【作者】 蒋璐；

【导师】 冯永；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2011， 博士

【摘要】 第一章：两个Waardenburg综合征Ⅳ型家系的临床特征及相关基因突变研究目的：收集了两个Waardenburg综合征Ⅳ型家系（WSO1家系和WS02家系）,拟对其进行临床诊断分析和基因突变检测研究。方法：对两个家系成员进行了病史采集、临床听力学分析及体格检查,并进行了Waardenburg综合征Ⅳ型相关基因（EDNRB.EDN3.SOX10基因）的外显子测序以筛查致病突变。结果：WS01家系一共两代5人,先证者与其大姐分别为Waardenburg综合征Ⅳ型和Waardenburg综合征Ⅱ型患者,两位患者均表现为左眼虹膜异色,先证者伴有先天性巨结肠；WS02家系先证者为1岁女童,Waardenburg综合征Ⅳ型患者,双侧虹膜异色,伴有先天性巨结肠。三位患者听力学检查均提示双耳极重度感音神经性聋,眼底检查呈现晚霞状改变,无皮肤毛发颜色异常及内眦移位。WSOl家系的先证者及其大姐均检测到有SOX10基因的2号外显子上存在c.254G>A （P. Trp85X）杂合突变,而先证者的母亲（正常人）SOX10基因的2号外显子上也检测到有c.254G>A杂合突变,多次测序后发现突变峰的高度仅为正常峰高度的1/3；WS02家系的先证者SOX10基因的4号外显子测序发现一个剪接位点的杂合突变c.698-2A>T,患者的父母未携带此突变。用直接测序法对100例不相关的正常人进行SOX10基因突变c.254G>A和c.698-2A>T的检测,结果没有检测到相同突变。结论：1.本研究所报道的Waardenburg综合征Ⅳ型病例属国内首次报道；2.SOX10基因突变位点c.254G>A（P. Trp85X）和c.698-2A>T属于新发突变位点,目前国内外尚无此突变位点的报道；3.SOX10基因可能存在镶嵌体。第二章：一个常染色体显性遗传性耳聋大家系的致病位点定位研究目的：为明确一来自湖南张家界地区非综合征型常染色体显性遗传耳聋家系的基因诊断,对此家系进行了初步的定位及候选基因筛查工作方法：收集了一4代人的常染色体显性遗传非综合征型耳聋家系,家系中一共有41人,由于家系发病年龄在20-30岁左右,故将未到达发病年龄的家系成员排除在研究外。本研究共对家系中14名成员（其中包括7名患者和7名正常人）进行了临床诊断分析、遗传基因定位及基因筛查研究。首先,根据家系耳聋患者的临床表型及遗传方式,对家系成员进行了最常见的非综合征型显性耳聋基因COCH、EYA4、GJB2、GJB3基因的直接测序排查及与家系表型相似的耳聋致病位点的排除定位分析。排除分析后应用Illumina linkage芯片对该家系进行了全基因组扫描以初步定位致病区间,在初步定位的区间内挑选MARKER进行精细定位。最后,对精细定位区间内的已知耳聋基因进行直接测序筛查家系致病突变。结果：家系临床特征表现为：语后聋,20-30岁左右发病,发病初期表现为耳鸣及高频听力下降,40岁左右进行性发展为双耳重至极重度聋。排除COCH、EYA4、GJB2、GJB3基因及相关耳聋致病位点后进行了全基因组扫描及微卫星精细定位,经过多点Lod值的计算及单体型分析最终将该家系的致病区间定位在chr1:P34.2-P34.3 （D1S2656;D1S3721）约8.21cM的区间,在D1S2729和D1S2892marker得到了最大多点lod值3.1,此区间与已定位的DFNA2位点重叠,对DFNA2位点上两个己克隆耳聋基因GJB3和KCNQ4进行直接测序筛查后未发现与家系相关的致病突变。结论：DFNA2位点上可能存在其他的基因参与该家系耳聋的发生。第三章：Illumina Goldengate 384k高通量耳聋基因筛查芯片的设计、研发及其对遗传性耳聋家系的检测目的：设计、制备Illumina Goldengate 384K高通量耳聋基因筛查芯片,采用此芯片以期在更广的范围内对已知耳聋突变位点进行筛查和对耳聋大家系进行己定位耳聋位点的排除定位。方法：芯片设计原则：由于芯片的引物探针需间隔60bp以上才能确保准确的杂交,故我们在找出所有己知耳聋相关突变位点的基础上,首先挑选出目前文献报道有2篇以上的突变位点,再依据60bp距离尽可能多的将剩余的突变位点设计在芯片上,一共选取了240个耳聋相关突变位点,包括77个显性突变位点及163个隐性突变位点。另外,我们在亚洲人群中报道的己克隆耳聋相关基因中,选取了144个SNP位点,整张芯片共384个检测点。该芯片可进行已知耳聋相关突变位点的高通量筛查和耳聋相关家系的初步排除定位。我们通过对465例DNA样本进行Illumina GoldenGate 384K高通量耳聋基因芯片筛查,统计芯片Call rate、对其准确性进行了验证、对芯片中144个SNP位点的等位基因频率进行了统计分析及对芯片的排除定位连锁分析进行了验证。对90个耳聋家系的135例DNA样本的芯片检测结果进行分析。结果：1.芯片总的Call rate为96.32%,Call rate达到100%的位点有110个；2.对芯片上的GJB2₂35delC突变检测点进行了准确性验证后发现芯片此检测点的假阳性率为3/97（3.1%）,假阴性率为0；3.芯片中有8个基因的19个SNP位点的最小等位基因频率小于0.1（10%）；4.芯片对本研究中一耳聋家系的定位位置与传统的微卫星定位扫描定位的位置相同,认为该芯片能较准确的进行耳聋相关基因的排除定位连锁分析；5.在此次患者样本检测中共有12个基因的31个突变检测位点在耳聋家系患者中有检出,剩余有189个检测位点在此次检测中无检出。这189个耳聋突变位点在中国耳聋家系人群中则可能属于罕见的突变位点。对90个耳聋家系的135例DNA样本的芯片检测结果进行分析后发现GJB2₂35delC、SLC26A4_IVS7-2A>G等突变位点与目前国内报道的突变检出率相似,PJVK₉88delG （57.36%）、SLC26A4_Gly497Ser （7.14%）等既往报道中的非热点突变在此次耳聋家系患者中也具有很高的突变检出率。结论：研究所设计的高通量耳聋基因筛查芯片可以更广范围内对一些不常检测的耳聋相关突变位点进行检测及可对耳聋家系进行初步排除定位。PJVK₉88delG （57.36%）、SLC26A4_Gly497Ser （7.14%）等既往报道中的非热点突变在耳聋家系患者中具有很高的突变检出率,其是否是多肽或是与检测人群不同有关还是芯片检测的准确性问题?我们将在下一步工作中对有检出的突变位点进行准确性验证,并对芯片设计内容进行调整,剔除突变频率较低、Call rate及准确性低的耳聋相关突变位点,加入由于突变位点引物设计要求60bp间距而在此次设计中未加入芯片的耳聋相关突变位点,剔除最小等位基因频率<10%的SNP位点,并选择同一位置的其他SNP位点进行替换,进一步完善芯片。更多还原

【Abstract】 Chapter 1:Analysis of the clinical features and family-related gene mutations for two pedigrees of typeⅣWaardenburg syndromeObjective:Analysis of the clinical features and family-related gene mutation for the two pedigrees of typeⅣWaardenburg syndrome （WSIV） which were collected （WSO1 and WS02） in the last year.Methods:After History taking, clinical analysis, haring test and physical examination for the two pedigrees, EDNRB、EDN3、SOX1O sequencing was taken for the two pedigrees to identify the Pathogenic mutation.Results:There were 5 persons from a 2 generations pedigree of WS01.The proband was a WSIV patient and his eldest sister was WSII. Both of them showed heterochromia iridis of left eye. Hirschsprung disease was found in the proband of WS01. Proband of WS01, a 1 year old girl, is a WSIV patient showed heterochromia iridis of eyes, hirschsprung disease. All of the patients presented bilateral profound hearing loss, a generalized decrease in retinal pigment with a focal hypopigmented lesion in the eyes with blue irides, without dystopia canthorum and pigmentary abnormalities of hair and skin. Heterozygous mutation Trp85X（c.254G>A） was detected in the proband, eldest sister and mother of WS01 pedigree. But the height of mutation peak was One third lower than that of normal one in the mother who was a healthy subject; Heterozygous mutation c.698-2A>T was detected in the proband of WS02 pedigree. However the mutation was not detected in the parents of the proband. All the mutations identified in these patients were not seen in any other unaffected family members and 100 unrelated control subjects.Conclusions:1.This was the first report of WSIV in Chinese patients.2. P.Trp85X （c.254G>A） and c.698-2A>T were the novel mutation for SOX 10 gene.3. SOX 10 gene was suspected of having mosaicism.Chapter 2:Localization for an autosomal dominant non-syndromic deafness family of ChinaObjective:We collected a four-generation family from the southern part of China with autosomal dominant sensorineural hearing impairment. In order to identify the responsible pathogenic mutations of the family, we set out to identify the locus and to sequentially analyze the candidate genes in the identified region.Methods:After family ascertainment and clinical analysis, exclusive analysis was performed. And then a Genome-wide scan was performed by using Illumina Linkage-12 DNA Analysis Kit （average spacing 0.58 cM）. Fine-mapping markers were genotyped to identify the locus. Finally, we performed haplotype analyses, and candidate gene DNA sequencing for the family.Results:The known genetic loci and genes were not associated with our family. The genome-wide scan and haplotype analyses traced the disease to chromosome 1p34.2-p34.3 with maximum multi-point LOD score of 3.2 which overlaps with DFNA2. We failed to identify any of the known or novel variants within KCNQ4, a voltage-gated potassium channel gene, and GJB3, a gene that encodes the gap junction protein connexin 31, which were the cloned deafness gene in DFNA2. Conclusions:There could be another candidate gene in DFNA2 which be responsible for hearing loss phenotype.Chapter 3:Designing and development of Illumina Goldengate 384k high-throughput BeadArrey for deafness gene mutations screening, and screening for patients in deafness pedigreesObjective:Designing and development of Illumina Goldengate 384k high-throughput BeadArrey. Screen for patients in deafness pedigrees and exclude locus for large deafness pedigrees by use of the BeadArrey.Methods:Design principles:Interval of 60bp for primer probe was needed to ensure accurate cross over on BeadArrey. So we selected mutations which have been reported twice, and then put the other mutations on the BeadArrey according the design priciple. Totally 240 mutations were selected, including 77 dominant mutations and 163 recessive mutations. On the other side, we selected 144 SNPs in deafness genes which were reported in Asian populations. Therefore, the BeadArrey we designed could screen for the deafness gene mutations and exclude locus for large deafness pedigrees. We conducted a call rate statistics and accurate verification for the BeadArrey. An allele frequency analysis of the 144 SNPs and exclusive location analysis were carried on. Finally, we analyzed the test results of BeadArrey for 135 DNA samples of 90 deafness pedigrees which were collected by ENT clinic of Xiangya Hospital and State Key Laboratory of Medical Genetics in 1997-2010.Results:1. Total Call rate of the BeadArrey was 96.32%,110 of 384 test points’ Call rate were 100%.2. False negative rate was 3/97 （3.1%） and false positive rate was 0 for test point GJB2₂35delC.3. Minimum allele frequencies of 19 SNPs in 8 genes were less than 0.1 （10%）.4. The region located by BeadArrey was similar to which was located by traditional micro-satellite scan.5. We detected 31 mutations of 12 genes in the BeadArrey screening for 135 patients.189 mutations which might be the rare mutations in Chinese deafness were not detected. Detection rate of GJB2 1 BP DEL 235C and SLC26A4 IVS7-2A>G were the same as previous reports. But mutations like PJVK（DFNB59）_1BP_DEL₉88G （57.36%） and SLC26A4_Gly497Ser （7.14%） which were not the hot mutations had a high detection rate.Conclusions:Illumina Goldengate 384k high-throughput BeadArrey could test more mutations for the patients and exclude locus for large deafness pedigrees. PJVK（DFNB59）_1BP_DEL₉88G （57.36%） and SLC26A4_Gly497Ser （7.14%） et al which were not the hot mutations had a high detection rate. To find the reason whether they are snps or the low accuracy, in the next step, we will conduct a accurate verification for more test point and remove the points with low call rate, mutation frequency and accuracy, add in the other mutation points which did not add in the BeadArrey as a result of the principle of 60bp Interval. We will remove the SNPs which allele frequencies are less than 10% and add in the other SNPs in the similar position.更多还原