Molecular Genetic Analysis Of Waardenburg Syndrome And Non-syndromic Hearing Loss

With the development of molecular biology and genomics,it's a research hotspot to explore the causes of hereditary deafness at the genetic level.Since the majority of Chinese hereditary deaf people had been confirmed to be caused by GJB2,SLC26A4 and mtDNA gene mutations,genetic testing of these three genes can not only make clear about cause of deafness but also provide theoretical evidences for genetic counseling.Furthermore,it makes prenatal diagnosis possible for deaf families.Now,many provinces has investigated the molecular epidemiology of the common deafness genes including GJB2,SLC26A4 and mtDNA A1555G mutations.But no similar research has been carried out in Hunan province.Meanwhile,there has been remarkable progress being made on syndromic hearing loss.Waardenburg syndrome is one of the most common dieases of SHL.So far,more than 100 gene mutations have been reported in Western WS patients,which disclosed its possible molecular mechanisms.But few WS patients had been reported in China,and very few reports had been observed on the molecular genetic background of WS in Chinese population.The purpose of the present study,through systematic study on clinical features and molecular genetics of patients with SHU,NSHL or maternally inherited nonsyndromic deafness,is to explore the molecular genetic background and relationship between genotype and phenotype of WS in Chinese population;To investigate the prevalence and hot spots of GJB2 and PDS genes and to estimate the incidence of the mitochondrial 12S ribosomal RNA A1555G mutation in subjects with NSHL in Han people of Hunan province,and try to disclose the mechanism of deafness associated with these genes and provide some theories or suggestions for preventing and curing this kind of hearing loss;Also to study the molecular etiology of a large family with maternally inherited nonsyndromic deafness and then to explore the relationship between mtDNA gene mutations and NSHL.We carried out our research as the following three parts:Part one:The clinical features and gene mutations in patients with Waardenburg syndrome.We analyzed the clinical features of 19 WS patients came from central south in China,in which 5 were diagonosed as WS type 1,while other 14 were WS type 2.All the WS1 patients were screened mutations for PAX3 gene and WS2 for MITF,SNAI2 and SOX10 gene mutations.We observed obvious phenotypic variability in Chinese WS patients.All the patients showed heterochromia iridis,and 89.5%patients displayed bilateral, congenital sensorineural hearing loss which was more common in WS2. We also observed 4 WS2 patients(28.6%) manifested much brown freckles on the skin,while other symptoms such as white forelock and premature greying could only be seen on a few patients.At molecular level,totally 10 mutations(3 mutations in PAX3 and MITF genes respectively,and 4 in SOX10) were identified and 8(H80D and H186fs in PAX3,R217I and T192fs in MITF,G38fs,R43X,E248fs and G37fs in SOX10) of them were novel.All the WS1 patients carried PAX3 gene mutations,while 35.7% and 42.9%of WS2 cases were detected MITF and SOX 10 gene mutations respectively.These results have provided important data to depicted the mutational spectrum in Chinese WS and the starting point to discover the underlying molecular mechanism of the disease.Part two:Mutation analysis of common deafness genes in non syndromic hearing loss patients.We carried out mutation screening of GJB2,PDS genes and mtDNA A1555G for 139 patients with NSHL in Han people of Hunan province by direct seqence,DHPLC and PCR-RFLP respectively.The study showed 43.9%patients carried at least one of the common genes mutations.The mutation detection rate of GJB2,PDS genes and mtDNA A1555G were 23%,18.7%and 3.6%respectively.Totatlly 6 GJB2 and 13 PDS gene mutations were identified,among them 1 GJB2(F115C) and 3 PDS(S8X, A227P,Cys565fs) gene mutations were novel.89.1%of GJB2 gene mutations was 235delC and 46.5%of PDS was IVS7-2A＞G,which implied 235delC and IVS7 -2A＞G were the most common mutations in the two genes.We made clear about the molecular etiology of 35.3%patients through this study,which supplied important data for further step to genetic counseling,gene diagnosis and prenatal diagnosis and also supplied instructions for clinical medication.Part three:Clinical characterization and molecular genetic study of a large family with maternally inherited non-syndromic deafness.We evaluated the clinical features of a large family with maternally inherited nonsyndromic deafness,and carried sequence analysis of the mitochondrial genome from the proband.The results showed 21 of 36 maternal members(58.3%) exhibited symmetric bilateral sensorineural hearing loss especially in high frequency,with a wide range of severity and ages at onset.Deafness of some maternal members was closely related to aminoglycoside antibiotic.Totally 23 variations were found by molecular analysis in the proband.18 maternal relatives harbored the homoplasmic mutation A1555G in thel2SrRNA gene,also a novel mutation T1541C.5 spouses and paternal relatives had nomal hearing and were negative of A1555G and T1541C mutations.Conclusions:(1) There are obvious variations in clinical features of WS,WS2 is more common than WS1;Heterochromia iridis and sensorineural hearing loss are the most common symptoms,while brown freckles may be a special type of pigmentary disturbance of the skin.Genetically,PAX3 mutations are mainly associated with WS1.The mutation detection rate of MITF and SOX10 genes in Chinese WS2 patients are higher than in European population,and SOX10 gene mutations are more common than MITF.Totally 10 different gene mutations were identified and 8 of them are novel,which implies the unique mutational spectrum in Chinese WS. No mutations were found in 3 WS2 patients,which means some other unknown genes related to WS2.(2) There are about 43.9%patients with NSHL carried the common deafness gene mutations,which indicates high incidence of hereditary deafness in Han people of Hunan province.GJB2 gene mutations are the most common cause of NSHL,then PDS gene mutations.235delC, IVS7-2A＞G and A1555G are the hot spots in GJB2,PDS and mtDNA genes mutations respectively,which account for 71.2%of all three gene mutations.We can use the hot spot mutations to develop fast screening for the common deafness genes.The etiology of 35.3%patients in this study have been determined.1 GJB2 and 3 PDS novel gene mutations multiplied Chinese mutation map.(3) The chacterization of maternally inherited nonsyndromic deafness is mainly symmetric bilateral sensorineural hearing loss especially in high frequency,with a wide range of severity and ages at onset.Mitochondrial mutation A1555G is mainly responsible for the disorder in this family. Environmental determinants and nuclear background may play a role in the clinical expression of deafness,T1541C is a novel mutation in Chinese population,and it may influence the penetrance of deafness due to A1555G mutation.