本文關(guān)鍵詞： 全外顯子組測(cè)序 家族性泛發(fā)性雀斑樣痣 色素性皮膚病 二代測(cè)序 色素性皮膚病 色素異常癥 遺傳性對(duì)稱性色素異常癥 ADAR1基因 基因突變　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景：雀斑樣痣在臨床上較常見,表現(xiàn)為大小不一的棕褐色斑點(diǎn)或斑片,可以作為綜合征的皮膚表現(xiàn)出現(xiàn),也可以作為疾病單獨(dú)出現(xiàn)。家族性泛發(fā)性雀斑樣痣臨床罕見,呈常染色體顯性遺傳,皮疹表現(xiàn)為泛發(fā)全身的雀斑樣痣,而缺乏系統(tǒng)受累的表現(xiàn),本病相關(guān)致病基因及致病機(jī)理尚不完全明確。全外顯子組測(cè)序技術(shù)自2005年商業(yè)化后即廣泛應(yīng)用于罕見病致病基因的檢測(cè),具有數(shù)據(jù)量相對(duì)全基因組測(cè)序小而診斷陽(yáng)性率較高的特點(diǎn)。研究目的：本研究擬通過全外顯子組測(cè)序技術(shù)探索一例三代3例患者的家族性泛發(fā)性雀斑樣痣家系的未知致病基因,并對(duì)候選致病基因在皮膚組織中的表達(dá)進(jìn)行研究。實(shí)驗(yàn)方法：本研究收集了一個(gè)家族性泛發(fā)性雀斑樣痣家系的臨床資料和家系資料,并繪制家系圖譜。對(duì)家系中2例患者和1例正常對(duì)照進(jìn)行了全外顯子組測(cè)序,重疊2例患者共同的變異,除外正常對(duì)照的良性變異,通過蛋白功能預(yù)測(cè)和與數(shù)據(jù)庫(kù)對(duì)比篩選得到一系列候選致病基因,通過家系共分離驗(yàn)證得到本病的候選致病基因,在2例散發(fā)的泛發(fā)性雀斑樣痣患者中進(jìn)行該基因的驗(yàn)證,并在200個(gè)隨機(jī)的正常人中篩查是否有檢出的突變。通過免疫組化、免疫熒光染色和Western Blot明確該致病基因在正常組織和病變組織中的表達(dá)。實(shí)驗(yàn)結(jié)果：EPHB1基因的突變?cè)诙鄠�(gè)蛋白功能預(yù)測(cè)軟件中預(yù)測(cè)有害,在多個(gè)基因數(shù)據(jù)庫(kù)中均無突變相關(guān)數(shù)據(jù),且與疾病共分離,在200個(gè)正常人中也未檢測(cè)到該突變,免疫組化,免疫熒光染色和Western Blot均證實(shí)EPHB1基因在患者皮損中的表達(dá)高于正常對(duì)照組。但該突變?cè)贓xAc數(shù)據(jù)庫(kù)中頻率高于疾病發(fā)病率,且在2例散發(fā)的不伴有系統(tǒng)受累的泛發(fā)性雀斑樣痣患者中均未檢出該基因的突變。結(jié)論：EPHB1基因不能明確是家族性泛發(fā)性雀斑樣痣的致病基因,EPHB1基因可能與色素性疾病的發(fā)病相關(guān)。研究背景：色素異常癥是一組疾病的統(tǒng)稱,臨床上較為常見,其皮疹表現(xiàn)為同時(shí)出現(xiàn)的色素增加和色素減退斑,一般不伴有系統(tǒng)癥狀。遺傳性對(duì)稱性色素異常癥和遺傳性泛發(fā)性色素異常癥是其中兩種經(jīng)典類型,兩者的臨床和組織病理表現(xiàn)相似,但兩者的致病基因不同,基因診斷在鑒別不典型病例中有一定優(yōu)勢(shì)。基于目標(biāo)區(qū)域捕獲的二代測(cè)序技術(shù)以其低成本,高通量的特點(diǎn),適用于異質(zhì)性疾病的基因篩查,但關(guān)于其在色素性疾病篩查診斷中的報(bào)道尚較少。研究目的：本研究擬通過基于目標(biāo)區(qū)域捕獲的二代測(cè)序技術(shù),批量檢測(cè)臨床診斷為泛發(fā)性色素異常癥的患者的致病基因,進(jìn)而驗(yàn)證二代測(cè)序技術(shù)在色素性皮膚病基因診斷中的應(yīng)用。實(shí)驗(yàn)方法：本研究收集了臨床診斷為遺傳性泛發(fā)性色素異常癥的7個(gè)家系的8名患者的臨床資料和全血標(biāo)本。因?yàn)檫z傳性對(duì)稱性色素異常和遺傳性泛發(fā)性色素異常皮損容易混淆,將目標(biāo)區(qū)段設(shè)定為ADAR1基因和ABCB6基因,提取患者DNA后通過超聲打斷,末端修復(fù),鏈接接頭等完成文庫(kù)制備,通過Nimblegen序列捕獲芯片完成目標(biāo)區(qū)段的雜交捕獲,并通過Hiseq2500測(cè)序儀進(jìn)行上機(jī)測(cè)序,最后通過信息分析和數(shù)據(jù)解讀獲得突變信息。實(shí)驗(yàn)結(jié)果：在收集到的8例臨床表現(xiàn)為泛發(fā)性色素異常癥的患者中,來自兩個(gè)家系的3例患者檢出了致病基因突變。本研究總結(jié)了檢出突變的患者的臨床表現(xiàn)及其家系圖。這3例患者都有全身彌漫分布的色素增加和色素減退斑,臨床均診斷為泛發(fā)性色素異常癥,其中1例患者檢出了ABCB6基因的突變(c.1270TC),而另外2例患者檢出了ADAR1基因的突變(c.1325CG)。結(jié)論：不典型的遺傳性對(duì)稱性色素異常皮疹可以不局限于四肢末端,也可累及軀干�；谀繕�(biāo)捕獲的二代測(cè)序技術(shù)降低了測(cè)序的成本和周期,適用于色素性疾病的臨床檢測(cè)。研究背景：遺傳性對(duì)稱性色素異常癥是一種罕見的遺傳性色素性皮膚病。本病表現(xiàn)為常染色體顯性遺傳,臨床上皮疹表現(xiàn)為以四肢伸側(cè)為主的網(wǎng)狀色素增加和色素減退斑。本病致病基因已知。研究目的：本研究擬通過Sanger測(cè)序的方法,檢驗(yàn)臨床收集到的5例遺傳性對(duì)稱性色素異常癥患者的已知致病基因,明確其致病突變。實(shí)驗(yàn)方.法：收集患者的臨床及家系資料,采集外周血,提取外周血DNA, PCR擴(kuò)增所有ADAR1基因的外顯子及其側(cè)翼的調(diào)控序列。Sanger法進(jìn)行直接測(cè)序。實(shí)驗(yàn)結(jié)果：在5例臨床診斷遺傳性對(duì)稱性色素異常的家系中均檢測(cè)到ADAR1的基因突變,突變均位于外顯子區(qū),影響蛋白結(jié)構(gòu)和功能。結(jié)論：檢出的突變?yōu)檫z傳性對(duì)稱性色素異常癥的致病突變。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R758.5

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