【作者】 吴宏；

【导师】 冯永；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一部分Ⅳ型Bartter综合征遗传学分析背景：Bartter综合征（Bartter syndrome, BS）是一组临床表现为肾性失盐、低钾血症、代谢性碱中毒、肾素-血管紧张素-醛固酮系统活性增强的遗传性疾病,主要为常染色体隐性遗传。根据不同的临床特点,可将BS分为3型：经典型BS（包括Gitelman综合征）、新生儿型BS、新生儿型BS伴感音神经性耳聋。迄今为止,已发现6个与BS有关的常染色体基因：NKCC2、ROMK、CLCNKB、 BSND、CLCNKA、 NCCT。根据遗传学的特征,可将BS分为6型：Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型、Ⅴ型、Ⅵ型,其中Ⅳ型、Ⅴ型为新生儿型伴感音神经性耳聋,Ⅵ型为Gitelman综合征。目的：本研究小组收集了一个Bartter综合征的散发病例,患者同时伴有感音神经性耳聋,拟通过分析临床特征与基因突变检测寻找病因。方法：对患者及父母进行了病史采集、体格检查及辅助检查,并选取100名正常人群作为对照组,进行了GJB2、BSND、CLCNKA、 CLCNKB等四个基因的外显子筛查。结果：患儿的临床表现为：新生儿期发病,肾素-血管紧张素-醛固酮系统活性增高,低钾血症、低氯血症,生长发育迟缓,神经性耳聋,肾脏影像学无明显异常,具有羊水过多和早产史,符合IV型Bartter综合征的典型症状。同时,患儿的BSND基因第一个外显子存在纯和突变c.22C>T和c.127G>A, GJB2基因存在复合杂合突变即c.235delC和c.109G>A。而患儿父母BSND基因均存在杂合突变c.22C>T和c.127G>A,父亲GJB2基因存在杂合突变c.109G>A,母亲GJB2基因存在杂合突变c.235delC。患儿及父母均未发现CLCNKA和CLCNKB基因的变异。正常对照组中37例存在BSND c.127位杂合等位基因G/A,2例存在BSND基因c.127位纯和等位基因A/A。结论：1.本研究所报道的Ⅳ型Bartter综合征患者同时携带BSND与GJB2基因变异属世界首次报道；2.患儿Ⅳ型BS是由BSND基因纯和突变c.22C>T导致,而患儿GJB2基因的复合杂合突变可能在耳聋发生过程中有一定的作用；3.患儿携带的BSND基因变异c.127G>A为单核苷酸多态性。第二部分Goldengate耳聋基因芯片的优化设计背景：耳聋是最常见的感觉神经性疾病,它不仅是造成听力残疾、影响人类身心健康的疾病,而且给患者的生活、家庭、工作和学习中的交流带来极大的不便。与此同时,耳聋也给社会带来了沉重的负担。据报道,全球有2.5亿人患有中度以上听力损失。耳聋遗传学研究表明,人类23对染色体中的22对都分布有耳聋遗传位点,线粒体基因也与耳聋相关。约有半数的先天性耳聋与遗传因素有一定联系。随着遗传学的发展,逐渐衍生出一些基因诊断手段,然而这些方法无法同时具备高通量、覆盖面广、低成本、高准确性的特征,因此对遗传性耳聋的基因诊断需要进行革命性的改革。在本课题组的前期工作中,设计了具有384个位点的Goldengate耳聋基因芯片。其中包括240个耳聋相关突变位点（77个显性突变位点及163个隐性突变位点）和144个SNP位点。应用该耳聋基因芯片对465个DNA模板进行筛查,统计分析了芯片Call rate、SNP位点的等位基因频率。结果显示,芯片总Call rate为96.32%; GJB2₂35delC检测位点的假阳性率为3.1%,假阴性率为0；对一个非综合征型显性遗传耳聋家系进行SNP位点的连锁分析,其结果与传统的微卫星定位扫描方法的定位结果相似,认为该芯片具备初步进行耳聋相关基因的排除定位连锁分析的能力。然而,芯片结果存在目前不能解释的疑点：第一,初步应用过程中发现部分不常见突变位点在检测过程中出现较高的阳性检测率,如PJVK₉88delG为57.36%、SLC26A4_Gly497Ser为7.14%；第二,部分位点的call rate较低；第三,部分患者为多种基因突变位点的携带者或纯合子基因型；第四,部分正常人群的检测结果存在一种或多种突变,但并未有耳聋的表现。基于以上原因,为了获取具有更高准确性、更具合理性的芯片,本课题小组拟将芯片进行进一步验证,并根据验证结果优化芯片中的耳聋突变检测位点及SNP位点。目的：验证第一版Goldengate耳聋基因芯片的准确性。在符合临床特点及应用的基础上,根据第一版芯片的经验优化Goldengate耳聋基因芯片,使其符合中国耳聋人群遗传学特点,具有应用性与合理性。方法：第一版Goldengate耳聋基因芯片共有384个位点,随机挑选芯片中19个突变检测位点,应用Sanger测序法验证其敏感性和特异性。结合验证结果优化第一版芯片位点。通过文献检索法统计目前国内外已报道的所有耳聋相关突变位点,筛选位点时结合临床应用,首要原则为：世界范围内突变位点报道次数大于2次,以中国人群报道次数较多的位点为主。在满足第一原则的基础上,其他辅助原则为：（1）相邻两个位点至少间隔60碱基以上；（2）尽量选择探针评分大于0.6的位点,热点突变位点探针评分在0.5-0.6之间也可纳入芯片；（3）点突变优先于长缺失及插入突变；（4）排除第一版芯片中call rate低于70%的位点。SNP位点的替换原则如下：（1）SNP位于亚洲人群中报道的已克隆耳聋相关基因；（2）与以上选取的耳聋致病突变位点间隔60bp及以上；（3）SNP位点探针评分大于0.6；（4）点突变优先于长缺失及插入突变；（5）剔除第一版芯片中call rate分数低于0.6的SNP位点。将优化设计后的384个位点的探针评分与前期芯片的评分进行t检验比较,初步判断芯片位点选择的合理性。结果：1.第一版Goldengate耳聋基因芯片位点的敏感性从0%-100%不等,特异性均为100%；2.优化后的第二版耳聋芯片中包含200个非综合症耳聋位点、40个常见综合征耳聋位点,以及144个SNP位点；3.耳聋突变位点包含中国人群最常见的8个热点突变,如下：GJB2_1BP_DEL₃5delG、GJB2_1BP_DEL₂35C、 GJB2_2BP_DEL₂99₃00at、GJB3_Arg180Term[C/T] SLC26A4_Asn392Tyr[A/T]、SLC26A4_IVS7₂[A/G] SLC26A4_His723Arg[A/G]、线粒体12rsRNA1555[A/G];4.第二版芯片覆盖综合征和非综合征型耳聋的46个核基因与线粒体基因；5.两版芯片探针设计评分进行t检验,P<0.05,统计学上具有显著性差异；6.第二版芯片位点采用自主性命名,达到实用性强、易解读、易判断芯片结果的作用。结论：1.第一版芯片的特异性高,而敏感性高低不等,其原因可来自位点设计、模板质量等多方面,需进行进一步优化；2.第二版Goldengate耳聋基因芯片涵盖了中国人群常见的非综合征耳聋突变热点,并且包含了常见的4种综合征耳聋基因的突变位点,具有与临床特点紧密结合、覆盖面广、应用性能强的特点。更多还原

【Abstract】 Part1:Genetic analysis of type IV Bartter syndromeBackground:Bartter syndrome is an autosomal recessive disease, with a group of manifestations such as renal loss of salt, hypokalemia, metabolic alkalosis, enhanced renin-angiotensin-aldosterone system activity. Bartter syndrome has traditionally been classified into3main clinical variants:neonatal （or antenatal） Bartter syndrome, classic Bartter syndrome, and neonatal （or antenatal） Bartter syndrome with sensorineural deafness. Advances in molecular diagnostics have revealed that Bartter syndrome results from mutations in numerous genes:NKCC2, ROMK, CLCNKB, BSND, CLCNKA and NCCT。According to the genetic features, BS can be divided into six types:type Ⅰ, type Ⅱ, type Ⅲ, type Ⅳ, type Ⅴ and typeⅥ. Type Ⅳ BS and type Ⅴ BS are neonatal （or antenatal） Bartter syndrome with sensorineural deafness. Type Ⅵ BS is Gitelman syndrome.Objective:A sporadic case with Bartter syndrome and was collected. We intended to analyze clinical features and carry out gene mutation detection to find out the etiology for this patient.Methods:The patient and her parents’history, physical examination, and laboratory examinations were carried out.100normal people were selected as the control group. And then, the exons of GJB2, BSND, CLCNKA and CLCNKB were detected.Results:The patient was neonatal-onset, and the clinical features were increased renin-angiotensin-aldosterone system activity, hypokalemia, hypochloremia, growth retardation, deafness polyhydramnios, preterm birth and normal kidney imaging. This was in line with the typical symptoms of type IV Bartter syndrome. At the same time, the patient had homozygous mutations c.22C>T and c.127G>A in BSND, and also compound heterozygous mutations c.235delC and c.109G>A in GJB2. Both parents had heterozygous mutations c.22C>T and c.127G>A in BSND. Father’s had a heterozygous mutation c.109G>A in GJB2, and mother had heterozygous mutation c.235delC in GJB2. Otherwise, CLCNKA and CLCNKB mutations were not found out for the patient and parents.37persons of control group had heterozygous c.127G>A in BSND, and2persons had homozygous c.127G>A.Conclusions:1. It was first reported around the world that the patient with type IV Bartter syndrome carried both BSND and GJB2mutations;2. The patient with type IV BS was caused by homozygous mutation c.22C>T in BSND, and compound heterozygous mutations in GJB2might take a role in the process of deafness;3. The mutation BSND c.127G>A carried by the patient was single nucleotide polymorphism. Part2:Optimization of Goldengate deafness microarrayBackground:Hearing loss is the most common sensory nerve disease, which not only causes hearing disability, but also affects mental health. At the same time, the deafness bring the society a heavy burden. It is reported that250million people worldwide suffer from moderate to severe hearing loss. Genetics studies have shown that22of23pairs of chromosomes and mitochondrial genes are distributed by deafness loci. About half of congenital deafness associated with genetic factors. Gene diagnostic tools are derived with the development of genetics. However, these methods are not characterized by high-throughput, wide coverage, low-cost and high-accuracy. Previously, we designed GoldenGate deafness gene chip with384sites, including240deafness mutation detection sites（77dominant mutation sites and163recessive mutation sites） and144SNPs.465DNA templates were screened by this chip. And then SNP allele frequency and call rate of the chip were analysized. The results showed that the chip total call rate was96.32%; false positive rate of GJB2₂35delC was3.1%, and false negative rate was0; linkage analysis of a dominant hereditary non-syndromic deafness pedigree had similar result with traditional micro satellite positioning scanning method. However, several items could not be explained:First, some mutation sites had high positive detection rates, such as57.36%for PJVK₉88delG,7.14%for SLC26A4_Gly497Ser; Second, some sites had low call rate; Third, some patients carried a variety of homozygous gene mutations; Fourth, some normal persons carried one or more mutations, but were not deafness. For these reasons, our research team intended to verify the chip and optimize it in order to obtain a more reasonable chip with higher accuracy.Objective:To verify the accuracy of the first edition GoldenGate deafness microarray and optimize it on the basis of the clinical features, making its application in line with the genetic features of Chinese deafness crowd.Methods:There were384sites in the Goldengate gene chip,19of which were randomly selected to verify sensitivity and specificity by Sanger sequencing method. And then, optimization of the first version chip sites was based on verification. The records of deafness related mutation sites were made by retrieving literature, which was followed by selecting the sites according to several rules. The paramount principle was that the mutation sites was reported more than2times, giving privacy to the sites reported in Chinese population. The secondary principles were following:（1） adjacent sites were separated by over60bp;（2） probe scoring was over0.6（some hotspot mutations were exceptional）;（4） elimating long-missing and insertional variants;（5） excluding the sites whose call rate were less than70%in the first version chip. SNP exchanging principles were as follows:（1） SNP-related genes have been cloned in the Asian population;（2） adjacent sites were separated by over60bp;（3） probe scoring was over0.6;（4） elimating long-missing and insertional variants;（5） excluding the SNP whose call rate were below0.6in the first version chip.Result:1. Sensitivity of the first edition GoldenGate deafness gene chip ranged from0%to100%and specificity was100%;2. Optimized chip covered200deafness nonsyndromic deafness detection sites,40syndromic deafness detection sites and144SNP;3. Optimized chip covered eight most common hot spot mutations in China, as follows: GJB2_1BP_DEL₃5delG, GJB2_1BP_DEL₂35C, GJB2_2BP_DEL₂99₃00at, GJB3_Arg180Term[C/T], SLC26A4_Asn392Tyr[A/T], the SLC26A4_IVS7₂[A/G], SLC26A4_His723Arg [A/G], mitochondrial12rsRNA1555[A/G];4. Optimized chip included46deafness related nucleus genes and mitochondria gene;5. Probe design rating of two editions chip were compared by t test, P<0.05, which meant statistically significant difference;6. Optimized chip sites were named to interpret and determined the results easily.Conclusions:1. The first version chip had high specificity, but the sensitivity varies, owing to process of design and the quality of templates. Hence, the chip was needed for further optimization.2. Optimized GoldenGate deafness gene chip covered common non-syndromic deafness mutation hot spots, and several common syndromic deafness gene mutation sites, which was closely integrated with clinical features.更多还原