【作者】 杨晓林；

【导师】 许政敏； 陈文霞； 吴冰冰；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 硕士

【摘要】 第一部分：无创性GJB2基因与mtDNA A1555G突变检测体系的建立第—章：口拭子刮取口腔颊粘膜脱落细胞用于聋病基因检测可行性研究目的：探讨口拭子刮取口腔颊粘膜脱落细胞无创、便捷DNA采样方式用于聋病基因检测的可行性。方法：随机收集门诊聋儿98例,分别采用无创的口拭子刮取口腔颊粘膜脱落细胞和抽取2ml静脉血两种方式采集标本,提取基因组DNA后遵循盲法原则,分别对常见聋病基因GJB2,线粒体基因1555A>G(mtDNA A1555G)进行检测,对检测结果通过Kappa检测进行一致性分析。结果：无创的口拭子刮取口腔颊粘膜脱落细胞和有创的抽取静脉血两种方式采样后24h内提取基因组DNA一次成功率分别为98.98%和100%,DNA获得量分别为2.16±0.44μg和21.47±4.59μ(500μl血),OD值260/280分别为1.82±0.13和1.79±0.11.口拭子采样24h后和1周后提取得率有明显差异,t=2.52,p=0.01,血样则无统计学差异,而血样在提取1周和1月后得率差异有统计学意义,t=2.87, p=0.01;GJB2及mtDNA A1555G基因检测结果一致(kappa=1.00,P=0.00),共发现GJB2突变的占27.56%(27/98),mtDNA A1555G占4.08%(4/98)。结论：口拭子刮取口腔颊粘膜脱落细胞可以用于聋病基因GJB2及mtDNA A1555G检测,以该方法可靠、无创、便捷,可以在大规模聋病基因检测特别是小年龄检测对象中推广应用,特别是适用于新生儿早期聋病基因筛查。第二章：建立高分辨率熔解曲线分析结合基因测序二步法GJB2基因检测体系目的：建立以高分辨率熔解曲线分析法(High Resolution Melt,HRM)结合基因测序法为基础的GJB2基因检测体系,实现廉价、快速、结果可靠的检测目标。方法：从复旦大学附属儿科医院耳鼻喉科门诊患儿中随机选择感音神经性聋儿121例和听力正常新生儿72名,平均年龄分别为2岁4月和1.1月,遵循盲法原则分别以HRM结合基因测序二步法与直接测序法进行检测,并采用Kappar分析比较两种方法检测结果的一致性。结果：HRM结合基因测序二步法检测结果为：聋儿组和听力正常组阴性样品分别有24例和58例,可疑突变样品分别有97例和14例,对可疑突变样品进行基因测序,最终确定各样品基因型共发现聋儿组中GJB2双链突变的占46.28%(56/121),单链突变占37.19%(45/121)野生型占22.31%(27/121),听力正常组中单链突变占9.72%(7/72),野生型占90.28%(65/72),未见双链突变者;直接测序法检测结果：对两组样品盲法编号后,直接测序法进行检测,结果与前者一致,Kappa值为1,p=0.00。HRM成本约为PCR基础上增加不足0.5元,而基因测序费用为增加20-25元,检测耗时分别为15分钟和10h左右,HRM结合基因测序二步法使病例组19.83%(24/121)和对照组80.56%(58/72)仅通过HRM检测而无需进行基因测序即可完成检测。结论：HRM法结合基因测序法检测GJB2基因具有廉价、快速、结果准确可靠的特点,可应用于GJB2基因大规模临床检测,为明确耳聋病因、降低聋儿出生率提供一种技术支持。第三章：定量连接酶连反应在mtDNA A1555G点突变检测中的应用目的：建立以定量连接酶连反应(quantitative ligase chain reaction, Q-LCR)为基础的mtDNA A1555G点突变诊断技术,实现廉价、快捷、准确的检测目标。方法：从复旦大学儿科医院耳鼻喉科门诊中随机选择感音神经性聋儿121例及听力正常患儿30例,针对mtDNA A1555G点突变设计相应的探针和引物,建立稳写的检测体系,遵循双盲法原则分别以Q-LCR法和酶切法进行检测,用Kappa分析比较两种方法的检测结果。结果：Q-LCR法检测结果为：聋儿组中携带mtDNA A1555G点突变的占4.01%(5/121),听力正常组中未见携带mtDNA A1555G点突变者;酶切法检测后结果与Q-LCR法一致：经Kappa分析两种检测方法结果完全一致,未发现假阳性和假阴性。结论：所建立的Q-LCR法检测mtDNA A1555G点突变具有价格便宜,操作快捷,结果准确可靠的特点,可应用于我国mtDNA A1555G突变的大规模筛查,为明确聋儿病因,降低聋生出生率,预防药物性耳聋的发生提供一种很好的方法。第二部分：先天性聋儿GJB2及mtDNA A1555G突变分子流行病学研究目的：明确GJB2基因及mtDNA A1555G突变在上海及周边地区先天性聋儿和新生儿重症监护病房(Neonatal intensive care unit NICU)新生儿中的突变率及突变热点分布情况。方法：收集复旦大学儿科医院耳鼻喉科门诊发病年龄小于1岁的聋儿183例,NICU入院大于48h的新生儿484例,口拭子刮取口腔颊粘膜脱落细胞取得DNA样本,用HRM结合基因测序二步法检测GJB2基因,用Q-LCR法检测mtDNA A1555G突变,对检测结果进行统计分析以明确GJB2基因和mtDNA A1555G在聋儿组和NICU对照组中的分布情况。结果：聋儿组183例中共检测出148例GJB2基因有改变,占80.87(148/183),共检测出12种突变方式,其中致病突变7种(235delC、299-300delAT、571T>C、605ins46、223C>T、224G>A、232G>A),多态性突变2种608T>C、79G>A,致病性有争议的突变3种(79G>A+341A>G、109G>A、509A>G),携带GJB2致病性纯合突变或双重杂合致病性突变(含3种有争议的突变类型)的杂合个体共有58例,占31.69%,仅携带一条GJB2致病性突变的有74例,占40.44%。不携带GJB2突变或仅携带多态性突变的有51例,占27.87%；NICU对照组484例中共检测出207例GJB2基因序列有改变,占42.77%,共检测出4种突变方式,其中致病突变有1种(235delC),多态性突变1种79G>A,致病性有争议的突变2种(79G)A+341A>G和109G>A),79G>A+341A>G纯合突变5例,占1.03%,仅携带一条GJB2致病性突变的有169例,占34.92%。不携带GJB2突变或仅携带多态性突变的有310例,占64.05%。mtDNA A1555G突变在聋儿组中有5例携带者,占2.73%,而NICU组中未发现突变阳性者。结论：本研究发现在中国特别是在上海及周边地区的先天性聋儿中,235delC/ 299-300delAT/109G>A/79G>A+341A>G等为常见的突变热点；支持109G>A、79G>A+341A>G这两个有争议的突变形式为耳聋的致病性突变；首次发现在聋病患者中发现509A>G同时伴235delC/223C>T同时伴79G>A这两种新型突变方式,另外在中国非显性遗传家系中首次发现了以往认为是显性遗传的突变方式224G>A和232G>A；mtDNA A1555G在本研究中发现其发生率约为2.73%,低于已报道的平均水平,提示该突变作为一种药物易感性突变可能在先天性耳聋不起主要作用；本研究为开展GJB2基因和mtDNA A1555突变临床检测提供了依据。第三部分：GJB2及mtDNAA1555G突变致聋早期听力学特征研究目的：探讨GJB2与mtDNAA1555G基因突变致聋早期的听力学特征,为聋病的临床诊断,预防和干预提供依据。方法：随机收集复旦大学儿科医院耳鼻喉科门诊中感音神经性聋儿183例,年龄范围在0-4岁,发病年龄在1岁以内。对照组30例年龄范围在0-3岁。收集入选对象病史资料,听力学信息(声阻抗、耳声发射、短声听性脑干反应、多频稳态反应等)以及GJB2、mtDNAA1555G基因检测情况,按GJB2致聋组、mtDNAA1555G致聋组、非GJB2/mtDNAA1555G致聋组以及正常对照组进行分组研究,分析不同致病基因所引起的听力损伤早期的听力学特征。结果：GJB2致聋组、mtDNA A1555G致聋组及非GJB2/mtDNA A1555G致聋组发病年龄分别为1.03±2.32月、2.54±9.32月、2.43±4.32月GJB2致聋组发病早,t=2.43,p(0.01；三组患儿生后3天初筛通过率分别为6.03%,10.00%,12.08%,42天复筛通过率分别为4.13%、10.00%、3.75%；GJB2致聋组中听力损失在中度及中度以下者有19.83%(23/116),而mtDNA A1555G致聋组仅有10.00%(1/10),其它组有30.84%(74/240)；GJB2致聋组51耳在刺激声强度为109.6dBnHL时能引出清晰Ⅰ、Ⅲ、Ⅴ波,6月内Ⅰ、Ⅲ波潜伏期及Ⅰ—Ⅴ波间期延长,6-12月时各波及波间期均延长,此类聋儿早期听力呈进行性下降；各组聋儿在ASSR250Hz、500Hz、1000Hz、2000Hz、4000Hz各频率引出率高于ABR V波,GJB2致聋组在低频率引出率低于其它组,提示此类聋儿早期听力损失累及各个频率。结论：本研究表明GJB2基因致聋的患儿听力损失发生早,听力损失以重度、极重度多见,在一岁以内有进行性下降趋势,病变累及各个频率,对于此类患儿早期进行适当的干预非常重要；mtDNA A1555G引起的耳聋除出生后接触耳聋性药物后引起的迟发性耳聋外,有一小部分患儿在出生时听力损失就已经发生提示可能与孕期母亲功能状态有关,对此类突变携带者的孕妇在早期就应该给予关注。更多还原

【Abstract】 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.更多还原