De novo variants in neurodevelopmental disorders with epilepsy

Transcription

1 SUPPLEMENTARY INFORMATION Analysis In the format provided by the authors and unedited. De novo variants in neurodevelopmental disorders with epilepsy Henrike O. Heyne 1,2,3,4 *, Tarjinder Singh 2,4, Hannah Stamberger 5,6,7, Rami Abou Jamra 1, Hande Caglayan 8, Dana Craiu 9, Peter De Jonghe 5,6,7, Renzo Guerrini 10, Katherine L. Helbig 11, Bobby P. C. Koeleman 12, Jack A. Kosmicki 2,4, Tarja Linnankivi 13, Patrick May 14, Hiltrud Muhle 15, Rikke S. Møller 16,17, Bernd A. Neubauer 18, Aarno Palotie 2, Manuela Pendziwiat 15, Pasquale Striano 19, Sha Tang 20, Sitao Wu 20, EuroEPINOMICS RES Consortium 21, Annapurna Poduri 22, Yvonne G. Weber 23, Sarah Weckhuysen 5,6,7, Sanjay M. Sisodiya 24,25, Mark J. Daly 2,4, Ingo Helbig 11,15, Dennis Lal 2,4,26 and Johannes R. Lemke 1 * 1 University of Leipzig Hospitals and Clinics, Leipzig, Germany. 2 Program in Medical and Population Genetics, and Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA. 3 Integrated Research and Treatment Center (IFB) Adiposity Diseases, University of Leipzig Hospitals and Clinics, Leipzig, Germany. 4 Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA. 5 Neurogenetics Group, Center for Molecular Neurology, VIB, Antwerp, Belgium. 6 Laboratory of Neurogenetics, Institute Born Bunge, University of Antwerp, Antwerp, Belgium. 7 Division of Neurology, University Hospital Antwerp, Antwerp, Belgium. 8 Department of Molecular Biology and Genetics, Bogaziçi University, Istanbul, Turkey. 9 Carol Davila University of Medicine Bucharest, Department of Clinical Neurosciences (No. 6), Pediatric Neurology Clinic, Alexandru Obregia Hospital, Bucharest, Romania. 10 Pediatric Neurology and Neurogenetics Unit and Laboratories, A. Meyer Children s Hospital University of Florence, Florence, Italy. 11 Division of Neurology, Children s Hospital of Philadelphia, Philadelphia, PA, USA. 12 Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands. 13 Department of Pediatric Neurology, Children s Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. 14 Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg. 15 Department of Neuropediatrics, University Medical Center Schleswig Holstein, Christian Albrechts University, Kiel, Germany. 16 Danish Epilepsy Centre, Dianalund, Denmark. 17 Institute for Regional Health Services, University of Southern Denmark, Odense, Denmark. 18 Department of Pediatric Neurology, University Hospital Giessen, Giessen, Germany. 19 Pediatric Neurology and Muscular Diseases Unit, Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, and Maternal and Child Health, University of Genoa G. Gaslini Institute, Genoa, Italy. 20 Division of Clinical Genomics, Ambry Genetics, Aliso Viejo, CA, USA. 22 Epilepsy Genetics Program, Department of Neurology, Division of Epilepsy and Clinical Neurophysiology, Boston Children s Hospital, Boston, MA, USA. 23 Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany. 24 Department of Clinical and Experimental Epilepsy, NIHR, University College London Hospitals Biomedical Research Centre, UCL Institute of Neurology, London, UK. 25 The Epilepsy Society, Chalfont-St-Peter Bucks, UK. 26 Cologne Center for Genomics (CCG), Cologne, Germany. 46 A list of members and affiliations appears at the end of the paper * hheyne@broadinstitute.org; johannes.lemke@medizin.uni-leipzig.de Nature Genetics Nature America Inc., part of Springer Nature. All rights reserved.

2 Supplementary Figure 1 Schematic Venn diagram of neurodevelopmental disorders (NDD) and epilepsy. Per definition, all individuals with EE have epilepsy and NDD. Thus, we group individuals with EE as NDD EE and individuals with NDD with unspecified epilepsy as NDD ue representing two phenotype groups within NDD with epilepsy. We therefore define NDD as an umbrella term for the following disorders: - ASD (autism spectrum disorder, blue circle, we include only ASD with ID) - ID (intellectual disability, yellow circle), used synonymously to DD (developmental delay), as approximately 81.7% of individuals with DD have ID1 - NDD EE (NDD with epileptic encephalopathy, brown circle) - NDD ue (NDD with unspecified epilepsy, intersection of red/yellow and partly blue circle)

3 A1 0.3 A2 0.3 frequency frequency DDD controls_ssc Lelieveld_et_al SSC Epi4k_E2 AmbryGenetics de_ligt_et_al EuroEPIN._RES Rauch_et_al DFG_EE Hamdan_et_al Leipzig_Uni DDD controls_ssc Lelieveld_et_al SSC Epi4k_E2 AmbryGenetics EuroEPIN._RES DFG_EE Hamdan_et_al Leipzig_Uni B 1.00 cohort C 0.6 cohort frequency frequency Supplementary Figure 2 DDD controls_ssc Lelieveld_et_al SSC Epi4k_E2 AmbryGenetics EuroEPIN._RES DFG_EE Hamdan_et_al Leipzig_Uni cohort DDD controls_ssc Lelieveld_et_al SSC Epi4k_E2 AmbryGenetics EuroEPIN._RES DFG_EE Hamdan_et_al Leipzig_Uni cohort Frequencies of DNV in different cohorts A, DNV synonymous; B, DNV mis; C, DNV trunc. In Figure A1 all DNV are shown. In Figures A2, B, and C, DNV have been restricted to regions with consistently high coverage across different capture solutions (see Online Methods). Error bars represent the 95%-CI of the point estimates. n (cases) = 6753; n (controls) = 1911.

4 rate of epilepsy SSC (no ID) DDD n = 1693 n = 4293 med.iq 94 ID 81.7% SSC (ID) n = 732 IQ<70 Lelieveld n = 820 IQ<70 cohort Rauch n = 51 IQ<60 Hamdan n = 41 IQ<50 de Ligt n = 100 IQ<50 Supplementary Figure 3 Rate of epilepsy is increasing with severity of intellectual disability (ID). Rates of epilepsy in all cohorts comprising individuals with epilepsy as well as without epilepsy. Cohort size is given by total number of individuals (n). Cohorts were sorted after severity of ID (according to IQ cutoffs, percentage or median for IQ in each cohort as written underneath each bar). Error bars represent the 95%-CIs of the point estimates.

5 A 0.3 DDD SSCnoid SSC Hamdan_et_al Rauch_et_al de_ligt_et_al Lelieveld_et_al frequency DNV in EE genes AmbryGenetics Leipzig_Uni Epi4k_E2 DFG_EE EuroEPIN._RES controls_ssc B 0.3 DDD SSCnoid SSC Hamdan_et_al Rauch_et_al de_ligt_et_al Lelieveld_et_al 0.2 frequency DNV in EE genes AmbryGenetics Leipzig_Uni Epi4k_E2 DFG_EE EuroEPIN._RES controls_ssc Supplementary Figure 4 DNV in 50 dominant EE genes across all cohorts. Frequency of DNV (A DNV trunc B DNV mis) in 50 dominant EE genes across twelve cohorts as well as healthy controls. Bars of cohorts with epilepsy are colored red, cohorts without epilepsy blue. Error bars represent the 95%-CIs of the point estimates. Cohorts are sorted after size and disorder. DNV have been restricted to regions with consistently high coverage across different capture solutions

6 (see Online Methods).

7 A 20 seizure phenotype febrile B 20 phenotype female focal male generalized 15 spasms 15 counts KCNQ2 SCN2A SCN1A CHD2 SYNGAP1 STXBP1 SCN8A MEF2C SLC6A1 DNM1 EEF1A2 CDKL5 DYRK1A SMC1A GABRB3 KIAA2022 ASXL3 WDR45 ARID1B GNAO1 ALG13 KCNH1 GRIN2B HNRNPU PURA GABRB2 COL4A3BP MECP2 FOXG1 ANKRD11 SNAP25 DDX3X IQSEC2 KCNQ2 SCN2A SCN1A CHD2 SYNGAP1 STXBP1 SCN8A MEF2C SLC6A1 DNM1 EEF1A2 CDKL5 DYRK1A SMC1A GABRB3 KIAA2022 ASXL3 WDR45 ARID1B GNAO1 ALG13 KCNH1 GRIN2B HNRNPU PURA GABRB2 COL4A3BP MECP2 FOXG1 ANKRD11 SNAP25 DDX3X IQSEC2 gene gene C 20 phenotype NDD_woE DD EE ID counts D 30 phenotype NDD_woE DD EE ID 15 ASD E_NDD 20 ASD E_NDD counts KCNQ2 SCN2A SCN1A CHD2 SYNGAP1 STXBP1 SCN8A MEF2C SLC6A1 DNM1 EEF1A2 CDKL5 DYRK1A SMC1A GABRB3 KIAA2022 ASXL3 WDR45 ARID1B GNAO1 ALG13 KCNH1 GRIN2B HNRNPU PURA GABRB2 COL4A3BP MECP2 FOXG1 ANKRD11 SNAP25 DDX3X IQSEC2 gene KCNQ2 SCN2A SCN1A CHD2 SYNGAP1 STXBP1 SCN8A MEF2C SLC6A1 DNM1 EEF1A2 CDKL5 DYRK1A SMC1A GABRB3 KIAA2022 ASXL3 WDR45 ARID1B GNAO1 ALG13 KCNH1 GRIN2B HNRNPU PURA GABRB2 COL4A3BP MECP2 FOXG1 ANKRD11 SNAP25 DDX3X IQSEC2 gene Supplementary Figure 5 Different phenotypes per gene Phenotype counts per gene in individuals with NDD EE+uE that have a DNV mis+trunc in a gene with exome-wide DNV burden. A, Seizure phenotypes per gene. Multiple seizure phenotypes per individual were allowed. B, Distribution of sex (female/male) per gene. C,D: counts of diagnoses per gene. C, DNV mis, D, DNV trunc. Total diagnoses with epilepsy: EE (n=585), DD (n=856), ID (n=265), NDD ue (n=164), ASD with ID (n=124), diagnoses without epilepsy (NDD woe): 4811.

8 A 1.00 B 250 gene ALG13 GRIN2B ASXL1 HNRNPU 200 CACNA1E IQSEC CDKL5 KCNQ3 DNM1 KMT2A Frequency 0.50 count DYNC1H1 FOXG1 GABRB3 GNAO1 KCNQ2 SCN1A WDR45 MECP2 PURA SNAP25 SCN2A CSNK2A SCN8A STXBP1 KIAA2022 SMARCA2 CHD2 SYNGAP1 CHD4 NA EEF1A2 IS LGS EE MAE diagnosis dravet NLES IS LGS EE MAE diagnosis dravet NLES Supplementary Figure 6 DNV per gene in different EE syndromes. Counts of DNV mis+trunc in DNV-enriched genes per EE syndrome. Numbers of individuals without DNV in DNV-enriched genes is plotted as NA. No DNV mis+trunc in DNV-enriched genes were found in ESES (n=42).

9

10 Supplementary Figure 7 Comparing genes with DNV burden in cases, >= 2 DNV in cases and >= 2 DNV in controls 33 genes with DNV burden in NDD EE+uE (red) and 115 genes with at least two DNV but no significant DNV burden (orange) have A, higher brain expression (two-sided t-tests) and are more constraint for B, missense (missense z-score2, two-sided t-test) and C, truncating variants (pli score 2, empirical p-value, see Online Methods) compared to 70 genes with at least two DNV in controls (healthy siblings of children with ASD: yellow). Violins are plotted to have the same maximum width. Bottom, middle and top of boxplots within violins show the 1 st, 2 nd and 3 rd quartile of the data; whiskers maximally extend to 1.5 x interquartile range. DNV refers to DNV mis+trunc not reported in ExAC 2.

11 age (months) A ANKRD11 ARID1B ASXL3 CHD2 COL4A3BP DDX3X DYNC1H1 DYRK1A SCN8A SLC6A1 SMARCA2 STXBP1 SYNGAP1 WDR45 GNAO1 GRIN2B KCNQ2 KIF1A MED13L PPP2R5D PURA SCN2A age of recruitment - epilepsy age of recruitment - no epilepsy age at seizure onset B age (months) CHD2 GABRB3 KCNQ2 SCN1A SCN2A SCN8A SLC6A1 STXBP1 SYNGAP1 Supplementary Figure 8 Age at recruitment positively associated with presence of epilepsy. Age of individuals with DNV mis+trunc in genes with DNV burden in NDD EE+uE. Genes were plotted if they were mutated in at least 3 patients with or without epilepsy (total n= 175). A, Patients without epilepsy (yellow) had significantly lower ages at time of recruitment than patients with epilepsy (blue), three-year OR 1.11, 95%-CI 1.04 to 1.18, p-value = 3x10-3, logistic regression. B, Age of seizure onset (red) is plotted next to age at recruitment. For clarity, the upper bound of age was set to 180 months. All violins are plotted to have the same maximum width. Bottom, middle and top of boxplots within violins show the 1 st, 2 nd and 3 rd quartile of the data; whiskers maximally extend to 1.5 x interquartile range.

12 A B DNVmis frequency, NDDuE DNVtrunc frequency, NDDuE SCN1A DNV mis frequency, NDD EE DNV trunc frequency, NDD EE Supplementary Figure 9 DNV frequencies in NDD ue versus NDD EE in genes with exome-wide DNV burden. DNV frequencies in NDD ue (n=1413) versus NDD EE (n=529) in genes with DNV burden in NDD with epilepsy (NDD EE+uE); A, DNV mis, B, DNV trunc. Genes with different DNV frequencies between NDD ue and NDD EE are labeled and colored (red: significant after multiple testing correction, method: two-sided Fisher s exact test). The dotted line represents equal frequency of DNV in NDD ue and NDD EE.

13 A counts KCNQ2 SCN2A SCN1A CHD2 SYNGAP1 STXBP1 SCN8A MEF2C DNM1 EEF1A2 SLC6A1 CDKL5 DYRK1A SMC1A GABRB3 KIAA2022 ASXL3 WDR45 ARID1B GNAO1 ALG13 KCNH1 GRIN2B HNRNPU PURA GABRB2 COL4A3BP MECP2 FOXG1 ANKRD11 SNAP25 DDX3X IQSEC2 B 60 gene counts PRRT2 CHRNA2 CHRNA4 SCN9A SYN1 EFHC1 CACNB4 DNAJC5 SRPX2 ATP6AP2 CHRNA7 CPA6 TREX1 CLCN2 MAPK10 CACNA1H CASR KCNJ11 SHH KCNH5 ARHGEF15 GPR98 SCN5A SIX3 UBE2A CRH AFG3L2 CBL CCL2 CDON DRD2 FOXH1 GFAP GLI2 GNE HEPACAM HSD17B10 IFIH1 JRK KCNAB1 NODAL NOL3 NPRL2 NPRL3 RAB39B SGCE SYP TGIF1 VANGL1 ZDHHC9 gene Supplementary Figure 10 Comparing the findings of this study to 24 commercial and academic providers of diagnostic gene panels for epileptic encephalopathy or comprehensive epilepsy. A, This barplot shows to what proportion the 33 genes with significant excess of DNV in NDD EE+uE were covered by 24 diagnostic gene panels of different providers. CDKL5, SCN1A, SCN2A, SCN8A, SPTAN1 and STXBP1 were contained in all 24 designs. No DNV in SPTAN1 was found in our cohort. Genes are sorted by number of DNVmis+trunc in this study. B, 50 genes on diagnostic panels failed two of the three criteria: at least two DNVmis+trunc in the study cohort, infant brain gene expression, genic constraint. Twenty-four of these 50 genes also had no, limited, or conflicting evidence for disease association as defined by ClinGen 3 (Supplementary Table 14, red bars). Genes with moderate to definitive evidence for association with other phenotypes than NDD with epilepsy are shown as yellow bars, genes with at least moderate evidence for association with NDD with epilepsy as blue bars.

14 Supplementary Information Supplementary Notes Among the genes with significant DNV burden, we highlight the following genes with so far limited evidence of disease association. SNAP25 The 25-KD synaptosomal-associated protein encoded by SNAP25 is part of the t-snare protein complex, which is involved in the molecular regulation of neurotransmitter release by mediating synaptic vesicle fusion for exocytosis 4 and interacts with the known epilepsy genes STXBP1 5 and STX1B 6. SNAP25 shows genic constraint (missense z-score 3.16, pli 0.96) 2. So far, two independent cases with DNV in SNAP25 have been published: p.(val48phe) in a patient with NDD 7 ue (included in our study) and p.(ile67asn) in a patient with congenital myasthenia, cortical hyperexcitability, cerebellar ataxia, and ID 8 (not included in our study). In our study, both DNV mis and DNV trunc appear to be associated with NDD with epilepsy [p.(gln174*) as well as p.(lys40glu), p.(gly43arg), p.(val48phe); ENST /NM_130811], which is in line with observations in mice, where missense and truncating mutations of Snap25 have been associated with seizures and cognitive dysfunction The three DNV mis from our study cluster within amino acid positions 40 to 48, a region completely lacking missense variants in ExAC 2 and GnomAD ( indicating purifying selection and were predicted damaging by PolyPhen and Sift. All four DNV mis reside in a t-snare coiled-coil homology domain (amino acid positions 19 to 81 of SNAP25, source important for the process of vesicular fusion with target membranes (interpro ID IPR000727, For p.(ile67asn), an inhibition of synaptic vesicle exocytosis has been confirmed by functional investigations 8. In summary, these findings support pathogenicity of the DNV mis+trunc in SNAP25 identified in our cohort. GABRB2 GABRB2 encodes the β2 subunit of the gamma-aminobutyric acid (GABA) A receptor, a multisubunit chloride channel that mediates inhibitory synaptic transmission in the central nervous system. Several other subunits of the GABA A receptor (GABRG2, GABRA1, GABRB1 and GABRB3) have previously been associated with NDD EE+nsE. GABRB2 shows genic constraint (missense z- score 3.05, pli 0.94). There have been two case reports of patients (not included in our study) with DNV mis [p.(m79t) 12 and p.(thr287pro) 13 ] in GABRB2 with NDD EE+nsE : p.(m79t) was found in a patient with ID and epilepsy and p.(thr287pro) was identified in a patient with early myoclonic encephalopathy 13. In our study, we identified additional five DNV mis in GABRB2 [p.(asp125asn), p.(tyr183his), p.(pro252ala), p.(leu277ser), p.(tyr301cys)], all of which were absent in ExAC and GnomAD and were predicted damaging by PolyPhen and Sift. Two individuals with GABRB2 DNV mis [p.(m79t) 12 and p.(tyr183his)] presented with fever-sensitive encephalopathy, a phenotype that had been associated with SCN1A, CHD2, GABRA1, STX1B, HCN1 but also GABRB3 14, which is a paralog of GABRB2. All of these seven novel and reported DNV mis 1

15 were associated with epilepsy, however, one additional individual from SSC 15 [p.(leu17ser)] did not (yet) display seizures. Functional studies of p.(thr287pro) 13 in human embryonic kidney cells showed a poor trafficking of both β2 and γ2 subunits to the cell membrane and surface. Moreover, peak amplitudes of currents from the mutant GABA A receptors were decreased compared to wild type. In summary, these findings support pathogenicity of the DNV mis in GABRB2 identified in our cohort. CACNA1E CACNA1E encodes the α-1e subunit of a voltage-gated calcium channel 16,17 and has thus far not been associated with human disease. CACNA1E shows significant genic constraint (missense z- score 6, pli 1.0). In our study, we identified nine individuals with DNV mis and two with DNV trunc. Ten of eleven DNV mis+trunc in CACNA1E were not present in ExAC. Eight of nine DNV mis were predicted damaging by PolyPhen and Sift. Four of eleven DNV mis+trunc were associated with epilepsy (three DNV mis, one DNV trunc ). Two of them had IS 18,19. Voltage-gated calcium channels mediate the entry of calcium ions upon activation and are composed of multiple subunits. The channel activity is directed by the pore-forming α-1 subunit, of which the α-1e subunit is predominantly expressed in neuronal tissue, similar to its paralog CACNA1A, that already had been associated with early-onset NDD EE 20. Within our study, the spectrum of DNV mis+trunc and presence of epilepsy in CACNA1E was similar to CACNA1A, where five of nine individuals with DNV mis+trunc had epilepsy and two of nine DNV in CACNA1A were DNV trunc. Further studies with deep phenotyping are needed to further characterize the phenotypic spectrum of CACNA1E. KCNQ3 KCNQ3 encodes for the voltage-gated potassium channel subfamily KQT member. In neuronal cells, KCNQ3 together with other potassium subunits, including the known epilepsy gene KCNQ2, (either as homomultimers or heteromultimers) represent the molecular basis of the M-current, a potassium selective, non-inactivating, and slowly activating/deactivating current 21. KCNQ3 shows genic constraint (missense z-score 2.86, pli 0.99). Missense variants and one single truncating variant in KCNQ3 have been associated with benign, self-limiting, familial neonatal or infantile seizure syndromes 21,22 There have been few reports on patients with NDD associated with KCNQ3 mutations 23,24 but only recently the DDD study presented for the first time significant statistical evidence for association of KCNQ3 with NDD 1 in a meta-analysis of different NDD cohorts most of which are also analyzed here. We replicate and further discuss their findings with respect to epilepsy. Seven DNV mis were found in the meta-analysis of this gene, six of them were absent in ExAC 2 and all predicted to have deleterious effects with Sift and PolyPhen. Five out of the six DNV mis were located in the fourth transmembrane, voltage sensor domain (amino acid positions 226 to 247 of KCNQ3, transcript ENST , source Of these, three were recurrent DNV mis p.(arg230cys). Two individuals with the recurrent variant were described with seizures, one of them was diagnosed with the EE syndrome LGS. The recurrent p.(arg230cys) missense mutation has been functionally studied and shown to exert a gain of function effect 24. Its homologous position in KCNQ2 p.(arg201cys) is described as a pathogenic variant according to ACMG-criteria 25 segregating in three family members with EE 26 ( 2

16 SCN1A SCN1A, a well-known epilepsy gene, encodes the voltage gated sodium channel subunit alpha. Mutations in SCN1A were first identified in large families with generalized epilepsy with febrile seizures plus (GEFS+) 27. Later, SCN1A was recognized as the most frequently mutated gene in DS, usually presenting as a clinically recognizable EE entity 28,29. Because of the strong genotype phenotype correlation, individuals with DS are often screened for mutations in SCN1A prior to exome sequencing. Hence, in our cohort, 9/12 DNV in SCN1A were recruited as non-ds EE (see Supplementary Figure S8) and 19% (3/16) of DS patients in our cohort present a DNV in SCN1A in contrast to >80% in DS cohorts without pre-screening 30. There were no differences between NDD ue and NDD EE for DNV mis for any gene including SCN1A. However, SCN1A was the only gene in our cohort with significantly different frequencies of DNV trunc in NDD EE (8/529 individuals) and NDD ue (0/1413 individuals). This implies that DNV trunc in SCN1A may lead more often to specific epilepsy syndromes than to NDD ue. Gene set enrichment analyses We aimed to explore, whether DNV in NDD with and without epilepsy might be associated with distinct biological pathways. We performed gene set enrichment analyses in genes with DNV in our dataset with 1,766 curated gene sets previously described 31. Briefly, gene sets were derived from public pathway databases and genome-wide screens and experiments related to ID, ASD, and other NDD. We performed logistic regression investigating the effect of having a DNV in each individual gene set on epilepsy. We used the number of DNV per individual as a covariate to control for mutation rate differences per gene and between individuals with and without epilepsy. We performed three separate tests for DNV mis, DNV trunc including DNV synonymous as negative control. Accordingly, we used a p-value threshold of 1.9x10-6 (Bonferroni correction for 1766 x 3 tests) as a significance cutoff. We restricted the analyses to regions with high coverage across capture kits (see online methods) and constraint genes (pli > 0.9 or missense z-score >3.09) as genes with DNV burden in NDD were significantly more constraint than genes enriched for DNV in controls (see Supplementary Figure S9). We observed significant differences between individuals with and without epilepsy in 62 gene sets for DNV mis, in two gene sets for DNV trunc and no gene sets for DNV synonymous. 59 of these gene sets were enriched and three were depleted in individuals with epilepsy. 41 of the gene sets that were enriched in epilepsy were directly or indirectly related to ion channels (e.g. GO CC [Gene Ontology Cellular Component]: ion channel complex, OR epilepsy = 4.0, p-value ). Other gene sets enriched in epilepsy mostly related to parts (e.g. axon) or pathways (e.g. synaptic transmission) of neuronal cells. The three gene sets with a significant depletion in epilepsy were related to CHD8 (e.g. CHD8-targeted promoters in the human brain 32, OR no_epilepsy 2.3, p-value 6x10-7 ) one of them significant in DNV mis and two in DNV trunc. For all gene sets see Supplementary Table 15. In conclusion, gene set enrichment analyses support the role of ion channels in pathological processes leading to epilepsy 33 while other processes in synaptic transmission or other neuronrelated tasks may also play a role. Genes experimentally associated with CHD8 are more frequently mutated in individuals without epilepsy. CHD8 interacting genes involved in 3

17 chromatin modification have repeatedly been linked to autism 32,34,35. It may therefore be plausible, that these processes may be more specifically involved in autism or other NDD while less specifically leading to epilepsy. However, potential issues with gene set enrichment analyses include substantial overlap of genes between different gene sets 31 as well as publication biases in annotation of genes in gene sets 36. Therefore, biological conclusions from these analyses should be drawn with caution and warrant further validation. 4

18 Supplementary Tables Supplementary Table 1. Description of Cohorts analyzed in this study. Please see supplementary Excel document. Supplementary Table 2. List of all DNV mis, DNV trunc, DNV synonymous of all NDD cohorts (n=6753) and controls (n=1911) analyzed in this study. Please see supplementary tab-separated text file. Supplementary Table 3. List of 50 dominant and X-linked known EE genes Please see supplementary tab-separated text file. Supplementary Table 4. Genes with at least two DNV mis+trunc in NDD EE+uE (n=1942). Columns per gene include expected and observed synonymous, missense and truncating (in header lof ) DNV, number of DNV not present in ExAC 2 (in header NoExAC ), ExAC constraint scores (pli, missense z-score), infant brain gene expression. Analysis was done with denovolyzer 37 using Poisson Exact tests. Please see supplementary tab-separated text file. Supplementary Table 5. Genes with at least two DNV mis+trunc in all NDD (NDD EE+uE+woE, n=6753). Columns per gene include expected and observed synonymous, missense and truncating DNV, number of DNV not present in ExAC 2, ExAC constraint scores (pli, missense z-score), brain expression. Analysis was done with denovolyzer 37 using Poisson Exact tests. Please see supplementary tab-separated text file. Supplementary Table 6. Significantly enriched HPO terms in 33 genes with DNV burden in NDD with epilepsy. Analysis was done with g:profiler 38 using hypergeometric tests. Please see supplementary tab-separated text file. Supplementary Table 7. Evaluating genes with at least two DNV mis+trunc in NDD with epilepsy for therapeutic consequences. Only genes with Centre for Evidence-Based Medicine level of evidence of II or higher, are shown. Please see supplementary Excel document. 5

19 Supplementary Table 8. Gene sets significantly enriched (Odds Ratio > 1) or depleted (Odds Ratio < 1) for DNV in epilepsy compared to no epilepsy (see Supplementary Note) were identified using logistic regression analyses. There are three separate sheets for DNV mis, DNV trunc and DNV synonymous. For the three classes of DNV, the Odds Ratio (OR), Standard Error, z-value and p-value for enrichment of DNV in individuals with epilepsy is given per gene set. The last two columns of each table show the number of DNV in the respective class and gene set in individuals with (n= 1874) and without epilepsy (n= 4728). Please see supplementary tab-separated text file. Supplementary Table 9. DNV in epilepsy vs. no epilepsy. We compared the frequency of DNV mis+trunc in individuals with epilepsy (NDD EE+uE, n=1942) to individuals without epilepsy (NDD woe, n=4188) in genes with DNV burden in NDD (NDD EE+uE+woE, n=6753) using two-sided Fisher s Exact tests. Effect sizes are given as Odds ratios with columns conf.int1 and conf.int2 representing its 2.5% and 97.5% confidence intervals, respectively. Supplementary Table 9A: DNV mis. Supplementary Table 9B: DNV trunc. Please see supplementary tab-separated text files. Supplementary Table 10. DNV in NDD ue versus NDD EE. We compared the frequency of DNV mis+trunc in individuals with NDD EE (n=529) vs. individuals with NDD ue (n=1413) in genes with DNV burden in NDD with epilepsy (NDD EE+uE, n=1942) using two-sided Fisher s Exact tests. Effect sizes are given as Odds ratios with columns conf.int1 and conf.int2 representing its 2.5% and 97.5% confidence intervals, respectively.. Supplementary Table 10A: DNV mis. Supplementary Table 10B: DNV trunc. Please see supplementary tab-separated text file. Supplementary Table 11. Diagnostic sequencing panels from 24 different academic and commercial providers. Please see supplementary tab-separated text file. Supplementary Table dominant/x-linked genes in sequencing panels from 24 different academic/commercial providers with three criteria for disease association in NDD with epilepsy (DNV burden, constraint, brain expression). Please see supplementary tab-separated text file. 6

20 Supplementary Table 13. Evaluating 50 genes lacking features of DNV-enriched genes (DNV enrichment, constraint, brain expression) for published evidence for disease association using guidelines from the ClinGen Gene Curation Workgroup 3. Please see supplementary tab-separated text file. 7

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