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 = 44) followed by targeted sequencing of 35 known epilepsy genes (n = 8) or whole-exome sequencing (WES) of familial trios (n = 18) to search for rare inherited or de novo mutations. aCGH analysis revealed de novo variants in 7% of patients (n = 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 = 1/14) and STXBP1 (n = 1/8). In contrast, sequencing of a panel of 35 known epileptic encephalopathy genes (n = 8) did not identify further mutations. Finally, WES (n = 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.

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 (48), chromosomal anomalies (6,915), neurocutaneous disorders (16) or cerebral malformations (1720). 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) (2224) 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. 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

Clinical characteristics

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 non-focal cortical and subcortical atrophy (n = 21, 47%), delayed myelination (n = 15, 34%), thinning of the corpus callosum (n = 8, 18%) and/or basal ganglia T2 hyperintensities (n = 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 = 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 = 35, 80%). The majority of patients had global DD (n = 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 = 18/25, 72%). Clinical data for the entire cohort are summarized in Table 1.

Table 1.

Clinical characteristics

 N (%) 
Total 44 
Gender 
 Males 22 (50) 
 Females 22 (50) 
Ethnicity 
 Caucasian 34 (77) 
 Arabic 7 (16) 
 Asian 2 (5) 
 American Indian 1 (2) 
Age onset 
 (Mean, months) 5.5 
Additional seizure types 
 Focal 26 (59) 
 Tonic 11 (25) 
 GTC 6 (14) 
 Atonic 2 (5) 
 Myoclonic 7 (16) 
# AED 
 (Mean) 
Development 
 Normal 4 (9) 
 Global delay 40 (91) 
MRI 
 Normal 11 (26) 
 Atrophy/ISAS 21 (50) 
 Myelination delay 15 (34) 
 CC anomaly 8 (18) 
 BG Hyperintensities 3 (7) 
 N (%) 
Total 44 
Gender 
 Males 22 (50) 
 Females 22 (50) 
Ethnicity 
 Caucasian 34 (77) 
 Arabic 7 (16) 
 Asian 2 (5) 
 American Indian 1 (2) 
Age onset 
 (Mean, months) 5.5 
Additional seizure types 
 Focal 26 (59) 
 Tonic 11 (25) 
 GTC 6 (14) 
 Atonic 2 (5) 
 Myoclonic 7 (16) 
# AED 
 (Mean) 
Development 
 Normal 4 (9) 
 Global delay 40 (91) 
MRI 
 Normal 11 (26) 
 Atrophy/ISAS 21 (50) 
 Myelination delay 15 (34) 
 CC anomaly 8 (18) 
 BG Hyperintensities 3 (7) 

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

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 = 3/44) and a rare hemizygous variant in 2% of patients (n = 1/44). These variants are likely pathogenic as detailed below. In addition, we found rare inherited variants in 14% of patients (n = 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 = 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.

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) Targeted sequencing WES 
IS, F, T, M H, M G, ID-M ISS Del 2q22.2q21.3 (d.n.  
C/Ar 17 IS, AT, F G, F, M G, ID-S MD Tetrasomy 15q11.1q13.1(d.n.  
IS, T, AT G, ID-S ISS, TCC Dup 16p11.2 (d.n.  
C/A 0.5 IS, F H, M G, ID-M ISS, TCC Dup Xp11.23 (mat)
Dup Xp11.23p11.22 (mat
  
IS ARX  
0.2 IS, CPC G, ID-M ISS, MD STXBP1  
0.25 IS, F AT, BGH  STXBP1 
IS, F H, M G, ID-S AT Del 9p24.2 (mat ALG13 
As 36 IS G, ID-S AT  CASK 
0.02 IS, M AT, ISS Dup 3q13.12q13.13 (mat PNPO 
        Del 8q23.2q23.3 (pat  
IS, F H, G G, ID-M  ADSL 
Gender Origin Age DX (months) Seizures RX EEG Dev. MRI CGH (inheritance) Targeted sequencing WES 
IS, F, T, M H, M G, ID-M ISS Del 2q22.2q21.3 (d.n.  
C/Ar 17 IS, AT, F G, F, M G, ID-S MD Tetrasomy 15q11.1q13.1(d.n.  
IS, T, AT G, ID-S ISS, TCC Dup 16p11.2 (d.n.  
C/A 0.5 IS, F H, M G, ID-M ISS, TCC Dup Xp11.23 (mat)
Dup Xp11.23p11.22 (mat
  
IS ARX  
0.2 IS, CPC G, ID-M ISS, MD STXBP1  
0.25 IS, F AT, BGH  STXBP1 
IS, F H, M G, ID-S AT Del 9p24.2 (mat ALG13 
As 36 IS G, ID-S AT  CASK 
0.02 IS, M AT, ISS Dup 3q13.12q13.13 (mat PNPO 
        Del 8q23.2q23.3 (pat  
IS, F H, G G, ID-M  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.

Table 3.

CNVs revealed by aCGH analysis

 Position Genes included Prediction 
De novo CNVs 
Del 2q22.2q21.3 (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 
Tetr 15q13.1 Identified on karyotype GABRA5, GABRB3, MAGEL2, SNRPN, UBE3A 
Dup 16p11.2 (hg 18: 28,518,059-28,970,054) SULT1a1, EIF3C, NPIPL1, ATXN21, TUFM, SH2B1, ATP2A1, RABEP2, CD19 
Hemizygous CNVs 
Dup Xp11.23 (hg 18: 48,696,007-49,031,414) OTUD5, KCND1, GRIPAP1, TFE3, PRAF2, WDR45, PLP2, PRICKLE3, SYP,CACNA1F, FOXP3, CCDC120, GPKOW, MAGIX, CCDC22, PPP1R3F 
Dup Xp11.23p11.22 (hg 18: 49,463,797-49,721,060) PAGE4, CLCN5, LOC158572, USP27X, MIR532, MIR188, MIR500A, MIR362, MIR501, MIR500B, MIR660, MIR502 
Inherited autosomal CNVs 
Del 1p31.1 (hg 18: 71,145,303-71,230,018) interrupts PTGER3 
Dup 3q13.12q13.13 (hg18: 109,468,131-109,925,023) HHLA2, MYH15, KIAA1524, DZIP3 
Del 8q23.2q23.3 (hg 18: 110,830,845-114,121,392) KCNV1, CSMD3, MIR2053 
Del 9p24.2 (hg 18: 4,398,900-4,548,776) interrupts SLC1A1 
Dup 15q13.3 (hg 18: 29,816,893-30,226,405) interrupts CHRNA7 
Dup 15q26.1 (hg18: 91,326,934-91,392,664) interrupts RGMA, CHD2 
Unconfirmed inheritance 
Dup 12p11.21 (hg 18: 32,683,821-32,726,913) interrupts FGD4, DNM1L 
Dup 22q11.21 (hg 18: 17,429,152-17,575,845) DGCR2, DGCR14, TSSK2, GSC2, SLC25A1, CLTCL1 
 Position Genes included Prediction 
De novo CNVs 
Del 2q22.2q21.3 (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 
Tetr 15q13.1 Identified on karyotype GABRA5, GABRB3, MAGEL2, SNRPN, UBE3A 
Dup 16p11.2 (hg 18: 28,518,059-28,970,054) SULT1a1, EIF3C, NPIPL1, ATXN21, TUFM, SH2B1, ATP2A1, RABEP2, CD19 
Hemizygous CNVs 
Dup Xp11.23 (hg 18: 48,696,007-49,031,414) OTUD5, KCND1, GRIPAP1, TFE3, PRAF2, WDR45, PLP2, PRICKLE3, SYP,CACNA1F, FOXP3, CCDC120, GPKOW, MAGIX, CCDC22, PPP1R3F 
Dup Xp11.23p11.22 (hg 18: 49,463,797-49,721,060) PAGE4, CLCN5, LOC158572, USP27X, MIR532, MIR188, MIR500A, MIR362, MIR501, MIR500B, MIR660, MIR502 
Inherited autosomal CNVs 
Del 1p31.1 (hg 18: 71,145,303-71,230,018) interrupts PTGER3 
Dup 3q13.12q13.13 (hg18: 109,468,131-109,925,023) HHLA2, MYH15, KIAA1524, DZIP3 
Del 8q23.2q23.3 (hg 18: 110,830,845-114,121,392) KCNV1, CSMD3, MIR2053 
Del 9p24.2 (hg 18: 4,398,900-4,548,776) interrupts SLC1A1 
Dup 15q13.3 (hg 18: 29,816,893-30,226,405) interrupts CHRNA7 
Dup 15q26.1 (hg18: 91,326,934-91,392,664) interrupts RGMA, CHD2 
Unconfirmed inheritance 
Dup 12p11.21 (hg 18: 32,683,821-32,726,913) interrupts FGD4, DNM1L 
Dup 22q11.21 (hg 18: 17,429,152-17,575,845) DGCR2, DGCR14, TSSK2, GSC2, SLC25A1, CLTCL1 

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

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.

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.

Of the de novo variants identified, one patient with moderate-to-severe ID and refractory seizures carried a de novo 7.9 Mb deletion encompassing 2q21.3-q22.2 (Supplementary Material). Deletions in this region have been associated with intellectual disability, behavioral difficulties and autism spectrum disorder (ASD) (2931) 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 (3235). 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 α-neurexin-binding 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 well-controlled 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 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 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 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.

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 = 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 (5559).

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 = 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 (6064).

Sequencing of the CDKL5 gene in 20 girls with IS was non-contributory. 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 (6669) 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 = 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 = 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 = 0.00) and POLYPHEN-2 (score = 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 well-controlled 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 = 0.00) but potentially benign according to POLYPHEN-2 (score = 0.146). This patient had elevated succinyladenosine urinary 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

We identified rare de novo non-synonymous variants (not inherited from the parents) in the majority of families (n = 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 = 4/19 versus 1146/18 331, χ2 with Yates correction = 4.783, P = 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 = 6/19, versus 1917/18 331, χ2 with Yates correction = 6.915, P = 0.0085, two-tailed).

Table 4.

Rare de novo variants revealed through WES

ID Chr. Position Ref. Mut. Variant Gene Detailed annotation SIFTa PolyPhen v2b 
1 10 928 268 SNV ALG13 NM_001099922:exon3:c.A320G:p.N107S 0.538 
1 79 247 941 SNV SQSTM1 NM_003900:exon1:c.C5T:p.A2V 0.01 0.289 
19 17 312 954 SNV MYO9B NM_001130065:exon28:c.G4678A:p.V1560M 
 92 921 132 SNV NR2F1 NM_005654:exon1:c.C403A:p.R135S 1.0 
44 579 758 SNV NPC1L1 NM_013389:exon2:c.C238T:p.R80C 0.01 0.957 
1 52 222 651 SNV TNFAIP6 NM_007115:exon3:c.T314C:p.F105S 0.18 0.953 
1 30 430 439 SNV STXBP1 NM_003165:exon10:c.G875A:p.R292H 0.00 1.0 
 1 34 371 230 SNV PRRC2B NM_013318:exon31:c.G6659A:p.R2220Q 0.57 0.026 
10 36 301 467 SNV EIF2C4 NM_017629:exon13:c.G1597A:p.G533S 0.001 0.972 
 10 29 747 509 SNV SVIL NM_021738:exon37:c.G6412A:p.V2138I 0.07 0.778 
 1 67 689 699 SNV TENM2 NM_001122679:exon29:c.G8182A:p.G2728R 0.00 0.999 
11 1 24 774 668 SNV HEG1 NM_020733:exon1:c.C67A:p.L23M 0.00 0.999 
 1 29 704 291 SNV LAMA2 NM_000426:exon35:c.G4984A:p.E1662K 0.05 0.618 
12 19 14 160 107 CCCCT fs_del. IL27RA NM_004843:exon10:c.1384_1387del:p.462_463del n/a n/a 
 1 22 640 860 SNV SEMA5B NM_001256348:exon11:c.C1416A:p.S472R 0.003 0.94 
14 41 712 458 stopgain CASK NM_001126054:exon2:c.C82T:p.R28X n/a n/a 
15 2 07 941 154 SNV CD46 NM_172359:exon8:c.C932T:p.A311V 0.4 0.0 
16 22 188 503 SNV HSPG2 NM_005529:exon38:c.C4846T:p.P1616S 0.012 0.998 
 20 62 221 927 SNV GMEB2 NM_012384:exon10:c.G1108T:p.A370S 0.55 0.567 
ID Chr. Position Ref. Mut. Variant Gene Detailed annotation SIFTa PolyPhen v2b 
1 10 928 268 SNV ALG13 NM_001099922:exon3:c.A320G:p.N107S 0.538 
1 79 247 941 SNV SQSTM1 NM_003900:exon1:c.C5T:p.A2V 0.01 0.289 
19 17 312 954 SNV MYO9B NM_001130065:exon28:c.G4678A:p.V1560M 
 92 921 132 SNV NR2F1 NM_005654:exon1:c.C403A:p.R135S 1.0 
44 579 758 SNV NPC1L1 NM_013389:exon2:c.C238T:p.R80C 0.01 0.957 
1 52 222 651 SNV TNFAIP6 NM_007115:exon3:c.T314C:p.F105S 0.18 0.953 
1 30 430 439 SNV STXBP1 NM_003165:exon10:c.G875A:p.R292H 0.00 1.0 
 1 34 371 230 SNV PRRC2B NM_013318:exon31:c.G6659A:p.R2220Q 0.57 0.026 
10 36 301 467 SNV EIF2C4 NM_017629:exon13:c.G1597A:p.G533S 0.001 0.972 
 10 29 747 509 SNV SVIL NM_021738:exon37:c.G6412A:p.V2138I 0.07 0.778 
 1 67 689 699 SNV TENM2 NM_001122679:exon29:c.G8182A:p.G2728R 0.00 0.999 
11 1 24 774 668 SNV HEG1 NM_020733:exon1:c.C67A:p.L23M 0.00 0.999 
 1 29 704 291 SNV LAMA2 NM_000426:exon35:c.G4984A:p.E1662K 0.05 0.618 
12 19 14 160 107 CCCCT fs_del. IL27RA NM_004843:exon10:c.1384_1387del:p.462_463del n/a n/a 
 1 22 640 860 SNV SEMA5B NM_001256348:exon11:c.C1416A:p.S472R 0.003 0.94 
14 41 712 458 stopgain CASK NM_001126054:exon2:c.C82T:p.R28X n/a n/a 
15 2 07 941 154 SNV CD46 NM_172359:exon8:c.C932T:p.A311V 0.4 0.0 
16 22 188 503 SNV HSPG2 NM_005529:exon38:c.C4846T:p.P1616S 0.012 0.998 
 20 62 221 927 SNV GMEB2 NM_012384:exon10:c.G1108T:p.A370S 0.55 0.567 

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:

aSIFT: <0.05, damaging, >0.05, tolerated.

bPolyPhen v2 score: >0.85, probably damaging, >0.15, possibly damaging, ≤0.15, benign.

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 = 17/19 variants, 89%), and were identified in 61% of patients (n = 11/18). Most of these variants were expected to be damaging by SIFT (n = 10/17, 59%) and POLYPHEN-2 (n = 12/17, 71%). In addition, we identified two clearly detrimental mutations in 11% of our patients (n = 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 = 9/18 versus n = 4264/16 957 genes with IVS scores, χ2 with Yates correction = 4.651, P = 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.genemania.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.

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.

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.

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).

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).

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 (7780) 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 = 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.

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 = 7) or inherited (n = 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 (8286), 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 well-defined and clinically homogeneous epileptic encephalopathy 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 pyridoxal-phosphate responsive epileptic encephalopathy (PNPO gene) and ADSL deficiency (ADSL gene), with significant impact on genetic counseling.

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 = 139 cases of sporadic IS), we found that only STXBP1 (n = 6), ALG13 (n = 2) and CDKL5 (n = 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 whole-genome 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-of-function 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 glucocorticoid-induced 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 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 (3235). Furthermore, CASK encodes a calcium/calmodulin-dependent serine protein kinase, which interacts with α-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 cell-based 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 Montréal. 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 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

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 = 8) (109) or through exome sequencing of familial trios (n = 18) as detailed below.

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 = 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.

FUNDING

This work was supported by funds from The Scottish Rite Charitable Foundation of Canada, The Savoy Foundation, the Réseau de Médecine Génétique du Québec (RMGA), Genome Quebec and Genome Canada. E.R. is a Clinician-Scientist I of the Fonds de Recherche du Québec—Santé. J.L. is a National Scientist of the Fonds de Recherche du Québec—Santé.

ACKNOWLEDGEMENTS

We wish to thank all families that participated in this study. Furthermore, we thank the members of the bioinformatic analysis team of Réseau de Médecine Génétique Appliquée du Québec (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.

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