Identification Of Molecular Etiology In Pedigrees With Inherited Hearing Loss And Study Of Noninvasive Prenatal Testing Of Hearing Loss

Hearing loss is a major public health problem in the globe.As the most populous country in the world,China is faced with the same serious situation.According to the second survey in China in 2006,there were 27.8 million people with hearing loss and speech impairment which accounted for 33.5%of the total number of people with disabilities in China.Among the hearing loss persons,there are about 137,000 children aged 0 to 6 years,and about 30 to 40 thousand children with congenital hearing impairments are newly added every year.At the same time,there are about 30,000 children who have drug-induced deafness or delayed deafness in China every year.Deafness has seriously affected the population quality of our country.It is estimated that about 60%deafness are hereditary.Hearing loss could be divided into nonsyndromic hearing loss（NSHL）and syndromic hearing loss（SHL）according to whether it is combined with other systemic diseases,the former accounting for about 70%,the latter accounting for 30%.There are four main types of inheritance:autosomal dominant（15-20%）,autosomal recessive（80%）,sexual linkage（1%）,and mitochondrial inheritance（1%）.Due to the high heterogeneity of deafness genetics,there are still a large number of patients with deafness whose molecular etiology is unknown.In recent years,the development of second-generation sequencing technology has further broaden the deafness gene mutation spectrum.In the prevention and control of deafness,it is clear that the molecular etiology is a prerequisite,and prenatal diagnosis is an important means.This study identified the molecular etiology of three dominant hereditary hearing loss and explored non-invasive prenatal testing of deafness.PartⅠ Identification of pathogenic mutation and phenotypic differences in a X-linked dominant non-syndrome deafness familyWe have collected a family with X-linked dominant non-syndromic hearing loss（M60）,some family members showed progressive acquired sensorineural hearing loss.A novel mutation of the SMPX c.29insA（p N10fs）was identified in the pedigree using targeted gene capture of 139 known deafness genes and the second-generation sequencing.Both IV3 and IV4 carries SMPX c.29insA,but IV3 showed hearing loss and IV4 had normal hearing.In the X-linked dominant inherited deafness,the phenotypic differences in female carriers have not been reported in detail.Our research has indicated that X-chromosome inactivation is related to phenotypic differences in female carriers.This result can provide genetic counseling and guidance for family members,and provide a reference for the analysis of X-linked dominant deafness.PartⅡ Identification of pathogenic mutations of the two common syndromic hearing lossThe early diagnosis is particularly important to syndromic hearing loss because it is accompanied by abnormalities of other organs,and the treatment is more difficult.We have identified the mutations in the more common Van der Hoeve syndrome and Branchio-oto syndrome through the the targeted gene capture and Sanger sequencing.The mutations in two families of VH syndrome are c.1342A>T（p.K448X）and c.124C>T（p.Q42X）of COL1A1 gene,and both were novel mutations.The mutations in two families of BO syndrome are c.GTCC1075₁078GAT（p.G359fs M365X）and c.1319G>A（p.R440Q）in EYA1 gene,and the former has not been reported.The above two syndromic hearing loss are autosomal dominant inheritance,and prenatal diagnosis is recommended because the probability of genetic mutations in the offspring of the patient is 50%.Our study has cleared the molecular causes of hearing loss in the families and further confirmed the diagnosis of the syndrome.This played a monitoring and early warning role to the patients’ other system diseases,and it could better provide genetic counseling for family members.Part III Study of noninvasive prenatal testing of hearing lossThe maturity and improvement of the molecular diagnosis of hereditary deafness is the basis for the prevention of deafness,and prenatal diagnosis is an important means of preventing genetic deafness.The traditional prenatal diagnosis is to obtain fetal tissues for genetic analysis by invasive methods,which has a certain risk to the fetus and pregnant women.Fetal genetic material required for noninvasive prenatal testing could be obtained from cell-free fetal DNA（cffDNA）and fetal cells from the peripheral blood of pregnant women.This study explored noninvasive testing of common deafness genes（GJB2 and SLC26A4）in both cffDNA and fetal cells.We applied the capture kit to capture and enrich the cffDNA and perform the second generation sequencing.According to the difference in mutation frequencies of the parental deafness gene mutations in plasma and leukocytes,combined with the fetal free DNA concentration in the plasma to comprehensively determine the fetal genotype.Compared with invasive prenatal diagnosis,25 of 30 fetuses（83.3%）were correctly genotyped by our method.Three cases of heterozygous mutation were misdiagnosed as normal,one case of compound heterozygous mutation was misdiagnosed as a single heterozygous mutation,and one case of heterozygous mutation was misdiagnosed as homozygous mutation.The sensitivity and specificity of the method for correctly diagnosing hearing loss of GJB2 and SLC26A4 were 75%and 96.2%,respectively.In this study we have also attempted to use fetal nucleated red blood cells（FNRBCs）for noninvasive prenatal testing.The target cells isolated and enriched by density gradient centrifugation,antibody sorting and single-cell micromanipulation.Amplification by multiple annealing and looping-based amplification cycles（MALBAC）and Sanger sequencing to find the deafness gene mutations,but the stability and accuracy of the results need to be improved.The capture strategy of FNRBCs and optimization of single cell whole genome amplification method will directly affect the purity and yields of the fetal cell,and the accuracy of the gene diagnosis results.It is necessary to achieve technological breakthrough in follow-up research.