遗传性眼前段疾病相关候选基因定位研究

发布时间：2018-01-04 13:16

本文关键词：遗传性眼前段疾病相关候选基因定位研究　出处：《浙江大学》2013年硕士论文　论文类型：学位论文

【摘要】：研究背景遗传性眼病是人类主要的致盲因素之一,严重危害人类的健康,至今仍无有效治疗方法。随着分子生物学和遗传学技术的不断发展,遗传性眼病的基因背景正不断被揭示,根据遗传方式不同,可分为单基因遗传、多基因遗传和染色体畸变三大类,其中以单基因遗传病最多见,单基因遗传性眼病又可表现为常染色体显性、常染色体隐性和性染色体遗传三种遗传方式。有些眼病的遗传方式不止一种,临床表型各异,症状轻重不一,如目前已经鉴定和克隆的与先天性白内障相关的基因有26个,突变型已达40余种。研究表明,约有60%的遗传性眼病的分子基础较复杂,其发病机理尚未阐明,有待进一步研究。因此,较全面地研究分析遗传眼病致病相关位点和基因型的分子基础,对进一步阐明相应遗传眼病的发病机理,指导临床诊断有重要意义；为今后开展相关遗传眼病的基因诊断和产前诊断具有重要意义；并为进一步开展基因功能及基因治疗研究奠定了基础。本文就我们收集的我国浙江地区的两个先天性白内障家系、一个角膜营养不良家系和一个伴晶状体脱位的Marfan相关疾病家系共四个遗传病家系进行了基因定位与基因突变研究。研究方法对收集的家系成员进行详细的家族史及临床资料的采集,并对所有的家系成员进行全面的眼科检查,包括视力、裂隙灯、散瞳眼底检查等。抽取外周血样本,提取基因组DNA,应用聚合酶链反应(PCR)扩增疾病相关候选基因的所有编码区,利用直接测序法确定致病的突变基因及位点,借助生物信息学方法(Polyphen-2and Align-GVGD)对突变基因的结构和功能进行分析。结果 1、临床检查表明2个遗传性白内障家系的表型分别为后囊下白内障和全白内障,遗传方式均为常染色体显性遗传。针对家系候选基因进行突变筛查,在家系1中,对CRYAB、CRYBA1/A3、CRYBB2、GJA3、GJA8、CHMP4B、PITX3和EPHA2八个候选基因进行测序,发现在EPHA2基因编码区的c.2668CT的杂合改变,该变化导致了保守的精氨酸被半胱氨酸置换(p.R890C),该杂合突变不存在于200名健康对照成员中,PolyPhen-2和Align-GVGD分数均提示R890C为影响蛋白质结构和功能的有意义的突变。在家系2中,未发现CRYAA、CRYAB、CRYBA1/A3、 CRYBB1、CRYBB1、CRYBB2、CRYGC、CRYGD、CRYGS、GJA3、GJA8和MIP十二个已知致病基因的致病性突变； 2、家系3：角膜营养不良家系遗传方式为常染色体显性遗传。家系中所有患者均10岁之前发病,反复发生角膜上皮糜烂,视力逐渐下降；裂隙灯检查可见患者双眼角膜中央灰白色混浊,大小、数目不一,混浊呈点、圆圈状,混浊间的角膜透明,角膜各层均有受累,不同患者间角膜累及层次和深度不同,即使同一患者,双眼混浊累及深度也有不同。虽然裂隙灯下该家系患者患者角膜混浊形态与典型的颗粒状角膜营养不良I相符,但典型的颗粒状角膜营养不良I型患者多10岁以后发病,混浊首先并主要累及角膜基质层,随病情发展,上皮层有不同程度的受累,而该家系中患者发病年龄较早,最小患者只有5岁,混浊主要累及上皮层,伴少量混浊位于角膜浅基质层,并且有的患者混浊只累及上皮层,这些与典型的颗粒状角膜营养不良Ⅰ是不同的；候选基因测序发现家系中所有患者TGFBI基因1663位碱基发生CT碱基置换(c.1663CT),导致第555位精氨酸突变为色氨酸(p.R555W),家系中参与研究的正常人及100名健康对照组成员均未检测到此突变存在。 3、家系4：该家系患者诊断为伴晶状体脱位的Marfan相关疾病,其遗传方式符合常染色体显性遗传,家系中所有患者均表现为双眼晶状体脱位且不伴有骨骼系统和心血管系统异常,对家系致病候选基因FBN1的65个外显子、内含子和外显子交界区进行突变筛查,未发现该病的致病突变。结论 1、本文收集到常染色体显性遗传性白内障家系2个,研究报道了1个国内外未见报道的与先天性后囊下白内障有关的EPHA2的基因突变：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为该先天性白内障家系的致病基因； 3、在颗粒状角膜营养不良家系中,发现了由TGFBI基因经典型突变R555W引起的非典型颗粒状角膜营养不良Ⅰ型的家系,拓展了颗粒状角膜营养不良Ⅰ型的表型； 4、在伴晶状体脱位的Marfan相关疾病家系中,排除了已知候选基因FBN1的致病性突变,提示可能存在与该病相关的未知致病基因。
[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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R776.1

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