Targeted next generation sequencing as a diagnostic tool in epileptic disorders

Abstract

Summary Purpose: Epilepsies have a highly heterogeneous background with a strong genetic contribution. The variety of unspecific and overlapping syndromic and nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents straightforward genetic testing. Knowing the genetic basis of a patient’s epilepsy can be valuable not only for diagnosis but also for guiding treatment and estimating recurrence risks. Methods: To overcome these diagnostic restrictions, we composed a panel of genes for Next Generation Sequencing containing the most relevant epilepsy genes and covering the most relevant epilepsy phenotypes known so far. With this method, 265 genes were analyzed per patient in a single step. We evaluated this panel on a pilot cohort of 33 index patients with concise epilepsy phenotypes or with a severe but unspecific seizure disorder covering both sporadic and familial cases. Key Findings: We identified presumed disease‐causing mutations in 16 of 33 patients comprising sequence alterations in frequently as well as in less commonly affected genes. The detected aberrations encompassed known and unknown point mutations (SCN1A p.R222X, p. E289V, p.379R, p.R393H; SCN2A p.V208E; STXBP1 p.R122X; KCNJ10 p.L68P, p.I129V; KCTD7 p.L108M; KCNQ3 p.P574S; ARHGEF9 p.R290H; SMS p.F58L; TPP1 p.Q278R, p.Q422H; MFSD8 p.T294K), a putative splice site mutation (SCN1A c.693A> p.T/P231P) and small deletions (SCN1A p.F1330Lfs3X [1 bp]; MFSD8 p.A138Dfs10X [7 bp]). All mutations have been confirmed by conventional Sanger sequencing and, where possible, validated by parental testing and segregation analysis. In three patients with either Dravet syndrome or myoclonic epilepsy, we detected SCN1A mutations (p.R222X, p.P231P, p.R393H), even though other laboratories had previously excluded aberrations of this gene by Sanger sequencing or high‐resolution melting analysis. Significance: We have developed a fast and cost‐efficient diagnostic screening method to analyze the genetic basis of epilepsies. We were able to detect mutations in patients with clear and with unspecific epilepsy phenotypes, to uncover the genetic basis of many so far unresolved cases with epilepsy including mutation detection in cases in which previous conventional methods yielded falsely negative results. Our approach thus proved to be a powerful diagnostic tool that may contribute to collecting information on both common and unknown epileptic disorders and in delineating associated phenotypes of less frequently mutated genes.

Pitfalls in genetic testing: the story of missed SCN1A mutations

“…Accession number for SCN1A : RefSeq NM_001165963.1, NP_001159435.1. Seven patients have previously been reported.aThese eight patients are part of the EuroEPINOMICS‐RES cohort.bLemke et al., 2012.cBayat et al., 2015.dCarvill et al., 2014.eGaily et al., 2013.fBased on the SCN1A database (http://www.gzneurosci.com/scn1adatabase/index.php) and the published papers mentioned in this manuscript.…”

“…Seven patients have previously been reported.aLemke et al., 2012.bBayat et al., 2015.cCarvill et al., 2014.dGaily et al., 2013.…”

“…described two mutations in a cohort of 33 patients with diverse epilepsy phenotypes (Lemke et al. 2012), Bayat et al. mentioned two patients with DS who initially tested negative upon SCN1A screening (Bayat et al.…”

Lessons learned from gene identification studies in Mendelian epilepsy disorders

“…This leads to a more accurate estimation of the success rate, which ranges from 10 to 48%. [8][9][10][11] Most of the early reports are, however, enriched for patient samples prescreened for established epilepsy genes, lowering the calculated success rate. Similar to our own experiences, the more recent NGS studies -including patients without preceding molecular investigations − provide a molecular diagnosis for~30% of the investigated epilepsy patients.…”

Unusual Course of Lafora Disease

“…It enables screening for pathogenic variants associated with PMEs, with results available in 4 weeks. Costs are comparable to those of sequencing three individual genes 11, 12. Here, we describe a 42‐year‐old male patient, initially diagnosed with JME, who appeared to have Lafora disease.…”