Study On Molecular Epidemiology And Pathogenesis Of Severe To Profound Hearing Loss In China

Deafness is an etiologically heterogeneous disorder with many known or unknown genetic, environmental causes or a combination thereof. The identification of 130 loci for deafness has provided new insights into the pathophysiology process of hearing. However, recent findings indicate that a large proportion of both syndromic and nonsyndromic forms of deafness are caused by a small number of genes. This study focused on analyzing GJB2, GJB3, GJB6, SLC26A4, mtDNA12SrRNA and mtDNAtRNASer^(UCN) mutations in Chinese deaf population to investigate the molecular pathogenesis and molecular epidemiology.Part IStudy on Molecular Epidemiology and Pathogenesis of Severe to Profound Hearing Loss in ChinaChapter I : Etiology Analysis of Deaf Students in SpecialEducational School of Chifeng Area, Inner MongoliaIn this chapter, we performed sequence analysis of GJB2, GJB3, GJB6, SLC26A4, mtDNA12SrRNA and mtDNAtRNASer^(UCN) in 134 subjects with hearing loss from ChiFeng Educational School. Individuals carrying SLC26A4 mutation were given further temporal bone CT scan. We investigated the etiology of deafness by analyzing the genes testing results and questionaires. GJB2 mutations were responsible for approximately 17.16% of the cases in ChiFeng area. GJB2 was the most common cause of deafness in ChiFeng area. By screening SLC26A4 followed by temporal bone CT scan, we diagnosed 20 cases of EVA and/or other inner ear malformation. SLC26A4 mutations wereresponsible for 14.93% of the cases and it was the second common gene causing deafness in this area. The aminoglycoside-related mtDNA 1555A>G mutation accounted for 0.76% of the cases in ChiFeng area. We provided genetic diagnosis for 33.58% (45 cases). We found that the ratio of hearing loss related to genetic factors in this population was 60.44% (81 cases). Our data suggested that gene testing had important meaning in clarifying etiology for hearing loss population. We established a new strategy that detects SLC26A4 mutations prior to the temporal bone CT scan to find EVA patients. This model had unique advantage in epidemiologic study in large-scale deaf population.Chapter II: Mutation Analysis of SLC26A4 in Chinese Deaf PopulationRecessive mutations of SLC26A4 can cause DFNB4 characterized by EVA and Pendred syndrome characterized by EVA, congenital severe to profound sensorineural hearing loss and euthyroid goiter.To investigate the molecular epidemiology of SLC26A4 mutations in Chinese deaf population, we screened the SLC26A4 hotspot mutation areas of exon 7+8, exon 19 and exon 10 in 2352 cases with severe to profound sensorineural hearing loss from 27 different areas in China. We finished the whole gene screening in individuals carrying a single heterozgyous mutation in exon 7+8, exon 19 and exon 10. Eighty-five SLC26A4 mutations were found, and 58 of them were novel. Hotspot mutations were identified, IVS7-2A>G and 2168A>G. The total detecting ratio of SLC26A4 mutations in Chinese hearing loss population was 14.54%. The detecting ratio of homozygous mutations was 4.93%, compound heterozygous mutations 5.61% and single heterozygous mutations 4.00%. As for the Han nationality, the major nationality in China, the total mutation detecting ratio of SLC26A4 was 16.55%, the detecting ratio of homozygous mutations was 5.68%, compound heterozgyous mutations 6.46% and single heterozygous mutations 4.41%. The mutation spectrum of SLC26A4 were distinct in various nationalities of China. And the mutation detecting ratio of SLC26A4 was different in various nationalities and in different areas. By screening exon 7+8, exon 19 and exon 10 of SLC26A4, we estimated that in the Han nationality, the initial ratio of EVA in the deaf population was at least 11.54%.In this chapter, we established a preliminary strategy for screening SLC26A4 in deaf population. We made a map of the SLC26A4 mutation and SNP spectrum with nationality and geographical information. This work provided important evidence for genotype-phenotype correlation and developing SLC26A4 chips.Chapter III: GJB6 Mutation Analysis in Chinese Deaf PopulationGJB6 encodes Connexin30, a gap junction protein expressing in the cochlea and is thought to be important for recycling potassium ions that flow into sensory hair cells as part of the transduction current. The most common mutation in GJB6 is a 342kb deletion, which causes NSHL when homozygous, or when present on the opposite allele of a GJB2 mutation.In this chapter, we investigated GJB6 mutations in Chinese deaf population. Three hundred and seventy-two patients with severe to profound hearing loss (of whom 295 cases had no definite etiology diagnosis and 77 cases carried one heterozygous GJB2 pathogenic mutation respectively) and 182 normal controls were first tested for GJB6 del(GJB6-D13S1830) using specific PCR primers. Then sequencing for GJB6 coding region was applied in all patients and normal controls. We found none of the patients and normal controls carried GJB6 del(GJB6-D13S1830). Two single base pair changes were detected. One was T135K detected in the patient group and the other was A149V detected in the control group. Our preliminary data suggested that mutation of GJB6 is not frequent in Chinese deaf population. Screening for GJB6 can be lined as unconventional in China for the time being.Chapter IV : Sequence Analysis of GJB3 in Chinese Deaf Population Carrying One Heterozygous GJB2 Pathogenic MutationGJB3 was first identified by a Chinese scientist, XiaJiahui, in 1998. GJB3 encodes Connexin31 and it has been reported to be responsible for both DFNA and DFNB.It has been suggested that GJB2 and GJB6 interact to produce the diseasephenotype, which is a digenic mode of inheritance. In this chapter, we investigated whether GJB3 and GJB2 interact to produce the deafness phenotype in a digenic mode of inheritance. One hundred and eight patients with severe to profound hearing loss carrying one heterozygous GJB2 pathogenic mutation and 100 normal controls were sequenced for GJB3 coding region. Three GJB3 missense variants, V84I, A194T and N166S, were detected in five patients. We considered V84I and A194T as polymorphisms in Chinese population. N166S probably was pathogenic since it was not detected in the controls. The patient carrying N166S mutation in one allele carried GJB2 235delC mutation in the other allele. We thought GJB3 and GJB2 might interact to produce deafness in a digenic mode of inheritance, but this point of view needs verification and the mechanism needs further studying.Part IIEstablishment of Real-Time Taqman Probe Technique Systems Detecting the MtDNA 1555A>G and 1494OT MutationMitochondrial DNA mutations have been found to be associated with sensorineural hearing loss. In particular, the mtDNA 1555A>G mutation has been shown to be a hotspot for aminoglycoside induced hearing loss and nonsyndromic hearing loss in different population from all over the world. The mtDNA 1494OT is a currently found mutation associated with aminoglycoside induced hearing loss.This part focused on the establishment of Real-time Taqman probe technique systems to detect the mtDNA 1555A>G and 1494OT mutation respectivly in deaf population. We successfully established two technique systems detecting mtDNA 1555A>G and 1494OT mutation using Real-time Taqman probes. The technique posseses the merits of accuracy, conveniency, good sensitivity, good specificity, intuitionistic results, etc. Importantly, the Real-time Taqman probe technique only needs 1.5 hours to detect the 1555A>G mutation and saves 4.5 hours for one reaction compared with the Hae III restriction enzyme method widely used at present. For detecting the 1494OT mutation, the Real-time Taqman probe technique needs 1.5 hours and saves 10.5 hours for one reaction compared withthe sequencing method popularly used nowadays. The technique systems of detecting mtDNA 1555A>G and 1494C>T mutation are reliable. They are suitable for large-scale detecting and preventive diagnosis of mtDNA 1555A>G and 1494C>T mutation.