Abstracts

Rationale: ADNFLE is a rare seizure disorder that follows an autosomal dominant pattern of inheritance. Mutations in the CHRNA2, CHRNA4 and CHRNB2 genes have been shown to cause the disease in some families, however, other families exist in which the gene mutations are not yet discovered. We are attempting to identify the causative mutation in a small nuclear family with ADNFLE using NGS. None of the affected relatives (n=3) harbor any of the known mutations for ADNFLE and the family is too small (n=4) to employ traditional linkage and positional cloning strategies.Methods: Blood (DNA) samples were collected from a small nuclear ADNFLE family consisting of a single affected parent and three children, two of whom are affected. The samples were subjected to whole genome sequencing (WGS) in an attempt to identify the causative mutation. All four individuals were sequenced using the ABI SOLID G4 platform at 10x coverage. In addition, two individuals are being subjected to high coverage (50X) whole exome sequencing to compare results with lower coverage WGS, reduce error rate and further narrow the candidate list. Raw data was aligned to the human reference sequence using the SOLID BioScope software. Variation calling was performed using diBayes and GATK programs. Variant annotation and filtering was performed using ANNOVAR and alignment visualization using UCSC custom track, IGV and SAMtools. Results: Preliminary findings indicate that the affected individuals are heterozygous for gene coding region variation (nonsynoymous SNPs) in 72 genes. Candidates are being prioritized based on expression patterns in brain and biological function. Variations are being systematically confirmed by traditional Sanger sequencing. Compelling candidates include genes related to neurotransmission and developmental biology, including but not limited to GABARAP, SNAP23, CDH1, WHSC1 and PTPN2.Conclusions: Taken together, these results show that NGS can be used to identify putative causal mutations in small families where traditional linkage and positional cloning strategies are not possible. Expanded pedigree data and DNA samples are available for this family and are being acquired. Functional studies on the top priority variations will help identify the causative mutation in this family and should elucidate mechanisms of pathology for ADNFLE specifically and human epilepsy in general.