Association of MASP2 levels and MASP2 gene polymorphisms with systemic lupus erythematosus

Abstract Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder. MASP2 is a mediator that plays an important role in complement system. As dysregulation of the complement system has been demonstrated to correlate with SLE pathogenesis, the role of MASP2 in lupus has not been widely discussed. In the present study, serum levels of MASP2 were evaluated in 61 lupus patients and 98 healthy controls by training cohort, and then a validation cohort including 100 lupus, 100 rheumatoid arthritis, 100 osteoarthritis, 100 gout, 44 Sjogren's syndrome, 41 ankylosing spondylitis patients confirmed the findings. Receiver operating characteristic (ROC) curve analysis determined the discriminatory capacity for serum MASP2. PCR methods tested the association of MASP2 gene polymorphisms (rs7548659, rs17409276, rs2273346, rs1782455 and rs6695096) and SLE risk. Impact of polymorphism on MASP2 serum levels was evaluated as well. Results showed that serum levels of MASP2 were significantly higher in lupus patients and correlated with some clinical, laboratory characteristics in the training cohort, and were much higher as compared to that in different rheumatic diseases patients in the validation cohort. Serum MASP2 showed a good diagnostic ability for lupus. Genotype frequencies and allele frequency of polymorphisms rs7548659, rs2273346 were strongly related to SLE risk, and genotypes of rs17409276, rs1782455, rs76695096 were significantly correlated with lupus genetic susceptibility. Interestingly, patients carrying GA genotype of rs17409276, TT, TC genotype of rs6695096 showed higher levels of serum MASP2. The findings suggested that MASP2 may be a potential disease marker for lupus, and correlate with SLE pathogenesis.

immune system. 2 It is known that complement is able to clear up immune complexes, apoptotic cells and efficiently regulate pro-inflammatory components production in response to pathogens. 3 Lectin pathway activates the complement, which will be further activated via mannose-binding lectin (MBL)-associated serine protease (MASPs). MASPs are the enzymatic constituents of the lectin pathway of the complement system. The MASPs family has three subgroups, including MASP1, MASP2 and MASP3. MASP1 was discovered as a bactericidal factor with structural similarities to C1s, while the biological function of MASP3 has not been clearly elucidated to date. 4 The MASP2 gene is located on chromosome 1p36.3-36.2, encoding MASP2 and MAp19. There are 12 exons for MASP2 gene. It is notable that exon 2 encodes the signal peptide. Along with exon 3, both of exon 2 and 3 encode CUB1 domain. Exon 4 encodes the epidermal growth factor-like domain, and exon 12 encodes serine protease domain, 3' UTR region. 5 MASP2 binds to MBL, ficolins, forming a homodimer, which autoactivates and initiates the lectin pathway. 6 In addition, MASP2 cleaves prothrombin, further leading to the covalent bind of cross-linked fibrin on bacterial surfaces. 7 As numerous studies indicated that the lectin pathways are dysregulated in SLE patients, evidence about MASP2 protein expression in SLE patients and association of MASP2 gene polymorphisms and SLE risk is limited. Therefore, in the present study, we discussed serum levels of MASP2 in lupus patients with large samples and revealed the SLE genetic susceptibility with MASP2 single nucleotide polymorphism (SNP).

| Measurement of MASP2 by Enzyme-linked immune sorbent assay (ELISA)
Concentrations of serum MASP2 were determined by ELISA for both patients and controls. The kits for human MASP2 were purchased from Cusabio 15 and evaluated the protein according to the manufactory.

| Statistics
All the data were analysed by SPSS 16.0 and STAT 11.0. If the data were normally distributed, mean ± standard deviation (SD) was used to describe the data, and Student's t test, ANOVA was appropriately used to test the differences between groups. Otherwise, median and inter-quartile range were selected and non-parametric test determined the differences between groups. Spearman's rank vande rbilt.edu/Power Sampl eSize). P value less than .05 was recognized significant.

| Increased serum levels of MASP2 in validation cohort
As the serum levels of MASP2 were higher in SLE patients and re-

| MASP2 gene polymorphisms with lupus patients' genetic susceptibility
In the present study, a total of 250 lupus patients and 385 age,

| Correlation between MASP2 gene polymorphisms with lupus clinical, laboratory parameters
As discussed above, the five polymorphisms in MASP2 gene were significantly related to SLE genetic susceptibility. To further evaluate the association of MASP2 gene polymorphisms and lupus clinical, laboratory parameters, subgroup analysis was tested for each polymorphism. Results showed that lupus patients with thrombocytopenia having increased frequencies of TT genotype, allele T as compared to the patients without the parameter for rs7548659 (P = .010, P = .004, respectively). Patients with pyuria also revealed increased frequencies of TT genotype, allele T as compared to the patients without the parameter (P = .039, P = .022) ( Table 3). For rs17409276, patients with the clinical, laboratory characteristics including pleuritis, pericarditis, hypocomplementemia, positive anti-dsDNA, pyuria showed much higher frequencies of genotype GG, allele G than that in lupus patients did not have the characteristics (Table 3). Similarly, frequencies of genotype TT, allele T for rs2273346 were higher in patients with rash, and frequencies of genotype TT, allele T for rs1782455 were increased in lupus patients having fever, thrombocytopenia, pyuria when compared with patients without the characteristics, respectively (Tables 3 and 4). Furthermore, patients had oral ulcer, hypocomplementemia showed elevated frequencies of genotype TT + TC, allele T as compared to that in patients without oral ulcer, hypocomplementemia for rs6695096 (Table 4).

| D ISCUSS I ON
+ rs1782455(C)) up-regulated susceptibility to RA, and allele T of rs72550870, allele C of rs12085877 correlated with articular symptoms in RA patients. 19 Genotype TC at rs2273346 and rs6695096 of MASP2 genes were more prevalent in the tuberculosis patients in Canada than the healthy controls. 20  Some limitations in this study should be realized. First, gene polymorphisms were not examined in other group of patients. Second, functional role of MASP2 involves in SLE pathogenesis should be discussed in the future. Third, a larger sample size to evaluate association of MASP2 gene polymorphisms with SLE risk will be better to reveal the genetic susceptibility of SLE.
In conclusion, this study found that serum levels of MASP2 may correlate with SLE pathogenesis, and MASP2 gene polymorphisms related to SLE genetic susceptibility in a Chinese Han population.

ACK N OWLED G EM ENTS
The study was sustained with National Natural Science Foundation of China (81701606), Sichuan Provincial Science and Technology Program (2019YJ0540).

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
Datasets are available from the corresponding author on reasonable request.