| Part 1 Genomics study of systemic sclerosis in Chinese Han populationObjective:Systemic sclerosis(SSc)is a rare systemic autoimmune disease characterized by thickening of the skin,fibrosis,and immune response.The pathogenesis of SSc is still elusive,but it is generally believed that genetic factors have played an important role.This study conducted a genomics study on the SSc of the Chinese Han population.Methods:(1)We performed a candidate gene association analysis enrolling 342 SSc patients and 694 healthy controls in Chinese Han population to investigate the association between genetic susceptibility variants and SSc;(2)Whole exome sequencing(WES)was conducted to study 40 cases of SSc patients in Chinese Han population and 20 cases of healthy controls.And then we performed Sanger sequencing and multiplex polymerase chain reaction(PCR)in 241 healthy controls and 258 SSc patients for verification.Results:(1)ARID3A rs10415976-G allele frequency was lower in SSc group than in the healthy control group,and the rs350146-TT genotype frequency was higher than in the healthy control group.ARID3A rs10415976-G was associated with SSc in a Chinese Han population in additive genetic model,AR1D3A rs62132345-T was associated with SSc in Chinese Han population in dominant model,and ARID3A rs350146-T was associated with SSc in Chinese Han population in recessive model.CXCR5 rs77871618-T allele frequency was lower in SSc group than in the healthy control group.In dominant model,CXCR5 rs77871618-T and CXCR5 rs2077579-G were related to SSc in Chinese Han population.GTF2I rs117026326 and rs73366469 were closely related to SSc.GTF2I rs117026326-T allele frequency and TT genotype frequency were significantly higher in SSc group than in the healthy control group.GTF2I rs73366469-C allele frequency and CC genotype frequency were significantly higher in SSc group than in the healthy control group.In three genetic models,GTF2I rs117026326-T and rs73366469-C were associated with SSc in Chinese Han population.NFKB1 rs1599961-AG genotype frequency was slightly higher in SSc group than in the healthy control group.In dominant model,NFKB1 rs1599961-A was related to SSc in Chinese Han population.TNFSF8 rs1555457-T allele frequency and TC genotype frequency were significantly higher in SSc group than in the healthy control group.In dominant and recessive genetic model,TNFSF8 rs1555457-T was related to SSc in Chinese Han population.No significant association was found between the rs2304256 locus of the TYK2 gene and SSc in Chinese Han population.(2)The results of WES suggest that the following mutations were related to SSc in Chinese Han population:the chr12:58220166 A>C locus of CTDSP2 gene,the chr17:27047791 G>T locus of RPL23A gene,the chr15:93595516 C>A locus of the RGMA gene,the chr16:2348431 A>G locus of the ABCA3 gene,the chr8:30969288 A>T locus of the WRN gene,the chr1:43131706 G>T locus of the PPIH gene,the chr4:115589309 G>A locus of the UGT8 gene,the chr6:150005366 G>C locus of the LATS1 gene,the chr9:100367851 G>C locus of the TSTD2 gene,the chr16:58538497 A>G locus of the NDRG4 gene,the chr17:29253861 G>A locus of the ADAP2 gene,the chr1 8:10736603 T>C locus of the PIEZO2 gene,the chr19:38907780 G>T locus of the RASGRP4 gene,the chr19:50097777 T>C locus of the PRR12 gene and the chr20:5905646 T>C locus of the CHGB gene.Conclusions:ARID3A rs10415976,CXCR5 rs77871618,GTF2I rs117026326 and rs73366469,NFKB1 rs1599961 and TNFSF8 rs1555457 were susceptibility variants for SSc in Chinese Han population.The mutations in the fifteen genes CTDSP2,RPL23A,RGMA,ABCA3,WRN,PPIH UGT8,LATS1,TSTD2,NDRG4,ADAP2,PIEZO2,RASGRP4,PRR12,and CHGB were rare mutations that may potentially cause the onset of SSc.It is speculated that these genes mutations have a greater impact on gene function and can be used as important target genes for subsequent research.Part 2 Study on autoantibodies related to systemic sclerosis in Chinese Han populationObjective:Autoantibodies are of great value in the diagnosis of systemic sclerosis(SSc).This study evaluated the value of known SSc-related autoantibodies in the diagnosis of SSc,and used the Huprot array method containing 21,148 human proteins to discover new SSc-related autoantibodies.Methods:(1)We detected 13 SSc-related autoantibodies in the serum of 320 SSc patients,100 connective tissue disease(CTD)controls and 30 healthy controls by using a commercial immunoblot assay.Enzyme-linked immunosorbent assay(ELISA)was used to detect anti-RNA polymerase Ⅲ(RNAP Ⅲ)antibody in the serum of 236 SSc patients,125 CTD controls and 166 healthy controls.And the correlation with clinical manifestations was evaluated.(2)A two-stage screening strategy was used to discover SSc-related autoantibodies.In the first stage,the Huprot array was used to screen candidate SSc-related autoantigens in the serum of 40 SSc patients,30 CTD controls and 20 healthy controls.In the second stage,a customized SSc-focused array was used for large sample verification,including 400 SSc patients,200 disease controls and 100 healthy controls.And the selected autoantigen was finally confirmed by Western blot.Results:(1)When compared with CTD control group,the prevalences of anti-RP11,anti-CENPB,anti-CENPA and anti-Scl-70 antibodies were higher in the SSc group.When compared with the healthy control group,the prevalences of anti-Ro-52,anti-CENPB,anti-CENPA and anti-Scl-70 antibodies were higher in the SSc group,and the differences were statistically significant.The prevalence of anti-Scl-70 antibodies were higher in the SSc with interstitial lung disease(ILD)group than in the SSc without ILD group,indicating that they have the value of predicting the occurrence of ILD in SSc patients.When compared with CTD and healthy controls,the prevalence of anti-RNAP Ⅲ antibody detected by ELISA was higher in the SSc group.The incidence of renal crisis in patients with anti-RNAP Ⅲ antibody was higher than in patients without anti-RNAP Ⅲ antibody.(2)In the first stage,the Huprot array screened out 113 candidate SSc-related autoantigens.In the second stage,the SSc-focused array and Western blot verified a novel SSc-related autoantibody—anti-SNRPA.The prevalence of anti-SNRPA was significantly higher in the SSc group than in the disease control and healthy control groups,and the difference was statistically significant.Conclusions:Anti-RP11,anti-CENPB,anti-CENPA and anti-Scl-70 antibodies can be used to diagnose SSc in Chinese Han population.Anti-RNAP Ⅲ antibody detected by ELISA has diagnostic value for SSc and has predictive value for kidney diseases related to SSc.Anti-SNRPA antibody is a novel autoantibody associated with SSc in Chinese Han population and has potential clinical application value,especially as a good supplementary marker in the clinical applications of anti-Scl-70 antibody. |
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