本文選題：先天性無虹膜　切入點(diǎn)：基因定點(diǎn)捕獲測(cè)序　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的先天性無虹膜是一種世界范圍內(nèi)罕見的雙眼發(fā)病可累及全眼多個(gè)組織結(jié)構(gòu)的遺傳疾病,遺傳方式通常為常染色體顯性遺傳(autosomal dominant),全球人群發(fā)病率約1/40000-1/100000,其中約2/3為家族性、1/3呈散發(fā)。經(jīng)典的先天性無虹膜癥為單純的眼部病變,主要表現(xiàn)為雙側(cè)眼的虹膜部分或完全缺失,可伴有包括角膜、前房、晶狀體、視網(wǎng)膜和視神經(jīng)等在內(nèi)的部分或全部眼組織發(fā)育異常或功能障礙。而上述先天性無虹膜表現(xiàn)可作為并發(fā)癥可伴發(fā)于WAGR綜合癥(約13%)和極為罕見的Gillespie綜合征(常染色體隱性遺傳,約占2%)。遺傳學(xué)研究表明,PAX6基因變異可解釋90%的先天性無虹膜的發(fā)生,隨著近年來第二代測(cè)序技術(shù)的普及和前房角發(fā)育相關(guān)基因研究的深入,ABCB6、FOXC1、PITX2、等多個(gè)基因的變異被報(bào)道發(fā)現(xiàn)于先天性無虹膜患者的基因序列中,且有動(dòng)物實(shí)驗(yàn)建立了相關(guān)模型證實(shí)了部分基因變異可導(dǎo)致虹膜發(fā)育異常。本研究旨于利用第二代測(cè)序技術(shù)聯(lián)合目標(biāo)基因定點(diǎn)捕獲技術(shù)篩查中國(guó)漢族一先天性無虹膜家系致病突變位點(diǎn)并通過家系驗(yàn)證和遺傳分析探索其與疾病的關(guān)聯(lián)。資料與方法采集2016年3月就診于鄭州大學(xué)第一附屬醫(yī)院眼二科一中國(guó)漢族先天性無虹膜家系,3代共9名成員,其中Ⅰ代1號(hào)已故(原因不明,排除無虹膜及相關(guān)系統(tǒng)性疾病病史),在世8名成員中3名患者、5名表型正常者。另召集100名無三代以內(nèi)直系及旁系親緣關(guān)系且無眼部及全身系統(tǒng)性基礎(chǔ)疾病的志愿者作為正常對(duì)照。本研究嚴(yán)格遵守赫爾基新宣言,經(jīng)鄭州大學(xué)第一附屬醫(yī)院倫理學(xué)委員會(huì)批準(zhǔn)(2016年科研第51號(hào)),所有參與實(shí)驗(yàn)的成年人(滿18歲)及未成年人(未滿18歲)監(jiān)護(hù)人均閱讀并簽署參與實(shí)驗(yàn)知情同意書。使用Tiangen DNA提取試劑盒提取家系成員及對(duì)照組血液樣本中的基因組DNA,根據(jù)已知文獻(xiàn)報(bào)道的無虹膜相關(guān)基因定制目標(biāo)序列捕獲測(cè)序Panel,利用二代測(cè)序結(jié)合定點(diǎn)捕獲技術(shù)對(duì)先證者基因組DNA進(jìn)行目標(biāo)序列捕獲測(cè)序,通過結(jié)果分析處理與基因庫進(jìn)行對(duì)比篩選出含有可疑突變的候選基因。根據(jù)上下游序列針對(duì)篩查后鎖定的突變位點(diǎn)設(shè)計(jì)引物,對(duì)全體家系成員行DNA定向PCR擴(kuò)增后應(yīng)用Sanger法進(jìn)行家系驗(yàn)證,最終確定該家系的致病位點(diǎn)。結(jié)果本家系先天性無虹膜遺傳模式符合常染色體顯性遺傳,患者均為雙眼發(fā)病,共同的眼部異常表現(xiàn)為:雙眼低視力(矯正無提高),高眼壓,虹膜缺如,角膜緣不規(guī)則增厚、基質(zhì)渾濁,眼球水平震顫,中心凹發(fā)育不良;不同患者特異性眼部表現(xiàn)分別為:Ⅱ-2患左眼上瞼下垂,右眼先天性白內(nèi)障,Ⅲ-2患雙眼先天性白內(nèi)障,雙側(cè)晶狀體向上不全脫位。分析目標(biāo)序列定點(diǎn)捕獲測(cè)序結(jié)果,經(jīng)基因庫對(duì)比后發(fā)現(xiàn)先證者目標(biāo)基因擁有13個(gè)可疑突變位點(diǎn),通過有害分析預(yù)測(cè)篩選出PAX6基因第6號(hào)外顯子上堿基替換c.183CA唯一與疾病高度相關(guān)聯(lián),Sanger測(cè)序驗(yàn)證該家系中所有患者均含有此突變,該突變屬無義突變,導(dǎo)致DNA轉(zhuǎn)錄翻譯提前終止,與此同時(shí)家系表型正常者及健康對(duì)照者均不存在此突變。查閱NCBI snp、dbsnp、1000genomes數(shù)據(jù)庫發(fā)現(xiàn)此突變對(duì)應(yīng)堿基替換人群發(fā)生率極低,不屬于snp,曾被一例西方文獻(xiàn)報(bào)道為散發(fā)突變但未有臨床資料證實(shí)。結(jié)論1.通過對(duì)先證者的目的基因二代測(cè)序篩選出該無虹膜家系的致病基因位點(diǎn)(PAX6基因c.183CA,p.Y61X),該堿基替換導(dǎo)致無義突變,DNA轉(zhuǎn)錄翻譯提前終止(PTC),雜合突變致使PAX6基因表達(dá)單倍劑量不足,Sanger測(cè)序家系內(nèi)驗(yàn)證該突變與表型共分離,且不存在于健康對(duì)照中,為本家系致病突變。2.本研究報(bào)道的基因突變位點(diǎn)為全球發(fā)現(xiàn)第二例并且是黃種人中的第一例,以家系方式出現(xiàn),證明該位點(diǎn)的突變可存在于不同種族人群中,且突變對(duì)應(yīng)表現(xiàn)型在白種人和黃種人中均為先天性無虹膜。3.而家系患者為直系親子關(guān)系,眼部表型各有不同,本研究未能發(fā)現(xiàn)可解釋眼部特異性表型的基因變異,為日后該疾病的表觀遺傳學(xué)研究提供了資源與參考。
[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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R773.1

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