Tip-link Related Genes And Hereditary Deafness

Background:Hearing loss or hearing impairment refers to a partial inability to hear sounds in one or both ears,while a deaf person has no ability to hear at all.The Global Burden of Disease Study measured the years lived with this disability and showed that hearing loss was the fourth leading cause of global disability.There are 466 million persons in the world live with disabling hearing loss(6.1%of the world’s population).Risk factors for hearing loss include genetic factors,aging,a noisy environment,ear trauma or infection,birth complications,certain medications,and toxins.To date,there are hundreds of gene mutations that lead to hearing loss either as the exclusive clinical feature or in combination with extra-auditory symptoms as part of a syndrome.The non-syndromic forms of deafness account for 70%of these cases,of which approximately 85%are inherited in an autosomal recessive manner.In most cases,deafness is frequently caused by single gene mutations.Among them,CDH23 provides a striking example.Deafness caused by CDH23 has been found in many populations worldwide,including African-American,Dutch,European,German,Pakistani,Turkish,Korean,Japanese and Chinese populations.Genetic variants of cadherin 23(CDH23)have been associated with Usher Syndrome type 1D(USH1D)and non-syndromic hearing loss(DFNB12),in a recessively transmitted manner.Moreover,p.P240L(c.C719T)was likely to be the most common cause of CDH23-associated hearing loss in Asian populations.It is now known that tip-link consists of proteins encoded by the CDH23 and PCDH15genes.CDH23 variants could induce disorganization of the stereocilia in the hair cells of the inner ear in the mouse model of the USH1D,waltzer.CDH23 expressed in the neurosensory epithelium during early hair-cell differentiation,while stereocilia organization was disrupted in waltzer.These results indicate a crucial role of CDH23 in hair cell differentiation,yet how CDH23 functions to dictate hair cell differentiation remains poorly understood.Objects:Using meta-analysis,immunology,molecular biology,bioinformatics analysis,as well as other methods,our research aims at the following aspects:(1)the association between p.P240L and non-syndromic hearing loss;(2)the role of CDH23 variants in Chinese patients with non-syndromic hearing loss;(3)searching for genes expressed in a similar pattern with CDH23 during inner ear development;(4)enhancing our understanding of how the hair cells normally develop and function.Methods:(1)Literatures that reported prevalence rates of CDH23 were identified using Pub Med,EMBASE,OVID,Cochrane library,Web of Science,Chinese National Knowledge Infrastructure,Wanfang Databases.Random and fixed effects models were used to generate pooled ORs and I~2values.(2)Clinical information and peripheral blood samples were acquired for DNA extraction and DNA sequencing focusing on nine gene loci(GJB2:35delG,176del16,235delC,299delAT,GJB3:538C>T,SLC26A4:2168A>G,and IVS7-2A>G,mitochondrial12SrRNA:1494C>T and 1555A>G).A customized exome enrichment kit was designed to capture genes that cause hearing loss.The quality control assessment and variants calling were processed.(3)Likely damaging variants were further screened for quality against the 1000Genomes public variant databases,the Single Nucleotide Polymorphism database,SIFT,and Polyphen-2.Polymerase chain reaction(PCR)based Sanger sequencing was performed to validate the variants.Structural analysis and visualization were processed for predicted molecular structure models of the extracellular domain.(4)We analyzed the dataset generated by microarray analysis to identify genes correlated with tip-link related genes expression patterns.The confirmation of tip-link related genes was performed on a set of samples from different development stages using real-time polymerase chain reaction(RT-PCR)and immunocytochemistry.The bioinformatics analysis was used to construct a network of associations between tip-link related genes to explore the function of these genes during the inner ear development.Results:(1)A total of four relevant studies were included in the meta-analysis.The results of meta-analysis indicated that the p.P240L variant was correlated with the risk of non-syndromic hearing loss in Asian populations(OR=10.17,95%CI=2.74-37.82,P=0.001).The T allele of p.P240L was associated with a 12-fold higher risk of NSHL than the C allele(OR=11.68;95%CI=3.16-43.24,P<0.001).Specifically,p.P240L heterozygotes(OR=8.49;95%CI=2.28-31.59,P=0.001),had a significantly higher risk of non-syndromic hearing loss.(2)The data revealed a heterozygous mutation of cadherin 23(CDH23)in both patients after DNA sequencing(i.e.,one of the two shifts caused by a transition CDH23:c.791A>T in exon 9).This transition results in the substitution of a valine with an aspartate at position 264(p.D264N),while the other variant was CDH23:c.5853T>A in exon 43.This resulted in the substitution of a glutamate residue with an aspartate at position 1951(p.D1951E).(3)Two novel mutations of CDH23(p.D264V and p.D1951E)were identified in the coding region,both of which are missense variants.The results of Polyphen-2 and SIFT predicted the impact of these two changes and implied that they might pathogenically affect protein structure or function.3D models of these variants indicated that mutated EC domains,containing the residue aspartate,contained the highly conserved calcium-binding motifs,EC3 and EC18,in the location of the linking cadherin repeats.(4)There were differences in the expression levels of tip-link related genes(CDH23,PCDH15,MYO7A,SANS)in different developmental stages of the cochlea(E15 to P7)(P<0.0001).Moreover,during the same cochlea development period,the expression levels of the above four genes were also different(P<0.0001).There was a correlation between the expression levels of tip-link related genes in the cochlea and ellipsoids in the GSE60019 dataset.Further,there was a correlation between the expression levels of tip-link related genes found in the fresh cochlear tissue.Protein protein interaction network showed that tip-link related genes played an important role in the development of inner ear,mediating electromechanical transduction of cochlear hair cells.Conclusions:(1)The p.P240L variant increased the risk of non-syndromic hearing loss in Asian populations,suggesting a remarkable ethnic specificity linked with susceptibility to this mutation.(2)We identified two novel CDH23 variants in one Chinese family with autosomal recessive non-syndromic hearing loss.(3)The CDH23 variants may contribute to the hearing impairment of these two patients and that there was a correlation between CDH23 mutations and disease pathogenicity.(4)By reanalyzing archived genetic profiling data,we identified a list of genes possibly involved in hair cell differentiation.