Application Of Targeted Genes Capture And High-throughput Sequencing On Hereditary Hearing Loss

Hearing loss is a common disabled disorder resulting in verbal communication disability, the cause of which has not yet been fully elucidated. Hereditary hearing impairment is a common type of hearing loss. Since the first non-syndromic hearing loss gene POU3F4 was cloned,85 non-syndromic deafness genes and more than 60 syndromic deafness genes have been identified (http://hereditaryhearingloss.org/). However, current clinical applications focus on the detection of common deafness genes, with the diagnosis rate of about 30%, leaving more genetic deafness needs further study.In recent years, with the rapid development of genomic detection technology, next-generation sequencing technology are widely used. Its increasing depth, accuracy provides an unprecedented opportunity for identifying hereditary hearing loss genes. Exome sequencing has a significant advantage of high throughput, high speed, low price, benefiting especially for the analysis of single gene disorders such as hereditary hearing loss, providing efficient tool for cloning deafness genes. This study was based on the rich genetic deafness resources of National Gene Bank deafness sub-library, combining abundant resources and high-throughput technology. The thesis is divided into two main parts to present the results of this study.Part 1:The Efficiency of Targeted Genes Capture and Next generation sequencing (NGS) on Genetic Diagnosis of Hereditary Hearing Impairment1. The efficiency verification of Targeted genes capture and NGS by samples with known gene mutationsObjective To establish the diagnostic platform of hereditary hearing impairment by combining targeted sequence capture with high-throughput sequencing technology, and verify the efficiency of this method, laying the methodological foundation of further analysis to cover a variety of genetic hereditary hearing impairment samples to determine the proportion of genetic causes of hearing loss preliminary, as well as to analyze the incidence of each gene in various hearing loss population. Methods In this study, mutation screening of 307 nuclear genes and mitochondrial genome responsible for human or mouse hearing impairment were performed in the eighty three patients with 39 known mutations to verify the validity of our methods. After extracting the genomic DNA of the eighty three chosen samples and building the DNA library, universal primers for PCR amplification were adopted, then sequencing of the PCR products were carried out on Illumina HiSeq2000. The high-throughput sequencing results were compared with the initial Sanger sequencing by self-controlled and double-blind method. Results Among the 83 samples with known pathogenic mutations, the test results of 81 cases were consistent with sanger sequencing, including 11 mutations of GJB2, two common disease-causing mutations of 12SrRNA,30 mutations of SLC26A4, two mutations of PJVK gene, one mutation of OTOF, and one mutation of POU3F4 respectively, the mutation types of which were missense mutations and indel (insert and deletion) mostly.2 cases of autosomal recessive were detected another mutation, deciphering the genetic etiology of these cases. Conclusion Targeted genes capture and high-throughput sequencing is an effective diagnostic method of hereditary hearing impairment, which can compensate the high-cost, time-consuming insufficient of traditional sequencing. This study laid the basis of clinical application, establishment of molecular epidemiological data, and selection of hotspot mutations, promoting the research progress of genetic diagnosis of hereditary hearing loss.2. The Application of Targeted Genes Capture and NGS on Genetic Hereditary Hearing Impairment without known genes mutationsTwenty five probands, who have been performed common genes screening, were recruited in this study. To further search for the causative genes in these families, we performed targeted region capture and high-throughput sequencing on the probands. The results of five cases were negative, without finding out positive variants. Among the 7 ADNSHL cases with complete clinical information, candidate genes KCNQ4, TJP2, MYH14, MYO7A were identified in five probands, leaving two probands no positive results. The results of thirteen cases were unknown clinical significance, needing further verification. This study identified five candidate pathogenic genes in the 25 families, with the detection rate of 20%; the thirteen cases with unknown clinical significance need further verification in the families members and population controls; the seven cases with negative results will be conducted in-depth phenotype analysis, further selecting among whole exome sequencing, whole genome sequencing or micro array comparative genomic hybridization method to dig disease-causing reasons. Part 2:Identification the disease-causing genes mutations for autosomal dominant hereditary hearing loss families.1. Targeted high-throughput sequencing identifies pathogenic mutations in KCNQ4 in two large Chinese families with autosomal dominant hearing lossIn this study, we identified a novel KCNQ4 mutation in a five generation Chinese family with 84 members and a known KCNQ4 mutation in a six generation Chinese family with 66 members. Patients in the two families showed post-lingual, progressive, symmetric sensorineural hearing loss and affected high frequencies initially, which are consistent with the DFNA2 hearing loss. Mutation screening of 30 non-syndromic autosomal dominant genes were performed in a cohort of 30 probands from Chinese families with ADNSHL by targeted region capture and high-throughput sequencing. In two probands from large Chinese families with ADNSHL we found two candidate variants of KCNQ4 gene. The candidate variants and the co-segregation of the phenotype were verified by polymerase chain reaction (PCR) amplification and Sanger sequencing in all ascertained family members, proving that the variant p.W275R and p.G285S in KCNQ4 at DFNA2 were co-segregated with the phenotype of these two families respectively. Thus, we have identified a novel KCNQ4 mutation p.W275R in exon 5 and a known KCNQ4 mutation p.G285S in exon 6 in two large Chinese families with ADNSHL. This is the first report of KCNQ4 mutation in Chinese mainland population, and we presumed that KCNQ4 is likely to be a common pathogenic gene in Chinese patients with non-syndromic autosomal dominant hearing loss.2. Identification of Two disease-causing genes TJP2 and GJB2 in a Chinese autosomal dominant hearing loss familyIn this part, we identified the causal genes of TJP2 and GJB2 for the hereditary hearing loss family 686 using the strategy of combination of traditional linkage analysis, pheno type-genotype correlation analysis, Sanger sequencing, and targeted high-throughput sequencing. To decipher the genetic code of a Chinese family (Family 686) with ADNSHL, we performed a series of studies using samples obtained from the family members over a period of approximately 10 years. We identified a probably pathogenic missense mutation, c.2081G>A (p.G694E), in TJP2, a gene that plays a crucial role in apoptosis and age-related hearing loss (ARHL). Compound heterozygosity for p.Y136* and p.G45E in the GJB2 gene was identified in another patient who presented with a phenotype different from that of the other affected individuals in the family. In conclusion, we identified the co-occurrence of two genetic causes for ADNSHL in Family 686. The possible disease-causing missense mutation of TJP2 in Family 686 presents an opportunity for further investigation into ARHL. In addition, we summarize the results of the studies that we performed on Family 686, concluding that no single technique is adequate for addressing all the problems associated with studies of the pathogenesis of ARHL, and it is therefore necessary to address these difficulties with an open mind and to use a combination of various methods.