DNA was extracted from the index patients, as well as from other affected and unaffected family members, using the FlexiGene DNA kit (QIAGEN, Hilden, Germany). Sanger sequencing of PCR products was performed using specific primers designed using the Primer3 software and the UCSC website (
Supplementary Table S1).
33 Whole exome sequencing (WES) analysis of 11 families (including at least two affected and one unaffected individuals per family) was performed at the Otogenetics Corporation using a paired-end sample preparation kit (NimbleGen V2, 44.1 Mbp; Roche, Basel, Switzerland) and HiSeq2000 (Illumina, San Diego, CA, USA) at a 50× and 100× coverage (ranging from 0 to 524 reads per nucleotide). Sequence reads were aligned to the human genome reference sequence (build hg19) and variants were called and annotated using the DNAnexus software package (
http://drorsharon1.wixsite.com/the-sharon-lab/data, in the public domain). Dataset files, including the annotated information, were analyzed using ANNOVAR software according to the dbSNP database (build 135) with the following filtering steps (see
Supplementary Table S2): (1) variant type, only the following variant types were included; missense, nonsense, insertions and deletions within the coding region, and splice-site; (2) variants found within segmental duplications were excluded; (3) variants with a minor allele frequency (MAF) greater than 0.05 in the ExAC Project were excluded (
http://exac.broadinstitute.org/, in the public domain); (4) variants with a SIFT (
http://sift.jcvi.org/, in the public domain) with a score less than 0.05 were excluded; (5) variants with a PolyPhen2 (
http://genetics.bwh.harvard.edu/pph2/, in the public domain) score less than 0.85 were excluded; and (6) single heterozygous variants that do not fit an autosomal recessive inheritance pattern were excluded. Suspected pathogenic mutations were verified using Sanger sequencing. All WES samples were analyzed for copy number variants, but no homozygous deletion was identified. In addition, whole genome single-nucleotide polymorphism (SNP) analysis was performed on DNA samples from 13 families (20 patients) using a SNP array (Affymetrix, Santa Clara, CA, USA) 10K, 250K, and 6.0 systems, and data analysis was performed using HomozygosityMapper (
http://www.homozygositymapper.org/, in the public domain). The frequency of novel missense variants was tested in a dataset of 408 exomes of Israeli individuals.