The Establishment For Detecting GJB2 And MtDNA A1555G And The Relation Between Genotype And Phenotype

PARTⅠ:To Establish Noninvasive Detecting System for GJB2 and mtDNA A1555GChapterⅠ:Feasibility Study of Deafness Genes Analysis Using Genomic DNA Obtained from Buccal Mucosa CellObjective:To study the feasibility of deafness genes analysis using genomic DNA obtained from buccal mucosa cells, which is noninvasive and rapid.Methods:98 children with hearing impairment were enrolled in this study. Their genomic DNAs were extracted from buccal mucosa cells which is sampled non-invasively and blood cells invasively. The GJB2 and mtDNA A1555G were detected, the consistency of result from different DNA was compared by Kappa test.Results:The one-time success rate of extracting genomic DNAs from buccal mucosa cells which is noninvasive and blood cells which is invasive were 98.98% and 100%,the DNA extraction rate were 2.16±0.44μg and 21.47±4.59μg(from 500μl blood),the OD rate were 1.82±0.13 and 1.79±0.11.Buccal mucosa cells placed for 24 hours and 1 week, the DNA extraction rate from them had obvious differences (t=2.52,p=0.01), while blood cells had no.Blood cells placed for 1 week and 1 month ,the DNA extraction rate from them had obvious differences(t=2.87, p=0.01);The result of GJB2 and mt DNA A1555G test indicates that they were consistent,27.56%(27/98) and 4.08%(4/98)were diagnosed carrying mutations of GJB2 and mt DNA A1555G.Conclusions:The genomic DNAs from buccal mucosa cells could be used in the detected of GJB2 and mtDNA A1555G, which is reliable, noninvasive and convenient. the new method could be applied to the large-scale detecting of GJB2 and mtDNA A1555G for low-age objects,especially for newborns.ChapterⅡ:To establish a two-step approach using high resolution melt and DNA sequencing to detect GJB2 mutationsObjective:To establish a two-step approach using high resolution melt and DNA sequencing to detect GJB2 mutations. The technique possesses the merits of low-cost, convenience,accuracy.Methods:The two-step approach using High Resolution Melt and DNA sequencing to detect GJB2 mutations were established.The reliability of the technique was tested in 121 patients and 72 children with normal hearing, who were randomly selected from the ENT in Children's Hospital of Fudan University. Following a double-blind numbered, those subjects were detected by two-step approach and Direct Sequencing at the same time.Finally the results by the above two ways were compared by Kappar test.Results:The results of two-step approach revealed:24 and 58 cases in deafness group and normal controls were wild type,97 and 14 doubtful positive sample were detected by DNA sequencing. In group of deafness 46.28%(56/121)double-stranded mutation, 37.19%(45/121)heterozygous mutation and 22.31%(27/121)wild genotypes were identified.In normal controls 9.72%(7/72)heterozygous mutation,90.28%(65/72) wild genotypes and zero double-stranded mutation were identified;The results of Direct Sequencing revealed result coincided with the results from sequencing by kappa test; The cost of HRM increased no more than 0.5RMB by PCR, but DNA sequencing 20-25RMB, time consuming were 15mins and about 10 hours respectively.The two-step approach could detect 19.83%(24/121)in deafness group and 80.56%(58/72)in normal control without DNA sequencing.Conclusions:The two-step technique system using High Resolution Melt and DNA sequencing possesses the merits of low-cost,convenience,accuracy.It is suitable for large-scale detecting and preventive diagnosis of GJB2 mutations in deafness.ChapterⅢ:To Establish a Quantitative Ligase Chain Reaction Technique System to Detect mtDNA A1555GObjective:To establish a quantitative ligase chain reaction (Q-LCR) technique system to detect mtDNA A1555Gin Chinese deafness. The technique possesses the merits of low-cost,convenience,accuracy.Methods:Primers and probes for mtDNA A1555G were designed and synthesized. The technique system for this mutation was established, Then the reliability of the technique was tested in 121 patients and 30 children with normal hearing, who were randomly selected from the ENT in Children's Hospital of Fudan University. Following a double-blind numbered, those subjects were detected by Q-LCR and restriction enzyme digestion at the same time.Finally the results by the above two ways were compared by Kappa test.Results:The results revealed 5 cases carrying mutations in 121 deafness children,and no cases carrying heterozygous mutation in 30 normal controls.these findings coincided with the results from sequencing by kappa test.Both the false positive rate and the false negative rate were zero.Conclusions:The Q-LDR technique system established possesses the merits of low-cost,convenience,accuracy.It is suitable for large-scale detecting and preventive diagnosis of mutations in deafness.PARTⅡ:Molecular Epidemiological Study on GJB2 and mtDNA A1555G in Congenital DeafnessObjective:To investigate the differences in GJB2 gene mutation frequency and hot spots of mutations between the children of congenital hearing loss and the children in Neonatal Intensive Care Unit.Methods:183 patients with hearing loss,0-1 years old, were enrolled from Fudan university children hospital ENT, and 484 children from NICU, all of them were born in shanghai or surrounding area. Their genomic DNAs were extracted from buccal mucosa cells, GJB2 were detected by Two-step approach of HRM and DNA sequencing, and mtDNA A1555G were detected by Q-LCR. According the gene test result to investigate gene mutation frequency and hot spots of mutationsResults:In deafness groups:148 cases with mutations of GJB2 gene was found in 183 cases 32.18%,12 different patterns of mutation were found including of 7 pathogenic mutation(235delC,299-300delAT,571T>C, 605ins46,223C>T,224G>A,232G>A),2 polymorphisms(608T>C,79G>A) and 3 contentious mutations(79G>A+341A>G,109G>A,509A>G),58(31.69%)cases carry homozygous or double-heterozygous pathogenic mutations(including 3 contentious mutations),74(40.44%)carry heterozygous mutations,51(27.87%)carry wild types or polymorphisms;In NICU controls:207 cases with mutations of GJB2 gene was found in 484cases 42.77%,4 different patterns of mutation were found including of 1 pathogenic mutation(235delC),1 polymorphisms(79G>A) and 2 contentious mutations (79G>A+341A>G,109G>A),5(1.03%)cases carry 79G>A+341A>G homozygous,169(63.43%)carry heterozygous including 2 contentious mutations),310(64.05%)carry wild types or polymorphisms;5 cases in deafness groups carry mtDNA Al 555G,and no case in NICU controls.Conclusions:The results revealed 235delC,299-300delAT,109G>A,79G>A+ 341A>G were hot spots of mutations in shanghai and surrounding area,we supported 109G>A,79G>A+341A>G were pathogenic mutation; 509A>G with 235delC and 223C>T with 79G>A were new gene types found first in this study; 224G>A and 232G>A,which had been considered dominant inheritance, were first found in recessive inheritance families; The mutation rate of mtDNA A1555G was 2.73%,lower than previous dates. This mutation type may not be the major factor of congenital hearing loss.PARTⅢ:Clinical Audiologic Features of Deafness Carrying GJB2 and mtDNAA1555GObjective:To investigate the clinical audiology Character of deafness genes caused by GJB2 or mtDNA A1555G mutations, providing the theoretical basis for the clinical diagnosis,prevention and intervention.Methods:183 children with hearing impairment aged 0-4 years, whose age of onset less than 1 year old were enrolled, and 30 cases were enrolled as controls.To collect the information of all cases, including of the disease history data, audiology data and the data of GJB2 or mtDNAA1555G.To analysis the audiology character of the four groups, grouped by GJB2, mtDNA A1555G,non-GJB2/mtDNA A1555G and controls.Results:The ages of onset in GJB2 group, mtDNA A1555G group and non-GJB2/mtDNA A1555G group were 1.03±2.32 months,2.54±9.32 months,2.43±4.32 months separately, the GJB2 groups is younger with the statistical difference(t=2.43,p<0.01);The normal rate of first newborn hearing screening for each groups were 6.03%,10.00%,12.08%,and the normal rate of second newborn hearing screening were 4.13%,10.00%,3.75%;19.83%(23/116) ears in GJB2 group were less than 71 dBnHL,10.00%(1/10) ears in mtDNA A1555G group, and 30.84%(74/240) in non-GJB2/mtDNA A1555G group.Ⅰ,Ⅲ,Ⅴwaves could be identified in 51 ears in GJB2 group at 109.6dBnHL acoustic stimulation, and latency ofⅠwaves andⅤwaves were delayed before 6 months, latency ofⅠ,Ⅲ,Ⅴand each wave duration were all delayed between 6 and 12 months, The degree of hearing loss in this group showed a progressive decrease. In every groups the extraction ratio of ASSR in 250Hz,500Hz,1000Hz,2000Hz,4000Hz were more than the Vwaves in ABR separately, The extraction ratio of GJB2 group in low frequency (250Hz,500Hz) were less than other groups, which showed the injury of hearing involved in every frequency.Conclusions:The results had important significance in the inventation of deafness at early stage:GJB2 mutation could cause earlier ages of onset. The degrees of hearing loss in this group were mainly severe and extremely severe,which had a progressive decrease.The injury of hearing involved in every frequency. We also found the hearing loss for some mtDNA A1555G carriers appeared in pregnancy, and the pregnancy should be given more attations.