系统性红斑狼疮风险位点单核苷酸多态性rs9268832与易感基因人类白细胞抗原-DRB1表达调控的研究

The regulation between risk loci single nucleotide polymorphism rs9268832 and susceptibility gene human leukocyte antigen DRB1 in systemic lupus erythematosus

目的研究系统性红斑狼疮(SLE)风险位点单核苷酸多态性(SNP)rs9268832与易感基因HLA-DRB1之间的调控关系,增加对SLE遗传易感性的认知。方法①收集2017年9月至2019年9月威海市立医院门诊及住院223例SLE患者及2017年9月至2020年12月2323名健康对照者,通过全基因组关联性研究筛选出与SLE显著相关的SNP位点。②利用测序技术对2019年12月至2020年12月威海市立医院门诊及住院SLE患者100例及100例健康对照者进行基因分型,间接免疫荧光及化学发光法检测100例SLE患者ANA和抗dsDNA抗体水平,实时定量PCR法测定100例SLE患者PMBCs和淋巴细胞HLA-DRB1 mRNA表达。③采用单因素方差分析,组间比较采用SNK-q检验或Tamhane′sT2检验。结果①全基因组关联性研究分析筛选出多个与SLE患者相关的SNP位点,其中位于易感基因HLA-DR区域的rs9268832与ANA显著相关。②100名SLE患者中,41例为rs9268832的TT基因型,32例为CT基因型,27例为CC基因型;而100名健康者中,10名为rs9268832 TT基因型,59名CT基因型,31名CC基因型;T等位基因在SLE患者中的分布频率高于健康者(χ2=27.58,P=0.020)。间接免疫荧光及化学发光法结果显示,与CC基因型ANA(23.5±1.2)U/ml、抗dsDNA抗体(16.6±0.9)U/ml相比,CT基因型ANA(40.2±2.5)U/ml、抗dsDNA抗体(36.2±1.8)U/ml增加(q=5.35,P=0.004;q=4.23,P=0.002),TT基因型ANA(56.3±3.1)U/ml、抗dsDNA抗体(52.5±2.9)U/ml增加(q=8.21,P<0.001;q=7.59,P<0.001)。④PCR结果显示:与CC基因型HLA-DRB1 mRNA表达量[PMBCs(402±8)×103,淋巴细胞(462±7)×103]比较,CT基因型HLA-DRB1 mRNA[PMBCs(572±11)×103,淋巴细胞(470±8)×103]表达增加(q=1.11,P=0.300;q=1.03,P=0.400),TT基因型HLA-DRB1 mRNA表达[PMBCs(1052±16)×103,淋巴细胞(856±6)×103]增加(q=4.27,P=0.007;q=3.05,P=0.010)。结论SNP rs9268832的T等位基因可能为SLE的风险位点,不同基因型与易感基因HLA-DRB1的表达之间可能存在调控关系。

ObjectiveTo study the regulatory relationship between the loci single nucleotide polymorphism(SNP)rs9268832 and susceptibility gene human leukocyte antigen(HLA)DRB1 in systemic lupus erythematosus(SLE),and to enhance the understanding of genetic susceptibility to SLE.Methods①A total of 223 out-and inpatients with SLE and 2223 healthy controls were collected from Weihai Municipal Hospital from September 2017 to September 2019,and the SNP sites that might significantly associated with SLE were screened by genome-wide association study.②Sequencing technology was used to genotype 100 out and-in patients with SLE and 100 healthy controls in Weihai Municipal Hospital from December 2019 to December 2020,the levels of anti-nuclear antibody and anti-double-stranded DNA antibody were detected by indirecte immunofluorescence and chemiluminescence immunoassay.Real-time quantitative PCR was used to determine HLA-DRB1 mRNA expression in peripheral blood mononuclear cells and B lymphocytes of 100 patients with SLE.③One-way analysis of variance was used in this study,and SNK-q test or Tamhane′s T 2 test were used for comparison between groups.Results①A number of SNPs associated with SLE were identified by genome-wide association studies.Rs9268832,located in the HLA-DR region of susceptibility genes,was significantly associated with antinuclear antibodies.②Of the 100 SLE patients,41 had TT genotype of rs9268832,32 had CT genotype,and 27 had CC genotype.In the 100 normal subjects,10 had TT genotype of rs9268832,59 had CT genotype,and 31 had CC genotype.The distribution frequency of T allele in SLE patients is higher than in normal people(χ2=27.58,P=0.020).③Indirecy immunofluorescence and Chen iluminescence results showed that compared with CC genotype[antinuclear antibody(23.5±1.2)U/ml,anti-double-stranded DNA antibody(16.6±0.9)U/ml],CT genotype[antinuclear antibody(40.2±2.5)U/ml,anti-double-stranded DNA antibody(36.2±1.8)U/ml]were increased(q=5.35,P=0.004;q=4.23,P=0.002),TT genotype[antinuclear antibody(56.3±3.1)U/ml,anti-double-stranded DNA antibody(52.5±2.9)U/ml]were increased(q=8.21,P<0.001;q=7.59,P<0.001).PCR results showed that:compared with CC genotype[monocyte(402±8)×103,lymphocyte(462±7)×103],CT genotype[monocyte(572±11)×103,lymphocyte(470±8)×103]increased HLA-DRB1 mRNA expression(q=1.11,P=0.030;q=1.03,P=0.040),TT genotype[monocytes(1052±16)×103,lymphocytes(856±6)]increased HLA-DRB1 mRNA expression(q=4.27,P=0.007;q=3.05,P=0.010).ConclusionThe T allele of SNP rs9268832 may be a risk locus for SLE,and there may be a regulatory relationship between different genotypes and the expression of susceptibility gene HLA-DRB1.