De novo mutations in epileptic encephalopathies

Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown. Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies: infantile spasms (n5 149) and Lennox–Gastaut syndrome (n5 115). We sequenced the exomes of 264 probands, and their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the 4,000 genes that are the most intolerant to functional genetic variation in the human population (P5 2.93 10). Among these are GABRB3, with de novomutations in four patients, and ALG13, with the same de novo mutation in two patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P5 4.13 10 and P5 7.83 10, respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR and NEDD4L. Finally, we show that the de novo mutations observed are enriched in specific gene sets including genes regulated by the fragile X protein (P, 10), as has been reported previously for autism spectrum disorders. Genetics is believed to have an important role in many epilepsy syndromes; however, specific genes have been discovered in only a small proportion of cases. Genome-wide association studies for both focal and generalized epilepsies have revealed few significant associations, and rare copy number variants explain only a few per cent of cases. An emerging paradigm in neuropsychiatric disorders is the major impact that de novo mutations have on disease risk. We searched for de novo mutations associated with epileptic encephalopathies, a heterogeneous group of severe epilepsy disorders characterized by early onset of seizures with cognitive and behavioural features associatedwith ongoing epileptic activity.We focused on two ‘classical’ forms of epileptic encephalopathies: infantile spasms and Lennox– Gastaut syndrome, recognizing that some patients with infantile spasms progress to Lennox–Gastaut syndrome. Exome sequencing of 264 trios (Methods) identified 439 putative de novomutations. Sanger sequencing confirmed 329 de novomutations (Supplementary Table 2), and the remainder were either false positives, a result of B-cell immortalization, or in regions where the Sanger assays did not work (Supplementary Table 3). Across our 264 trios, we found nine genes with de novo single nucleotide variant (SNV) mutations in two or more probands (SCN1A, n5 7; STXBP1, n5 5; GABRB3, n5 4; CDKL5, n5 3; SCN8A, n5 2; SCN2A, n5 2; ALG13, n5 2; DNM1, n5 2; and HDAC4, n5 2). Of these, SCN1A, STXBP1, SCN8A, SCN2A andCDKL5 are genes that have a previously established association with epileptic encephalopathy. To assess whether the observations in the other genes implicate them as risk factors for epileptic encephalopathies, we determined the probability of seeing multiple mutations in the same gene given the sequencespecific mutation rate, size of the gene, and the number and gender of patients evaluated in this study (Methods). The number of observed de novomutations inHDAC4 and DNM1 are not yet significantly greater than the null expectation. However, observing four unique de novo mutations in GABRB3 and two identical de novo mutations in ALG13 were found to be highly improbable (Table 1 and Fig. 1). We performed the same calculations on all of the genes withmultiple de novomutations observed in 610 control trios and found no geneswith a significant excess of de novo mutations (Supplementary Table 4). Although mutations in GABRB3 have previously been reported in association with another type of epilepsy, and in vivo mouse studies suggest that GABRB3 haploinsufficiency is one of the causes of epilepsy in Angelman’s syndrome, our observations implicate it, for the first time, as a single-gene cause of epileptic encephalopathies and provide the strongest evidence to date for its association with any epilepsy. Likewise, ALG13, an X-linked gene encoding a subunit of the uridine diphosphate-N-acetylglucosamine transferase, was previously shown to carry a novel de novo mutation in amalepatientwith a severe congenital glycosylationdisorderwithmicrocephaly, seizures and early lethality. Furthermore, the same ALG13 de novo mutation identified in this study was observed as a de novo mutation in an additional female patientwith severe intellectual disability and seizures. Each trio harboured on average 1.25 confirmed de novomutations, with 181 probands harbouring at least one. Considering only de novo SNVs, each trio harboured on average 1.17 de novo mutations (Supplementary Fig. 1). Seventy-two per cent of the confirmed de novo SNVmutations weremissense and 7.5%were putative loss-of-function (splicedonor, splice acceptor, or stop-gainmutations).Compared to rates of these classes of mutations previously reported in controls (69.4%missense and 4.2% putative loss-of-function mutations), we observed a significant excess of loss-of-function mutations in patients with infantile spasms and Lennox–Gastaut syndrome (exact binomial P5 0.01), consistent with data previously reported in autism spectrum disorder. A framework was recently established for testing whether the distribution of de novomutations in affected individuals differs from the general population. Here, we extend the simulation-based approach of ref. 8 by developing a likelihood model that characterizes this effect and describes the distribution of de novo mutations among affected individuals in terms of the distribution in the general population, and a set of parameters describing the genetic architecture of the disease. These parameters include the proportion of the exome sequence that can carry disease-influencingmutations (g) and the relative risk (c) of the mutations (Supplementary Methods). Consistent with what was reported in autism spectrum disorder, we found no significant deviation in the overall distribution of mutations from expected (c5 1 and/or g5 0). It is, however, now well established that some genes tolerate protein-disrupting mutations without apparent adverse phenotypic consequences, whereas others do not. To take this into account, we used a simple scoring system that uses polymorphismdata in the human population to assign a tolerance score to every considered gene (Methods). We then found that genes with a known association with epileptic encephalopathy rank among the most intolerant genes using this scheme (Supplementary Table 8). We therefore evaluated the distribution of de novo mutations within these 4,264 genes that are within the 25th percentile for intolerance and found a significant shift from the null distribution (P5 2.93 10). Themaximum likelihood estimates of g (percentage of intolerant genes involved in epileptic encephalopathies) was 0.021 and c (relative risk) was 81, indicating that there are 90 genes among the intolerant genes