In this study, WES was applied to search for molecular etiology in 12 patients from six unrelated Chinese families with non-syndromic deafness. These families showed obvious autosomal recessive heritance based on at least two affected individuals with normal parents. After excluding the common GJB2 and SLC26A4 mutations, we analyzed the WES results of 76 non-syndromic autosomal recessive HL-related genes. Overall, 3 reported and 7 novel variants in 3 distinct deafness genes (COL11A2, CDH23, and MYO15A) were identified and cosegregated with HL. The corresponding amino acid
changes included 4 missense mutations, 3 frameshift indel mutations, 1 in-frame mutation, and 2 splicing-site mutations.
In family HL01, the patients carried a reported homozygous variant c.966dupC (p.T323Hfs*19) in the COL11A2 gene, and both parents were carriers of the heterozygous variant. Remarkably, this was also the only family to present moderate bilateral deafness. The COL11A2 encoding type XI collagen is critical for the integrity and development of the skeleton . Mutations in COL11A2 (OMIM 120290) are associated with several conditions that include autosomal dominant (DFNA13, OMIM 601868) or recessive NSHL (DFNB53, OMIM 609706), as well as Stickler syndrome (OMIM 184840), otospondylmegaepiphyseal dysplasia (OMIM 215150), fibrochondrogenesis (OMIM 614524), and Weissenbacher-Zweymuller syndrome (OMIM 277610). Hitherto, 61 variants have been reported to be pathogenic or likely pathogenic, of which, 12 are associated with sensorineural hearing loss (http://www.hgmd.cf.ac.uk/ac/). In addition, c.966dupC (p.T323Hfs*19) has also been detected in the probands from consanguineous Iranian families that show profound  or moderate  sensorineural hearing loss. COL11A2 is considered a “rare gene” responsible for deafness, especially in China. Only a few cases of variants have been reported in the hearing-loss patients from China. Ji et al.  reported 4 copy number variations (CNVs) in 11/79 sporadic patients clinically diagnosed with sensorineural hearing loss. Among these, only 1 patient carried two types of CNVs, while the remaining 10 had only one heterozygous form of CNV without any other form of variation in the COL11A2 gene. Chen et al.  detected p.Pro445Ser in 116 Chinese patients with hereditary hearing loss using next-generation sequencing that examined 60 genes relevant to hearing loss. Thus, COL11A2 gene is not the main cause of deafness and could be easily missed by the conventional sequencing approach.
Homozygous or compound heterozygous variants in MYO15A gene were identified in the affected members from families HL02, HL04, HL05, and HL06, who presented similar phenotypes showing a bilateral congenital severe hearing loss. Compound heterozygous c.8240_8241delAC (p.Q2749Efs*91)/c.10419_10423delCAGCT (p.S3474Pfs*41), compound heterozygous c.4875+1G>T/c.3943G>A (p.G1315R), homozygous c.8582C>T (p.F2861S), and c.5964+3G>A/c.10245-10247delCTC (p.3417delS) were identified as causative pathogenic etiology in the patients from HL02, HL04, HL05, and HL06, families, respectively. MYO15A is the corresponding gene of DFNB3 deafness (OMIM: 600316) . Moreover, the variants of MYO15A gene are the third or the fourth most common causes of autosomal recessive hereditary hearing loss . To date, 244 MYO15A pathogenic or likely pathogenic mutations contribute to hearing loss (http://www.hgmd.cf.ac.uk/ac/). Such mutations are identified in the sequence encoding all the domains of myosin XVa protein . In this study, we identified 7 variants in MYO15A gene, including 2 reported pathogenic mutations: c.5964+3G>A and c.10245-10247delCTC (p.3417delS) [19-21]. Five novel variants included 2 missense mutations (p.G1315R and p.F2861S), 2 protein-truncating mutations (p.Q2749Efs*93 and p.S3474Pfs*43), and 1 canonical + 1 splicing-site mutation (c.4875+1G>T). p.G1315 and c.4875+1G>T, occurring in exons 7 and 15, respectively, located within the motor domain, the most important functional unit in myosin XV protein for the dysfunction of the motor domain leads to short stereocilia with a severe deafness phenotype . p.Q2749Efs*93 and p.S3474Pfs*43, leading to early termination of its coding peptide chain, might affect the normal formation of the FERM domain of myosin XVA. p.F2861S variant is also harbored within the conserved FERM domain. The substitution of phenylalanine with serine changed the hydrophobicity and altered the structure of the protein. Earlier findings suggested that mutations, which affect both isoforms of MYO15A, would result in profound deafness [29, 30]. According to ACMG, we hypothesized that these variants occurring in the above exons were the causative pathogenic variants.
In family HL03, we identified two novel deafness co-segregating variants c.G3890T (p.C1297F) and c.A6649G (p.K2217E) in CDH23 gene. The two affected sisters, carrying the heterozygous compound variants, showed bilateral congenital profound hearing loss. According to the parents, the individual II:3 had a normal hearing but was not available for testing, neither clinical nor genetic. Mutations in CDH23 are the pathogenic cause for Usher syndrome 1D (USH1D, OMIM: 601067), autosomal recessive NSHL (DFNB12, OMIM: 601386), and pituitary adenoma 5 (OMIM: 617540). A total of 373 disease-related forms of variations are included in the HGMD database, of which 351 were associated with deafness or Usher syndrome. More than half of the variants were nonsense, splice-site, and frameshift types in the patients with USH1D, causing severe sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa, whereas individuals with DFNB12, presenting only as autosomal recessive moderate-severe sensorineural hearing loss, usually carried missense variants in any domain of CDH23 gene (data summarized from the website: http://www.hgmd.cf.ac.uk/ac/). However, only a few of these variants were detected in the Chinese population . In the present study, two variants were identified in the CDH23 gene by WES in two patients with severe hearing loss. These two variants were reported for the first time and were contained in the highly conserved domain of the CDH23 gene. Other than deafness, neither of the patients had visual problems or vestibular dysfunction at the time of this report. Nonetheless, we cannot definitely rule out that they would not develop retinopathy later in their lives, since they are currently only 11- and 5-years-old, respectively. Thus, these two novel mutations expanded the CDH23 mutation spectrum.
Next, we identified 2 homozygous variants in the affected members from HL01 and HL05 families. HL01 family came from the Ningxia Hui Autonomous Region in China. All the members were of Hui nationality, and the parents of the patients were remote cousins. Homozygous variants accounted for a high rate of diagnoses in close and presumed consanguineous multiplex families with recessive inherited disease , suggesting us to consider the segregation of homozygous variants. The normal parents of HL05 were not consanguineously married and were of Han nationality from Shaanxi province in China. All the other members were normal, except the deaf offspring. The homozygous variant detected in MYO15A indicated its high prevalence in the Chinese population and suggested the necessity of screening MYO15A in the recessive inherited families with multiple affected individuals.