| Background: SLE(systemic lupus erythematosus,OMIM 152700)is a kind of inflammatory connective tissue diseases with multiple autoantibodies in SLE patients’ circulatory system,skin,heart,kidney,joint,neuropsychiatric system and so on are also injured during their lifetime.Genetic changes,abnormal immunity,environmental factors(UV radiation,drug and infection)as well as endocrine imbalance all devote to the pathogenesis of SLE.Nowadays people tend to regard SLE as when genetically susceptible individual undergos genetic changes and stimulated by environment followed by hormones undulation,bodies occur innate and acquired immune responses,produce autoantigens and autoantibodies,and then damage self-immune tolerance in the end.With the theory and technology development of molecular genetics,scholars all over the world used have carried out amount of studies with SLE patients among nations using linkage analysis,candidate gene method,Genome-wide association study(GWAS),GWAS-Meta and so on.After America carried out the first SLE GWAS in 2008,our team conducted the first Chinese Han SLE population GWAS and discovered nine new SLE susceptibility genes/regions in 2009.Till now total about 80 susceptibility genes/loci have been identified at home and abroad,mainly during T cell signaling pathway,B cell signaling pathway,TLR/IFN signaling pathway,NF-κB signaling pathway,antigen processing and presenting signaling pathway as well as immune complex clearing and so on.Gradually GWAS becomes the most conventional genetic method and have been widespread used in the genetic studies of many complex diseases and achieved a series of results.Although GWASs have identified multiple susceptibility genes/loci,most variations are located within noncoding regions which are hard to explain why SLE patients suffered from multiple organs injury and lab abnormality.What’s more,GWAS just covers only about 15% of all the susceptibility genes/loci related with complex diseases and characters(phenotype)which explain only a part of the genetic risk but can’t help to study complex genetic characteristics.Moreover,human HLA possesses haplotype inheritance,high polymorphism and wide linkage disequilibrium,which further increase the difficulty and complexity notablely.Also the microarray dentisy in GWAS MHC study are usually low so that it can’t locate SNPs to functional variations to discover more suaceptibility loci.Imputation means using the full genotypes in reference group to build linkage haplotype,and then to fill up the missing genotype loci for the target group.Taking SNP genotype datas as reference panel,Imputation can calculates the genotype of ungenotyped SNP close to genotyped SNP.Imputation means using the full genotypes in reference group to build linkage haplotype,and then to fill up the missing genotype loci for the target group.Imputation can increase the usage rate and value of sequencing datas almost at no cost,fill in the missing genotypes due to combining different genotype genotying platforms and realize the combination analysis and Meta analysis.Till now several groups at home and abroad have carryed out many researches of GWAS data analysis based on combined Imputation datas that achieved a lot of SNPs,classical HLA alleles and amino acid polymorphisms associated with diseases.Because the sample size of existing reference panel is small,the effect of Europe reference panel in Chinese Han population is limited,and tranditional microarray detection method can only cover certain loci(about 7,889)rather than total MHC regions,there are still no Chinese Han SLE Imputation till now.In 2016 we sequenced more than 10,000 Chinese Han normal samples and arrested most of the MHC variations(260,000)in total.The results covered more than 70% rare and low frequency variations based on which we built Han-MHC reference panel specific for Chinese Han population.This study permits finemapping susceptibility variation in HLA region especial functional variations,and contributes to Chinese Han complex diseases susceptibility researches.Objective:(1)to Imputation datas of GWAS HLA regions in Chinese Han SLE samples,and verified with Imputation of Hong Kong Han SLE GWAS HLA regions datas as well as meta analysis between mainland and Hog Kong SLE MHC imputation datas;(2)to identify the SNP,alleles variation and amino acids;(3)to forecast the spatial structure of amino acid polymorphism;(4)to analyse genotype-phenotype for independent signals.Methods:(1)Carrying out GWAS analysis for 1,047 Chinese Mainland Han SLE patients and 1,205 controls and extract SNPs locating on HLA regions.(2)Using Imputation to fill up GWAS HLA region datas.(3)Analysing the Imputation results by stepwise regression analysis to identify SNPs,classical allele and amino acids polymorphism in HLA region.(4)Carrying out GWAS and stepwise regression analysis for Hong Kong Han SLE population(612 cases,2,193 controls),and meta analysis for the results of both Chinese mainland and Hong Kong SLE population.(5)Analysing amino acid spatial structure with SWILL-MODEL.(6)Concluding the clinical characterizes,such as age of onset,severity,lesion type,family history,and parts of laboratory indexes such as ANA,anti-ds DNA and analysing genotype-phenotype with Plink 1.07 software.Results:(1)Total 493,955 SNPs were obtained via GWAS on Chinese mainland SLE population and 473,565 SNPs in Hong Kong SLE population.(2)After Imputation in mainland Han SLE population,we acquired 22,589 SNPs,68two-digit classical HLA alleles,108 four-digit classical HLA alleles and 673 amino acid polymorphism.The most significantly SNP was rs32632601(P = 5.18×10-16,OR = 1.65,95%CI = 1.46-1.87),most significantly amino acid polymorphism was HLA-DQβ1amino acid 87(Phe,F)(P = 7.81×10-17,OR = 1.79,95%CI = 1.56–2.05),and most significantly allele was HLA-DQB1*03(P = 6.91×10-15,OR = 0.62,95%CI = 0.55 –0.70).(3)In stepwise regression analysis,we found one independent signal,that is,HLA-DQB1*0301(P = 1.43×10-7,OR = 0.63,95% CI = 0.53 – 0.75)in mainland Han SLE population.(4)After Imputation and in Hong Kong Han SLE population,we acquired 21,523 SNPs,64 two-digit classical HLA alleles,96 four-digit classical HLA alleles and 641 amino acid polymorphism.SNP rs32632601(P = 2.89×10-8,OR = 1.44,95%CI = 1.27-1.64)and HLA-DQβ1 amino acid 87(Phe,F)(P = 3.79×10-7,OR = 1.39,95%CI = 1.26 –1.58)all reached statistical significance.The most significantly allele was HLA-DQB1*03(χ2 = 60.62,OR = 0.62,95%CI = 0.55 – 0.70).We further carried out meta analysis on both imputation results of mainland and Hong Kong Han SLE,and the result were that both HLA-DQβ1 amino acid 87 and HLA-DQB1*0301 all reached statistical significance,and HLA-DQβ1 amino acid 87haven’t been reportedn in SLE till now.(5)Genotype-phenotype analysis identified the association between the SLE photosensitivity and HLA-DQβ1 amino acid 87(P = 7.81×10-17,OR = 1.79,95%CI =1.56–2.05).Conclusion: During carrying out Imputation finemapping in HLA region among Chinese mainland Han SLE population and verification in Hong Kong Han SLE population as well as meta analysis between the two cohorts in our research,we identified two independent loci in HLA region in Han SLE patients,and found that HLA-DQβ1 amino acid 87 was associated with photosensitivity.Our study proved that HLA plays certain role in the pathogenesis of SLE,and may provide genetic evidence for SLE clinical forecast,diagnosis and therapy. |
PDF Full Text Request