Whole exome sequencing (WES) and bioinformatic analysis

WES genetic analysis were designed on offspring-parents trios, including one affected individual (II:1) and two unaffected parents (I:1 and I:2). Genomic DNA was extracted from peripheral blood using a blood DNA extraction kit according to the manufacturer’s instructions (TianGen, Beijing, China). DNA was sheared, ligated to adaptors, extracted, amplified by ligation-mediated PCR. 1 μg DNA library was mixed with Buffer BL and GenCap probe (MyGenostics, Beijing, China) for enrichment. Each captured library was loaded onto the Illumina NovaSeq 6000 platform. After filtering out low-quality and duplicate reads, clean data were aligned to the human reference genome hg19 using the Burrows-Wheeler Aligner. After alignment, variants were called using four types of software (SOAPsnp, GATK, Samtools and Platypus), merged into variant call format files and annotated by ANNOVAR and associated with multiple databases, including those with minor allele frequencies (MAF < 0.05), in public databases, including gnomAD, Inhouse database (MyGenostics), HGMD, and predicted by SIFT, PolyPhen-2, MutationTaster, GERP +  + . Under the assumption of an autosomal-recessive or autosomal-dominant (de novo) pattern of inheritance, only variants that were homozygous or compound heterozygous in the affected boy and heterozygous in their parents, and de novo variant were selected as candidates. VarSome was used for a comprehensive interpretation of the variants [16]. Manually classification of those variants was conducted based on American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines for genetic hearing loss [17]. Potential pathogenic variants finally screened by these analyses were confirmed using Sanger sequencing.