Subjects and Clinical Evaluations
The study was approved by the Ethics Committee of Shahrekord University of Medical Sciences (IR.SKUMS.REC.1397.008), Iran. Two Iranian families from Hamedan Province with hearing impairments with no additional symptoms were thus studied. Moreover, informed written consent was taken from both families and the proband from each family was further subjected to clinical evaluations of the inner ear accompanied by pure-tone audiometry (PTA).
Molecular Analysis
Genomic deoxyribonucleic acid (DNA) was extracted from whole peripheral blood of each study subject utilizing DNA Extraction Kit DNP (Sinacolon, Iran) according to the manufacturer’s instructions. Purity and concentration of DNA samples were further measured via Thermo Scientific NanoDrop 2000c Spectrophotometer.
DNA samples from each pedigree’s proband (Figure 1A, V-4 in family 1; Figure 2A, II-3 in family 2) were then subjected to WES at Macrogen Online Sequencing Order System (Seoul, South Korea) on Genome Analyzer/HiSeq 2000 (Illumina, San Diego, CA, USA, 151-bp paired-end reads). It should be noted that the library had been prepared through SureSelect XT Library Prep Kit (Agilent Technologies, CA, USA). Data analysis was correspondingly performed using an in-house developed pipeline, adopted from Genome Analysis Tool Kit v3.6 and ANNOVAR software.[6] Homozygous missense, start codon change, splice site, nonsense, stop loss, and indel variants with minor allele frequency <1% were further filtered in dbSNP (version 138), 1000 Genomes Project, Exome Aggregation Consortium, and NHLBI GO Exome Sequencing Project (ESP). Based on autosomal recessive inheritance, the homozygosity region of samples was determined using homozygosity mapping algorithms.
Several online prediction software including MutationTaster2, FATHMM, PANTHER, SIFT, PROVEAN, MetaLR, PolyPhen-2, CADD, and ConSurf were also used to evaluate the pathogenic effect of the variants. Next, the variants were investigated in the Human Gene Mutation Database and the related literature to survey their association with a phenotype and novelty of the variants.
Besides, candidate variant segregation from exome data was evaluated through polymerase chain reaction (PCR)-based Sanger sequencing. Therefore, the following primers were synthesized: 5ʹ-GAACTACATCGTGCAGAAGG-3ʹ and 5ʹ-CCTATCCAGTCCCACTCACT-3ʹ for human MYO15A c.T6442A variant and 5ʹ-CCACCATTCGGCCTTCCA-3ʹ and 5ʹ-CTGCCTCCTCTTAGTGTCCTC-3ʹ for human MYO15A c.10504dupT variant.