Clinical and genetic study of twelve Chinese Han families with non-syndromic deafness

BACKGROUND: Non-syndromic hearing loss is clinically and genetically heterogeneous. In this study, we characterized the clinical features of twelve Chinese Han deaf families in which mutations in common deafness genes GJB2 , SLC26A4 and MT-RNR1 were excluded. RESULTS: Targeted next-generation sequencing of 147 known deafness genes was performed in probands of ten families, while whole-exome sequencing was applied in those of the rest two. Pathogenic mutations in a total of 11 rare deafness genes, OTOF , CDH23 , PCDH15 , PDZD7 , ADGRV1 , KARS , OTOG , GRXCR2 , MYO6 , GRHL2 , and POU3F4 , were identified in all 12 probands, with 17 mutations being novel. Intrafamilial co-segregation of the mutations and the deafness phenotype were confirmed by Sanger sequencing. CONCLUSIONS: Our results expanded the mutation spectrum and genotype-phenotype correlation of non-syndromic hearing loss in Chinese Hans and also emphasized the importance of combining both next-generation sequencing and detailed auditory evaluation to achieve a more accurate diagnosis for non-syndromic hearing loss.


Background
Non-syndromic hearing loss (NSHL) is one of the most common sensory defects in humans and is a remarkably complex and heterogeneous disease with variable phenotypes (1). Genetic components contribute significantly to the cause of hearing loss (http://hereditaryhearingloss.org), with mutations in a great variety of deafness genes being reported in the Chinese Han population (2)(3)(4)(5)(6). In recent years, next-generation sequencing (NGS) technology including both targeted and whole-exome sequencing has provided an easier and more cost-effective approach for identifying causative mutations (2)(3)(4)(5)(6). It provides crucial information for diagnosis, intervention and treatment of hearing disorders (6). In this light, we recruited a series of Chinese Han deaf families that were pre-excluded from mutations in common deafness genes GJB2, SLC26A4 and MT-RNR1. Targeted NGS for known deafness genes or whole-exome sequencing (WES) were performed on the probands of each family to search for pathogenic mutations.

Clinical evaluation
Patients from twelve deaf families were enrolled through the Department of Otolaryngology, Affiliated Hospital of Nantong University, Nantong, China. Comprehensive clinical evaluations, imaging examination results, audiograms, and other relevant clinical information were collected for the probands. All affected individuals were evaluated through detailed audiological evaluations as described previously (5,6). The probands had no obvious syndromic symptoms other than the hearing loss. All subjects or their family members gave written, informed consent to participates in this study. This study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University.

Genetic Analysis
Genomic DNA from the family members were extracted from the blood samples using the Blood DNA kit (Tiangen Biotech, China). Pre-screening of mutations in GJB2, SLC26A4 and MT-RNR1 was performed in all probands by Sanger sequencing. Among probands of the 12 deaf families, ten were subjected to targeted NGS of 147 deafness-related genes (Supplementary file 1) and rest two to WES.
Targeted gene capturing, data processing, bioinformatic analysis, and filtering against multiple databases for SNPs were performed as previously reported (2)(3)(4). Intrafamilial co-segregate of the candidate variants and the deafness phenotype was confirmed by Sanger sequencing in all available family members.

Clinical Manifestations
Patients in the twelve Chinese families(Supplementary file 3), aged from 11 months to 87 years, exhibited bilateral, symmetrical, sensorineural hearing loss with the variable developing course and degree of severity, ranging from stable to progressive and from mild to profound(Supplementary file 4). The age at onset of HL in these patients ranged from at birth to 44 years. Patients with X-linked deafness in Families NT-42 and NT-43, carrying mutations in POU3F4 as subsequently revealed, showed characteristic inner ear radiological features (Supplementary file 5) compatible with incomplete partition type3 (IP3), including absent modiolus and lamina spiralis but preserved interscalar septum in a normal-sized cochlea and abnormal dilatation of the lateral end of the internal auditory canal (IAC). Through physical examination, no other abnormalities, such as retinal pigment degeneration or other optic defects, vestibular, neurologic or systemic abnormalities, were detected in any of the patients, suggesting that the hearing loss is non-syndromic.

Genetic Findings
The 12 Chinese probands have been previously excluded for mutations in common deafness genes GJB2, SLC26A4 and MT-RNR1 by Sanger sequencing. To detect possible causative mutations by targeted NGS or WES, non-synonymous variants with minor allele frequencies lower than 0.005 were filtered through as previously described (2)(3)(4)(5)  Mutations in CDH23 and PCDH15 may lead to both NSHL (DFNB12 and DFNB23, respectively) and Usher syndrome type 1 (USH1D and USH1F, respectively) characterized by both congenital hearing loss and childhood retinitis pigmentosa (5,10,11 The previous report has associated PDZD7 mutations with digenic Usher syndrome(13) and DFNB57 (14,15). To our knowledge, our study is the third report to identify PDZD7 as a causative gene for autosomal recessive non-syndromic hearing loss (ARNSHL) in the Chinese population (14,15). We also ascertained two Chinese families with an autosomal dominant form of progressive NSHL. For Family NT-49, we identified a novel missense p.T197I mutation inMYO6. Mutations in MYO6 have been associated with dominant and recessive non-syndromic hearing loss DFNA22 and DFNB37 (17,18).
The hearing loss in affected members of Family NT-49 was progressive, mid-life onset, and mild to severe, affecting high frequencies to the greatest degree. The hearing impairment gradually progressed to all frequencies later and eventually became severe in the seventh decade. For Family NT-50, A nonsense p.R426X mutation in GHRL2 was found responsible for autosomal dominant hearing loss DFNA28. To date, only two mutations in GRHL2 have been described (19,20). Phenotypic characterization of Family NT-50 shows that the p.R426 X mutation in GRHL2 resulted in progressive, bilateral hearing loss with a typical onset in middle adulthood, which was consistent with the phenotype reported for the other two DFNA28 families (19,20). Our clinical data supported the emerging genotype-phenotype correlation for DFNA22 and DFNA28.
In this study, our targeted NGS analysis identified mutations in nine rare deafness genes in the aforementioned 10 families. In recent years, whole-exome sequencing (WES) has become a powerful tool for both new gene discovery and molecular diagnosis in hereditary hearing loss (3,4). Here we used proband-WES approach to successfully identify novel compound heterozygous mutations p.G145S/c.2117-6C>T in OTOG and a homozygous p.K22R mutation in GRXCR2 in Family NT-51 and NT-52, respectively. To our knowledge, this is the first reported OTOG mutation associated with hearing loss in China (21,22). In Family NT-51, the patient had experienced progressive and steeply sloping high-frequency hearing loss without any vestibular impairment. She also reported troublesome tinnitus. GRXCR2 mutations are rare causes of recessive deafness as there is only one report worldwide (23). The 72-year-old proband in NT-52 had a moderate sensorineural hearing loss affecting primarily high frequencies, resulting in a downsloping audiometric configuration. Her hearing loss started during her mid-40s and followed by steady and gradual progression. The proband had a less severe hearing loss as compared to the previous study (23), suggesting a variable genotypephenotype correlation.

Conclusion
In this report, we performed a comprehensive mutation screening by targeted NGS or WES in twelve Chinese families with NSHL. Our results revealed a number of novel or recurrent mutations in rare deafness genes and supported the heterogeneity of the genetic and phenotypic spectrum of NSHL in Chinese Hans. Our study also showed that combining NGS-based molecular diagnosis and detailed clinical evaluation can achieve a more accurate diagnosis for NSHL patients.

Competing interests
The authors declare that they have no competing interests.
LPZ conceived and designed the experiments. DW, WYH, SL, J Z, XHC, YT, JHQ, ZXW, and ZHX contributed to the gathering and interpretation of clinical data. DW and XCD performed NGS screening and bioinformatics analyses. LPZ acted as a head surgeon for cochlear implantation performed in this study. All authors read and approved the final manuscript and collected data.

Funding
This research was supported by grants from the National Science Foundation of China (81870725 to LPZ and 81900949 to XCD) and the Postgraduate Innovation Program from Nantong University(KYCX19 2081 to SL).

Data Availability Statement
The data relating to the findings of this study are available from the corresponding author.

Ethics approval and consent to participate
Approval was obtained from the Ethics Committee of the Affiliated Hospital of Nantong University and written informed consent was obtained from each studied family member.

Consent for publication
Consent for publication of individuals' details were obtained from each subject or their family members. 0000-0003-1745-081X