【摘要】： 研究背景 銀屑病是一種臨床常見(jiàn)的以紅斑鱗屑為基本表現(xiàn)的慢性炎癥性皮膚疾病。該病發(fā)病原因不清,可能和遺傳、免疫以及各種環(huán)境因素共同作用有關(guān)。家系研究及人群流行病學(xué)研究證實(shí)了銀屑病的遺傳學(xué)發(fā)病基礎(chǔ),早期的連鎖分析、候選基因研究及全基因組關(guān)聯(lián)分析研究(Genome-Wide Association Study ,GWAS)發(fā)現(xiàn)銀屑病的多個(gè)易感基因位點(diǎn),其中多數(shù)未能被證實(shí)。目前發(fā)現(xiàn)的遺傳信號(hào)不能完全解釋銀屑病的發(fā)病機(jī)制,提示可能尚有其他遺傳因素的存在。此外,中國(guó)和高加索人群GWAS結(jié)果的差異提示該病在不同人群中的易感等位基因或位點(diǎn)存在異質(zhì)性。 人類基因組計(jì)劃(Human Genome Project,HGP)和國(guó)際單體型圖計(jì)劃(International HapMap Project ,Hap2Map)的完成,為復(fù)雜疾病的易感基因研究提供了重要的生物信息和分析工具。GWAS是近幾年興起的一種搜尋復(fù)雜疾病易感基因的強(qiáng)效有力的方法,被科學(xué)界認(rèn)為是目前最為有效的搜尋重大疾病易感基因的研究方法之一。 本研究小組前期首次對(duì)中國(guó)人群銀屑病易感基因進(jìn)行了大規(guī)模GWAS研究。除證實(shí)高加索人群兩個(gè)已知的易感位點(diǎn):MHC和IL12B,還發(fā)現(xiàn)了位于染色體1q21上一個(gè)新的銀屑病易感位點(diǎn)LCE基因簇。通過(guò)銀屑病、系統(tǒng)性紅斑狼瘡、白癜風(fēng)等多個(gè)皮膚復(fù)雜疾病易感基因的GWAS研究,目前已建立了較完整的復(fù)雜疾病易感基因研究的GWAS操作平臺(tái)和統(tǒng)計(jì)分析方法,并通過(guò)建立的遺傳資源庫(kù)收集了大量的銀屑病和對(duì)照樣本。 本研究是在前期銀屑病易感基因GWAS研究的基礎(chǔ)上,采取六階段驗(yàn)證方法,對(duì)來(lái)自中國(guó)的8,312個(gè)病例和12,919個(gè)對(duì)照進(jìn)行驗(yàn)證試驗(yàn),同時(shí)對(duì)來(lái)自歐洲的3,293個(gè)病例和4,188個(gè)正常對(duì)照以及254個(gè)核心家系進(jìn)行驗(yàn)證,以進(jìn)一步搜尋漢族人銀屑病其他可能的易感基因位點(diǎn)。 目的 進(jìn)一步搜尋中國(guó)漢族人銀屑病相關(guān)的易感基因位點(diǎn);確定漢族人相關(guān)的銀屑病遺傳危險(xiǎn)因素;比較中國(guó)人群與高加索人群的銀屑病遺傳學(xué)異質(zhì)性。 方法 (一)樣本來(lái)源: 驗(yàn)證1(4,610個(gè)病例和5,373名對(duì)照)和驗(yàn)證2(2,024例患者和5,495名對(duì)照)的樣本來(lái)源于全國(guó)多家合作醫(yī)院收集的漢族人群。驗(yàn)證3(539名病例和824名健康對(duì)照)的樣本,來(lái)自新疆維吾爾自治區(qū)的維吾爾族人群。驗(yàn)證4(823個(gè)病例和1,840名對(duì)照)的樣本來(lái)自德國(guó)。驗(yàn)證5(2,470名患者和2,348名對(duì)照)的樣本及驗(yàn)證6的254個(gè)同胞對(duì)核心家系來(lái)自美國(guó)。每一階段的驗(yàn)證樣本中,病例和對(duì)照均來(lái)源于同一地區(qū)和同一種族。 (二)驗(yàn)證階段SNP的選取: 本研究采用了兩種挑選SNP的方法。第一種方法,在1,139病例和1,227對(duì)照的關(guān)聯(lián)分析結(jié)果中挑選了21個(gè)P10-5的SNP。第二種方法:將3496例其他自身免疫性相關(guān)疾病作為擬對(duì)照,同1,227例正常對(duì)照一起共5173例,作為共同的對(duì)照標(biāo)本,統(tǒng)計(jì)分析,參與選點(diǎn),從中選取P10-5的40個(gè)SNP參與進(jìn)一步驗(yàn)證。最終選取61個(gè)SNP參與驗(yàn)證實(shí)驗(yàn)。 驗(yàn)證2共有35個(gè)SNP被選取分型,其中包括:1)從驗(yàn)證1中挑選的20個(gè)P 0.05的SNP; 2)從高加索人群中報(bào)道銀屑病相關(guān)的8個(gè)位點(diǎn)中選取的15個(gè)SNP。 (三)GWAS中基因分型和質(zhì)量控制: 基于前期GWAS(包括1,139銀屑病患者和1,132對(duì)照的620,901 SNP和CNV探針)的分型數(shù)據(jù),加入95例新的正常對(duì)照基因分型數(shù)據(jù)以解釋數(shù)據(jù)質(zhì)控。采用Cochran-Armitage trend檢驗(yàn)法計(jì)算基因型-表型的關(guān)聯(lián)性。 (四)驗(yàn)證階段的基因分型和質(zhì)量控制: 驗(yàn)證1-3采用Sequenom MassArray系統(tǒng)(San Diego, USA)和Biosystems TaqMan assays (USA)進(jìn)行基因分型。MALDI-TOF MS檢測(cè)等位基因。MassARRAY TYPER軟件(Sequenom公司)完成質(zhì)譜圖分析。 驗(yàn)證4和5的基因分型采用TaqMan assays (Applied Biosystems)完成。數(shù)據(jù)的質(zhì)量控制通過(guò)PLINK 1.05完成。 驗(yàn)證6的標(biāo)本運(yùn)用Sequenom MassArray system完成。 (五)統(tǒng)計(jì)分析: 采用統(tǒng)計(jì)分析軟件R繪制染色體圖和Q-Q圖。PCA分析法評(píng)估有異常偏離樣本及人群分層存在。病例和對(duì)照進(jìn)行PCA分析,經(jīng)過(guò)質(zhì)量控制后,SNPs分型數(shù)據(jù)在GWAS階段進(jìn)行分析。 在驗(yàn)證1-5中,分別采用Cochran-Armitage trend檢驗(yàn)計(jì)算其關(guān)聯(lián)性。在多階段驗(yàn)證研究的合并分析中,采用固定效應(yīng)模型或隨機(jī)效應(yīng)模型分析進(jìn)行分析。家系的關(guān)聯(lián)研究采用半似然函數(shù)進(jìn)行病例對(duì)照關(guān)聯(lián)分析。 多重logistic回歸分析用于檢測(cè)區(qū)域內(nèi)的信號(hào)的獨(dú)立性。 在病例中采用將臨床亞型作為輸出變量的logistic回歸分析檢測(cè)與臨床亞型有關(guān)的SNP。 采用異質(zhì)性檢驗(yàn)方法檢測(cè)不同組間的遺傳異質(zhì)性。 結(jié)果 一、第一階段驗(yàn)證中,在漢族人群的4,610例患者和5,373例對(duì)照中對(duì)61個(gè)SNP進(jìn)行基因分型。綜合GWAS和驗(yàn)證1的結(jié)果顯示其中的4個(gè)SNP達(dá)到了全基因組顯著性標(biāo)準(zhǔn)(P5.0×10-8),這四個(gè)SNP分別是位于5q33.1(TNIP1/ANXA6)區(qū)域的rs3762999和rs999556(P=1.1×10-12,OR=1.24;P=4.3×10-13,OR=1.24);位于5q33.3(PTTG1)區(qū)域的rs2431697(P=1.1×10-8,OR=1.20)和位于13q12.11(GJB2)區(qū)域的rs3751385(P=1.7×10-10,OR=1.18)。 二、對(duì)GWAS,驗(yàn)證1和驗(yàn)證2的綜合分析結(jié)果中發(fā)現(xiàn)7個(gè)區(qū)域的9個(gè)SNP達(dá)到了全基因組顯著性水平,它們分別是:位于5q15 (ERAP1)的rs151823,PCombined=9.3×10-9;位于5q33.1(TNIP1/ANXA6)的rs999556和rs3762999, PCombined分別是3.8×10-21和4.6×10-18;位于5q33.3 (PTTG1)的rs2431697,PCombined=1.1×10-8;位于8p23.2 (CSMD1)的rs10088247和rs7007032,PCombined分別是4.5×10-9和3.8×10-8;位于13q12.11 (GJB2)的rs3751385,PCombined=8.6×10-8(PGWAS+Replication1=1.7×10-10);位于18q22.1 (SERPINB8)的rs514315,PCombined=5.9×10-9;以及位于19q13.41 (ZNF816A)的rs9304742,PCombined=2.1×10-9。 三、對(duì)維吾爾人群(驗(yàn)證3)、德國(guó)人群(驗(yàn)證4)、美國(guó)人群(驗(yàn)證5)及254個(gè)核心家系(驗(yàn)證6)的分型數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,結(jié)果顯示位于13q12.11 (GJB2)的rs3751385以及位于5q15 (ERAP1)的rs151823分別在德國(guó)人群及中國(guó)維吾爾人群中與銀屑病相關(guān)(P=3.6×10-3,OR=1.26;P=2.9×10-5,OR=1.45)。ZNF816A在漢族人群(P=2.1×10-9,OR=0.88)、中國(guó)維吾爾人群(P=1.7×10-3,OR=0.77)和德國(guó)人群中(P=7.9×10-3,OR=0.84)均顯示一致的關(guān)聯(lián)性,在美國(guó)人群(驗(yàn)證5)中提示關(guān)聯(lián)性證據(jù)(P=1.6×10-2,OR=0.90)。對(duì)高加索人群(驗(yàn)證4和5)的合并分析,顯示新發(fā)現(xiàn)的SNP位點(diǎn)在高加索人群中均與銀屑病不相關(guān)。 四、漢族人合并分型數(shù)據(jù)提示ZNF816A(rs9304742)和ERAP1(rs151823)可能與早發(fā)型銀屑病有關(guān)聯(lián)性(P=1.5×10-3,OR=0.85;P=6.5×10-3,OR=0.88)。 結(jié)論 在中國(guó)漢族人群中發(fā)現(xiàn)了六個(gè)新的銀屑病易感位點(diǎn),同時(shí)證實(shí)了高加索人群報(bào)道的易感位點(diǎn)5q33.1也在漢族人群中發(fā)揮作用。ZNF816A和GJB2在德國(guó)人群中與銀屑病相關(guān)聯(lián)。ERAP1和ZNF816A與漢族人I型銀屑病的發(fā)生有顯著的相關(guān)性。研究結(jié)果豐富了銀屑病的遺傳危險(xiǎn)因素,提示遺傳因素可能導(dǎo)致該病發(fā)病年齡的差異,并發(fā)現(xiàn)銀屑病遺傳易感因素在中國(guó)人與歐洲人群中存在異質(zhì)性,為銀屑病的發(fā)病機(jī)制提供了新的潛在的生物學(xué)證據(jù)。
[Abstract]:Research background
Psoriasis is a common chronic inflammatory skin disease characterized by erythema scales. The cause of the disease is not clear and may be associated with the combination of genetic, immune and various environmental factors. Family studies and population epidemiological studies have confirmed the genetic basis of psoriasis, early linkage analysis, candidate bases. Genome-Wide Association Study (GWAS) has found multiple susceptibility loci of psoriasis, most of which have not been confirmed. The present genetic signals can not fully explain the pathogenesis of psoriasis, suggesting the presence of other genetic factors. In addition, GW in China and the Caucasus population. The difference of AS results suggests that there are heterogeneity in susceptible alleles or loci in different populations.
The completion of the Human Genome Project (HGP) and the international mono plograph plan (International HapMap Project, Hap2Map) provides an important biological information and analysis tool for the study of the susceptible genes of complex diseases..GWAS is a powerful and powerful method to search for the susceptible genes of complex diseases in recent years. The academic community believes that it is currently one of the most effective methods to search for susceptible genes of major diseases.
A large GWAS study of psoriatic susceptibility genes in Chinese people was conducted for the first time. In addition to two known predisposing sites in the Caucasus, MHC and IL12B, a new LCE gene cluster of psoriatic susceptibility loci on chromosome 1q21 was found. The GWAS study of the susceptible genes for complex diseases has now established a complete GWAS operating platform and statistical analysis method for the study of susceptible genes for complex diseases, and a large number of psoriasis and control samples have been collected through the establishment of a genetic resource pool.
This study was based on the preliminary study of the predisposing gene GWAS of psoriasis. The six stage validation method was used to test 8312 cases and 12919 controls from China, and 3293 cases from Europe, 4188 normal controls and 254 core families were tested to further search for psoriasis in Han people. His possible locus of susceptibility.
objective
To further search for the susceptibility loci related to psoriasis in Chinese Han people, to determine the genetic risk factors associated with psoriasis in Han people, and to compare the genetic heterogeneity of psoriasis between Chinese and Caucasus.
Method
(I) sample sources:
Samples of 1 (4610 cases and 5373 controls) and 2 (2024 patients and 5495 controls) were collected from the Han population collected from multiple cooperative hospitals throughout the country. A sample of 3 (539 cases and 824 healthy controls) from the Uygur population from the Xinjiang Uygur Autonomous Region. A sample of 4 (823 cases and 1840 controls) was tested. From Germany. A sample of 5 (2470 patients and 2348 controls) and 6 of 254 siblings from the core family were from the United States. In each stage of the validation sample, both the case and the control were derived from the same area and the same race.
(two) the selection of SNP in the verification phase:
Two methods of selecting SNP were used in this study. The first method selected 21 P10-5 SNP. second methods in the correlation analysis of 1139 cases and 1227 controls: 3496 cases of other autoimmune related diseases as comparison, and 5173 cases with 1227 normal controls as a common control specimen, statistical analysis, and participation. The 40 SNP of P10-5 was selected for further verification. Finally, 61 SNP were selected to participate in the verification test.
A total of 35 SNP types were selected for validation 2, including: 1) 20 P 0.05 SNP selected from verification 1; 2) from the Caucasus, 15 SNP. of the 8 loci related to psoriasis were selected.
(three) genotyping and quality control in GWAS:
Based on the typing data of early GWAS (including 1139 psoriasis patients and 1132 controls 620901 SNP and CNV probes), 95 new normal control genotyping data were added to explain the data quality control. The genotype phenotype correlation was calculated by Cochran-Armitage trend test.
(four) genotyping and quality control at the stage of verification:
Verification 1-3 uses the Sequenom MassArray system (San Diego, USA) and Biosystems TaqMan assays (USA) to carry out the alleles of the genotyping.MALDI-TOF MS detection allele and perform the mass spectrogram analysis.
The genotypes of 4 and 5 were verified by TaqMan assays (Applied Biosystems). Data quality control was completed by PLINK 1.05.
6 of the specimens were verified by Sequenom MassArray system.
(five) statistical analysis:
The statistical analysis software R was used to draw the chromosome map and Q-Q map.PCA analysis to evaluate the abnormal deviation samples and the population stratification. The cases and the controls were analyzed by PCA. After quality control, the SNPs typing data were analyzed in the GWAS stage.
In the verification 1-5, Cochran-Armitage trend test was used to calculate its relevance respectively. In the combined analysis of multi stage validation study, the fixed effect model or random effect model was used to analyze. The family association study was analyzed by semi likelihood function for case control correlation.
Multiple logistic regression analysis is used to detect the independence of signals in the region.
In the case, SNP. was used to detect the clinical subtype of logistic by using the clinical subtype as the output variable.
Heterogenous test was used to detect genetic heterogeneity among different groups.
Result
First, in the first stage, 4610 patients and 5373 controls of the Han population were genotyping. The results of integrated GWAS and verifying 1 showed that 4 SNP reached the whole genomic significance standard (P5.0 x 10-8), and these four SNP were rs3762999 and rs999556 (P=1.1 * 10-12, OR) located in the 5q33.1 (TNIP1/ANXA6) region. (P = 1.24; P = 4.3 x 10-13, OR = 1.24); rs2431697 (P = 1.1 x 10-8, OR = 1.20) in 5q33.3 (PTTG1) and rs3751385 (P = 1.7 x 10-10, OR = 1.18) in 13q12.11 (GJB2).
Two, the comprehensive analysis of GWAS, verification 1 and verification 2 found that 9 SNP in the 7 regions reached the level of the whole genome, they were rs151823, PCombined=9.3 x 10-9 in 5q15 (ERAP1), rs999556 and rs3762999 in 5q33.1 (TNIP1/ANXA6), PCombined 3.8 * 10-21 and 4.6 x 10-18, respectively. 2431697, PCombined=1.1 x 10-8; rs10088247 and rs7007032 in 8p23.2 (CSMD1), PCombined 4.5 * 10-9 and 3.8 x 10-8, rs3751385, PCombined=8.6 * 10-8 (PGWAS+Replication1=1.7 * 10-10) in 13q12.11 (GJB2); Combined=2.1 x 10-9.
Three, the Uygur population (verification 3), German population (verification 4), American population (verification 5) and 254 core families (verification 6) were analyzed statistically. The results showed that rs3751385 in 13q12.11 (GJB2) and rs151823 in 5q15 (ERAP1) were associated with psoriasis in German and Chinese Uygur populations (P=3.6 x 10-3, O). R=1.26; P=2.9 * 10-5, OR=1.45).ZNF816A showed a consistent association in the Han population (P=2.1 * 10-9, OR=0.88), Chinese Uygur population (P=1.7 x 10-3, OR=0.77) and German population (P=7.9 x 10-3, OR=0.84). The associated evidence (P=1.6 * 10-2, OR=0.90) was suggested in the United States population (5). Combined analysis of the Caucasus population (verification 4 and 5), The newly discovered SNP locus is not associated with psoriasis in Caucasian populations.
Four, the Han population combined with typing data suggested that ZNF816A (rs9304742) and ERAP1 (rs151823) may be associated with early onset psoriasis (P=1.5 * 10-3, OR=0.85; P=6.5 x 10-3, OR=0.88).
conclusion
Six new susceptibility loci of psoriasis were found in the Chinese Han population, and it was confirmed that the susceptible locus 5q33.1 reported by the Caucasus also played a role in the Han population..ZNF816A and GJB2 were associated with psoriasis in the German population..ERAP1 and ZNF816A were significantly related to the occurrence of I psoriasis in the Han people. The results were abundant. The genetic risk factors of psoriasis suggest that the genetic factors may lead to the difference in the age of the disease, and that the genetic susceptibility to psoriasis is heterogeneous in the Chinese and European population, and provides new potential biological evidence for the pathogenesis of psoriasis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R758.63

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 羅軼波;楊利蓉;王婷婷;陳吉輝;;延續(xù)護(hù)理對(duì)銀屑病患者的影響探究[J];中國(guó)地方病防治雜志;2014年S2期

相關(guān)碩士學(xué)位論文 前1條

1 孫海波;CSMD1基因和BRG1基因與喉鱗狀細(xì)胞癌發(fā)生的相關(guān)性研究[D];吉林大學(xué);2012年

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本文編號(hào)：2160412