【作者】 梁燕华；

【导师】 张学军； 杨森； 周育文；

【作者基本信息】 安徽医科大学 ， 皮肤病与性病学， 2007， 博士

【摘要】 〖白癜风连锁区域及其内易感基因的发现〗白癜风(OMIM:#193200)是一种常见的色素脱失性皮肤疾病,全球人群患病率大约为0.5%,主要表现为皮肤、粘膜及毛发的黑素细胞选择性减少或消失导致的白斑、灰/白发,其病因和发病机制尚不明了。目前已有以下学说:自身免疫学说、遗传学说、黑素细胞自身破坏学说及生物化学学说等。而近年来,国内外大量研究表明遗传因素与白癜风发病密切相关,很可能是一种多基因遗传病,同时还发现其它各种学说均存在其遗传学基础。目前已有文献报道白种人中的4个白癜风易感位点—17p13(SLEV1)、1p31(AIS1)、7q(AIS2)、8p(AIS3)。2005年,美国白癜风研究小组采取候选基因研究策略,对1p31(AIS1)易感区域内的9个候选基因进行了检测,结果发现FOXD3基因启动子区域的-639G/T与家族型白癜风相关;2007年,通过对17p13(SLEV1)位点的精细定位和基于SNP的相关性研究发现,17p13区域内存在与白癜风及/或其他自身免疫性疾病密切相关的易感基因NALP1,由于研究对象50%以上白癜风患者伴发其他自身免疫性疾病,影响严重,该重大发现以长篇论著发表在医学领域最权威的《New England Journal of Medicine》(IF: 44.016/2005),从另一侧面说明白癜风的自身免疫学基础。2004年,本课题组对106个中国汉族人白癜风家系进行了全基因组扫描,在4q13-q21区域获得最强连锁信号(NPL =4.62, p=0.000003),被OMIM收录并命名为AIS4 (#609400);另外,发现5个提示性连锁信号,分别为22q12(NPL=3.49,p=0.0003)、6p21-p22(NPL=3.16,p=0.00092)、6q24-q25(NPL=2.98,p=0.0016)、1p36(NPL=2.37,p=0.0093)、14q12-q13(NPL =1.4,p=0.077)。〖研究目的〗借鉴从AIS1和SLEV1易感位点内发现易感基因的定位候选策略及成功经验,本课题拟对中国汉族人白癜风肯定连锁位点AIS4进行大规模高通量相关研究,搜寻候选基因(该重大课题正在进行中,本文不作阐述);同时,对5个提示性连锁位点进行精细定位,进一步明确这些提示连锁位点是否真正蕴藏着白癜风的易感基因,同时缩小易感区域(本文的研究内容)。〖研究材料和方法〗用覆盖5个提示易感位点的40个微卫星标记,对143个白癜风家系共736个体(包括369名患者和367名无白癜风表型者)进行基因组扫描研究。以Merlin软件进行基因分型校订,以GENEHUNTER软件(2.0 Version)对基因分型结果进行参数和非参数连锁分析。〖精细定位研究结果〗1.肯定连锁位点:(1) 22q12:前期研究发现D22S280-D22S283区域存在提示连锁信号(NPL=1.75,p = 0.039),对该区域(D22S280-D22S283)及其邻近范围共增加5个微卫星标记以尽可能覆盖连锁区间,所有微卫星位点平均信息量达到79%( D22S1167-D22S1163-D22S1150-D22S280-D22S1265-D22S283-D22S272),平均遗传距离为2.7cM。通过对全扫时使用的106个家系、共7个微卫星标记进行基因分型,整合先前全扫分析的数据一起进行分析,多点非参数连锁分析揭示在D22S1167-D22S1150位点处获得最大的NPL值为3.49(p=0.0003),在显性遗传模式下,在同样位置获得最大的HLOD值2.28和较高的连锁家系比例(α=35%),较全扫时106个家系仅对D22S280和D22S283的分析结果有明显提高,表明前期研究微卫星标记的密度不够;结合新增37个家系数据,对143个家系进行基因分型后统计分析,多点非参数连锁分析揭示在D22S1167-D22S283位点处获得最大的NPL值4.14(p=0.000015);多点参数连锁分析在隐性遗传模式下,在相同位置获得最大的HLOD值为3.26 (α= 37.3% ) ,遗传距离跨越在22q22.1-22q22.3(0-7.59cM)区域内的7.6cM;(2) 6p21-p22:前期6p21-p22区域研究所用标记密度很高,针对上述12个微卫星标记,本次研究在新增加的37个家系中进行基因分型,结合全扫时106个家系数据进行连锁分析。143个家系两点连锁分析揭示在D6S289位点处获得最大的NPL值为2.9(p =0.0016);且在相同的位点在显性遗传模式下、外显率为99%时获得最大的HLOD值为3.54(α=49.4%);多点连锁分析揭示在D6S289-D6S1584位点处获得最大的NPL值为4.1(p =0.000018);且在相同参数设置获得最大的HLOD值为3.73(α=42.3%)。两点和多点参数和非参数连锁分析结果均明显提高,达到肯定连锁标准;2.否定连锁位点14q12-q13:在14q12-q21区域内,通过在D14S70附近增加3个微卫星标记,对143个家系进行基因分型,两点、多点的参数和非参数连锁分析均未提供连锁信息,因此,可以排除该位点与白癜风的连锁关系,前期结果可能为假阳性结果;3.提示连锁位点:(1) 6q24-q25:对累积的143个家系进行了两点、多点参数和非参数连锁分析,均比106个家系全基因扫描时的结果明显提高。HLOD值最高为1.95(α=17.1%);NPL最高为3.33,对应p值为0.00038。以上结果提示该区域与白癜风发病提示性连锁的易感基因位点;(2) 1p36:对全扫时提示连锁区域D1S2734-D1S234区域及其附近共6个微卫星标记进行两点、多点的参数和非参数连锁分析,结果发现:多点连锁分析最高NPL值为2.59(p=0.004)和HLOD值0.78(α=10%),两点和多点参数和非参数连锁分析结果均较全扫时有所升高,支持1p36提示连锁;〖结论〗1. 22q12和6p21-p22证实为肯定连锁位点,可能存在白癜风的易感基因;2. 6q24-q25和1p36提示连锁信号进一步增强,但仍未达到肯定连锁标准;3.否定14q12-q13为白癜风易感基因所在位点。中国汉族人白癜风遗传因素复杂,存在多个易感基因位点。更多还原

【Abstract】 〖Mapping and Identification of vitiligo Susceptibility Genes〗Vitiligo (OMIM: #193200) is an acquired depigmentation disorder of the skin and hair caused by the selective destruction of melanocytes from the skin and hair that gives rise to well-defined white patches. About 0.5% of the world’s population is affected by the disease, regardless of age, gender or skin color. However, relatively little is known of its pathogenesis. Several theories have been proposed to explain the destruction of melanocytes in vitiligo. These include melanocyte self-destruction, biochemical hypothesis, neural hypothesis, and autoimmune mediated melanocyte elimination. Strong evidences from twin and family studies underline the importance of genetic factors in the development of vitiligo, with polygenic features. And genetic evidences are also consistent with the basis of other theories.Up to date, four susceptibility loci has been reported on different chromosomes, 17p13 (SLEV1), 1p31 (AIS1), 7q (AIS2) and 8p (AIS3). By association analysis for 9 candidates in the 1p31 (AIS1), -639G/T in the promoter region of FOXD3 gene showed significantly related with vitiligo; Variants in or around NALP1 on chromosome 17p13 gene showed an association with vitiligo alone, with an extended autoimmune and autoinflammatory disease phenotype, or with both. The NALP1 findings are discussed on《New England Journal of Medicine》(IF: 44.016/2005), also indicating the autoimmunity basis on another view.In 2004, we have performed a genome-wide scan for 106 Chinese Han vitiligo families. Linkage results provided significant evidence for a major vitiligo susceptibility locus 4q13-q21 (max NPL=4.62, p=0.000003), which was later named AIS4 with OMIM#609400, and suggestive evidences for linkage on other regions, 22q12(max NPL=3.49, p=0.0003), 6p21-p22(max NPL=3.16, p=0.00092), 6q24-q25(max NPL=2.98, p=0.0016), 1p36(max NPL=2.37, p=0.0093), 14q12-q13(max NPL =1.4, p=0.077).〖Objectives〗Regarding the successful identification of susceptibility genes from the AIS1 and SLEV1 with mapping and functional candidate strategy, we aim to search susceptibility genes from the significant region AIS4 with large scale and high-through method (still being on the process). To make clear whether susceptibility genes are really covered in the suggestive loci, we would like to finely map the 5 suggestive loci with more families and high density markers (the major contents of this paper).〖Materials and Methods〗We have finely scanned 143 families of 736 individuals (369 affected and 367 unaffected) for the 5 suggestive loci with 40 microsattellites. Genotypes were corrected with Merlin software. Parameter or nonparameter linkage analysis were made with GENEHUNTER software 2.0 Version.〖Results〗1. significant linkage findings:(1) 22q12:As we reported elsewhere, a suggestive signal was indicated by a maximal nonparametric linkage score of 1.75 (p = 0.039) by two marks (D22S280 and D22S283). In this study, we saturate this region with additional 5 markers. Hence, a total of 7 markers (D22S1167-D22S1163-D22S1150-D22S280-D22S1265-D22S283-D22S272) were included to maximize the information content. The average information of markers at 22q12 reached 79%, with average genetic distance of 2.7 cM. To determine the extent of allele sharing at chromosome 22q12, we have genotyped the joint 143 vitiligo families. With higher marker intensity studied here, finer mapping of the same 106 families cohort used in our previous genome-wide scan has improved the linkage results, respectively by multipoint NPL score from 1.75 (p=0.039) to 3.49(p=0.0003), and HLOD from 0.72(α=19%) to 2.28(α=35%). Multipoint nonparametric linkage analysis of the combined 143 families yielded a maximum NPL score of 4.14 (p=0.000015) at the marker position D22S1163. Once the evidence of linkage at 22q12 was found, we performed parametric multipoint analyses, employing simple dominant and recessive models. A significant evidence of linkage is found by assuming genetic heterogeneity (HLOD=3.26) under a dominant model, and about 37.3% families are estimated to be linked with this locus. These summary data achieved genome-wide criteria for highly significant linkage, providing 22q12 as a vitiligo susceptibility locus that contributes to vitiligo.(2) 6p21-p22:Similar analysis was performed in the 143 families on 6p21-p22, another region designated as“suggestive linkage locus”in previous report. The marker density of original 12 markers was saturated with an interval distance of 1.9 cM. In this view, we have genotyped the same 12 markers in the new 37 families. two-point analysis showed the max NPL of 2.9(p =0.0016)and a dominant HLOD of 3.54(α=49.4%), which are consistent with multipoint parameter and nonparameter score (NPL=4.1, p=0.000018;HLOD=3.73,α=42.3%). Both two and multiple point linkage analysis provided significant linkage signals on 6p21-p22.2. negative linkage signal (14q12-q13):With respect to the suggested vitiligo susceptibility locus on chromosome 14q, no suggestive indications of linkage were obtained either with nonparametric or parametric analyses for 4 microsatellite markers flanking D14S70. The previous signal seems to be a false positive linkage locus by the present negative results;3. Suggestive linkage signals: (1) 6q24-q25:Both two and multiple point linkage analysis provided stronger linkage signals on 6q24-q25 than what we got from the 106 families in the genome-wide scanning, with max NPL of 3.3 3(p=0.00038) and HLOD of 1.95(α=17.1%), which also indicated suggestive signal;(2) 1p36:Fine mapping of the 1p36 region, flanked by D1S2828 (50.1 cM) and D1S449 (60.9 cM), was performed with 6 original STRs. When the 37 new families were analyzed in combination with the previous family, we obtained a summary multipoint nonparametric LOD score of 2.59 (P=0.0042) at the 1.1 cM interval D1S234-D1S2885 .The recessive heterogeneity LOD (HLOD) at this interval was 0.78 withα=10%. These summary data still achieve genome-wide criteria for suggestive linkage;〖Conclusions〗1. Identification 22q12 and 6p21-p22 as significant susceptibility loci,which potentially contain the susceptibility genes;2. Stronger linkage signals on 6q24-q25and1p36, but still in suggestive threshold;3. Exclusion of 14q12-q13 as susceptibility locus in Chinese Han vitiligo families;更多还原