Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by production of autoantibodies that mainly recognize the self nuclear antigens. As antigen-presenting cells present the epitopes of nucleosome that are major products from apoptotic cells, lupus T-helper cells are improperly activated and proliferated. We aimed to test whether susceptibility to SLE is associated with 26 candidate genes that are located in an SLE-linkage locus and shows the different expression level in T-helper T cells between SLE patients and unaffected individuals. Genomic DNAs from 476 Korean SLE patients and 704 controls recruited in the Hanyang University Hospital for Rheumatic Diseases were genotyped for 170 candidate SNPs located in the promoter region of the candidate genes by using MassARRAYTM system. We analyzed the disease association and Hardy-Weinberg equilibrium by χ2 tests. Linkage disequilibrium patterns and frequencies of haplotypes were analyzed by using Haploview program v3.2. As the result of individual genotyping, CDKN1A and P2RX5 are associated with SLE susceptibility, respectively. First, the two SNPs in promoter and 5’UTR of P2RX5 showed the significant association in a small LD (P = 0.0093) and the most frequent haplotype (CC) constructed the associated SNPs was associated with increased SLE susceptibility in comparison with the other haplotypes (P = 0.0007). Second, one promoter SNP in CDKN1A showed a significant allelic association (P = 0.0069) and dominant genotypic association (P = 0.00059) with susceptibility to SLE. Furthermore, genotypes of the associated SNP in the patient group were not in Hardy-Weinberg equilibrium (P = 0.030). In contrast, the other four SNPs were not associated with SLE susceptibility and were not linked to the associated SNP as r2 values between the SNPs were <0.30, raising a possibility that the associated SNP itself has a functional effect on the gene transcription. We found that the SNP is located in a c-Myb binding site and non-risk allele has higher affinity to c-Myb in an electrophoretic mobility shift assay. Finally, the two promoter variants each carrying the risk allele or non-risk allele of the SNP were tested in c-Myb-expressing cells such as U937 and HeLa by luciferase assays. The non-risk allele variant of promoter showed significantly higher luciferase activity (P = 0.016) than the risk allele variant. These results suggest the reduced expression of CDKN1A by the risk allele of the SNP is a novel risk factor for SLE susceptibility.
전신성 홍반성 낭창(루푸스)은 다인성 자가 면역 질환으로 DNA, 히스톤등과 같은 핵 항원을 인식하는 자가항체를 주로 생성하여 전반적으로 환자의 염증반응과 손상을 일으켜 다양한 증상을 유발한다. 이러한 과정에서 T-helper 세포는 자가에 대한 활성, 낮은 자극에 대한 활성과 성장을 보인다.
본 연구는 루푸스의 원인 유전자를 발굴을 위해 T helper 세포의 환자 특이적 발현을 보이는 유전자 중 루푸스 감수성 염색체부위에 위치한 26개의 유전자가 루푸스와 연관성 유무를 검증하기 위해 26개 후보유전자 내 170 SNP의 유전형을 대량의 유전체 샘플(476명의 루푸스 환자와 704명의 대조군)에 대하여 분석하였다. 그 결과, CDKN1A와 P2RX5 유전자가 루푸스 질환과 유의미한 연관성을 보였다.
사이클린 의존 인산화효소 억제제인 CDKN1A 유전자의 프로모터 -899 위치에 있는 SNP가 루푸스 유병과 연관 있음을 (P = 0.00059) 발견하였고, 프로모터 -899 SNP가 전사인자 c-Myb의 결합력을 변화시켜 발현에 영향을 준다는 사실을 발견하였다. 이것은 루푸스 환자에서는 낮았다는 과거의 보고들과 상통한다.
ATP 수용체인 P2RX5의 5’-비번역부위(5’-UTR) SNP과 프로모터 SNP이 루푸스 유병과 연관성이 있음을 (P = 0.009 및 0.029) 발견하였고, 이들 SNP로 만들어지는 CC 일배체형(haplotype)이 루푸스 유병과 연관성이 강함을 (P = 0.0007) 발견하였다.