Genotype Imputation For Genome-wide Association Data Identify Novel Loci Associated With SLE

Background: Systemic lupus erythematosus(SLE)is a chronic,many organs involved and complex prototypic autoimmune disease with multisystem influenced,such as skin,renal,immune system and so on.The severe cases can be life threatening.It brought great harm to their physical and mental health.As is known to all,women of child-bearing age comprise the majority of SLE and the incidence ratio between male to female is 1:9,so it also has serious impact on patients’ reproductive health.Previous epidemiological studies have found that the prevalence and incidence rates of Asian SLE patients were approximately 2 to 3 times higher than those of Caucasians.Moreover,the Asian SLE patients were reported with higher clinical severity,significantly higher mean and maximum SLE disease activity index(SLEDAI),increased susceptibility to renal involvement,persistently active disease,and higher proportion of autoantibody positivity than non-Asian SLE patients.Many multiethnic cohort studies also showed that Asian patients are more likely to have severe SLE in many aspects,including increased renal disease,autoantibody positivity,disease activity,and organ damage accrual.China,as the largest country in Asia-Pacific area,was reported with relatively higher prevalence rate as 97.5 to 100/100,000 when compared to other ethnicities(generally 20–70/100,000).For such a high prevalence,complicated pathogenesis and limited treatment,SLE disease brought tremendous harm to the marriage,employment and physical and mental health of patients,as well as posed a heavy financial burden on society.At present,the exact pathogenesis of SLE is still not clear.Most scholars believe that the environmental factors(physical damage such as UV radiation,infection factors such as bacteria and viruses,drug-induced lupus,environmental pollution,mental stress and so on)and genetic factors co-contribute to SLE disease,but the genetic factor plays an important role.The genetic model of SLE includes dominant,recessive and additive model.In the world,the first SLE GWAS included in GWAS Catalog was published in 2007.Until now,hundreds of SLE susceptible mutations have been detected in different ethnicity.These susceptible genes are involved in the mechanism of SLE through IFN signaling pathway,NF-κB signaling pathway,JAK / STAT signaling pathway and immune cell signaling pathway.Since2005,our group has conducted series researches to fully reveal the pathogenesis of SLE genetics in Han population,including genome-wide linkage analysis,GWAS,GWAS meta-analysis,genome-wide Exon-chip study and Lnc RNA study.In 2007,our group found that genetic degree of SLE is about 43.6% in a Chinese Han population epidemiological study.At present,more than 50 SLE susceptible genes are found in the Han population by GWAS.However,the susceptibility loci found by GWASs can only explain nearly 30% of the heritability for SLE,so there are still some genetic loci to be discovered in the future.As a complex disease of SLE,the role of each susceptibility gene has a weak effect;it can’t completely explain the genetic mechanism of SLE.In addition,the GWAS study is a research tool that requires an advanced instrument platform,considerable experimenters,financial and biological samples.These stringent requirements also limit the rapid and widespread development of SLE genetic study.In recent years,with the development of genotype imputation and the improvement of analytical method,genotype imputation has gradually been accepted and widely adopted by scholars.Compared with GWAS,genotype imputation can predict previously undetected SNPs(Single nucleotide polymorphisms)and increase the number of SNPs that can be tested in association study.In this way,we can bolster power and save work load and cost at the same time.It has been successfully applied in GWAS of type 2 diabetes,atherosclerosis and so on.Object: In order to determine the presence of novel susceptibility genes for SLE,we used imputed data from our previously published GWAS in Han Chinese population to generate candidate association signals and performed a replication study.At the same time,we aim to explain the function of the findings in this study by bioinformatics analysis,expression difference study and expression Quantitative Trait Loci(e QTL)study.Methods: GWAS data from 1,047 SLE cases and 1,205 controls were pre-phased using SHAPEIT and imputed using IMPUTE 2 with 1KG reference data(phase 1 integrated set,March 2012,build 37).After quality control and association analysis,we selected83 SNPs(P < 5.00 × 10-4)for further replication study in an independent cohort including 3,509 patients and 8,246 controls.We performed a meta-analysis of these studies.Moreover,we collected 135 SLE patients and 130 controls and isolated the peripheral blood mononuclear cells(PBMC)from these samples.Then Real-Time PCR(RT-PCR)and expression quantitative trait loci(e QTL)were used to determine gene expression differences and regulatory effect of SNPs in PBMC,respectively.In addition,we investigated the association between genes expression level and the indicators of clinical laboratory test in patients,including antinuclear antibodies(ANA),anti-double stranded DNA(anti-ds DNA),complement component C3(C3),C-Reactive Protein(CRP)and erythrocyte sedimentation rate(ESR).At the end,we did functional annotation and biological insights by database or software,such as Haplo Reg V.4.0,Regulome DB v1.1,Genotype-Tissue Expression Database(GETx Portal V7),DAVID6.8,Gene MANIA,SIFT and Bio GPS.Results: We identified three novel noncoding variants(rs2855772_C,rs13116227_T and rs10018951_T)are significantly associated with SLE in Chinese Han populations(Pmeta= 1.21×10-15,OR= 1.40,Pmeta=1.18×10-14,OR=1.31 and Pmeta=3.05×10-11,OR=1.34,respectively).We also replicated the associations of previously reported gene UHRF1BP1 encompassing a missense variant(rs13205210_C,Pmeat= 2.26 × 10-17,OR=1.41)in Chinese Hong Kong.We found the m RNA expression level of KIT,TRAPPC11 and UHRF1BP1 was decreased in SLE cases(P<1.00×10-4,P=9.50×10-2and P <1.00 × 10-4)by RT-PCR study.However,compared to controls,the expression level of GPR78 was increased(P=4.00 × 10-2).After Bonferroni test,the expression level of KIT and UHRF1BP1 are significantly different between SLE patients and controls(P<0.05/3).But after Bonferroni test,there are no significant results of e QTL study.There are also no positive results of the association between clinical parameters gene expression level.Through bioinformatics annotation,we detected that the identified SNPs overlapped with gene regulatory sites and has cis-e QTL effect on gene expression in several types of immune cells or skin,for example T and B cells.Conclusions: In this study,we applied imputation analysis strategy to reuse our GWAS data.We identified three novel susceptibility regions at KIT,GPR78 and TRAPPC11 for SLE.We also replicated the associations of previously reported region at UHRF1NP1.This discovery increases the number of established susceptibility loci for SLE in the Han Chinese population and provides new insights into the genetic and biological basis of SLE.