【摘要】：研究背景: 特應(yīng)性皮炎(atopic dermatitis, AD) ,是一種反復發(fā)作的慢性炎癥性皮膚病。其特征是皮膚瘙癢,皮損多形性并有滲出傾向,常伴有哮喘、過敏性鼻炎及血清IgE增高等,在兒童和青少年中較多見。近三十年來,AD發(fā)病率呈逐年上升趨勢,較之前增長達2-3倍之多,在兒童中已達到15%-30%,在成人中達到2%-10%。本病病因和發(fā)病機制尚不清楚。國內(nèi)外通過大量的人群、家系及雙生子研究均表明遺傳因素是構(gòu)成AD易感性的主要因素,屬多基因遺傳病。 多年來,國內(nèi)外學者主要應(yīng)用全基因組連鎖研究(Genome-wide linkage studies)、候選基因研究(Candidate genes studies)等研究策略搜尋AD的易感基因,并取得了一些進展。但這些研究均存在著一定的局限性,如全基因組連鎖研究應(yīng)用的微衛(wèi)星標記信息量有限,所定位區(qū)域往往較大,很難從其中找出真正的易感基因。同時,全基因組連鎖研究主要是針對家系研究,因而所發(fā)現(xiàn)的疾病易感位點是否能夠代表散發(fā)病例尚不清楚。對于候選基因研究,其選取候選基因主要根據(jù)研究者們認為最有可能導致疾病發(fā)生的易感基因,其分析的位點過少,故不可避免的存在著局限性,不可能全面搜尋到AD的易感基因。 2005年以后,隨著人類基因組計劃(HGP)和人類基因組單體型圖計劃(HapMap)的相繼完成,以及高通量基因分型技術(shù)的飛速發(fā)展和分型費用的降低,使得在大規(guī)模人群中開展全基因組關(guān)聯(lián)研究(genome-wide association study, GWAS)成為可能。這種研究方法對全基因組范圍內(nèi)的SNP(single nucleotide polymorphism)進行總體的分析,能夠更有效的發(fā)現(xiàn)和疾病有關(guān)聯(lián)的基因。GWAS是目前尋找復雜疾病易感基因的最有效方法之一。多項研究的成功有力證明了GWAS搜尋復雜疾病易感基因的有效性,也為AD易感基因的搜尋提供了新的思路和方法。2009年,Esparza-Gordillo等對德國人群進行了大規(guī)模的GWAS研究,發(fā)現(xiàn)11q13.5(C11orf30)為AD的易感位點。 AD在不同人種間存在遺傳異質(zhì)性,主要表現(xiàn)在發(fā)病率和患病率以及臨床表現(xiàn)存在一定的差異。由于多數(shù)AD研究主要是在歐洲高加索白人中進行的,因而這些AD遺傳易感基因/位點與中國人群的關(guān)聯(lián)性尚不清楚。因此對AD在中國人群中的易感基因研究很有必要。本研究首次在中國漢族人群中應(yīng)用GWAS方法在全基因組范圍內(nèi)對AD的易感基因進行篩查,以期發(fā)現(xiàn)AD的易感基因。 目的: 在中國漢族人群中,利用全基因組關(guān)聯(lián)分析研究方法在全基因組范圍內(nèi)搜尋與AD關(guān)聯(lián)的遺傳變異,鑒定中國漢族人AD的易感基因/位點。 方法: (1)初篩階段:在中國漢族人群中,利用Illumina Human 610-Quad全基因組SNP分型芯片對1012個AD病例和1362個對照進行SNPs分型。 (2)驗證階段:①驗證第一階段:經(jīng)過嚴格的數(shù)據(jù)質(zhì)控和統(tǒng)計分析,從初篩階段選擇最有意義的67個SNPs,使用Sequenom平臺在二個獨立的中國人群中進行驗證(北部人群:1119個病例和1203個對照;南部人群:2505個病例和6762個對照); ②驗證第二階段:從驗證第一階段中選取了19個SNPs,進一步加大樣本量使用Sequenom平臺在二個獨立的中國人群中進行驗證(北部人群:276個正常對照;南部人群:3956個正常對照)。之后,將上述附加的對照和之前已分型的對照數(shù)據(jù)及病例進行合并分析(驗證第一、二階段北部人群總計:1119個病例和1479個對照;驗證第一、二階段南部人群總計:2505個病例和10718個對照;GWAS初篩階段人群總計:1012個病例和1362個對照),以發(fā)現(xiàn)了達到全基因組顯著關(guān)聯(lián)性水平SNPs(P5×10~(-8)); ③驗證第三階段:將上一階段發(fā)現(xiàn)的有意義的SNPs進一步在德國的人群中使用TaqMan進行驗證(1,806個病例和3,256個正常對照)。 結(jié)果: (1)在驗證第一階段中,在中國漢族人群的3624例患者和7965例對照中對67個SNPs進行基因分型。將驗證第一階段的結(jié)果和GWAS結(jié)果合并分析后顯示,位于2個易感位點的3個SNPs達到了全基因組關(guān)聯(lián)顯著水平(P5×10~(-8)):①位于1q21.3(FLG)易感位點上的2個SNPs的rs3126085/rs11204971 (關(guān)聯(lián)性分別為P_(combined)=5.75×10~(-12), OR=0.83; P_(combined)=5.02×10~(-9), OR=0.86);②位于5q22.1(TMEM232 /SLC25A46)易感位點上的1個SNP rs7701890 (P_(combined)=4.33×10~(-8), OR=1.23); (2)在驗證第二階段中,對從驗證第一階段中選取的19個SNPs在附加的4232對照中進行分型,之后與第一階段的樣本進行合并分析,最后再與GWAS數(shù)據(jù)綜合分析后發(fā)現(xiàn)了位于3個易感位點區(qū)域(1q21.3, 5q22.1和20q13.33)內(nèi)的7個SNPs達到全基因組顯著水平:①位于1q21.3(FLG)易感位點上的2個SNPs rs3126085/rs11204971 (關(guān)聯(lián)性分別為P_(combined)=5.90×10~(-12), OR=0.82; P_(combined)=1.20×10~(-10), OR=0.85);②位于5q22.1(TMEM232/SLC25A46)易感位點上的4個SNPs rs7701890/rs10067777/rs13360927/rs13361382 (關(guān)聯(lián)性分別為P_(combined)=3.15×10~(-9), OR=1.24; P_(combined)=1.20×10~(-8), OR=1.24; P_(combined)=2.45×10~(-8), OR=1.22; P_(combined)=4.02×10~(-8), OR=1.22);③位于20q13.33(TNFRSF6B/ZGPAT)易感位點上的1個SNP rs6010620 (P_(combined)=3.0×10~(-8), OR=1.17)。此外,尚有1個提示性的易感位點,即位于10q21.2(ZNF365)易感位點上的SNP rs2393903 (P_(combined)=1.05×10~(-7), OR=1.15); (3)在驗證第三階段中,發(fā)現(xiàn)20q13.33(TNFRSF6B/ZGPAT)也與德國AD存在關(guān)聯(lián)性(rs6010620, P=2.87×10~(-5), OR=1.25)。 結(jié)論: 本研究通過大樣本量的全基因組關(guān)聯(lián)研究搜尋中國漢族人群AD易感基因,構(gòu)建了第一個中國漢族人群AD病例-對照的全基因組關(guān)聯(lián)分析數(shù)據(jù)庫。在中國人群中發(fā)現(xiàn)了2個新的AD易感基因/位點:5q22.1 (TMEM232/SLC25A46),20q13.33 (TNFRSF6 / ZGPAT);驗證出既往歐洲人群和亞洲人群報道的1q21.3(FLG)易感位點;發(fā)現(xiàn)了AD一個提示易感位點10q21.2(ZNF365)。此外,發(fā)現(xiàn)20q13.33 (TNFRSF6B / ZGPAT)與德國人群AD發(fā)病也存在關(guān)聯(lián)性。
[Abstract]:Research background:
Atopic dermatitis (AD) is a recurrent chronic inflammatory skin disease characterized by itching, pleomorphic skin lesions and exudative tendencies, often accompanied by asthma, allergic rhinitis and increased serum IgE, more common in children and adolescents. In the past 30 years, the incidence of AD has increased year by year, compared with the previous increase. As many as 2-3 times, it has reached 15-30% in children and 2-10% in adults. The etiology and pathogenesis of AD are still unclear.
Over the years, scholars at home and abroad have made some progress in searching for the susceptible genes of AD by using the strategies of Genome-wide linkage studies and Candidate genes studies. However, these studies have some limitations, such as the application of microsatellite markers in whole-genome linkage studies. It is not clear whether the susceptibility loci can represent sporadic cases. For candidate gene research, the candidate genes are selected mainly according to the researchers'opinion. The susceptibility genes most likely to lead to disease are too few to analyze, so there are inevitable limitations and it is impossible to search for the susceptibility genes of AD comprehensively.
Since 2005, with the completion of the Human Genome Project (HGP) and the HapMap, as well as the rapid development of high-throughput genotyping techniques and the reduction of typing costs, it has become possible to conduct genome-wide association studies (GWAS) in large populations. GWAS is one of the most effective methods to find susceptible genes for complex diseases. Successful studies have proved the effectiveness of GWAS in searching susceptible genes for complex diseases. In 2009, Esparza-Gordillo et al. conducted a large-scale GWAS study on the German population and found that 11q13.5 (C11orf30) was the susceptible site of AD.
The genetic heterogeneity of AD among different races is mainly manifested by differences in morbidity, prevalence and clinical manifestations. Since most AD studies have been conducted mainly among Caucasian Europeans, the association of these AD susceptibility genes/loci with Chinese populations is not yet clear. It is necessary to study the susceptibility genes of AD. GWAS method was used to screen the susceptibility genes of AD in Chinese Han population for the first time.
Objective:
In order to identify the susceptible gene/locus of AD in Chinese Han population, genome-wide association analysis was used to search for the genetic variation associated with AD in the whole genome.
Method:
(1) Preliminary screening stage: 1 012 AD cases and 1 362 controls were classified by using the Illumina Human 610-Quad genome-wide SNP typing chip.
(2) Verification phase: 1. Verification phase: After strict data quality control and statistical analysis, 67 most significant SNPs were selected from the initial screening phase and validated in two independent Chinese populations using the Sequenom platform (northern population: 1119 cases and 1203 controls; southern population: 2505 cases and 6762 controls);
(2) The second phase of validation: 19 SNPs were selected from the first phase of validation, and further enlarged the sample size using the Sequenom platform to validate in two independent Chinese populations (northern population: 276 normal controls; southern population: 3956 normal controls). Consolidated analysis (validation of 1,119 cases and 1,479 controls in the northern population of the first and second stages; validation of 2,055 cases and 10,718 controls in the southern population of the first and second stages; total of 1,012 cases and 1,362 controls in the GWAS preliminary screening stage) was performed to identify SNPs (P5 *10~(-8)) reaching the genome-wide significant association level.
(3) Verification Phase 3: The meaningful SNPs found in the previous phase were further validated with TaqMan in the German population (1,806 cases and 3,256 normal controls).
Result:
(1) In the first stage of validation, 67 SNPs were genotyped in 3 624 Chinese Han patients and 7 965 controls. The results of the first stage of validation and GWAS analysis showed that three SNPs located at two susceptibility loci reached a significant level of genome-wide association (P5 *10-8):1 at the 1q21.3 (FLG) susceptibility locus The rs3126085 / rs11204971 of the two SNPs were P_ (combined) = 5.75 * 10 ~ (- 12) and OR = 0.83; P_ (combined) = 5.02 * 10 ~ (- 9), OR = 0.86; and 1 SNP rs7701890 (P_ (combined) = 4.33 * 10 ~ (- 8), OR = 1.23, located at 5q22.1 (TMEM232 / SLC25A46) susceptibility site.
(2) In the second stage of validation, 19 SNPs selected from the first stage of validation were typed in an additional 4232 control, and then combined with the first stage of the sample analysis. Finally, comprehensive analysis with GWAS data revealed that 7 SNPs located in three susceptibility locus regions (1q21.3, 5q22.1 and 20q13.33) reached genome-wide significance. Levels: 1) Two SNPs at 1q21.3 (FLG) susceptibility locus rs3126085/rs11204971 (P_ (combined) = 5.90 *10 ~(-12), OR = 0.82; P_ (combined) = 1.20 *10 ~(-10), OR = 0.85); 2) Four SNPs at 5q22.1 (TMEM232/SLC25A46) susceptibility locus rs7701890/rs10067777/rs13360927/rs13362 (comb P_ 13362, respectively) (combined) =2.45 x 10 ~ (-8), OR = 1.22; P (combined) = = 2.45 x 10 ~ (-8), OR = 1.22; P (combined) = = 4.02 x 10 ~ (-8), OR = 1.22; P (combined) = = 4.02 x 10 ~ (-8), OR = 1.22); 3) 20 q13.33 (TNFRSF6B / ZGPAT) at the susceptibility site of 20q13.33 (TNFRSF6B / ZGPAT) susceptibility site of 1 SNP 6010 620 (combined) =3.0 (combined) =3.0 (combined) =3.0 ~ (-10 ~ (-8 Suggestive susceptibility locus, i.e. at 1 0q21.2 (ZNF365) SNP rs2393903 (P_ (combined) =1.05 * 10~ (-7), OR=1.15) on susceptible loci.
(3) In the third stage of verification, 20q13.33 (TNFRSF6B/ZGPAT) was also found to be associated with German AD (rs6010620, P = 2.87 x 10-5, OR = 1.25).
Conclusion:
In this study, we searched for AD susceptibility genes in Chinese Han population and constructed the first Chinese Han population case-control genome-wide association analysis database. Two new AD susceptibility genes/loci were found in Chinese population: 5q22.1 (TMEM232/SLC25A46), 20q13.33 (TNFRSF6/ZGPAT). In addition, 20q13.33 (TNFRSF6B/ZGPAT) was also found to be associated with AD in the German population.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R758.2

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