本文關(guān)鍵詞：遺傳性眼前段疾病相關(guān)候選基因定位研究　出處：《浙江大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 先天性白內(nèi)障 常染色體顯性遺傳 晶狀體脫位 顆粒狀角膜營(yíng)養(yǎng)不良 EPHA2 TGFBI 基因突變

【摘要】：研究背景 遺傳性眼病是人類主要的致盲因素之一,嚴(yán)重危害人類的健康,至今仍無(wú)有效治療方法。隨著分子生物學(xué)和遺傳學(xué)技術(shù)的不斷發(fā)展,遺傳性眼病的基因背景正不斷被揭示,根據(jù)遺傳方式不同,可分為單基因遺傳、多基因遺傳和染色體畸變?nèi)箢?其中以單基因遺傳病最多見(jiàn),單基因遺傳性眼病又可表現(xiàn)為常染色體顯性、常染色體隱性和性染色體遺傳三種遺傳方式。有些眼病的遺傳方式不止一種,臨床表型各異,癥狀輕重不一,如目前已經(jīng)鑒定和克隆的與先天性白內(nèi)障相關(guān)的基因有26個(gè),突變型已達(dá)40余種。研究表明,約有60%的遺傳性眼病的分子基礎(chǔ)較復(fù)雜,其發(fā)病機(jī)理尚未闡明,有待進(jìn)一步研究。因此,較全面地研究分析遺傳眼病致病相關(guān)位點(diǎn)和基因型的分子基礎(chǔ),對(duì)進(jìn)一步闡明相應(yīng)遺傳眼病的發(fā)病機(jī)理,指導(dǎo)臨床診斷有重要意義；為今后開(kāi)展相關(guān)遺傳眼病的基因診斷和產(chǎn)前診斷具有重要意義；并為進(jìn)一步開(kāi)展基因功能及基因治療研究奠定了基礎(chǔ)。本文就我們收集的我國(guó)浙江地區(qū)的兩個(gè)先天性白內(nèi)障家系、一個(gè)角膜營(yíng)養(yǎng)不良家系和一個(gè)伴晶狀體脫位的Marfan相關(guān)疾病家系共四個(gè)遺傳病家系進(jìn)行了基因定位與基因突變研究。 研究方法 對(duì)收集的家系成員進(jìn)行詳細(xì)的家族史及臨床資料的采集,并對(duì)所有的家系成員進(jìn)行全面的眼科檢查,包括視力、裂隙燈、散瞳眼底檢查等。抽取外周血樣本,提取基因組DNA,應(yīng)用聚合酶鏈反應(yīng)(PCR)擴(kuò)增疾病相關(guān)候選基因的所有編碼區(qū),利用直接測(cè)序法確定致病的突變基因及位點(diǎn),借助生物信息學(xué)方法(Polyphen-2and Align-GVGD)對(duì)突變基因的結(jié)構(gòu)和功能進(jìn)行分析。 結(jié)果 1、臨床檢查表明2個(gè)遺傳性白內(nèi)障家系的表型分別為后囊下白內(nèi)障和全白內(nèi)障,遺傳方式均為常染色體顯性遺傳。針對(duì)家系候選基因進(jìn)行突變篩查,在家系1中,對(duì)CRYAB、CRYBA1/A3、CRYBB2、GJA3、GJA8、CHMP4B、PITX3和EPHA2八個(gè)候選基因進(jìn)行測(cè)序,發(fā)現(xiàn)在EPHA2基因編碼區(qū)的c.2668CT的雜合改變,該變化導(dǎo)致了保守的精氨酸被半胱氨酸置換(p.R890C),該雜合突變不存在于200名健康對(duì)照成員中,PolyPhen-2和Align-GVGD分?jǐn)?shù)均提示R890C為影響蛋白質(zhì)結(jié)構(gòu)和功能的有意義的突變。在家系2中,未發(fā)現(xiàn)CRYAA、CRYAB、CRYBA1/A3、 CRYBB1、CRYBB1、CRYBB2、CRYGC、CRYGD、CRYGS、GJA3、GJA8和MIP十二個(gè)已知致病基因的致病性突變； 2、家系3：角膜營(yíng)養(yǎng)不良家系遺傳方式為常染色體顯性遺傳。家系中所有患者均10歲之前發(fā)病,反復(fù)發(fā)生角膜上皮糜爛,視力逐漸下降；裂隙燈檢查可見(jiàn)患者雙眼角膜中央灰白色混濁,大小、數(shù)目不一,混濁呈點(diǎn)、圓圈狀,混濁間的角膜透明,角膜各層均有受累,不同患者間角膜累及層次和深度不同,即使同一患者,雙眼混濁累及深度也有不同。雖然裂隙燈下該家系患者患者角膜混濁形態(tài)與典型的顆粒狀角膜營(yíng)養(yǎng)不良I相符,但典型的顆粒狀角膜營(yíng)養(yǎng)不良I型患者多10歲以后發(fā)病,混濁首先并主要累及角膜基質(zhì)層,隨病情發(fā)展,上皮層有不同程度的受累,而該家系中患者發(fā)病年齡較早,最小患者只有5歲,混濁主要累及上皮層,伴少量混濁位于角膜淺基質(zhì)層,并且有的患者混濁只累及上皮層,這些與典型的顆粒狀角膜營(yíng)養(yǎng)不良Ⅰ是不同的；候選基因測(cè)序發(fā)現(xiàn)家系中所有患者TGFBI基因1663位堿基發(fā)生CT堿基置換(c.1663CT),導(dǎo)致第555位精氨酸突變?yōu)樯彼?p.R555W),家系中參與研究的正常人及100名健康對(duì)照組成員均未檢測(cè)到此突變存在。 3、家系4：該家系患者診斷為伴晶狀體脫位的Marfan相關(guān)疾病,其遺傳方式符合常染色體顯性遺傳,家系中所有患者均表現(xiàn)為雙眼晶狀體脫位且不伴有骨骼系統(tǒng)和心血管系統(tǒng)異常,對(duì)家系致病候選基因FBN1的65個(gè)外顯子、內(nèi)含子和外顯子交界區(qū)進(jìn)行突變篩查,未發(fā)現(xiàn)該病的致病突變。 結(jié)論 1、本文收集到常染色體顯性遺傳性白內(nèi)障家系2個(gè),研究報(bào)道了1個(gè)國(guó)內(nèi)外未見(jiàn)報(bào)道的與先天性后囊下白內(nèi)障有關(guān)的EPHA2的基因突變：c.2668CT (p.R890C); 2、在家系2中,排除了CRYAA、CRYAB、CRYBA1/A3、CRYBB1、CRYBB2、 CRYBB3、CRYGC、CRYGD、CRYGS、GJA3、GJA8和MIP為該先天性白內(nèi)障家系的致病基因； 3、在顆粒狀角膜營(yíng)養(yǎng)不良家系中,發(fā)現(xiàn)了由TGFBI基因經(jīng)典型突變R555W引起的非典型顆粒狀角膜營(yíng)養(yǎng)不良Ⅰ型的家系,拓展了顆粒狀角膜營(yíng)養(yǎng)不良Ⅰ型的表型； 4、在伴晶狀體脫位的Marfan相關(guān)疾病家系中,排除了已知候選基因FBN1的致病性突變,提示可能存在與該病相關(guān)的未知致病基因。
[Abstract]:Research background
Hereditary eye disease is one of the major causes of blindness in humans, serious harm to human health, there is still no effective treatment. With the development of molecular biology and genetics, genetic background of genetic eye diseases are being discovered, according to the mode of inheritance, can be divided into single gene, genetic and chromosomal aberration of three the most common categories, with monogenic disease, single gene hereditary eye disease can be manifested as autosomal dominant, autosomal recessive and X-linked genetic three genetic ways. Some eye more than one, different clinical phenotypes, symptoms of varying severity, as has been the identification and cloning of related congenital cataract and 26 genes, the mutant has reached more than 40 species. The study shows that the molecular basis of genetic eye diseases about 60% of the more complex, its pathogenesis is not yet clear, further Study. Therefore, a comprehensive analysis of the molecular basis of genetic loci related ophthalmopathy and pathogenic genotype, to further clarify the pathogenesis of the corresponding genetic eye disease, have important significance to guide the clinical diagnosis; it has important significance for gene diagnosis and prenatal diagnosis in the future to carry out the relevant genetic eye disease; and laid the foundation for further research on gene function and gene therapy. Two congenital cataract pedigrees in this paper, we collected in the Zhejiang region in China, a corneal dystrophies and a disease associated with dislocation of the lens Marfan family a total of four genetic pedigrees studied mutation gene mapping and gene.
research method
Detailed clinical information and family history of family members to collect the collection, and to conduct a comprehensive eye examination of all family members including visual acuity, slit lamp, fundus examination. Peripheral blood samples, extract genomic DNA by polymerase chain reaction (PCR) amplification region encoding all related diseases the candidate gene, the mutant gene and pathogenic sites by direct sequencing method with biological information (Polyphen-2and Align-GVGD) on the structure and function of gene mutation analysis.
Result
1, the clinical examination showed that the phenotype of 2 hereditary cataract pedigrees were posterior subcapsular cataract and cataract, mode of inheritance are autosomal dominant. The candidate gene family mutation screening was carried out in 1 families, CRYAB, CRYBA1/A3, CRYBB2, GJA3, GJA8, CHMP4B, sequencing of eight a candidate gene PITX3 and EPHA2, found in the encoding region of EPHA2 gene of c.2668CT heterozygous change, this change led to a conserved arginine by cysteine (p.R890C), the heterozygous mutation does not exist in 200 healthy controls, PolyPhen-2 and Align-GVGD scores showed R890C effects of protein structure and function significant mutation in the family. 2, CRYAA was not found, CRYAB, CRYBA1/A3, CRYBB1, CRYBB1, CRYBB2, CRYGC, CRYGD, CRYGS, GJA3, pathogenicity of twelve known genes GJA8 and MIP mutation;
2, 3 families: corneal dystrophies autosomal dominant inheritance in the family before. All the patients were 10 years of age of onset, recurrent corneal erosion, visual acuity decreased gradually; slit lamp examination showed both eyes of patients with central corneal opacity gray, size, number one, a little cloudy, circle the corneal transparency, opacity, corneal layers were different between patients with corneal involvement, involving the level and depth of different, even the same patient, involving the depth of turbid eyes are also different. Although the slit lamp in the family of patients with corneal opacity and form typical granular corneal dystrophy I consistent, but the typical granular corneal malnutrition in patients with type I after more than 10 years of onset, turbid first and is mainly involved in corneal stroma, with the progression of the disease, the epithelial layer has a different degree of involvement, and the families of patients with early age of onset, the youngest patient Only 5 years old, mainly involving the turbid epithelial layer, with a small amount of opacities in the superficial corneal stroma, and some patients only in turbid epithelium. These typical granular corneal dystrophy 1 is different; candidate gene was found in all patients in the family of TGFBI gene 1663 nucleotide CT (c.1663CT), lead to base substitution 555th arginine mutation for tryptophan (p.R555W), normal subjects participated in the study in a family and 100 healthy controls were not detected this mutation.
3, 4 families: the diagnosis of this pedigree was related to Marfan disease with dislocation of the lens, the mode of inheritance was consistent with autosomal dominant inheritance. All patients showed abnormal binocular lens dislocation without skeletal system and cardiovascular system in the family, the exon of FBN1 gene candidate family 65. The intron and exon junction mutations, has been found in the disease causing mutations.
conclusion
1, 2 autosomal dominant cataract families were collected. 1 EPHA2 mutations were reported at home and abroad, which were related to congenital posterior subcapsular cataract: c.2668CT (p.R890C).
2, in family 2, CRYAA, CRYAB, CRYBA1/A3, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS, GJA3, GJA8 and GJA3 were excluded as the pathogenic genes of the congenital cataract family.
3, we found a family with atypical granular corneal dystrophy type TGFBI caused by classic R555W mutation in the granular corneal dystrophy family, and expanded the phenotype of granular corneal dystrophy type I.
4, in families with Marfan related diseases with lens dislocation, a pathogenic mutation of known candidate gene FBN1 was excluded, suggesting that there may be an unknown pathogenic gene associated with this disease.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R776.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李根林，，張士元;先天性白內(nèi)障形成的臨床特征[J];中國(guó)實(shí)用眼科雜志;1996年04期

本文編號(hào)：1378660