Association and functional studies of genetic polymorphisms of MFGE8 gene in systemic lupus erythematosus

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by impaired clearance of apoptotic cells. Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein connects between αvβ3 integrin of phagocytic macrophage and phosphatidylserine (PS) of apoptotic cell surface. Previous studies have reported that MFG-E8 deficiency causes lupus-like disease in murine models. However, MFG-E8 serum level was reported to be elevated in SLE patients. In this study, we determine whether genetic variation of MFGE8 gene and serum protein concentration are associated with SLE. MATERIALS AND METHODS: We collected the blood samples from SLE patitents (n = 280) and normal controls (NC, n = 260) at the rheumatology clinic of Ajou University Hospital. Single nucleotide polymorphisms (SNPs) were genotyped by three steps. At first, we used polymerase chain reaction (PCR) in 20 SLE patients and 20 normal controls (NC) for sequencing of a whole MFGE8 gene in Korean population. Then we screened twelve selected SNPs in 55 SLE patients and 30 NC. Furthermore we used Taq-man probe assay in 225 SLE patients and 230 NC for genotyping of targeted five SNPs. For the PCR assays, we extracted genomic DNA from whole blood. Also, we collected the all SLE patient's clinical characteristics, laboratory and medication data. Furthermore, we analyzed using sandwich ELISA and immunoblotting for comparing serum MFG-E8 concentration in SLE patients. All of results analyzed using SPSS 23.0 and R 3.2.5 statistical programs. RESULTS: Total five SNPs were identified with whole gene sequencing. All SLE patient's mean age was 35.7 ± 7.8 years and 92% were women, which is not different form NC (28.1 ± 7.4 years and 93%, respectively). rs2271715's C allele and rs3743388's G allele were shown higher frequency in SLE than NC (p-value = 0.036, p-value = 0.005, respectively). As the linkage disequilibrium test, rs1878326 and rs1878327 were shown high linkage (r2 score was 0.879). Three haplotypes were found by four SNPs (rs1878326, rs1878327, rs2271715, and rs3743388); AACG, CGCG, and CGTC. The CGCG haplotype was significantly higher in SLE patients compared with NC (p-value = 0.001, odds ratio = 2.312). rs4945 was associated with erythrocyte sedimentation rate, and rs1878326 was assiciated with C-reactive protein (CRP), andi-double stranded deoxyribonucleic acid (anti-dsDNA) antibody, systemic lupus erythematosus disease activity (SLEDAI) score and steroid cumulated dose, and rs1878327 was associated with alopecia, CRP, complement 3, anti-dsDNA antibody and SLEDAI score. rs2271715 and rs3743388 were associated with renal disease, steroid cumulated dose, cyclophosphamide and mycophenolate mofetil dose and rs3743388 also was associated with anti-dsDNA antibody. Serum MFG-E8 concentration was shown significantly higher in SLE than NC (2,030.3 ± 1,292.3 pg/mL vs 1,433.0 ± 946.3 pg/mL, p-value = 0.017). Also, MFGE8 gene's rs4945‘s A allele and rs2271715's C allele were shown higher MFG-E8 concentration in SLE patients than NC (5136.1 ± 2140.5 pg/mL vs 1254.8 ± 480.4 pg/mL, p = 0.035; 2460.2 ± 1158.3 pg/mL vs 1571.6 ± 461.1 pg/ml, p-value = 0.004). CONCLUSIONS: Our data suggest possibility that MFGE8 rs2271715 and rs3743388 SNP can be involved in susceptibility of SLE. Also, these SNPs are associated with renal disease and disease activity in SLE. Furthermore, rs4945, rs1878326 and rs1878327 polymorphisms may be a marker of disease activity.

Author dc:creator

• 우, 지민

Contributors dc:contributor

• 서, 창희
• 대학원 의생명과학과
• 201424572

• 전신홍반루푸스
• MFGE8
• 단일염기다형성
• 질병활성도
• 생체표지자

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023246
000000023246

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/13036