Genetic analysis
DNA was isolated from peripheral whole blood using the Qiagen (Germantown, MD) Gentra Puregene isolation kit. Linkage analysis was performed using the genotype data generated with Illumina HumanOmniExpress-24 chip for the mother and the four sibs. The Allegro module (Gudbjartsson et al., 2000) of easyLINKAGE software was used, assuming autosomal recessive inheritance and parents as third cousins. No deletion or duplications common to just the two affected brothers were detected using cnvPartition plug-in in Illumina Genome Studio v.1.02 software.
Exome sequencing was performed independently twice on one subject. Capture for whole exome sequencing was performed with NimbleGen SeqCap EZ Exome Library v1.0 kit (Roche, Indianapolis, IN). Captured regions were sequenced with Illumina HiSeq2000 instruments. Variants were filtered to remove common variants based on 1000 Genomes, Exome Sequencing Project, and Exome Aggregation Consortium databases, variants outside of identified linkage regions, variants not expected to change protein coding, and variants not following a recessive model of inheritance (Exome Aggregation Consortium (ExAC), 2015; Genomes Project Consortium et al., 2012; NHLBI Go Exome Sequencing Project, 2015).
PCR followed by Sanger sequencing was performed to validate the variant identified through exome sequencing and test for segregation within the family. The variant of interest was further examined in two separate collections of a total of 500 Turkish samples, and found absent.