中国一先天性无虹膜家系基因突变位点的测序及遗传分析

发布时间：2018-03-18 09:03

本文选题：先天性无虹膜　切入点：基因定点捕获测序　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的先天性无虹膜是一种世界范围内罕见的双眼发病可累及全眼多个组织结构的遗传疾病,遗传方式通常为常染色体显性遗传(autosomal dominant),全球人群发病率约1/40000-1/100000,其中约2/3为家族性、1/3呈散发。经典的先天性无虹膜症为单纯的眼部病变,主要表现为双侧眼的虹膜部分或完全缺失,可伴有包括角膜、前房、晶状体、视网膜和视神经等在内的部分或全部眼组织发育异常或功能障碍。而上述先天性无虹膜表现可作为并发症可伴发于WAGR综合症(约13%)和极为罕见的Gillespie综合征(常染色体隐性遗传,约占2%)。遗传学研究表明,PAX6基因变异可解释90%的先天性无虹膜的发生,随着近年来第二代测序技术的普及和前房角发育相关基因研究的深入,ABCB6、FOXC1、PITX2、等多个基因的变异被报道发现于先天性无虹膜患者的基因序列中,且有动物实验建立了相关模型证实了部分基因变异可导致虹膜发育异常。本研究旨于利用第二代测序技术联合目标基因定点捕获技术筛查中国汉族一先天性无虹膜家系致病突变位点并通过家系验证和遗传分析探索其与疾病的关联。资料与方法采集2016年3月就诊于郑州大学第一附属医院眼二科一中国汉族先天性无虹膜家系,3代共9名成员,其中Ⅰ代1号已故(原因不明,排除无虹膜及相关系统性疾病病史),在世8名成员中3名患者、5名表型正常者。另召集100名无三代以内直系及旁系亲缘关系且无眼部及全身系统性基础疾病的志愿者作为正常对照。本研究严格遵守赫尔基新宣言,经郑州大学第一附属医院伦理学委员会批准(2016年科研第51号),所有参与实验的成年人(满18岁)及未成年人(未满18岁)监护人均阅读并签署参与实验知情同意书。使用Tiangen DNA提取试剂盒提取家系成员及对照组血液样本中的基因组DNA,根据已知文献报道的无虹膜相关基因定制目标序列捕获测序Panel,利用二代测序结合定点捕获技术对先证者基因组DNA进行目标序列捕获测序,通过结果分析处理与基因库进行对比筛选出含有可疑突变的候选基因。根据上下游序列针对筛查后锁定的突变位点设计引物,对全体家系成员行DNA定向PCR扩增后应用Sanger法进行家系验证,最终确定该家系的致病位点。结果本家系先天性无虹膜遗传模式符合常染色体显性遗传,患者均为双眼发病,共同的眼部异常表现为:双眼低视力(矫正无提高),高眼压,虹膜缺如,角膜缘不规则增厚、基质浑浊,眼球水平震颤,中心凹发育不良;不同患者特异性眼部表现分别为:Ⅱ-2患左眼上睑下垂,右眼先天性白内障,Ⅲ-2患双眼先天性白内障,双侧晶状体向上不全脱位。分析目标序列定点捕获测序结果,经基因库对比后发现先证者目标基因拥有13个可疑突变位点,通过有害分析预测筛选出PAX6基因第6号外显子上碱基替换c.183CA唯一与疾病高度相关联,Sanger测序验证该家系中所有患者均含有此突变,该突变属无义突变,导致DNA转录翻译提前终止,与此同时家系表型正常者及健康对照者均不存在此突变。查阅NCBI snp、dbsnp、1000genomes数据库发现此突变对应碱基替换人群发生率极低,不属于snp,曾被一例西方文献报道为散发突变但未有临床资料证实。结论1.通过对先证者的目的基因二代测序筛选出该无虹膜家系的致病基因位点(PAX6基因c.183CA,p.Y61X),该碱基替换导致无义突变,DNA转录翻译提前终止(PTC),杂合突变致使PAX6基因表达单倍剂量不足,Sanger测序家系内验证该突变与表型共分离,且不存在于健康对照中,为本家系致病突变。2.本研究报道的基因突变位点为全球发现第二例并且是黄种人中的第一例,以家系方式出现,证明该位点的突变可存在于不同种族人群中,且突变对应表现型在白种人和黄种人中均为先天性无虹膜。3.而家系患者为直系亲子关系,眼部表型各有不同,本研究未能发现可解释眼部特异性表型的基因变异,为日后该疾病的表观遗传学研究提供了资源与参考。
[Abstract]:Background and objective: congenital aniridia is a rare disease in the world's eyes can be a genetic disease involving the whole eye more organization, genetic is usually autosomal dominant (autosomal dominant), the global incidence rate of about 1/40000-1/100000, which is about 3 2/ for familial, sporadic congenital classic 1/3. The aniridia is pure eye disease, mainly for bilateral partial or complete absence of the iris of the eye, can be accompanied by including the cornea, anterior chamber, lens, retina and optic nerve, all or part of the eye tissue growth retardation or dysfunction. The congenital aniridia manifestations as a complication may be associated with WAGR syndrome (about 13%) and extremely rare Gillespie syndrome (autosomal recessive, approximately 2%). Genetic studies have shown that congenital PAX6 gene mutations can explain 90% of the film with no rainbow, In recent years the popularity of the anterior chamber and the second generation sequencing technology development research angle related genes ABCB6, FOXC1, depth, PITX2, mutation of multiple genes was reported in gene sequence of aniridia, and animal experiment to establish the correlation model confirmed some of gene mutations can lead to abnormal development of the iris. The purpose of this research is to use the second generation sequencing technology combined with target gene screening Chinese designated capture Han pedigree with congenital aniridia mutations and through pedigree validation and genetic analysis to explore its association with disease. Materials and methods collected in the First Affiliated Hospital of Zhengzhou University from March 2016 two, a China eye congenital absence of Han the iris family, the 3 generation of a total of 9 members, of which the first generation 1 late (for unknown reasons, excluding non iris and related systemic disease), all 8 members of the 3 patients, 5 morphologically Normal. The other called 100 within three generations without immediate and extended kinship and no ocular and systemic disease volunteers as normal control. In this study, strictly abide by the Hull based new declaration, approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University (2016 research No. fifty-first), all the adults in the experiment (18 years old) and minors (under 18 years) guardians read and sign the informed consent to participate in the experiment. The genomic DNA group in blood samples and control using Tiangen DNA extraction kit according to family members, aniridia related gene sequences of known target custom literature reports to capture the sequencing of Panel, using the two generation sequencing combined with capture technology of the proband genomic DNA targeted sequencing results, through analysis and comparison of gene library screening candidate genes containing suspicious mutations. According to the downstream sequence for mutation primers after screening locking, pedigree validation on the application of Sanger to all family members for DNA directional PCR amplification method, and ultimately determine the disease locus in this family. The family congenital aniridia autosomal dominant inheritance, with both eyes disease, common eye abnormalities: bilateral low vision (no correction improved), high intraocular pressure, iris agenesis, corneal irregular thickening and matrix turbidity, nystagmus, foveal hypoplasia; different patient specific eye respectively: II -2 patients with left eye ptosis, eye congenital cataract, -2 III patients bilateral congenital cataract, bilateral lens subluxation. To analysis the target sequence designated capture sequencing results, the gene pool after comparison revealed that the proband has 13 suspicious target gene mutations, the harmful Analysis and prediction of screened PAX6 gene exon sixth c.183CA substitution only highly associated with disease, Sanger sequencing of the family and all the patients were with this mutation, the mutation is a nonsense mutation, resulting in early termination of transcription and translation of DNA at the same time, the family is often the phenotype of patients and healthy controls were not there refer to the NCBI mutation. SNP, dbSNP, 1000genomes database found this mutation corresponds to substitution incidence rate is extremely low, does not belong to the SNP, has been a case of Western literature as sporadic mutation but confirmed the clinical data before. Conclusion 1. by gene of the proband two generation sequencing screened pathogenic gene loci of the iris family the Department (PAX6 gene c.183CA, p.Y61X), the nucleotide substitution resulting in nonsense mutation, DNA transcription and translation termination (PTC), heterozygous mutation in PAX6 gene expression haploinsufficiency, Sanger sequencing within family experience The mutation cosegregated with the phenotype, and does not exist in healthy controls, for this family. This study reported pathogenic mutations in the.2. gene mutation was found in second cases for the global and yellow in the first case, appear in the family, the mutation site that can exist in different ethnic groups, and the mutation the corresponding phenotype for white and yellow are congenital aniridia and.3. pedigrees for immediate parentage, ocular phenotype is different, this study failed to find mutations could explain eye specific phenotype, provide a reference for genetic research resources and the disease apparent day.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R773.1

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