Abstract
The complement system comprises a large number of plasma proteins participating in pathogen defense, apoptotic cell removal, immune-complex clearance and inflammatory response of innate immunity; it also has regulatory potencies affecting B cell maturation and altering T cell function. Impaired complement function or complement deficiency is believed to induce defective clearance of apoptotic cells to drive the autoimmunity in systemic lupus erythematosus (SLE). Complement component 2 (C2), an early member of the classical complement pathway, is correlated with systemic manifestations of SLE. We hypothesize that C2 polymorphism may confer genetic susceptibility to complement dysfunction in SLE so we aim to investigate the clinical and serological associations of C2 variants in Taiwanese patients with SLE. The single-nucleotide polymorphisms (SNPs) were selected according to the NCBI dbSNP database and analyzed using the Genomatix software suite; we also performed computational calculations to estimate the linkage disequilibrium (LD) relationships for effective selection including three intronic SNPs (rs2844455, rs3130681 and rs2734335) and three exonic SNPs (rs1042663, rs1042664 and rs4151648). These SNP allele frequencies were tested in normal control subjects; upon comparison with the allele frequency in SLE patients, if the minor allele frequency (MAF) exceeded 5%, the SNPs were chosen for further study. The rs2844455 located in the GC-rich region of C2 gene was genotyped by direct sequencing in 95 SLE patients and 95 matched normal control subjects. The gene expression profiles were generated by quantitative real-time PCR (qPCR) and reverse transcription PCR (RT-PCR); the serum C2 levels were determined using commercial ELISA tests. Our results showed that SLE patients had significantly higher frequencies of the AA genotype (22.1% vs. 9.5%, P<0.05) and the A allele (40.5% vs. 28.4%, P<0.05) when compared with normal control subjects. The A allele was strongly associated with the occurrence of hair loss, photosensitivity and anticardiolipin antibodies; whereas the G allele was associated with lower frequencies of these clinical presentations. The relative expression levels of C2 mRNA were significantly lower in patients with the AA genotype [median: 18.86, interquartile range (IQR): 11.36-22.43, P=0.002] and the AG genotype (18.38, IQR: 13.13-28.82, P=0.004) than in those with the GG genotype (35.76, IQR: 19.33-49.71). Differential expression patterns of C2 gene were demonstrated while the gel showed that the banding patterns and their intensity were obviously related to the genetic variants in patients; however, serum protein assays showed no significant connection existed between rs2844455 genotypes and serum C2 level in SLE patients (n=10 for each genotype; ANOVA F =0.071, P>0.05). As expected, we confirmed the A allele as a risk factor for SLE development in a Taiwanese population, in contrast, the G allele might be a protective factor against the pathogenic autoantibody formation and cutaneous manifestations in SLE patients.