplained. By merging these two datasets (n ¼ 139 cases of sporadic IS), we found that only STXBP1 (n ¼ 6), ALG13 (n ¼ 2) and CDKL5 (n ¼ 2) contained de novo mutations in at least two patients, representing 7% of IS cases. This observation indicates that the study of much larger cohorts will be required to validate the involvement of the remaining de novo mutations. Furthermore, this finding suggests that unbiased investigation with whole-exome or wholegenome sequencing will remain more efficient than targeted re-sequencing of known epilepsy genes until a larger fraction of IS genes have been identified through sequencing of larger cohorts of patients. We were intrigued to find the same ALG13 mutation, c.320A.G (p.N107S) (NM_001099922.2), that has just been reported in three other girls with epileptic encephalopathy (48,81). No other mutation in ALG13 had been reported in girls to date. In contrast, another ALG13 mutation, c.280A.G (K94E) was reported in a male patient with global delay, refractory epilepsy, microcephaly, pyramidal and extrapyramidal signs, a bleeding diathesis and recurrent infections with early lethality (87). The recurrence of a single ALG13 mutation in sporadic cases of epileptic encephalopathy in girls is puzzling and could suggest either a dominant-negative or a gain-offunction effect. Although most patients with sporadic IS appear to carry ‘private mutations’ in a genetically heterogeneous fashion, some mechanistic insight regarding the pathophysiology of IS can be gained from the known or presumed function of these genes taken in the context of systems biology. For instance, by reviewing published annotations in UNIPROT and Pubmed, we found that 35% of the genes identified as carrying de novo variants affect gene transcription or protein translation, with a majority being involved in known neurodevelopmental genetic cascades. For instance, 35% of genes are involved in regulating neurogenesis, neuronal cell fate or differentiation: EIF2C4/ AGO4 regulates neuronal cell-fate (88), ARX is involved in neurogenesis (89), NR2F1 regulates cortical patterning and cortical pyramidal cell sub-type specification (90,91), HSPH2 (Perlecan) controls neurogenesis (92) and GMEB2 is a co-activator that regulates the expression of glucocorticoidinduced genes (93). Furthermore, 45% of genes with de novo variants were found to affect axonal guidance and/or cellular or neuronal migration, including ARX (89), NR2F1 (94,95), SVIL (96), TENM2 (97), SEMA5B (98) and CXCR4 (99). In addition, some of these genes were found to bind the actin cytoskeleton and affect dendritogenesis, spine dynamics or signaling through the Rho/Ras cascade, such as MYO9B (100) and SVIL (101). In addition, 25% of the genes with de novo variants are known to participate in cellular adhesion and/or synaptogenesis, including TENM2 (102), LAMA2 (103,104) and CASK (77,78), and 12% of the genes are involved in synaptic transmission, including STXBP1 (54) and CASK (79). Finally, 15% of genes with de novo variants are implicated in protein modification or degradation, including ALG13 (105) and SQSTM1 (106), and might affect a variety of neuronal developmental processes. Together, this data suggest that disruption of several

9

10


OS2.0 manufactured for Signature Genomic Laboratories (Spokane, WA, USA) by Roche NimbleGen, Madison, WI, USA; based on UCSC 2006 hg18 assembly), according to the manufacturer’s protocol. Genomic coordinates indicate the minimal size of the CNVs. When possible, fluorescence in situ hybridization (FISH) studies were performed in the probands to confirm CNVs and in both parents to investigate inheritance patterns. In cases where FISH was not possible, aCGH was performed in the parents. In cases where the CNV was inherited, the parental study was used as a confirmation. In de novo cases, the CNV was confirmed using a chromosome specific microarray (Roche NimbleGen). Targeted sequencing

Whole-exome sequencing WES of probands and parents was conducted in 18 familial trios with unexplained IS. Briefly, blood genomic DNA was captured with the Agilent SureSelect Human All Exon Capture V4 kit (Mississauga, ON) and sequenced (paired-end, 2 × 100 bp, three exomes/lane format) using the Illumina HiSeq2000 at the McGill University Genome Quebec Innovation Center (Montreal, Canada). Sequence processing, alignment (using a Burroughs– Wheeler algorithm, BWA), and variant calling were done according to the Broad Institute Genome Analysis Tool Kit (GATK v4) best practices, and variant annotation was done using Annovar (110). The exome target base average coverage was 117×, with 95% of the target bases being covered by at least 10 reads. Only the variants whose positions were covered at ≥8× and supported by ≥3 variant reads that constitute at least 20% of the total reads for each called position were retained. To identify rare potentially pathogenic variants, we filtered out: (1) synonymous variants or intronic variants other than those affecting the consensus splice sites; (2) variants seen in more than 2% of our in-house exomes dataset (n ¼ 1000) from unrelated projects; and (3) variants with a minor allele frequency .1.0% in either the 1000 genomes or NHLBI exome sequencing project (ESP) datasets (EVS). We identified de novo mutations by excluding all variants found in the proband that were transmitted by one of the parents. We confirmed all potential de novo mutations by Sanger re-sequencing. We cataloged all homozygous, compound heterozygous and hemizygous variants.

SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online.

We wish to thank all families that participated in this study. Furthermore, we thank the members of the bioinformatic analysis team of Re´seau de Me´decine Geńe´tique Appliqueé du Que´bec (Alexandre Dionne-Laporte, Dan Spiegelman, Edouard Henrion and Ousmane Diallo) for the bioinformatic analysis of the exome sequencing data. Conflict of Interest statement. None declared.

FUNDING This work was supported by funds from The Scottish Rite Charitable Foundation of Canada, The Savoy Foundation, the Re´seau de Me´decine Geńe´tique du Que´bec (RMGA), Genome Quebec and Genome Canada. E.R. is a Clinician-Scientist I of the Fonds de Recherche du Que´bec—Sante´. J.L. is a National Scientist of the Fonds de Recherche du Que´bec—Sante´.

REFERENCES 1. Carmant, L. (2002) Infantile spasms: West syndrome. Arch. Neurol., 59, 317– 318. 2. Riikonen, R. (2009) Current management of infantile spasms. In: Maria. E.B, ed. Current in Child Neurology, pp. 153– 158. 3. Osborne, J.P., Lux, A.L., Edwards, S.W., Hancock, E., Johnson, A.L., Kennedy, C.R., Newton, R.W., Verity, C.M. and O’Callaghan, F.J. (2010) The underlying etiology of infantile spasms (West syndrome): information from the United Kingdom Infantile Spasms Study (UKISS) on contemporary causes and their classification. Epilepsia, 51, 2168–2174. 4. Bennett, C.L., Chen, Y., Hahn, S., Glass, I.A. and Gospe, S.M. Jr. (2009) Prevalence of ALDH7A1 mutations in 18 North American pyridoxine-dependent seizure (PDS) patients. Epilepsia, 50, 1167–1175. 5. Mikati, M.A., Zalloua, P., Karam, P., Habbal, M.Z. and Rahi, A.C. (2006) Novel mutation causing partial biotinidase deficiency in a Syrian boy with infantile spasms and retardation. J. Child Neurol., 21, 978–981. 6. Naito, E., Ito, M., Yokota, I., Saijo, T., Ogawa, Y., Shinahara, K. and Kuroda, Y. (2001) Gender-specific occurrence of West syndrome in patients with pyruvate dehydrogenase complex deficiency. Neuropediatrics, 32, 295–298. 7. Shah, N.S., Mitchell, W.G. and Boles, R.G. (2002) Mitochondrial disorders: a potentially under-recognized etiology of infantile spasms. J. Child Neurol., 17, 369– 372. 8. Lev, D., Weigl, Y., Hasan, M., Gak, E., Davidovich, M., Vinkler, C., Leshinsky-Silver, E., Lerman-Sagie, T. and Watemberg, N. (2007) A novel missense mutation in the NDP gene in a child with Norrie disease and severe neurological involvement including infantile spasms. Am. J. Med. Genet. A., 143A, 921– 924. 9. Kajimoto, M., Ichiyama, T., Akashi, A., Suenaga, N., Matsufuji, H. and Furukawa, S. (2007) West syndrome associated with mosaic Down syndrome. Brain Dev., 29, 447–449. 10. Battaglia, A., Hoyme, H.E., Dallapiccola, B., Zackai, E., Hudgins, L., McDonald-McGinn, D., Bahi-Buisson, N., Romano, C., Williams, C.A., Brailey, L.L. et al. (2008) Further delineation of deletion 1p36 syndrome in 60 patients: a recognizable phenotype and common cause of developmental delay and mental retardation. Pediatrics, 121, 404– 410. 11. Bahi-Buisson, N., Guttierrez-Delicado, E., Soufflet, C., Rio, M., Daire, V.C., Lacombe, D., Heron, D., Verloes, A., Zuberi, S., Burglen, L. et al. (2008) Spectrum of epilepsy in terminal 1p36 deletion syndrome. Epilepsia, 49, 509– 515. 12. Tuysuz, B., Demirel, A., Uysal, S., Beyer, V. and Bartsch, O. (2008) Boy with seizures (West syndrome) and distal 7q duplication syndrome due to an unbalanced 7q;9p translocation. Genet. Couns., 19, 29–35. 13. Morimoto, M., An, B., Ogami, A., Shin, N., Sugino, Y., Sawai, Y., Usuku, T., Tanaka, M., Hirai, K., Nishimura, A. et al. (2003) Infantile spasms in a patient with Williams syndrome and craniosynostosis. Epilepsia, 44, 1459– 1462.


In children with negative aCGH results, Sanger sequencing of one or two candidate genes was performed on a clinical basis, according to presentation. ARX was tested in boys, CDKL5 in girls, STXBP1 in children with severe ID and other seizure types. Cases with unexplained IS following aCGH and targeted sequencing were then either investigated by re-sequencing of a panel of 35 known epileptic encephalopathy genes (Supplementary Material, Table S2) as described previously (n ¼ 8) (109) or through exome sequencing of familial trios (n ¼ 18) as detailed below.

ACKNOWLEDGEMENTS


33. Lopez-Bendito, G., Sanchez-Alcaniz, J.A., Pla, R., Borrell, V., Pico, E., Valdeolmillos, M. and Marin, O. (2008) Chemokine signaling controls intracortical migration and final distribution of GABAergic interneurons. J. Neurosci., 28, 1613– 1624. 34. Tanaka, D.H., Mikami, S., Nagasawa, T., Miyazaki, J., Nakajima, K. and Murakami, F. (2010) CXCR4 is required for proper regional and laminar distribution of cortical somatostatin-, calretinin-, and neuropeptide Y-expressing GABAergic interneurons. Cereb. Cortex, 20, 2810– 2817. 35. Wang, Y., Li, G., Stanco, A., Long, J.E., Crawford, D., Potter, G.B., Pleasure, S.J., Behrens, T. and Rubenstein, J.L. (2011) CXCR4 and CXCR7 have distinct functions in regulating interneuron migration. Neuron, 69, 61– 76. 36. Shepherd, A.J., Loo, L., Gupte, R.P., Mickle, A.D. and Mohapatra, D.P. (2012) Distinct modifications in Kv2.1 channel via chemokine receptor CXCR4 regulate neuronal survival-death dynamics. J. Neurosci., 32, 17725– 17739. 37. Missler, M., Hammer, R.E. and Sudhof, T.C. (1998) Neurexophilin binding to alpha-neurexins. A single LNS domain functions as an independently folding ligand-binding unit. J. Biol. Chem., 273, 34716–34723. 38. Missler, M., Fernandez-Chacon, R. and Sudhof, T.C. (1998) The making of neurexins. J. Neurochem., 71, 1339–1347. 39. Craig, A.M. and Kang, Y. (2007) Neurexin–neuroligin signaling in synapse development. Curr. Opin. Neurobiol., 17, 43– 52. 40. Missler, M., Zhang, W., Rohlmann, A., Kattenstroth, G., Hammer, R.E., Gottmann, K. and Sudhof, T.C. (2003) Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis. Nature, 423, 939– 948. 41. Crolla, J.A., Harvey, J.F., Sitch, F.L. and Dennis, N.R. (1995) Supernumerary marker 15 chromosomes: a clinical, molecular and FISH approach to diagnosis and prognosis. Hum. Genet., 95, 161–170. 42. Ungaro, P., Christian, S.L., Fantes, J.A., Mutirangura, A., Black, S., Reynolds, J., Malcolm, S., Dobyns, W.B. and Ledbetter, D.H. (2001) Molecular characterisation of four cases of intrachromosomal triplication of chromosome 15q11-q14. J. Med. Genet., 38, 26–34. 43. Bachmann-Gagescu, R., Mefford, H.C., Cowan, C., Glew, G.M., Hing, A.V., Wallace, S., Bader, P.I., Hamati, A., Reitnauer, P.J., Smith, R. et al. (2010) Recurrent 200-kb deletions of 16p11.2 that include the SH2B1 gene are associated with developmental delay and obesity. Genet. Med., 12, 641–647. 44. Tabet, A.C., Pilorge, M., Delorme, R., Amsellem, F., Pinard, J.M., Leboyer, M., Verloes, A., Benzacken, B. and Betancur, C. (2012) Autism multiplex family with 16p11.2p12.2 microduplication syndrome in monozygotic twins and distal 16p11.2 deletion in their brother. Eur. J. Hum. Genet., 20, 540–546. 45. Chung, B.H., Drmic, I., Marshall, C.R., Grafodatskaya, D., Carter, M., Fernandez, B.A., Weksberg, R., Roberts, W. and Scherer, S.W. (2011) Phenotypic spectrum associated with duplication of Xp11.22-p11.23 includes Autism Spectrum Disorder. Eur. J. Med. Genet., 54, e516 –e520. 46. Saitsu, H., Nishimura, T., Muramatsu, K., Kodera, H., Kumada, S., Sugai, K., Kasai-Yoshida, E., Sawaura, N., Nishida, H., Hoshino, A. et al. (2013) De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood. Nat. Genet., 45, 445–449, 449e1. 47. Haack, T.B., Hogarth, P., Kruer, M.C., Gregory, A., Wieland, T., Schwarzmayr, T., Graf, E., Sanford, L., Meyer, E., Kara, E. et al. (2012) Exome sequencing reveals de novo WDR45 mutations causing a phenotypically distinct, X-linked dominant form of NBIA. Am. J. Hum. Genet., 91, 1144– 1149. 48. Allen, A.S., Berkovic, S.F., Cossette, P., Delanty, N., Dlugos, D., Eichler, E.E., Epstein, M.P., Glauser, T., Goldstein, D.B., Han, Y. et al. (2013) De novo mutations in epileptic encephalopathies. Nature, 501, 217–221. 49. Shaikh, T.H., O’Connor, R.J., Pierpont, M.E., McGrath, J., Hacker, A.M., Nimmakayalu, M., Geiger, E., Emanuel, B.S. and Saitta, S.C. (2007) Low copy repeats mediate distal chromosome 22q11.2 deletions: sequence analysis predicts breakpoint mechanisms. Genome. Res., 17, 482– 491. 50. Shaikh, T.H., Kurahashi, H., Saitta, S.C., O’Hare, A.M., Hu, P., Roe, B.A., Driscoll, D.A., McDonald-McGinn, D.M., Zackai, E.H., Budarf, M.L. et al. (2000) Chromosome 22-specific low copy repeats and the 22q11.2 deletion syndrome: genomic organization and deletion endpoint analysis. Hum. Mol. Genet., 9, 489– 501. 51. Helbig, I., Mefford, H.C., Sharp, A.J., Guipponi, M., Fichera, M., Franke, A., Muhle, H., de Kovel, C., Baker, C., von Spiczak, S. et al. (2009) 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy. Nat. Genet., 41, 160–162.


14. Yamamoto, H., Fukuda, M., Murakami, H., Kamiyama, N. and Miyamoto, Y. (2007) A case of Pallister– Killian syndrome associated with West syndrome. Pediatr. Neurol., 37, 226– 228. 15. Hino-Fukuyo, N., Haginoya, K., Uematsu, M., Nakayama, T., Kikuchi, A., Kure, S., Kamada, F., Abe, Y., Arai, N., Togashi, N. et al. (2009) Smith– Magenis syndrome with West syndrome in a 5-year-old girl: a long-term follow-up study. J. Child Neurol., 24, 868– 873. 16. Tachi, N., Fujii, K., Kimura, M., Seki, K., Hirakai, M. and Miyashita, T. (2007) New mutation of the PTCH gene in nevoid basal-cell carcinoma syndrome with West syndrome. Pediatr. Neurol., 37, 363–365. 17. Rosser, T. (2003) Aicardi syndrome. Arch. Neurol., 60, 1471–1473. 18. Rosser, T.L., Acosta, M.T. and Packer, R.J. (2002) Aicardi syndrome: spectrum of disease and long-term prognosis in 77 females. Pediatr. Neurol., 27, 343– 346. 19. Parrini, E., Ferrari, A.R., Dorn, T., Walsh, C.A. and Guerrini, R. (2009) Bilateral frontoparietal polymicrogyria, Lennox–Gastaut syndrome, and GPR56 gene mutations. Epilepsia, 50, 1344–1353. 20. Masruha, M.R., Caboclo, L.O., Carrete, H. Jr., Cendes, I.L., Rodrigues, M.G., Garzon, E., Yacubian, E.M., Sakamoto, A.C., Sheen, V., Harney, M. et al. (2006) Mutation in filamin A causes periventricular heterotopia, developmental regression, and West syndrome in males. Epilepsia, 47, 211– 214. 21. Kato, M., Das, S., Petras, K., Sawaishi, Y. and Dobyns, W.B. (2003) Polyalanine expansion of ARX associated with cryptogenic West syndrome. Neurology, 61, 267–276. 22. Nemos, C., Lambert, L., Giuliano, F., Doray, B., Roubertie, A., Goldenberg, A., Delobel, B., Layet, V., N’Guyen, M.A., Saunier, A. et al. (2009) Mutational spectrum of CDKL5 in early-onset encephalopathies: a study of a large collection of French patients and review of the literature. Clin. Genet., 76, 357– 371. 23. Bahi-Buisson, N., Nectoux, J., Rosas-Vargas, H., Milh, M., Boddaert, N., Girard, B., Cances, C., Ville, D., Afenjar, A., Rio, M. et al. (2008) Key clinical features to identify girls with CDKL5 mutations. Brain, 131, 2647– 2661. 24. Scala, E., Ariani, F., Mari, F., Caselli, R., Pescucci, C., Longo, I., Meloni, I., Giachino, D., Bruttini, M., Hayek, G. et al. (2005) CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms. J. Med. Genet., 42, 103– 107. 25. Marshall, C.R., Young, E.J., Pani, A.M., Freckmann, M.L., Lacassie, Y., Howald, C., Fitzgerald, K.K., Peippo, M., Morris, C.A., Shane, K. et al. (2008) Infantile spasms is associated with deletion of the MAGI2 gene on chromosome 7q11.23-q21.11. Am. J. Hum. Genet., 83, 106– 111. 26. Otsuka, M., Oguni, H., Liang, J.S., Ikeda, H., Imai, K., Hirasawa, K., Tachikawa, E., Shimojima, K., Osawa, M. and Yamamoto, T. (2010) STXBP1 mutations cause not only Ohtahara syndrome but also West syndrome—result of Japanese cohort study. Epilepsia, 51, 2449– 2452. 27. Wallace, R.H., Hodgson, B.L., Grinton, B.E., Gardiner, R.M., Robinson, R., Rodriguez-Casero, V., Sadleir, L., Morgan, J., Harkin, L.A., Dibbens, L.M. et al. (2003) Sodium channel alpha1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms. Neurology, 61, 765– 769. 28. Ogiwara, I., Ito, K., Sawaishi, Y., Osaka, H., Mazaki, E., Inoue, I., Montal, M., Hashikawa, T., Shike, T., Fujiwara, T. et al. (2009) De novo mutations of voltage-gated sodium channel alphaII gene SCN2A in intractable epilepsies. Neurology, 73, 1046–1053. 29. Porfirio, M.C., Lo-Castro, A., Giana, G., Giovinazzo, S., Purper Ouakil, D., Galasso, C. and Curatolo, P. (2012) Attention-deficit hyperactivity disorder and binge eating disorder in a patient with 2q21.1-q22.2 deletion. Psychiatr. Genet., 22, 202 –205. 30. Mulatinho, M.V., de Carvalho Serao, C.L., Scalco, F., Hardekopf, D., Pekova, S., Mrasek, K., Liehr, T., Weise, A., Rao, N. and Llerena, J.C. Jr. (2012) Severe intellectual disability, omphalocele, hypospadia and high blood pressure associated to a deletion at 2q22.1q22.3: case report. Mol. Cytogenet., 5, 30. 31. Shanske, A.L., Edelmann, L., Kardon, N.B., Gosset, P. and Levy, B. (2004) Detection of an interstitial deletion of 2q21–22 by high resolution comparative genomic hybridization in a child with multiple congenital anomalies and an apparent balanced translocation. Am. J. Med. Genet. A., 131, 29– 35. 32. Li, G., Adesnik, H., Li, J., Long, J., Nicoll, R.A., Rubenstein, J.L. and Pleasure, S.J. (2008) Regional distribution of cortical interneurons and development of inhibitory tone are regulated by Cxcl12/Cxcr4 signaling. J. Neurosci., 28, 1085–1098.

11

12


70. Khayat, M., Korman, S.H., Frankel, P., Weintraub, Z., Hershckowitz, S., Sheffer, V.F., Ben Elisha, M., Wevers, R.A. and Falik-Zaccai, T.C. (2008) PNPO deficiency: an under diagnosed inborn error of pyridoxine metabolism. Mol. Genet. Metab., 94, 431–434. 71. Mills, P.B., Surtees, R.A., Champion, M.P., Beesley, C.E., Dalton, N., Scambler, P.J., Heales, S.J., Briddon, A., Scheimberg, I., Hoffmann, G.F. et al. (2005) Neonatal epileptic encephalopathy caused by mutations in the PNPO gene encoding pyridox(am)ine 5′ -phosphate oxidase. Hum. Mol. Genet., 14, 1077–1086. 72. Lundy, C.T., Jungbluth, H., Pohl, K.R., Siddiqui, A., Marinaki, A.M., Mundy, H. and Champion, M.P. (2010) Adenylosuccinate lyase deficiency in the United Kingdom pediatric population: first three cases. Pediatr. Neurol., 43, 351– 354. 73. Mi, H., Muruganujan, A. and Thomas, P.D. (2012) PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res., 41, D377– D386. 74. Petrovski, S., Wang, Q., Heinzen, E.L., Allen, A.S. and Goldstein, D.B. (2013) Genic intolerance to functional variation and the interpretation of personal genomes. PLoS Genet., 9, e1003709. 75. Najm, J., Horn, D., Wimplinger, I., Golden, J.A., Chizhikov, V.V., Sudi, J., Christian, S.L., Ullmann, R., Kuechler, A., Haas, C.A. et al. (2008) Mutations of CASK cause an X-linked brain malformation phenotype with microcephaly and hypoplasia of the brainstem and cerebellum. Nat. Genet., 40, 1065–1067. 76. Moog, U., Kutsche, K., Kortum, F., Chilian, B., Bierhals, T., Apeshiotis, N., Balg, S., Chassaing, N., Coubes, C., Das, S. et al. (2011) Phenotypic spectrum associated with CASK loss-of-function mutations. J. Med. Genet., 48, 741– 751. 77. Butz, S., Okamoto, M. and Sudhof, T.C. (1998) A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain. Cell, 94, 773–782. 78. Hsueh, Y.P. (2009) Calcium/calmodulin-dependent serine protein kinase and mental retardation. Ann. Neurol., 66, 438– 443. 79. Klemmer, P., Smit, A.B. and Li, K.W. (2009) Proteomics analysis of immuno-precipitated synaptic protein complexes. J. Proteomics, 72, 82–90. 80. Atasoy, D., Schoch, S., Ho, A., Nadasy, K.A., Liu, X., Zhang, W., Mukherjee, K., Nosyreva, E.D., Fernandez-Chacon, R., Missler, M. et al. (2007) Deletion of CASK in mice is lethal and impairs synaptic function. Proc. Natl. Acad. Sci. U. S. A., 104, 2525–2530. 81. de Ligt, J., Willemsen, M.H., van Bon, B.W., Kleefstra, T., Yntema, H.G., Kroes, T., Vulto-van Silfhout, A.T., Koolen, D.A., de Vries, P., Gilissen, C. et al. (2012) Diagnostic exome sequencing in persons with severe intellectual disability. N. Engl. J. Med., 367, 1921–1929. 82. Mefford, H.C., Muhle, H., Ostertag, P., von Spiczak, S., Buysse, K., Baker, C., Franke, A., Malafosse, A., Genton, P., Thomas, P. et al. (2010) Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS Genet., 6, e1000962. 83. Striano, P., Coppola, A., Paravidino, R., Malacarne, M., Gimelli, S., Robbiano, A., Traverso, M., Pezzella, M., Belcastro, V., Bianchi, A. et al. (2012) Clinical significance of rare copy number variations in epilepsy: a case– control survey using microarray-based comparative genomic hybridization. Arch. Neurol., 69, 322–330. 84. Ezugha, H., Anderson, C.E., Marks, H.G., Khurana, D., Legido, A. and Valencia, I. (2010) Microarray analysis in children with developmental disorder or epilepsy. Pediatr. Neurol., 43, 391– 394. 85. Mulley, J.C. and Mefford, H.C. (2011) Epilepsy and the new cytogenetics. Epilepsia, 52, 423– 432. 86. Tiwari, V.N., Sundaram, S.K., Chugani, H.T. and Huq, A.H. (2012) Infantile spasms are associated with abnormal copy number variations. J. Child Neurol, 28, 1191– 1196. 87. Timal, S., Hoischen, A., Lehle, L., Adamowicz, M., Huijben, K., Sykut-Cegielska, J., Paprocka, J., Jamroz, E., van Spronsen, F.J., Korner, C. et al. (2012) Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing. Hum. Mol. Genet., 21, 4151–4161. 88. Potenza, N., Papa, U. and Russo, A. (2009) Differential expression of Dicer and Argonaute genes during the differentiation of human neuroblastoma cells. Cell. Biol. Int., 33, 734–738. 89. Friocourt, G., Kanatani, S., Tabata, H., Yozu, M., Takahashi, T., Antypa, M., Raguenes, O., Chelly, J., Ferec, C., Nakajima, K. et al. (2008) Cell-autonomous roles of ARX in cell proliferation and neuronal migration during corticogenesis. J. Neurosci., 28, 5794–5805.


52. Consortium, I.S. (2008) Rare chromosomal deletions and duplications increase risk of schizophrenia. Nature, 455, 237– 241. 53. Lacaze, E., Gruchy, N., Penniello-Valette, M.J., Plessis, G., Richard, N., Decamp, M., Mittre, H., Leporrier, N., Andrieux, J., Kottler, M.L. et al. (2013) De novo 15q13.3 microdeletion with cryptogenic West syndrome. Am. J. Med. Genet. A, 161, 2582– 2587. 54. Ma, C., Su, L., Seven, A.B., Xu, Y. and Rizo, J. (2013) Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release. Science, 339, 421–425. 55. Saitsu, H., Kato, M., Mizuguchi, T., Hamada, K., Osaka, H., Tohyama, J., Uruno, K., Kumada, S., Nishiyama, K., Nishimura, A. et al. (2008) De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy. Nat. Genet., 40, 782 –788. 56. Hamdan, F.F., Piton, A., Gauthier, J., Lortie, A., Dubeau, F., Dobrzeniecka, S., Spiegelman, D., Noreau, A., Pellerin, S., Cote, M. et al. (2009) De novo STXBP1 mutations in mental retardation and nonsyndromic epilepsy. Ann. Neurol., 65, 748–753. 57. Mastrangelo, M., Peron, A., Spaccini, L., Novara, F., Scelsa, B., Introvini, P., Raviglione, F., Faiola, S. and Zuffardi, O. (2013) Neonatal suppression-burst without epileptic seizures: expanding the electroclinical phenotype of STXBP1-related, early-onset encephalopathy. Epileptic Disord., 15, 55–61. 58. Otsuka, M., Oguni, H., Liang, J.S., Ikeda, H., Imai, K., Hirasawa, K., Tachikawa, E., Shimojima, K., Osawa, M. and Yamamoto, T. (2011) STXBP1 mutations cause not only Ohtahara syndrome but also West syndrome—result of Japanese cohort study. Epilepsia, 51, 2449–2452. 59. Deprez, L., Weckhuysen, S., Holmgren, P., Suls, A., Van Dyck, T., Goossens, D., Del-Favero, J., Jansen, A., Verhaert, K., Lagae, L. et al. (2010) Clinical spectrum of early-onset epileptic encephalopathies associated with STXBP1 mutations. Neurology, 75, 1159– 1165. 60. Kitamura, K., Yanazawa, M., Sugiyama, N., Miura, H., Iizuka-Kogo, A., Kusaka, M., Omichi, K., Suzuki, R., Kato-Fukui, Y., Kamiirisa, K. et al. (2002) Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans. Nat. Genet., 32, 359 –369. 61. Bienvenu, T., Poirier, K., Friocourt, G., Bahi, N., Beaumont, D., Fauchereau, F., Ben Jeema, L., Zemni, R., Vinet, M.C., Francis, F. et al. (2002) ARX, a novel Prd-class-homeobox gene highly expressed in the telencephalon, is mutated in X-linked mental retardation. Hum. Mol. Genet., 11, 981 –991. 62. Kato, M., Saitoh, S., Kamei, A., Shiraishi, H., Ueda, Y., Akasaka, M., Tohyama, J., Akasaka, N. and Hayasaka, K. (2007) A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome). Am. J. Hum. Genet., 81, 361 –366. 63. Scheffer, I.E., Wallace, R.H., Phillips, F.L., Hewson, P., Reardon, K., Parasivam, G., Stromme, P., Berkovic, S.F., Gecz, J. and Mulley, J.C. (2002) X-linked myoclonic epilepsy with spasticity and intellectual disability: mutation in the homeobox gene ARX. Neurology, 59, 348– 356. 64. Sherr, E.H. (2003) The ARX story (epilepsy, mental retardation, autism, and cerebral malformations): one gene leads to many phenotypes. Curr. Opin. Pediatr., 15, 567–571. 65. Mari, F., Azimonti, S., Bertani, I., Bolognese, F., Colombo, E., Caselli, R., Scala, E., Longo, I., Grosso, S., Pescucci, C. et al. (2005) CDKL5 belongs to the same molecular pathway of MeCP2 and it is responsible for the early-onset seizure variant of Rett syndrome. Hum. Mol. Genet., 14, 1935–1946. 66. Fichou, Y., Bieth, E., Bahi-Buisson, N., Nectoux, J., Girard, B., Chelly, J., Chaix, Y. and Bienvenu, T. (2009) Re: CDKL5 mutations in boys with severe encephalopathy and early-onset intractable epilepsy. Neurology, 73, 77– 78; author reply 78. 67. Liang, J.S., Shimojima, K., Takayama, R., Natsume, J., Shichiji, M., Hirasawa, K., Imai, K., Okanishi, T., Mizuno, S., Okumura, A. et al. (2011) CDKL5 alterations lead to early epileptic encephalopathy in both genders. Epilepsia, 52, 1835– 1842. 68. Mirzaa, G.M., Paciorkowski, A.R., Marsh, E.D., Berry-Kravis, E.M., Medne, L., Alkhateeb, A., Grix, A., Wirrell, E.C., Powell, B.R., Nickels, K.C. et al. (2013) CDKL5 and ARX mutations in males with early-onset epilepsy. Pediatr. Neurol., 48, 367–377. 69. Elia, M., Falco, M., Ferri, R., Spalletta, A., Bottitta, M., Calabrese, G., Carotenuto, M., Musumeci, S.A., Lo Giudice, M. and Fichera, M. (2008) CDKL5 mutations in boys with severe encephalopathy and early-onset intractable epilepsy. Neurology, 71, 997 –999.


100. Long, H., Zhu, X., Yang, P., Gao, Q., Chen, Y. and Ma, L. (2012) Myo9b and RICS modulate dendritic morphology of cortical neurons. Cereb. Cortex, 23, 71– 79. 101. Crowley, J.L., Smith, T.C., Fang, Z., Takizawa, N. and Luna, E.J. (2009) Supervillin reorganizes the actin cytoskeleton and increases invadopodial efficiency. Mol. Biol. Cell., 20, 948– 962. 102. Silva, J.P., Lelianova, V.G., Ermolyuk, Y.S., Vysokov, N., Hitchen, P.G., Berninghausen, O., Rahman, M.A., Zangrandi, A., Fidalgo, S., Tonevitsky, A.G. et al. (2011) Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities. Proc. Natl. Acad. Sci. U. S. A., 108, 12113–12118. 103. Nasu, M., Takata, N., Danjo, T., Sakaguchi, H., Kadoshima, T., Futaki, S., Sekiguchi, K., Eiraku, M. and Sasai, Y. (2013) Robust formation and maintenance of continuous stratified cortical neuroepithelium by laminin-containing matrix in mouse ES cell culture. PLoS One, 7, e53024. 104. Tian, M., Hagg, T., Denisova, N., Knusel, B., Engvall, E. and Jucker, M. (1997) Laminin-alpha2 chain-like antigens in CNS dendritic spines. Brain Res., 764, 28–38. 105. Gao, X.D., Tachikawa, H., Sato, T., Jigami, Y. and Dean, N. (2005) Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation. J. Biol. Chem., 280, 36254–36262. 106. Ramesh Babu, J., Lamar Seibenhener, M., Peng, J., Strom, A.L., Kemppainen, R., Cox, N., Zhu, H., Wooten, M.C., Diaz-Meco, M.T., Moscat, J. et al. (2008) Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration. J. Neurochem., 106, 107–120. 107. Rossignol, E. (2011) Genetics and function of neocortical GABAergic interneurons in neurodevelopmental disorders. Neural. Plast., 2011, 649325. 108. Sebe, J.Y. and Baraban, S.C. (2010) The promise of an interneuron-based cell therapy for epilepsy. Dev. Neurobiol., 71, 107– 117. 109. Carvill, G.L., Heavin, S.B., Yendle, S.C., McMahon, J.M., O’Roak, B.J., Cook, J., Khan, A., Dorschner, M.O., Weaver, M., Calvert, S. et al. (2013) Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. Nat. Genet., 45, 825– 830. 110. Wang, K., Li, M. and Hakonarson, H. (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res., 38, e164.


90. Faedo, A., Tomassy, G.S., Ruan, Y., Teichmann, H., Krauss, S., Pleasure, S.J., Tsai, S.Y., Tsai, M.J., Studer, M. and Rubenstein, J.L. (2008) COUP-TFI coordinates cortical patterning, neurogenesis, and laminar fate and modulates MAPK/ERK, AKT, and beta-catenin signaling. Cereb. Cortex, 18, 2117– 2131. 91. Armentano, M., Chou, S.J., Tomassy, G.S., Leingartner, A., O’Leary, D.D. and Studer, M. (2007) COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas. Nat. Neurosci., 10, 1277– 1286. 92. Giros, A., Morante, J., Gil-Sanz, C., Fairen, A. and Costell, M. (2007) Perlecan controls neurogenesis in the developing telencephalon. BMC Dev. Biol., 7, 29. 93. Kaul, S., Blackford, J.A. Jr., Chen, J., Ogryzko, V.V. and Simons, S.S. Jr. (2000) Properties of the glucocorticoid modulatory element binding proteins GMEB-1 and -2: potential new modifiers of glucocorticoid receptor transactivation and members of the family of KDWK proteins. Mol. Endocrinol., 14, 1010–1027. 94. Alfano, C., Viola, L., Heng, J.I., Pirozzi, M., Clarkson, M., Flore, G., De Maio, A., Schedl, A., Guillemot, F. and Studer, M. (2011) COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression. Development, 138, 4685– 4697. 95. Armentano, M., Filosa, A., Andolfi, G. and Studer, M. (2006) COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth. Development, 133, 4151–4162. 96. Fang, Z., Takizawa, N., Wilson, K.A., Smith, T.C., Delprato, A., Davidson, M.W., Lambright, D.G. and Luna, E.J. (2010) The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility. Traffic, 11, 782–799. 97. Rubin, B.P., Tucker, R.P., Brown-Luedi, M., Martin, D. and Chiquet-Ehrismann, R. (2002) Teneurin 2 is expressed by the neurons of the thalamofugal visual system in situ and promotes homophilic cell–cell adhesion in vitro. Development, 129, 4697– 4705. 98. Browne, K., Wang, W., Liu, R.Q., Piva, M. and O’Connor, T.P. (2012) Transmembrane semaphorin5B is proteolytically processed into a repulsive neural guidance cue. J. Neurochem., 123, 135–146. 99. Stumm, R.K., Zhou, C., Ara, T., Lazarini, F., Dubois-Dalcq, M., Nagasawa, T., Hollt, V. and Schulz, S. (2003) CXCR4 regulates interneuron migration in the developing neocortex. J. Neurosci., 23, 5123– 5130.

13

Infantile spasms.

Treatment of infantile spasms.

Management of infantile spasms.

Surgical treatment for infantile spasms?

Unusual variants of infantile spasms.

Vigabatrin in infantile spasms.

CT findings of infantile spasms.

Psychiatric disorders following infantile spasms.

Infantile spasms and cytomegalovirus infection.

Infantile spasms and HLA antigens.

The ocular findings in infantile spasms.

Williams Syndrome with Infantile Spasms.

Cytomegalovirus infection and infantile spasms.

Treatment of infantile spasms: medical or surgical?

Neonatal hemangiomatosis presenting as infantile spasms.

Infantile spasms--evidence based medical management.

Infraslow EEG changes in infantile spasms.

The epidemiology and natural history of infantile spasms.

Letter: Infantile spasms and cytomegalovirus infection.

Choroid plexus papilloma and infantile spasms.

Simultaneous infantile spasms and partial seizures.

CPP-115, a vigabatrin analogue, decreases spasms in the multiple-hit rat model of infantile spasms.

Magnetic resonance imaging in infantile spasms: effects of hormonal therapy.

Etiology and Long-Term Outcomes of Late-Onset Infantile Spasms.