Improving molecular diagnosis in epilepsy by a dedicated high-throughput sequencing platform

Abstract

We analyzed by next-generation sequencing (NGS) 67 epilepsy genes in 19 patients with different types of either isolated or syndromic epileptic disorders and in 15 controls to investigate whether a quick and cheap molecular diagnosis could be provided. The average number of nonsynonymous and splice site mutations per subject was similar in the two cohorts indicating that, even with relatively small targeted platforms, finding the disease gene is not an univocal process. Our diagnostic yield was 47% with nine cases in which we identified a very likely causative mutation. In most of them no interpretation would have been possible in absence of detailed phenotype and familial information. Seven out of 19 patients had a phenotype suggesting the involvement of a specific gene. Disease-causing mutations were found in six of these cases. Among the remaining patients, we could find a probably causative mutation only in three. None of the genes affected in the latter cases had been suspected a priori. Our protocol requires 8-10 weeks including the investigation of the parents with a cost per patient comparable to sequencing of 1-2 medium-to-large-sized genes by conventional techniques. The platform we used, although providing much less information than whole-exome or whole-genome sequencing, has the advantage that can also be run on 'benchtop' sequencers combining rapid turnaround times with higher manageability.

Figures

Figure 1

Flow chart representing the strategy adopted to analyze sequencing data. Discrete filtering, prioritization, and re-evaluation steps are highlighted in blue, orange and green, respectively.

Figure 2

Alignments loaded on IGV 2.1(Integrative Genomics Viewer) for every causative mutation reported in bold in Table 2. Chromosomal view has the covered 41 bp delimitated in red, followed by genomic coordinates, relative coverage for single base pair, and alignments covering 101 bp in average. At the bottom of every image the reference sequence and the corresponding amino-acid sequence are visible.