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CR1 exon variants are associated with lowered CR1 expression and increased susceptibility to SLE in a Plasmodium falciparum endemic population

Background Complement receptor 1 (CR1) plays an important role in immune complex clearance by opsonisation and possibly protects subjects from development of autoantibodies. Lower CR1 expression has been associated with susceptibility to systemic lupus erythematosus (SLE). In contrast, subjects displaying lower CR1 expression are protected against severe manifestations of falciparum malaria. This study is the first of its kind to investigate the association of CR1 variants with development of SLE in a P. falciparum endemic population from Odisha, India. Methods CR1 polymorphisms (intron 27 (A>T), exon 22 (A>G) and exon 33 (G>C)) were typed by PCR and restriction length polymorphism in 297 cases of female patients with SLE and 300 age-matched and sex-matched healthy controls from malaria endemic areas in Odisha, India. CR1 expression on monocytes was quantified by flow cytometry. Results The homozygous mutants of CR1 exon 22 (GG) and exon 33 (GG) and their minor alleles were associated with susceptibility to SLE. Furthermore, patients with SLE who harboured the GG genotype of the exon 33 polymorphism had a 3.12-fold higher chance of developing lupus nephritis. CR1 exon (22 and 33) variants were associated with lowered CR1 expression on monocytes in patients with SLE and in healthy controls. Patients with lupus nephritis showed significantly diminished CR1 expression than those without renal involvement (p=0.01). Conclusions The results of the present study demonstrate that common CR1 exon variants are associated with diminished CR1 expression on monocytes and increased susceptibility to development of SLE and lupus nephritis in a malaria endemic area.


INTRODUCTION
Systemic lupus erythematosus (SLE) is an autoimmune disorder characterised by formation of autoantibodies, abnormalities in immune complex (IC) production, its clearance and deposition on various organ systems leading to tissue damage. 1 CR1 facilitates binding, transport and endocytosis of IC bound to complements. Lower levels of CR1 have been associated with SLE 2 3 and lupus nephritis (LN). [4][5][6] Furthermore, functional CR1 polymorphisms which are believed to alter its expression on cells have been associated with increased susceptibility to SLE in different population. 7 8 Deposition of IC on various tissues has been linked to pathogenesis of several diseases. 9 In primates, erythrocytes bind to IC and facilitate its safe disposal by transporting it to Kupffer cells of the liver for phagocytosis. 10 11 Complement receptor 1 (CR1, CD35, C3b/ C4b receptor) is the key molecule on the surface of erythrocytes which mediate binding with complement proteins C1q, C4b, C3b and C3bi found in IC. 12 CR1 is expressed on a wide range of cells viz. erythrocytes, eosinophils, monocytes, B lymphocytes, dendritic cells and kidney podocytes. 13 14 Presence of CR1 in plasma and urine has also been reported. 15 16 CR1 is a member of receptors for complement activation (RCA) located in the 'cluster RCA' region of chromosome 1 band 32. 17 Several single nucleotide polymorphisms (SNPs) have been reported in both coding and non-coding regions of the CR1 gene (ID 1378), but limited number of SNPs have functional relevance. The association of three common SNPs (intron 27: rs11118133, exon 22: rs2274567 and exon 33: rs3811381) have been well investigated in Plasmodium falciparum malaria 18 19 and autoimmune disorders 20 21 since they are believed to be associated with CR1 expression. CR1 seems to play an important role in pathogenesis of P. falciparum malaria by facilitating rosetting, a phenomenon by which plasmodium infected red blood cells (RBCs) bind to uninfected RBCs. 22 Rosetting is mediated by the parasite ligand Plasmodium falciparum erythrocyte membrane protein 1 on the surface of infected RBCs which binds to CR1 molecules. 23 Subjects who express higher CR1 are believed to develop severe pathology such as cerebral malaria due to enhanced rosette formation leading to blockage of blood flow in brain capillaries. 23 Association of CR1 variants (intron 27, exon 22 and exon 33) with severe P. falciparum malaria has been well characterised. In a recent case-control study and meta-analysis, we showed that the CR1 minor alleles associated with lower levels of expression of CR1 are associated with protection against severe Plasmodium falciparum malaria, and they are highly prevalent in malaria endemic areas. 18 In a murine model, Plasmodium yoelli infection decreased CR1 expression on monocytes and hindered IC internalisation which recovered after the clearance of parasites. 24 Furthermore, human subjects infected with P. falciparum or P. vivax also display lower CR1 expression on monocytes compared with uninfected individuals. 24 Differential CR1 expression has been reported based on malarial endemicity. 25 26 In view of the fact that in malaria endemic areas CR1 expression is reduced, and lowered CR1 expression is associated with improper clearance of IC, a feature observed in SLE, we hypothesised that this factor could predispose genetically susceptible subjects living in malaria endemic areas for development of SLE.

Subjects
Patients with SLE admitted to the Department of Medicine, under the unit of Clinical Immunology and Rheumatology of SCB Medical College, Cuttack, Odisha, were enrolled in the present study. The diagnosis of SLE was based on revised American College of Rheumatology classification criteria. 27 Since, SLE affects mostly women 28 only female patients from coastal districts of Odisha (Cuttack, Puri, Anugul, Khurdha and Dhenkanal), endemic to P. falciparum infection, 18 29 30 were included in the study. The average annual parasite index of malaria in these districts is 6.67. 31 After a detailed clinical examination and laboratory investigations, patients were categorised into various clinical phenotypes. The clinical profiles of 297 patients with SLE are summarised in table 1. Three hundred age-matched, healthy female subjects from similar geographical areas (costal districts) were included as healthy controls (HCs). None of the controls reported history of autoimmune disorder or malaria. Both, patients with SLE and HCs were free of active P. falciparum infection as shown by immunochromatography test and P. falciparum-specific PCR. 18 About 5 mL of blood was collected in EDTA tubes from each participant. The methods were carried out in accordance with approved guidelines. Informed consent was obtained from each patient and HC.
DNA isolation and genotyping of CR1 variants Genomic DNA was isolated from whole blood by the GenElute Blood Genomic DNA Kit (SIGMA). As described earlier, 32 samples were genotyped for the intron 27 HindIII A>T, exon 22 3650A>G and exon 33 5007 G>C genes by PCR-restriction length polymorphism (RFLP). For intron 27 and exon 22 the restriction fragment length polymorphism (RFLP) products were separated on 3.5% agarose and visualised by ethidium bromide staining under trans-UV illumination. To resolve restriction fragments of exon 33 polymorphism, 16% non-denaturing polyacrylamide gel was used. For quality control of genotyping results, about 25% of the randomly selected samples were directly sequenced for exon 22, exon 33 and about 15% were sequenced for intron 27 and the results were observed to be 100% concordant. Electropherograms of sequencing analysis are shown in online supplementary figure S1.
Quantification of CR1 expression on monocytes CR1 expression on monocytes was determined by flow cytometry using two-colour fluorescence in 35 patients with SLE and 33 HCs. Briefly, 50 μL of whole blood were directly stained for 30 min at 4°C with phycoerythrinlabelled anti-CR1 (eBiosciences) and fluoresceinisothiocyanate labelled anti-CD14 (eBiosciences). Appropriate isotype controls (eBiosciences) were also used. Erythrocytes were lysed using fluorescence-activated cell sorting (FACS) lysis solution (BD Biosciences, USA) and the cells were washed with washing buffer followed by centrifugation at 300×g for 5 min. The cell pellet was then resuspended in 500 μL of sheath fluid and analysed by flow cytometry in FACS calibre using CellQuest Pro software.

Association of polymorphisms with monocyte expression of CR1
Association of common CR1 polymorphisms (intron 27, exon 22 and exon 33) with expression of CR1 on erythrocytes has been demonstrated earlier. 19 25 26 32 34 35 We hypothesised that common CR1 polymorphisms may lead to its altered expression on monocytes which   may be a possible trigger in the development of SLE.
In patients with SLE, major alleles for exon 22 (AA) and exon 33 (CC) polymorphisms displayed significantly higher levels of CR1 on monocytes compared with their respective heterozygotes (exon 22: AG; exon 33: CG) and homozygous (GG) alleles ( figure 2D, F). Furthermore, CR1 expression on monocytes of the CG genotype (exon 33) was observed to be higher than the homozygous mutants (GG). Such difference between heterozygous (AG) and homozygous minor alleles (GG) was not observed in CR1 exon 22 polymorphism ( figure 2D, F). Expression of CR1 on monocytes of subjects with various genotypes of intron 27 polymorphisms was comparable ( figure  2B). Interestingly, similar genotype-phenotype association results were also observed in HCs ( figure 2A, C, E).

Distribution of CR1 polymorphisms in LN
Patients with LN display lowered levels of CR1 on monocytes and its expression was associated with common CR1 variants (exon 22 and exon 33), We analysed for possible association of CR1 polymorphisms with susceptibility/resistance to LN. As depicted in table 4, the GG genotype of CR1 exon 33 polymorphism was more frequent in LN+ patients compared with LN− patients. However, the difference was not statistically significant ( p=0.03, OR=3.12). Distributions of other polymorphisms (intron 27 and exon 22) in clinical phenotypes (LN + vs LN−) were comparable (table 4).

Association between CR1 expression and autoantibodies
Diminished CR1 expression is functionally associated with defective clearance of ICs which could be a possible trigger for autoantibody formation in SLE. We analysed the association between CR1 polymorphisms and levels of anti-dsDNA and the results are depicted in figure 3. The GG genotype of CR1 exon 22 and exon 33 polymorphisms displayed higher anti-dsDNA titres than their respective reference genotypes (exon 22: AA; exon 33: CC). However, the difference was not statistically significant. Anti-dsDNA titres were comparable among genotypes of the CR1 intron 27 polymorphism. Furthermore, we analysed correlation between CR1 expression on monocytes and levels of anti-dsDNA ( figure 3). A negative correlation was observed between CR1 expression and anti-dsDNA ( p=0.0007, Spearman's r=−0.54), suggesting a possible role of CR1 in facilitating increased production of anti-ds DNA.

DISCUSSION
The present study investigates, for the first time, an association between CR1 common variants with possible development of SLE in genetically susceptible individuals residing in areas endemic for P. falciparum malaria. It demonstrates that CR1 variants (exon 22 and 33 polymorphisms) are significantly associated with patients with SLE and the exon 33 polymorphism is associated with LN. CR1 exon polymorphisms (22 and 33) are also   significantly associated with reduced CR1 expression on monocytes, and patients with SLE display lower CR1 expression on monocytes compared with HCs. Interestingly, LN+ patients had lower CR1 levels compared with LN− patients. SNPs have been reported in the CR1 gene. 32 However, studies indicating their association with SLE susceptibility are limited. Most studies were focused on CR1 HindIII intron 27 polymorphism and the results were contradictory. A recent meta-analysis reported absence of association between the CR1 intron 27 polymorphism and SLE. 20 Our observations were similar with regard to the CR1 intron 27 variant. However, exon 22 and exon 33 polymorphisms showed significant association with SLE. Homozygous and minor alleles were more prevalent in patients with SLE compared with HCs, indicating that subjects with the GG genotype of exon 22 and exon 33 have 4.27-fold and 3.82-fold higher associations with SLE. Furthermore, CR1 exon 33 homozygous (GG) alleles were more frequent in LN+ patients compared with LN− patients. CR1 exon polymorphisms (22 and 33) were associated with CR1 expression on monocytes: homozygous (GG) alleles displayed lower levels of CR1 compared with their respective major alleles. The mechanism(s) related to reduced expression of CR1 in codon variants (22 and 33) is not known. It is possibly associated with formation of defective CR1 proteins that are quickly degraded resulting in lower expression on the cell surface. To the best of our knowledge, this is the first study to investigate an association between CR1 exon polymorphisms (22 and 33) with SLE in malaria endemic areas. However, there are several factors responsible for the development of SLE and mutation in the CR1 gene could be one among them.
CR1 expression on erythrocytes has been reported to be low in patients with SLE 3 and grossly reduced in LN+ patients. [4][5][6] In the present study, we observed diminished CR1 expression on monocytes of patients with SLE compared with HCs. Comparison of CR1 expression on different clinical manifestations revealed significantly lower levels of CR1 on monocytes in LN+ patients compared with LN− patients. The relationship between CR1 and clearance of IC has already been indicated earlier.
Reduced expression provides a basis for increased circulating IC that has the potential to facilitate development of autoantibodies in SLE. Lower CR1 expression has been associated with improper IC clearance in mice models. 24 Furthermore, CR1 expression was found to be inversely correlated with IC deposition in the kidney 24 increasing the possibility of renal damage.
The current study also corroborates our earlier observation 18 on CR1 polymorphisms in malaria endemic areas. Mutants of CR1 exon 22 and intron 27 were more prevalent in the studied population compared with their respective major alleles (exon 22: AG+GG=85%; intron 27: AT+TT=78%). The present study also revealed a higher prevalence of the CR1 exon 33 variant (CG +GG=85%) among the same population. A higher prevalence of the CR1 intron 27 minor allele has been reported in various malaria endemic areas viz., Thailand 34 and India 19 25 while in non-endemic areas, their frequency has also been found to be significantly low. 26 36 The other CR1 variant (exon 22) also showed a similar pattern: higher frequency of variants in endemic populations like Papua New Guinea, 26 Thailand, 34 India 19 25 and lower prevalence in non-endemic areas. 25 26 Similarly, the minor allele of the exon 33 polymorphism is more frequent in malaria endemic than non-endemic areas. 26 The occurrence of a higher frequency of CR1 minor alleles in malaria endemic areas has been attributed to their protective function against severe P. falciparum malaria and its mortality. 18 26 The association between SLE and CR1 variants in these areas has not been studied earlier.
The area in the current study is endemic to P. falciparum malaria. Earlier, we had reported a protective association between of CR1 variants (for mutations in exon 22) against severe P. falciparum malaria and mortality. 18 We had proposed that higher prevalence of CR1 variants in malaria endemic areas could have been a result of selection pressure. 18 Our current observation of increased prevalence of CR1 mutations ( possibly selected out by malaria) in patients with SLE suggests an interesting possibility that malaria could be a contributing factor in the development of SLE in genetically susceptible individuals in malaria endemic regions of the world. The prevalence of SLE has been reported to be threefold to fourfold higher in African-Americans compared with Caucasians, 37 and they are the people who have migrated from the malaria endemic regions of Africa. Validation of the observations reported in this study, in regions of high endemicity for P. falciparum infection, could provide novel insights into the natural history of SLE.

CONCLUSIONS
The results of the present study demonstrate that common CR1 exon variants are associated with diminished CR1 expression on monocytes and possibility of increased susceptibility to development of SLE and LN in malaria endemic areas.