遗传性色素性皮肤病基因突变及相关致病机理的研究

发布时间：2018-02-28 01:49

本文关键词： 全外显子组测序家族性泛发性雀斑样痣色素性皮肤病二代测序色素性皮肤病色素异常症遗传性对称性色素异常症 ADAR1基因基因突变　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：研究背景：雀斑样痣在临床上较常见,表现为大小不一的棕褐色斑点或斑片,可以作为综合征的皮肤表现出现,也可以作为疾病单独出现。家族性泛发性雀斑样痣临床罕见,呈常染色体显性遗传,皮疹表现为泛发全身的雀斑样痣,而缺乏系统受累的表现,本病相关致病基因及致病机理尚不完全明确。全外显子组测序技术自2005年商业化后即广泛应用于罕见病致病基因的检测,具有数据量相对全基因组测序小而诊断阳性率较高的特点。研究目的：本研究拟通过全外显子组测序技术探索一例三代3例患者的家族性泛发性雀斑样痣家系的未知致病基因,并对候选致病基因在皮肤组织中的表达进行研究。实验方法：本研究收集了一个家族性泛发性雀斑样痣家系的临床资料和家系资料,并绘制家系图谱。对家系中2例患者和1例正常对照进行了全外显子组测序,重叠2例患者共同的变异,除外正常对照的良性变异,通过蛋白功能预测和与数据库对比筛选得到一系列候选致病基因,通过家系共分离验证得到本病的候选致病基因,在2例散发的泛发性雀斑样痣患者中进行该基因的验证,并在200个随机的正常人中筛查是否有检出的突变。通过免疫组化、免疫荧光染色和Western Blot明确该致病基因在正常组织和病变组织中的表达。实验结果：EPHB1基因的突变在多个蛋白功能预测软件中预测有害,在多个基因数据库中均无突变相关数据,且与疾病共分离,在200个正常人中也未检测到该突变,免疫组化,免疫荧光染色和Western Blot均证实EPHB1基因在患者皮损中的表达高于正常对照组。但该突变在ExAc数据库中频率高于疾病发病率,且在2例散发的不伴有系统受累的泛发性雀斑样痣患者中均未检出该基因的突变。结论：EPHB1基因不能明确是家族性泛发性雀斑样痣的致病基因,EPHB1基因可能与色素性疾病的发病相关。研究背景：色素异常症是一组疾病的统称,临床上较为常见,其皮疹表现为同时出现的色素增加和色素减退斑,一般不伴有系统症状。遗传性对称性色素异常症和遗传性泛发性色素异常症是其中两种经典类型,两者的临床和组织病理表现相似,但两者的致病基因不同,基因诊断在鉴别不典型病例中有一定优势。基于目标区域捕获的二代测序技术以其低成本,高通量的特点,适用于异质性疾病的基因筛查,但关于其在色素性疾病筛查诊断中的报道尚较少。研究目的：本研究拟通过基于目标区域捕获的二代测序技术,批量检测临床诊断为泛发性色素异常症的患者的致病基因,进而验证二代测序技术在色素性皮肤病基因诊断中的应用。实验方法：本研究收集了临床诊断为遗传性泛发性色素异常症的7个家系的8名患者的临床资料和全血标本。因为遗传性对称性色素异常和遗传性泛发性色素异常皮损容易混淆,将目标区段设定为ADAR1基因和ABCB6基因,提取患者DNA后通过超声打断,末端修复,链接接头等完成文库制备,通过Nimblegen序列捕获芯片完成目标区段的杂交捕获,并通过Hiseq2500测序仪进行上机测序,最后通过信息分析和数据解读获得突变信息。实验结果：在收集到的8例临床表现为泛发性色素异常症的患者中,来自两个家系的3例患者检出了致病基因突变。本研究总结了检出突变的患者的临床表现及其家系图。这3例患者都有全身弥漫分布的色素增加和色素减退斑,临床均诊断为泛发性色素异常症,其中1例患者检出了ABCB6基因的突变(c.1270TC),而另外2例患者检出了ADAR1基因的突变(c.1325CG)。结论：不典型的遗传性对称性色素异常皮疹可以不局限于四肢末端,也可累及躯干。基于目标捕获的二代测序技术降低了测序的成本和周期,适用于色素性疾病的临床检测。研究背景：遗传性对称性色素异常症是一种罕见的遗传性色素性皮肤病。本病表现为常染色体显性遗传,临床上皮疹表现为以四肢伸侧为主的网状色素增加和色素减退斑。本病致病基因已知。研究目的：本研究拟通过Sanger测序的方法,检验临床收集到的5例遗传性对称性色素异常症患者的已知致病基因,明确其致病突变。实验方.法：收集患者的临床及家系资料,采集外周血,提取外周血DNA, PCR扩增所有ADAR1基因的外显子及其侧翼的调控序列。Sanger法进行直接测序。实验结果：在5例临床诊断遗传性对称性色素异常的家系中均检测到ADAR1的基因突变,突变均位于外显子区,影响蛋白结构和功能。结论：检出的突变为遗传性对称性色素异常症的致病突变。
[Abstract]:Background: lentigines is common in clinic, is the size of the brown spots or patches, can be used as a skin manifestation of syndrome, also can appear as a separate disease. Familial generalized lentigo clinical rare, autosomal dominant, skin rash for lentigo pan of the body, and the lack of involvement in the system, the disease related genes and pathogenic mechanism is still not completely clear. The detection of exon sequencing technology since 2005 after commercialization that widely used in rare disease genes, with the amount of data is relatively small and whole genome sequencing of the positive rate of diagnosis of higher. Objective: This study by whole exome sequencing technology to explore a three generation of unknown pathogenic gene in 3 cases of patients with idiopathic familial pan lentigo pedigrees, and candidate genes in skin tissue of the table As experimental research. Methods: This study collected a family of generalized lentigo pedigrees clinical and pedigree data, and draw the family patterns of the family. In 2 patients and 1 cases of normal controls were examined by whole exome sequencing, 2 cases of patients with common overlapping except normal variation, benign variation, the prediction of protein function and comparison with the database obtained a series of candidate genes, and verified the candidate genes of the disease by family were isolated, for verification of the gene in 2 sporadic generalized lentigo patients, and screening in 200 random normal people have mutations detected. By immunohistochemistry, immunofluorescence staining and Western Blot clear the virulence gene expression in normal tissues and tissues. Results: EPHB1 gene mutation in multiple protein function prediction software In the prediction of harmful, in multiple gene mutations were not found in the database of relevant data, and cosegregated with the disease, in 200 normal people did not detect the mutation, immunohistochemistry, immunofluorescence staining and Western Blot confirmed the expression of EPHB1 gene in patients with skin lesions than that in normal control group. But the mutation the frequency is higher than the incidence of a disease in the ExAc database, and in 2 cases of sporadic without involvement of generalized lentigo patients were not detected in the gene mutation. Conclusion: EPHB1 gene is not clear the pathogenic gene of familial generalized lentigo, pathogenesis of EPHB1 gene might be related with pigment disease. Background: hereditaria is referred to as a group of diseases, more common clinical manifestations of the rash, at the same time, hyperpigmentation and hypopigmented macules without symptoms. Dyschromatosis Symmetrica abnormal disease And Dyschromatosis univcrsalis hereditaria is one of the two classical types, clinical and histopathological manifestations of the two are similar, but the difference between the two genes, gene diagnosis has certain advantages in the differential diagnosis of atypical cases. The target area to capture the two generation sequencing technology based on the characteristics of its low cost, high throughput gene. Screening for disease heterogeneity, but the reports about its in diagnosis of pigmented diseases is still less. Objective: This study aimed to capture the two generation sequencing technology target area based on the patients with clinical diagnosis of mass detection pathogenic gene of Dyschromatosis pan, and then verify the application of the two generation sequencing technology in the gene diagnosis of pigmented skin disease. Methods: 7 families, this study collected the clinical diagnosis of Dyschromatosis hereditary pan 8 patients clinical data and blood samples. Hereditary Dyschromatosis symmetrical and Dyschromatosis univcrsalis hereditaria lesions are easily confused, the target region is set to the ADAR1 gene and ABCB6 gene, were extracted by ultrasonic DNA interrupt, end repair, link connector to complete library preparation, hybrid capture by Nimblegen sequence capture chip to complete the target section, and the sequencing by Hiseq2500 sequencing, finally get the mutation information through information analysis and interpretation of data. Results: Dyschromatosis pan in the clinical manifestations of 8 cases were collected, 3 cases from two families were found in the disease causing mutation. This study summarized the clinical manifestations and their family figure detection of mutation patients. The 3 patients have systemic diffuse hyperpigmentation and depigmentation spots, the clinical diagnosis of Dyschromatosis pan, among which 1 cases were detected by ABCB6 The gene mutation (c.1270TC), and the other 2 cases were detected the mutation of ADAR1 gene (c.1325CG). Conclusion: hereditary atypical Dyschromatosis symmetrical rash can not confined to the extremities, trunk may also be involved. The two generation sequencing technology target acquisition can reduce the cost and cycle sequencing based on clinical suitable for detecting pigmented diseases. Background: DSH is a rare hereditary pigmented skin disease. The disease is autosomal dominant, clinically characterized by rash in the extensor extremities mainly increased and reticular pigment depigmentation disease. The pathogenic gene is known. Objective: This study by Sanger sequencing method, 5 patients with DSH known pathogenic genes of clinical test and collected, clear the pathogenic mutation. The experiment. Method: collect clinical patients Data of bed and family, collect peripheral blood, extracting peripheral blood DNA and.Sanger regulatory sequence of ADAR1 gene by PCR amplification of all exons and flanking by direct sequencing. Results: in the families of 5 patients with a clinical diagnosis of hereditary symmetry pigment abnormalities were detected in the ADAR1 gene mutation. The mutations were located in exon region, the structure and function of protein. Conclusion: the detection of mutations for DSH mutations.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R758.5

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