Study On Construction Of Deafness Resource Bank And Strategy Of Genetic Diagnosis For Northwest Minorities Of China

Hearing loss seriously affects the patients’function of audition and speech, which leads to the barriers of social communication. The high morbidity makes it a research hot issue in medical field. Since the first deafness gene was cloned in1995, more than40NSHL related genes have been found and molecular mechanism of deafness has been revealed. China is a big family, which contains56ethnic groups and has rich genetic resources especially from ancient minority ethnic groups. Therefore, it is necessary to carry out the research of deafness genes of ethnic minorities in China. This study aims to collect the deafness resources from ethnic minorities in the northwest China, and build a clinical and genetic resource library to cover major ethnic minorities in the northwest. Based on this, three types of high-risk deafness genes’molecular epidemiology research are carried out to reveal the relations of these genes and deafness,to explore the strategies for developing the deafness gene diagnosis and to find the ways to prevent deafness through human intervention. The main content of the research is comprised of four parts:The first chapter:The construction of deaf resources library from ethnic minorities in northwest China.The genetic resourceof Northwest minority deaf population is extremely scarce, and has a decreasing trend. We first design the standard process of the construction of the resources library. Then we collect deafness sporadic cases and deaf family resources, conduct questionnaire surveys, physical examination, audiological examination and family mapping, sign informed consent form and extract the peripheral venous blood, extract genomic DNA to establish a full set of libraty for digital management. We have collected1673deafness sporadic cases and146hereditary deafness families from ethnic minorities. It has accumulated resources for further genetic studies.We believe that protection of minority ethnic group deafness resources needs to be further strengthened, and a scientific and perfect resource libraty management system needs to be established.The second chapter:Deafness related gene GJB2screening and diagnosis strategy research in the northwest ethnic minorities. GJB2gene is the most common molecular etiology in congenital deafness. GJB2gene codes gap junction protein Cx26, which plays an important role in the inner ear electrolyte exchange and information transmission between cells, and is closely associated with human auditory function.We carry out GJB2gene’s screening study in northwest minority deafness patients to analyze the epidemic condition of the gene, reveal the mutation hotspot of ethnic minorities and explore the genetic diagnosis strategy suitable for the deaf people.In this study, we finished1330ethnic minorities NSHL cases and457Han ethnic NSHL cases for GJB2gene mutation screening. The results showed that11mutation forms were found, including a new mutation:c.275C>G.202mutation or sequence change cases,75cases of homozygous mutations,92cases of heterozygous mutations and35cases of compound heterozygous mutations were found in ethnic minorities.The mutation frequency was15.19%(202/1330) in ethnic minorities and19.04%(87/457) in ethnic Han. It has no significant difference in allele frequency from ethnic minorities and ethnic Han. No differences were found between the ethnic groups except Uygur and Han.Frameshift mutation is the main form of GJB2gene in various ethnic groups. This gene type has determined that the hearing loss is either serious or profound.In ethnic minorities, c.235delC, c.35delG, c.109G>A and c.299-300delAT were the highest allele frequency forms. Different nations have different hotspots: Tibetan was c.235delC and c.109G>A; Dongxiang was c.299-300delAT and c.235delC; Hui ethnic was c.235delC and c.109G>A; Kazak was c.35delG; Uygur was c.235delC and c.35delG; Han ethnic was c.235delC, c.109G>A and c.299-300delAT.The research also showed that mutation frequency of each ethnic group was consistent with its ethnic background, especially in kazak and uighurs which has obvious Caucasus background.In this study, complete GJB2gene mutation spectrums of all ethnic groups were made, which could provide the methodology basis for personalized genetic diagnosis and widespread gene screening in all ethnic groups. Considering the structural characteristics and the dispersion of mutations, it is advisable to choose the diagnosis model of the whole coding sequence screening.The third chapter:SLC26A4geneand mtDNAA1555G mutation screeningof non-syndrome sensorineural deafness in the northwest ethnic minorities.Vestibular aqueduct expandingis the most common inner ear malformation.,SLC26A4gene which associated with vestibular aqueduct expanding is closely associated with deafness. mtDNAA1555G mutation associates with deafness caused by AmAn. The mutation detection is helpful to explore the relationship between drug deafness and environmental factors. In this study, we collected1330patients with NSHL from ethnic minorities in northwest China to carry out SLC26A4gene (8and9exons) and mtDNAA1555G mutation screening through PCR-RFLP or direct sequencing methods.In SLC26A4gene, three mutation forms have been found:919-2A>G, c.2168A>G and2162C>T. Seven genetypes were found in ethnic minorities, including26cases of homozygous,40cases of hybrid and5cases of compound heterozygous; six genetypes were found in ethnic Han, including15cases of homozygous,31cases of hybrid and8cases of compound heterozygous. The detection rates of homozygous and compound heterozygous mutations were2.33%(31/1330) and5.03%(23/457), respectively. The comparison of different ethnics showed that statistically significant differences had been found between ethnic Han and Tibetan, Dongxiang, Uighur. The difference also existed in ethnic Hui and Dongxiang and Uighur. The study also confirmed that919-2A>G allele frequencies had statistically significant differences between ethnic Han and Tibetan, Dongxiang, Uyghur; between Uighur and ethnic Hui, Kazak; and also between ethnic Hui and Dongxiang. In the study of mtDNA, we found that:28cases had mtDNAA1555G mutation in ethnic minorities, mutation frequency was2.11%(28/1330);32cases had mtDNAA1555G mutation in ethnic Han, mutation frequency was7.00%(32/457). Statistical processing showed that there was a significant difference between mutation frequency of ethnic minorities and ethnic Han.In106cases of AAID,9cases had mtDNAA1555G mutation and mutation frequency was8.49%.After statistical analysis, there is a statistically significant difference on mtDNAA1555G mutation frequency between Dongxiang and ethnic Han, and also between ethnic Hui and Uighur.The results showed that all the ethnic minorities have the unique spectrum of SLC26A4gene mutation, and the hotspots are not identical. We need to formulate genetic diagnosis strategy and choose the suitable methods of gene diagnosis and prenatal diagnosis for different ethnics; it is necessary to carry out the mtDNA1555A>G mutation genetic diagnosis in the region deafness people from ethnic minorities, which will help to slow down and reduce the occurrence of AAID.The fourth chapter:Analysis of candidate genes in an Autosomal dominant non-syndrome hearing loss pedigreeThere are many gene positional cloning methods for genetic disease. It is necessary to choose the appropriate research method according to the characteristics of the genetic resources. In this study, the purpose was to explore suitable and effective approach to the study of the hereditary deafness etiology.We carried out systematic research in clinical data and phenotypic characteristics of a hereditary deafness pedigree. Through candidate gene mutation screening, we made clear the pathogenic genes. Firstly, we defined the genetic way as autosomal dominant inheritance through the analysis of the onset of the characteristics of deafness patients. The phenotypic characteristics are summarized as:High frequency hearing loss was the first performance, and then the whole frequency hearing would be affected. Some patients had symptoms of vestibular function obstacle performance. Through the detailed analysis of the phenotype, candidate genes were confined to a handful of genes. And then we chose COCH gene which accord the most with the characteristics of phenotype to screen mutations in all exons. c.485G>A was found in exon8and mutated into pathogenic mutation. The mutation and deafness phenotype was in the line of separation.The study found that:the hearing loss penetrance was100%, which gradually aggravated with age; penetrance of vestibular dysfunction was22.22%, which was not associated with the degree of hearing loss. Judging from characteristics of audiological and vestibular function obstacle features, the family was not conforming to the clinical manifestations of Meniere’s disease. We believe that the hereditary deafness family gene mapping, preferred position cloning and candidate gene cloning methods can locate the position of deafness genes through linkage analysis.Candidate gene cloning methodis economical, fast and convenient. Collecting detailed clinical data and audiology data, analyzing fully of clinical characteristics are indispensable for choosing appropriate research method.