The Study On The Molecular Pathogenic Mechanism Of DDOD Syndrome And Molecular Etiology Analysis Of Nonsyndromic Hearing Impairment In Southwest Of China

Part Study on the pathogenic mechanism of DDODsyndromeBackgroundDDOD syndrome is a rare autosomal dominant genetic disease,which seriously affects the quality of patients’ life.ATP6V1B2is the main cause ofDDOD syndrome, while its pathogenic mechanism is not clear. In order to clarifythe molecular pathogenesis, we study on a DDOD syndrome family from Shanxiprovince. MethodsBy Sanger sequencing, we confirmed the pathogenic de novomutation in ATP6V1B2in the family. Through the relative quantitative PCR,research the change of transcription of ATP6V1B2. ResultsThe proband carryingATP6V1B2’ new nonsense mutation c.1516C>T, her parents and brother didn’tcarry the mutant. Relative quantitative PCR revealed there was not obviouschange between the proband and her parents’transcription level, and comparedwith younger brother, the charge is obvious. ConclusionATP6V1B2is the maincause of DDOD syndrome. And the gene mutation didn’t change thetranscriptional level. Partll Study on the deafness mechanism of ATP6V1B2BackgroundATP6V1B2encodedbyATP6V1B2is a subunit of ATPase, widelyexpressing in human organs.It has been confirmed that the ATP6V1B2mutation isthe major cause of DDOD syndrome and its pathogenesis is not clear. In order toclarify the role of ATP6V1B2in the auditory system,we studied on mouse cochleaof different developmental stages at mRNA and protein level. MethodsKunmingmice at different developmental stages （E17, E19, P1, P7, P14, P21, P30） wereused. We removed the cochlea and made frozen sections. The expressionofAtp6v1b2during the development of mouse inner ear was observed byimmunohistochemistry and immunofluorescence. In situ hybridization was usedto study the transcription level of Atp6v1b2during the development of mouseinner ear. The wild type and mutant ATP6V1B2expression vector wereconstructed and transfected into HEK293cells, to identify the ATPase’shydrolyzing activity and transferring function.ResultsATP6V1B2markedlyexpressed in the inner hair cells, outer hair cells and spiral ganglion neurons ofKunming mouse cochlear. ATP6V1B2expressed in the same position duringmouse inner ear development. With the proportion of mutant plasmid transfectionincreasing, ATPase hydrolysis activity of cells transfected was significantlydecreased and pH value in lysosome increased. ConclusionATP6V1B2mainlyexpressed in the inner and outer hair cells and spiral ganglion neuronsin mousecochlea.And the expression site was stable during the developmental processes inthe cochlea of mouse, indicating that ATP6V1B2has important function in theauditory system. The mutant plasmid can decrease the ATPase hydrolysis activityand ATPase proton pump transport activity. It is confirmed that the ATP6V1B2c.1516C>T mutation can affect the hydrolysis activity of V-ATPase, then the H+ion transport function,resulting in the decrease of acidity in lysosome. Part lll Molecular etiology analysis of nonsyndromichearing impairment in southwest of ChinaBackgroundEach year in China,30,000babies are born with congenital hearingimpairment. However, the molecular etiology of hearing impairment in theYunnan Province population where more than52minorities live has not beenthoroughly investigated. To provide appropriate genetic testing and counseling tothese families, we investigated the molecular etiology of nonsyndromic deafnessin this population.MethodsUnrelated students with hearing loss （n=235） whoattended Kunming Huaxia secondary specialized school in Yunnan enrolled in thisstudy. Three prominent deafness-related genes, GJB2, SLC26A4and mtDNA12SrRNA, were analyzed. High-resolution temporal bone computed tomography （CT）scan examinations were performed in100cases, including16cases withSLC26A4gene variants, and37minorities and47Han cases without anySLC26A4gene mutation. ResultsThe GJB2mutation was detected in16.67%（7/42） of minority patients and17.62%（34/193） of Chinese Han patients （P>0.05）.235delC was the hotspot mutation in nonsyndromic hearing loss （NSHL）patients, whereas35delG was not found. The431₄50del19mutation wasdetected for the first time in Han NSHL patients, which resulted in a prematurestop codon and changed the protein. The SLC26A4mutation was found in9.52%（4/42） of minority patients and9.84%（19/193） of Han Chinese patients （P>0.05）.The frequencies of mtDNA12S rRNA mutation in minority and Han Chinesepatients were11.90%（5/42） and7.77%（15/193; P>0.05）, respectively. Sixteen（16/23,69.57%） patients with SLC26A4mutations received temporal bone CTscan, and14patients were diagnosed with enlarged vestibular aqueducts （EVAs）;the other2patients had normal inner ear development. The ratio of EVA in theminorities was14.63%（6/41）. ConclusionsIn this study, a total of35.74%deaf patients showed evidence of genetic involvement, based on either geneticscreening or family history;17.45%,9.79%and8.51%of the patients weredetermined to have inherited hearing impairment caused by GJB2,SLC26A4andmtDNA1555A>G mutations. There was no significant difference in deafnessassociated gene mutational spectrum and frequency between the Yunnan minorityand Han patients.