Polymorphisms of MFGE8 are associated with susceptibility and clinical manifestations through gene expression modulation in Koreans with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by impaired clearance of apoptotic cells. Milk fat globule epidermal growth factor 8 (MFGE8) is a protein that connects αvβ3 integrin on phagocytic macrophages with phosphatidylserine on apoptotic cells. We investigated whether genetic variation of the MFGE8 gene and serum MFGE8 concentration are associated with SLE. Single nucleotide polymorphisms (SNPs) were genotyped and serum concentrations were analyzed. The rs2271715 C allele and rs3743388 G allele showed higher frequency in SLE than in healthy subjects (HSs). Three haplotypes were found among 4 SNPs (rs4945, rs1878327, rs2271715, and rs3743388): AACG, CGCG, and CGTC. CGCG haplotype was significantly more common in SLE than in HSs. rs4945 was associated with the erythrocyte sedimentation rate and rs1878327 was associated with alopecia, C-reactive protein, complement 3, anti-dsDNA antibody, and high disease activity. rs2271715 and rs3743388 were associated with renal disease, cumulative glucocorticoid dose, and cyclophosphamide and mycophenolate mofetil use. Serum MFGE8 concentrations were significantly higher in SLE than in HSs. Furthermore, the levels of MFGE8 were significantly higher in SLE than HSs of the rs2271715 CC genotype. In conclusion, MFGE8 genetic polymorphisms are associated not only with susceptibility to SLE but also with disease activity through modulation of gene expression.

Determination of MFGE8 genotypes in Koreans. DNA sequencing was carried out for the entire MFGE8 gene in 20 Korean patients with SLE and 20 HSs. We compared DNA sequences and single nucleotide polymorphisms (SNPs) reported in the National Center for Biotechnology Information (NCBI) database, and selected 12 SNPs with at least 5% minor alleles. Two SNPs (5306 C > T, 11743 T > C) were not reported in the NCBI database (Fig. 1). With the addition of 35 patients with SLE and 10 HSs, DNA sequencing was performed for 12 SNPs. Evaluation was performed in a total of 55 SLE patients and 30 HSs with the independent samples t-test. We selected 5 target SNPs (rs4945 C/A, rs1878326 C/A, rs1878327 A/G, rs2271715 T/C, and rs3743388C/G) with a P value < 3% (P < 0.03; Supplementary  according to the Hardy-Weinberg equilibrium (Table 1). Regression analysis showed that the CC genotype in rs2271715 C > A and the GG genotype in rs3743388 G > C were more common in patients with SLE than in HSs (P = 0.036 and P = 0.005, respectively). Linkage disequilibrium assessment of 5 SNPs showed that rs1878326 SNP and rs1878327 SNP were strongly associated (r 2 = 0.879) ( Supplementary Fig. 1). SHEsis software was used to identify 3 haplotypes among 4 SNPs 17 , and the CGCG haplotype showed a statistically significant association with SLE (P = 0.001; Table 2).

Analysis of MFGE8 protein expression in patients with SLE. MFGE8 protein expression was examined
in 48 SLE patients with SLEDAI scores >6 and 40 age-and sex-matched HSs (Supplementary Table 3). The serum level of MFGE8 was higher in patients with SLE than in HSs (2,030.6 ± 2,308.3 pg/mL vs. 1,433 ± 946.3 pg/mL, P = 0.017) (Fig. 2). correlation between MFGE8 gene Snp and protein expression. The level of MFGE8 protein was increased significantly in SLE patients with rs4945 CA or AA genotype than in SLE patients with rs4945 CC genotype (5,136.1 ± 2,140.5 pg/mL vs. 1841.8 ± 1,168.6 pg/mL, P < 0.001; Table 4). In addition, NCs with rs2271715 CC genotype had significantly higher MFGE8 protein levels (1,571.6 ± 461.1 pg/mL vs. 1407.7 ± 1,022.9 pg/mL, P = 0.037). When comparing the MFGE8 protein level according to genotype, SLE patients with rs4945 CA or AA genotype had higher levels than HSs with the same genotype (2454.8 ± 480.4 pg/mL, P = 0.035). Furthermore, in the rs2271715 CC genotype, patients with SLE showed higher MFGE8 protein levels than those in HSs (2460,2 ± 1,158.3 pg/mL vs. 1,571.6 ± 461.1 pg/mL, P = 0.004).

Discussion
This study identified 5 SNPs by searching the entire MFGE8 genome in Korean patients with SLE. The CC genotype of rs2271715 and the GG genotype of rs3743388 were more common in patients with SLE than in HSs. In haplotype analysis, the CGCG haplotype was found to have a 2.3-fold increased risk of SLE. In addition, these SNPs were associated with several disease activity markers and renal disease in SLE. Furthermore, the expression level of MFGE8 protein in SLE patients was significantly higher than in HSs with the same genotype.
The Human Genome Project and International Human HapMap Project have provided reference information on genetic variations associated with complex diseases, and advances in microarray technology have now allowed large numbers of SNP markers to be screened at once [18][19][20] . However, GWAS based on a common variant hypothesis have generated controversy 21 . Although new genetic markers have been discovered through use of GWAS, various indicators offer little explanation. Since GWAS cannot account for the association between a signalling pathway and a gene, the findings cannot be used to identify and prevent the causes of a disease 22 . Therefore, a candidate gene analysis is required for precise identification of an association.
MFGE8 is a bridging molecule interposed between apoptotic cells and phagocytes that plays an important role in the clearance of dying cells. The level of MFGE8 protein is thought to be associated with the pathogenesis of SLE. Research on MFGE8 has been reported from Japan, Taiwan, and the USA [23][24][25][26][27] ; however, no prior study has correlated MFGE8 polymorphism with function. Thus, this study evaluated the association between MFGE8 polymorphisms and SLE based on MFGE8 protein levels in Korean patients with SLE.
First, DNA sequencing was performed on the entire MFGE8 gene to compare SNPs reported to the NCBI with 5 verified SNPs showing minor alleles of 3% or more (rs4945, rs1878326, rs1878327, rs2271715, and rs3743388). Among these, rs4945 has also been studied in Taiwanese patients with SLE. When only genotypes were compared, the rs2271715 C allele and rs3743388 G allele were more common in patients with SLE than in HSs. According to data reported to the NCBI, the rs2271715 T allele and rs3743388 C allele were more common in Europeans, Chinese, Japanese, and Koreans. Thus, it was notable that the rs2271715 C allele and rs3743388 G allele frequencies were higher in patients with SLE. An examination of 3 haplotypes among 4 SNPs in the CGCG haplotype showed statistically significant differences between patients with SLE and HSs, (P = 0.001), with a (2019) 9 www.nature.com/scientificreports www.nature.com/scientificreports/ 2.3-fold increased risk of developing SLE. An analysis of the correlations between clinical characteristics in SLE and MFGE8 SNP showed that rs4945 showed an association with ESR, rs1878326 showed an association with CRP, anti-dsDNA antibody, SLEDAI score, and glucocorticoid dose, and rs1878327 showed an association with alopecia, CRP, complement C3, anti-dsDNA, and SLEDAI score. rs2271715 and rs3743388 showed associations with kidney disease, glucocorticoid dose, and use of cyclophosphamide and MMF. In addition, rs3743388 SNP showed an association with anti-dsDNA antibody. These results suggest that these 5 SNPs are associated not only with the occurrence of SLE but also with clinical manifestations.
There was a report that the level of MFGE8 in the blood of patients with type 2 diabetes was higher than HSs 24 . Reduction of expression of MFGE8 in diabetic mouse models alleviated atherosclerosis, a major complication of diabetes. Therefore, the expression level of MFGE8 is a very important factor in its function, and can be said to be related to the pathogenesis of autoimmune diseases including SLE. In addition, serum MFGE8 levels in Japanese patients with SLE were significantly higher than those in healthy individuals 23,28 . Moreover, the medical treatment of SLE resulted in significantly lower levels of MFGE8. In another study, MFGE8 overproduction in patients with SLE protected against lupus-related damage by reducing neutrophil migration and the development of neutrophil extracellular traps (NETosis) 29 .
We found that levels of serum MFGE8 were higher in patients with SLE than in HSs. This is consistent with the results of prior studies. Additionally, we examined the correlation between MFGE8 genotype and protein level. The level of MFGE8 protein was significantly higher in SLE patients with the rs2271715 CC genotype than in HSs with the same genotype. This suggests that the increased level of MFGE8 in SLE patients with the increased-risk genotype of rs2271715 is involved in the pathogenesis of SLE. In addition, high MFGE8 levels may cause competitive inhibition in the phagocytosis of apoptotic cells. This can result in incomplete elimination of apoptotic cells, in turn resulting in autoantigen exposure to immune cells in SLE. Unravelling the mechanisms of MFGE8 overexpression-driven SLE may increase knowledge about autoimmune disease, but additional studies are required.
Our results have shown that MFGE8 rs2271715 and rs3743388 SNP not only increase the risk of SLE by increasing MFGE8 protein but also influence SLE disease activity. Moreover, rs4945, rs1878326, and rs1878327 can be used as biomarkers of SLE disease activity. In addition, the level of MFGE8 protein was higher in patients with SLE than in HSs. The strength of our study is the inclusion of clinical manifestations of SLE and the determination of serum MFGE8 levels.
In relation to lupus nephritis and MFGE8 protein, it was reported that MFG-E8 genetic polymorphism studies showed glomerulonephritis in both MFGE8 deficient mice and SLE patients, but did not show significant differences in MFGE8 genotype distributions between SLE patients with or without lupus nephritis 26 . In our study showed that rs2271715 and rs3743388 showed associations with kidney disease and use of cyclophosphamide and MMF.
In conclusion, MFGE8 genetic polymorphisms are associated not only with susceptibility to SLE but also with disease activity through their influence on gene expression.

SLE patients and healthy subjects.
The study included 280 patients with SLE and 260 HSs. All patients satisfied at least 4 of the revised American College of Rheumatology (1997) criteria for the classification of SLE 30 . This study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMR-GEN-15-237), and informed consents were given by all subjects. All procedures for this study were carried out in accordance with the approved guidelines. The clinical features, serum test results, and medication status of patients were collected and registered in a database. Blood samples were collected from patients with SLE and HSs and immediately stored at −70 °C.
Genomic DNA extraction. Genomic DNA was extracted from whole blood of patients with SLE and HSs using a Total DNA Extraction Mini kit (Intron Biotechnology, Seoul, Korea), according to the manufacturer's instructions. The eluted genomic DNA samples were quantified individually using a spectrophotometer (Nanodrop Lite; Thermo, Waltham, MA, USA).
Identification of single nucleotide polymorphisms in MFGE8 genes. SNPs were genotyped in 3 steps. First, SNPs were identified as DNA sequences with at least 5% minor allele frequencies, compared to nucleotide sequences. The presence of previously reported SNPs was confirmed using dbSNP (http://www.ncbi. nlm.nih.gov/projects/SNP). We then designed forward and reverse primers (18 each) of the MFGE8 gene using the nucleotide sequence reported in the NCBI (http://www.ncbi.nlm.nih.gov) to determine the entire single nucleotide polymorphism of the MFGE8 gene (Supplementary Table 4). Polymerase chain reaction analysis was  www.nature.com/scientificreports www.nature.com/scientificreports/ performed on 20 genomic DNA samples from patients with SLE and HSs using these primers. We then selected 12 SNPs with at least 5% minor allele frequencies. Next, DNA sequencing of the MFGE8 gene was performed (Cosmogenetech, Seoul, Korea) using 36 primers. The results of the analysis were confirmed with a sequencer program (Gene Codes Corporation, Ann Arbor, MI, USA), and the nucleotide sequences of 55 patients with SLE and 30 HSs were compared with an independent samples t-test. Finally, we selected 5 target SNPs (rs4945 C/A, rs1878326 C/A, rs1878327 A/G, rs2271715 T/C, and rs3743388C/G) with a P value < 3%. enzyme-linked immunosorbent assay. The