The Mutations Of Heredity Deafness Gene GJB2, PDS And Mitochondrial DNA 1555A＞G And The Development Of A New Technique Detecting The Mutations

Part One The detection of GJB2, PDS and Mitochondrial DNA 1555A＞GChapter One The detection of GJB2 mutationsObjective: To explore the frequency, hot spot and correlation of phenotype with genotypeof the GJB2 mutation with PCR and sequencing. To accumulate the experience and samplesfor the new technique which is for the detection of gene mutation.Methods: The clinical materials and DNA samples were collected from the 50 patientsdiagnosed of congenital pre-lingual deafness. The results of pure tone audiometry andauditory brainstem response were recorded to evaluate the phenotype. GJB2 gene wasdetected with PCR and sequencing. 50 DNA samples form the people with normal hearingwere collected as the control group. Analyze the difference of the genotypic frequencieswith patients group and control group, x~2 test was uses as the statistics method.Results: 18 samples have mutations and 12 samples have polymorphisms among 50patients. 235delC is the commonest mutation, which account for 63.3% in totalmutations. The genotypic frequency is 24% of 235delC in patients group and 1% incontrol group. P＜0.05. Significant difference was found in the two groups. 75% patientswere profound hearing loss in 12 patients with 235delC mutation.Conclusions: Different types of GJB2 mutations were found in different races. 235delC isthe commonest mutation in Chinese people. Profound hearing loss is the commonestphenotype in patients with 235delC.Chapter Two The detection of PDS mutationsObjective: To explore the frequency, hot spot and correlation of phenotype with genotypeof the PDS mutation with DHPLC. To accumulate the experience and samples for the newtechnique which is for the detection of gene mutation.Methods: The clinical materials and DNA samples were collected from the 42 patientsdiagnosed of enlarged vestibular aqueduct (EVA). The results of pure tone audiometry andauditory brainstem response were recorded to evaluate the phenotype. PDS gene wasdetected with DHPLC and sequencing. Analyze the difference of phenotype between thegroups of diverse genotype, x~2 test was uses as the statistics method.Results: 37 samples have mutations among 42 patients. H723R is the commonest mutation,which account for 24.2% in total mutations. Two novel mutations were found at the splice site of No. 7 and No. 13 exon. The hearing loss of the patients with splicesite and frameshift mutation is severer than that with missense mutation,significant difference was found. (P＜0.05).Conclusions: splice site and frameshift mutation predict severer hearing lossthan missense mutation.Chapter Three The detection of mtDNA1555A＞GObjective: To detect the mtDNA1555A＞G in families and sporadic patients.Methods: 3 maternal transmitted pedigree and 12 sporadic patients were collected. Detectthe mtDNA1555A＞G with allele-specific PCR (ASPCR).Results: 15 samples were found positive mutation in family2 and family3. None ofmutation was detected in familyl. The genotypic frequency is 53.6%. 1 sample was foundpositive mutation in 12 sporadic patients. The genotypic frequency is 8.3%. The penetranceis 100% in family2 and 42.8% in family3. The results of ASPCR are as good as that ofsequencing after the experimental conditions being improved.Conclusions: The mtDNA1555A＞G is a vital factor of the aminoglycoside induced hearing loss withmaternal transmitted pedigree.Part Two A new technique: Multiple probes and ligase dependent assayObjective: To establish a new mutation detecting technique depending multiple probes andligase.Methods: Establish single and multiple probes system. 50 samples were detected by thenew technique and sequencing. Evaluate the sensitivity and specificity of the new technique.Experimenting with diverse amount of DNA, ration of probes/primers, hybridize time toexplore the most optimal condition.Results: The multiple probes system was established, which has the ability to detect 3genes and 7 mutations. The sensitivity is 75% and the specificity is 100%. The optimalexperimental condition including: DNA 200ng, hybridize time no less than 12 hour, theration of probe/primer more than 1:500.Conclusions: The multiple probes and ligase dependent new technique could be wellapplied for mutation detecting of deafness genes.