ExACtly zero or once

Objective: To assist the interpretation of genomic data for common epilepsies, we asked whether variants implicated in mild epilepsies in autosomal dominant families are present in the general population. Methods: We studied 12 genes for the milder epilepsies and identified published variants with strong segregation support (de novo germline mutation or $4 affected family members). These variants were checked in the Exome Aggregation Consortium (ExAC), a database of genetic variation in over 60,000 individuals. We subsequently evaluated variants in these epilepsy genes that lacked strong segregation support. To determine whether the findings in epilepsies were representative of other diseases, we also assessed the presence of variants in other dominant neurologic disorders (e.g., CADASIL). Results: Published epilepsy variants with strong segregation support (n 5 65) were absent (n 5 61) or present once (n5 4) in ExAC. By contrast, of 46 published epilepsy variants without strong segregation support, 8 occurred recurrently (2–186 times). Similarly, none of the 45 diseaseassociated variants from other neurologic disorders with strong segregation support occurred more than once in ExAC. Reanalysis using the larger ExAC V2 plus gnomAD reference cohort showed consistent results. Conclusions: Variants causing autosomal dominant epilepsies are ultra-rare in the general population. Variants observed more than once in ExAC were only found among reports without strong segregation support, suggesting that they may be benign. Clinicians are increasingly faced with the interpretation of genetic variants of unknown significance. These data illustrate that variants present more than once in ExAC are less likely to be pathogenic, reinforcing the valuable clinical role of ExAC. Neurol Genet 2017;3:e163; doi: 10.1212/NXG.0000000000000163 GLOSSARY ExAC5 Exome Aggregation Consortium;GEFS15 genetic epilepsy with febrile seizures plus; VOUS5 variants of unknown significance; WES 5 whole-exome sequencing. Recent advances in DNA technologies have increased the influence that genomic data have in epilepsy clinical practice. Finding a genetic cause can have important implications for diagnosis and prognosis, treatment and genetic counseling, as well as the psychological and financial benefits associated with ending the diagnostic journey. Clinical laboratories are understandably conservative in ascribing pathogenicity; however, the elevated reporting of variants of unknown significance (VOUS) makes interpretation complex and renders follow-up efforts, such as segregation analyses and functional studies, impractical at a large scale. Novel missense variants are particularly challenging. Establishing the frequency of a VOUS in control populations is currently among the most reliable considerations in variant interpretation. From the Department of Medicine (C.A.B., S.P., K.L.O., S.F.B.), Epilepsy Research Centre; and Department of Medicine (S.P.), Royal Melbourne Hospital, University of Melbourne, Victoria, Australia. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Neurology.org/ng Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology 1 The Exome Aggregation Consortium (ExAC) database contains exome data for more than 60,000 individuals compiled at the Broad Institute. For de novo mutations affecting established epilepsy genes, absence from ExAC is widely accepted as one supportive argument for pathogenicity and this is particularly true for epileptic encephalopathies and most neurodevelopmental disorders. The question is whether this utility extends to the milder epilepsies where negative selection may not have been as strong. The milder presentations and the observation of reduced penetrance within dominant families raise the possibility that some causative variants might be present in the general population at low frequencies. Here, to assess how informative ExAC might be to ascribing pathogenicity to inherited variants among milder epilepsies, we evaluated the frequency in ExAC of published epilepsy variants, where de novo status or segregation in large families supported pathogenicity. METHODS A list of 12 genes was generated for the “milder” epilepsies (table 1). We excluded PRRT2, as it is prone to alignment errors in next-generation sequencing. Literature search. We attempted an exhaustive PubMed search for missense and splice variants deemed causative for dominant familial epilepsies, published in English, on the basis of strong segregation support, which we defined as prior linkage analysis or familial segregation in at least 4 affected family members. We note that segregation in 4 individuals is inadequate to support gene discovery but, in the context of an established gene for an appropriate phenotype, such segregation provides considerable support for the role of the variant in the particular family. Sanger validated de novo variants found in our list of 12 epilepsy genes, among sporadic cases of “mild” epilepsies, were also included. We deliberately limited our search to the literature published prior to 2015, as this literature can realistically be considered to precede the launch of the ExAC database. A separate review was conducted for each of the 12 genes by searching the gene name and “epilepsy.” For genes associated with both milder and more severe phenotypes, such as genetic epilepsy with febrile seizures plus (GEFS1) and Dravet syndrome with SCN1A, only variants found among families or individuals with the milder phenotype were included. Variants reported exclusively among the epileptic encephalopathies were excluded. A second list of variants without strong segregation support was generated. Lack of strong segregation support was defined as segregation data based on less than 4 affected individuals or not performed at all. We hypothesized that this group of variants might be more enriched for background genetic variants in epilepsy genes. To determine whether findings in mild epilepsies were reflective of other autosomal dominant neurologic diseases, we evaluated variants with strong segregation support from 45 published families with familial hemiplegic migraine, CADASIL, autosomal dominant Alzheimer disease or frontotemporal dementia. The genes examined were ATP1A2, CACNA1A, SCN1A, NOTCH3, APP, PSEN1, PSEN2, GRN, MAPT, and TARDBP1. Finally, after the study was completed, the expanded ExAC V2 plus gnomAD was released and the analysis was rerun. This effectively doubled the size of the ExAC control cohort, with whole-exome sequencing (WES) and whole-genome sequencing data available on 123,136 individuals and 15,496 individuals, respectively. RESULTS Sixty-five missense or predicted splice variants for the milder familial epilepsies with strong segregation support were identified. Of these, 61 (94%) were absent from ExAC and 4 (6%) were present in ExAC once (a single allele was reported) (figure; table e-1 at Neurology.org/ng). By contrast, of the 46 missense or splice variants contained in the same publications but lacking the strong segregation support, 13 (28%) were present in ExAC. Of these, 8 variants were observed among 2–186 carriers (figure; table e-1). These 8 variants are listed in table 2. Of the 45 missense variants with strong segregation support for other neurologic disorders, 43 (96%) were absent from ExAC and 2 (4%) were present once (figure; table e-1). Reanalysis with ExAC V2 and gnomAD datafiles showed that the epilepsy variants in our current study with strong segregation support are present no more than once, with the exception of 2 variants in SCN1B associated with febrile seizures and GEFS1 which are present 3 and 4 times (table e-1). For variants without strong segregation support, their frequency in ExAC V2 approximately doubled, as expected, compared with the initial analysis and 3 new variants were observed multiple times in ExAC V2 (table 2). Table 1 Genes for autosomal dominant epilepsy syndromes Epilepsy syndrome Gene Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) CHRNA4