Abstract
Background
Functional polymorphisms of the solute carrier family 22, member 4 (SLC22A4), runt related transcription factor 1 (RUNX1) and small ubiquitin‐like modifier 4 (SUMO4) genes have been shown to be associated with several autoimmune diseases.
Objective
To test the possible role of these variants in susceptibility to or severity of systemic lupus erythematosus (SLE), on the basis that common genetic bases are shared by autoimmune disorders.
Methods
597 SLE patients and 987 healthy controls of white Spanish origin were studied. Two additional cohorts of 228 SLE patients from Sweden and 122 SLE patients from Colombia were included. A case–control association study was carried out with six single nucleotide polymorphisms (SNP) spanning the SLC22A4 gene, one SNP in RUNX1 gene, and one additional SNP in SUM04 gene.
Results
No significant differences were observed between SLE patients and healthy controls when comparing the distribution of the genotypes or alleles of any of the SLC22A4, RUNX1, or SUMO4 polymorphisms tested. Significant differences were found in the distribution of the SUMO4 genotypes and alleles among SLE patients with and without nephritis, but after multiple testing correction, the significance of the association was lost. The association of SUMO4 with nephritis could not be verified in two independent SLE cohorts from Sweden and Colombia.
Conclusions
These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analysed do not play a role in the susceptibility to or severity of SLE.
Keywords: systemic lupus erythematosus, nephritis, SLC22A4 gene, RUNX1 gene, SUMO4 gene
Systemic lupus erythematosus (SLE) is a chronic complex inflammatory disease that is thought to have an autoimmune origin. Although the precise aetiology of SLE is unknown, a strong genetic component is well established.1 The genetic background of systemic autoimmune diseases such as SLE is complex and probably involves multiple genes encoding proteins with significant functions in the regulation of the immune system.
The co‐localisation of susceptibility loci in genome‐wide scan studies have led to the hypothesis that common genes may contribute to the susceptibility to autoimmune diseases.2,3 In addition, case–control and family based association studies support this hypothesis. Recent findings have proposed the CTLA4 and PTPN22 genes, as well as HLA, as “general” autoimmune disease loci.4,5 Therefore, investigation of genes shown to be associated with autoimmune diseases, on the basis of an increased susceptibility to related autoimmune traits, seems a good way of studying the genetic basis of autoimmunity.
A recent study in a Japanese population reported an association between rheumatoid arthritis and a functional variant of the SLC22A4 (solute carrier family 22, member 4) gene, which encodes the organic cation transporter 1 (OCT1).6 This polymorphism disrupts a RUNX1 transcription factor binding site and affects the expression of SLC22A4. Furthermore, in the same study, an association between rheumatoid arthritis and a single nucleotide polymorphism (SNP) located in the RUNX1 gene was also found. Recently, regulatory polymorphisms mapping in RUNX1 binding sites have been independently reported to be associated with SLE and psoriasis.7,8 In addition, SLC22A4 polymorphisms have been reported to be associated with Crohn's disease in a white population.9 However, the physiological function of OCT1 is not as yet completely understood; therefore, the role it might play in the pathogenesis of autoimmunity remains uncertain.
Other papers have recently reported evidence for the association of a SUMO4 common non‐synonymous SNP, 163 A→G resulting in the amino acid substitution M55V, with susceptibility to type 1 diabetes in several white and Asian populations.10,11 SUMO4 protein conjugates to IκBα and negatively regulates NFκB transcriptional activity.10 NFκB activates transcription of different genes encoding proteins involved in the immune response. Thus the impaired ability to control NFκB function correctly may lead to the development of autoimmune inflammatory disorders. The SUMO4 M55V substitution has been shown to result in increase in NFκB transcriptional activity and in the expression of the IL12B gene.10 This SNP might therefore play an important role in the development of SLE because of the involvement of NFκB and interleukin 12.
Bearing in mind these recent findings, we sought to test the possible role of these SLC22A4, RUNX1, and SUMO4 polymorphisms in the susceptibility to and severity of SLE. Moreover, as SUMO4 is expressed at various levels in immune tissues, and particularly in the kidney,10 we hypothesised that the SUMO4 M55V variation might be implicated in the development of lupus nephritis.
Methods
Subjects
In the present study 597 Spanish SLE patients meeting the American College of Rheumatology (ACR) criteria for SLE12 were recruited from Hospital Universitario Virgen de las Nieves (Granada), Hospital Clínico Universitario San Cecilio (Granada), Hospital Xeral‐Calde (Lugo), Hospital Carlos Haya (Málaga), and Hospital Universitario Virgen del Rocio (Sevilla).
The mean (SD) age of the SLE patients at diagnosis was 43 (13.3) years and at the onset of SLE symptoms, 32 (15) years. The clinical manifestations studied were articular manifestations, renal involvement, cutaneous lesions, serositis, neurological disease, and haematological disturbances. In addition, clinical activity or severity was assessed every six months by the SLE disease activity index (SLEDAI) score. In all, 987 Spanish blood bank and bone marrow donors of the corresponding cities were included as healthy controls. All the subjects—patients and controls—were of white Spanish origin and were included in the study after giving their written informed consent. We obtained approval for the study from all local ethics committees of the corresponding hospitals.
We also included two independent cohorts of 228 Swedish and 122 Colombian SLE patients meeting the ACR criteria,12 in order to carry out a study of the SUMO4 polymorphism with respect to nephritis susceptibility in SLE patients. Colombian patients were collected from Clínica Universitaria Bolivariana (Medellín). Swedish SLE patients were recruited from the Karolinska University Hospital in the Stockholm area and from the southern city of Lund. All patients were of self reported white European origin or had great grandparents born in Sweden. Controls were recruited partly from the blood bank at the Uppsala University Hospital and from a population cohort in Lund.
Genotyping
DNA from patients and controls was obtained from peripheral blood using standard methods. SNPs were selected according to previous studies of autoimmune diseases including those studied in Japanese rheumatoid arthritis patients spanning the SLC22A4 region (rs(reference SNP ID)3763112 [slc2‐E1], rs1007602 [slc2‐1], rs3792876 [slc2‐F2], rs2073838 [slc2‐F1], and rs2269822 [slc2‐3]),6 and the SLC22A4 SNP associated with Crohn's disease in a white population (rs1050152 [SLC22A4*L503F]).9 In addition, we also tested the RUNX1 rs2268277 variant which has been reported to be associated with rheumatoid arthritis 6 and the SUMO4 163 A→G polymorphism previously shown to be associated with type 1 diabetes.10 Samples were genotyped for SLC22A4, RUNX1 and SUMO4 polymorphisms using a TaqMan 5′ allelic discrimination Custom TaqMan® SNP genotyping assays method (Applied Biosystems, Foster City, California, USA). Allele specific probes were labelled with the fluorescent dyes VIC and FAM, respectively. Polymerase chain reaction (PCR) was carried out in a total reaction volume of 8 μl with the following amplification protocol: denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, and finished with annealing and extension at 60°C for one minute. Post‐PCR, the genotype of each sample was attributed automatically by measuring the allelic specific fluorescence on the ABI PRIM 7000 sequence detection system using the SDS 1.1 software for allelic discrimination (Applied Biosystems).
Statistical analysis
Allelic and genotypic frequencies of all the genetic variants were obtained by direct counting. Statistical analysis to compare allelic and genotypic distributions was done using the χ2 test. Odds ratios (OR) and 95% confidence intervals (CI) were calculated according to Woolf's method. The software used was the Statcalc program (Epi Info 2002; Centers for Disease Control and Prevention, Atlanta, Georgia, USA). Probability (p) values less than 0.05 were considered significant. Uncorrected p values are presented in all the tables. For non‐parametric data analysis, the Mann–Whitney U test was used for ordinal variables and Fisher's exact test for dichotomous variables. For the haplotype analysis, pairwise linkage disequilibrium measures were investigated and haplotypes constructed by the expectation‐maximisation (EM) algorithm implemented in UNPHASED software.13
Sample sizes were estimated a priori by Quanto 0.5 software (Department of Preventive Medicine, University of Southern California) according to previously reported allele frequencies,6,9,10 so that each association study had at least 80% power to detect an association with the same odds ratio as detected in previous studies (odds ratios of 1.5 to 2.0) at the 5% significance level assuming a dominant inheritance model.
Results
SLC22A4 genotypes were in Hardy–Weinberg equilibrium in patients and controls. We observed that the SLC22A4 rs3792876 and rs2073838 SNPs were in complete linkage disequilibrium, as previously described in a Japanese population. No significant differences in the allele and genotype frequencies of the different SNPs tested in the SLC22A4 region were found between SLE patients and controls (table 1).
Table 1 Genotype and allele frequencies of different SLC22A4, RUNX1, and SUMO4 single nucleotide polymorphisms among Spanish patients with systemic lupus erythematosus and healthy controls.
| Genotypes | SLE patients (%) | Healthy controls (%) | p Value | OR (95% CI) | |
|---|---|---|---|---|---|
| and alleles | (n = 597) | (n = 987) | |||
| SLC22A4 rs3763112 | G/G | 181 (30.3) | 273 (27.6) | 0.14 | |
| (slc2‐E1) | G/A | 315 (52.8) | 509 (51.6) | ||
| A/A | 101 (16.9) | 205 (20.8) | |||
| G | 677 (56.7) | 1055 (53.4) | 0.07 | 1.14 (0.98 to 1.3) | |
| A | 517 (43.3) | 919 (46.6) | |||
| SLC22A4 rs1007602 | C/C | 227 (38) | 360 (36.5) | 0.78 | |
| (slc2‐1) | C/T | 301 (50.4) | 515 (52.2) | ||
| T/T | 69 (11.6) | 112 (11.3) | |||
| C | 755 (63.2) | 1235 (62.6) | 0.71 | 1.03 (0.88 to 1.20) | |
| T | 439 (36.8) | 739 (37.4) | |||
| SLC22A4 rs3792876 | C/C | 516 (86.5) | 862 (87.3) | 0.86 | |
| (slc2‐F2) | C/T | 78 (13) | 120 (12.2) | ||
| T/T | 3 (0.5) | 5 (0.5) | |||
| C | 1110 (93) | 1844 (93.4) | 0.62 | 0.93 (0.69 to 1.25) | |
| T | 84 (7) | 130 (6.7) | |||
| SLC22A4 rs2073838 | G/G | 516 (86.5) | 862 (87.3) | 0.86 | |
| (slc2‐F1) | G/A | 78 (13) | 120 (12.2) | ||
| A/A | 3 (0.5) | 5 (0.5) | |||
| G | 1110 (93) | 1844 (93.4) | 0.62 | 0.93 (0.69 to 1.25) | |
| A | 84 (7) | 130 (6.7) | |||
| SLC22A4 rs1050152 | C/C | 176 (29.5) | 313 (31.7) | 0.60 | |
| (SLC22A4*L503F) | C/T | 319 (53.5) | 504 (51.1) | ||
| T/T | 102 (17) | 170 (17.2) | |||
| C | 671 (56.2) | 1130 (57.2) | 0.56 | 0.96 (0.83 to 1.11) | |
| T | 523 (43.8) | 844 (42.8) | |||
| SLC22A4 rs2269822 | C/C | 472 (79) | 751 (76.1) | 0.11 | |
| (slc2‐3) | C/T | 113 (19) | 199 (20.2) | ||
| T/T | 12 (2) | 37 (3.7) | |||
| C | 1057 (88.5) | 1701 (86.2) | 0.06 | 1.24 (0.99 to 1.55) | |
| T | 137 (11.5) | 273 (13.8) | |||
| RUNX1 rs2268277 | C/C | 84 (14) | 147 (14.9) | 0.90 | |
| C/G | 302 (50.7) | 494 (50) | |||
| G/G | 211 (35.3) | 346 (35.1) | |||
| C | 470 (39.3) | 788 (39.9) | 0.75 | 0.98 (0.84 to 1.13) | |
| G | 724 (60.7) | 1186 (60.1) | |||
| SUMO4 163A→G | A/A | 153 (25.6) | 217 (22.0) | 0.25 | |
| G/A | 275 (46) | 481 (48.7) | |||
| G/G | 169 (28.4) | 289 (29.3) | |||
| A | 581 (48.7) | 915 (46.4) | 0.21 | 1.10 (0.95 to 1.27) | |
| G | 613 (51.3) | 1059 (53.6) |
CI, confidence interval; OR, odds ratio; SLE, systemic lupus erythematosus; SNP, single nucleotide polymorphism.
Of note, the frequencies of these SLC22A4 polymorphisms in our population differed significantly from those found in the Japanese population.6
We also estimated the SLC22A4 haplotype frequencies and evaluated associations between these variants with SLE (table 2). However, we found no association of SLC22A4 haplotypes with SLE in our population.
Table 2 Frequencies of the most common haplotypes (frequency >5%) formed by the SNPs rs3763112 (slc2‐E1), rs1007602 (slc2‐1), rs3792876 (slc2‐F2), rs2073838 (slc2‐F1), and rs2269822 (slc2‐3) in patients with systemic lupus erythematosus and healthy controls.
| Haplotype | SLE patients (%) | Healthy controls (%) | p Value | OR (95% CI) |
|---|---|---|---|---|
| (n = 597) | (n = 987) | |||
| GCCGC | 514 (43) | 830 (42) | 0.58 | 1.04 (0.9 to 1.21) |
| ATCGC | 454 (38) | 730 (37) | 0.56 | 1.05 (0.9 to 1.22) |
| ACCGC | 72 (6) | 138 (7) | 0.29 | 0.85 (0.63 to 1.16) |
| GCCGT | 60 (5) | 118 (6) | 0.26 | 0.83 (0.60 to 1.16) |
CI, confidence interval; OR, odds ratio; SLE, systemic lupus erythematosus.
With regard to the rs2268277 RUNX1 polymorphism, genotypes were in Hardy–Weinberg equilibrium in patients and controls. Similarly, no significant differences between SLE patients and healthy controls were observed when the distributions of the genotypes or alleles of this RUNX1 SNP were compared (table 1).
Genotype and allele frequencies of the SUMO4 163A→G SNP in SLE patients and healthy controls are shown in table 1. The genotype frequencies were not found to be significantly different from those predicted by Hardy–Weinberg equilibrium testing in healthy controls. The observed allele frequencies in our control population were in accord with those found in other white populations.10,11,14 However, they differ significantly from those described in Asian populations (Spanish v Taiwanese, p<10−7; Spanish v Chinese, p = 6.10−6; Spanish v Korean, p = 12.10−6).10 No significant differences in the distribution of the alleles or genotypes of the SUMO4 163A→G polymorphism were found when we compared SLE patients with the control group (table 1).
We then analysed the demographic and clinical characteristics of SLE patients (age at disease onset, sex, articular manifestations, renal involvement, cutaneous lesions, serositis, neurological disease, and haematological disturbance) according to their SLC22A4, RUNX1, and SUMO4 genotypes. However, no significant differences were observed except for the presence of renal involvement according to SUMO4 genotype (table 3).
Table 3 Distribution of SUMO4 163A→G genotypes and alleles in Spanish, Swedish, and Colombian patients with systemic lupus erythematosus, according to the presence of nephritis.
| SLE patients | SLE patients | p Value | OR (95% CI) | |
|---|---|---|---|---|
| with nephritis | without nephritis | |||
| Spanish | (n = 189) | (n = 351) | ||
| A/A | 39 (20.5) | 101 (28.8) | 0.04* | 0.64 (0.41 to 1.00) |
| G/A | 87 (46.0) | 164 (46.6) | 0.88 | 0.97 (0.67 to 1.41) |
| G/G | 63 (33.5) | 86 (24.6) | 0.03* | 1.54 (1.03 to 2.31) |
| A | 165 (43.5) | 366 (52.2) | 0.008* | 0.71 (0.55 to 0.92) |
| G | 213 (56.5) | 336 (47.8) | 0.008* | 1.41 (1.08 to 1.82) |
| Swedish | (n = 68) | (n = 160) | ||
| A/A | 22 (32.4) | 49 (30.6) | 0.8 | 1.08 (0.56 to 2.08) |
| G/A | 37 (54.4) | 83 (51.9) | 0.7 | 1.11 (0.60 to 2.04) |
| G/G | 9 (13.2) | 28 (17.5) | 0.42 | 0.72 (0.29 to 1.72) |
| A | 81 (59.6) | 181 (56.6) | 0.55 | 1.13 (0.74 to 1.74) |
| G | 55 (40.4) | 139 (43.4) | 0.55 | 0.88 (0.58 to 1.36) |
| Colombian | (n = 63) | (n = 59) | ||
| A/A | 17 (27) | 19 (32.2) | 0.52 | 0.78 (0.33 to 1.82) |
| G/A | 31 (49.2) | 28 (47.5) | 0.85 | 1.07 (0.5 to 2.32) |
| G/G | 15 (23.8) | 12 (20.3) | 0.64 | 1.22 (0.48 to 3.15) |
| A | 65 (51.6) | 66 (55.9) | 0.5 | 0.84 (0.49 to 1.43) |
| G | 61 (48.4) | 52 (44.1) | 0.5 | 1.19 (0.7 to 2.04) |
Values are n (%).
*Corrected p value non‐significant.
CI, confidence interval; OR, odds ratio; SLE, systemic lupus erythematosus.
The overall distribution of genotypes among SLE patients with and without nephritis yielded significant differences (p = 0.04 by χ2 test from a 2×3 contingency table). Also, the G/G genotype was found at a higher frequency (p = 0.03, OR = 1.54 (95% CI, 1.03 to 2.31)) and the A/A genotype at a lower frequency (p = 0.04, OR = 0.64 (95% CI, 0.41 to 1.00)) in SLE patients with nephritis compared to those without. In addition, we found a greater frequency of the SUMO4 G163 allele among SLE patients with nephritis compared with those without nephritis (p = 0.008, OR = 1.41 (95% CI, 1.08 to 1.82)). Nonetheless, these differences turned out to be non‐significant after Bonferroni correction for multiple testing. To test further the implications of the SUMO4 163A→G SNP in the development of nephritis in SLE patients, we studied two additional independent SLE cohorts, from Sweden and Colombia. When we compared the genotype and allele frequencies of the SUMO4 163A→G polymorphism between SLE patients with and without nephritis in both populations, we found no significant differences (table 3).
Discussion
In this study we have tried to establish for the first time the relation of SLC22A4 and RUNX1 polymorphisms previously associated with rheumatoid arthritis and Crohn's disease (in the case of SLC22A4) with a related autoimmune inflammatory disease, SLE. In addition, this study constitutes the first attempt to assess the potential implication of the functional variant 163 A→G of the SUMO4 gene—which has been shown to be associated with type 1 diabetes—with susceptibility to SLE. We found no evidence of an association of the alleles and genotypes of any of the polymorphisms under study with SLE.
With regard to SLC22A4, the lack of association in our study could have arisen because of a type 2 error (false negative). According to the a priori calculation, our sample size reached at least 80% power to detect the relative risk for the individual SNPs reported in the Japanese study at the 5% significance level. Although an association of the SLC22A4 gene with Crohn's disease has been reported in both Japanese15 and white populations,9 the associated disease polymorphism in the Japanese was the rs3792876, while in the Europeans it was rs1050152. It is possible that disease relevant genes or alleles may be specific for certain populations and vary among different ethnic groups.
With regard to the RUNX1 rs2268277 polymorphism, the lack of association with SLE was not caused by a lack of power. Thus our SLE sample size (597 patients) was large enough to reach >90% statistical power to detect a relative risk similar to the Japanese study at the 5% significance level. It will be of interest to analyse the influence of RUNX1 in Japanese patients with SLE, in addition to replicating the reported RUNX1 rheumatoid arthritis association in a white rheumatoid arthritis cohort.
No evidence of association of the SUMO4 163 A→G SNP with susceptibility to SLE was found. This lack of association is not attributable to the sample size because the power of our study to detect a difference considering OR = 1.5 at α = 0.05 was >99%. The allele and genotype frequencies observed in our study were similar to those described previously in other white populations.10,11,14 The reported association of the SUMO4 gene with type 1 diabetes is now under debate.16,17 Of note, Guo et al did not find association between the SUMO4 polymorphism and type 1 diabetes in a case–control study carried out in a Spanish population.10
However, our results show that the frequency of the SUMO4 163G allele, encoding Val55, is higher among SLE patients with nephritis than in the remaining SLE patients without nephritis, suggesting a role for this polymorphism in the development of renal disease in SLE. However, the differences in the distribution of the genotypes and alleles of SUMO4 between SLE with and without nephritis turned out to be non‐significant after multiple test correction. In addition, we were unable to confirm the association of lupus nephritis in two additional populations of SLE patients from Sweden and Colombia.
Several recent findings prompted us to speculate about a possible role of SUMO4 in the development of nephritis in SLE patients. SUMO4 mRNA has been detected mainly in the kidney.11 Furthermore, the 163G variant of the SUMO4 gene has been shown to produce a greater NFκB transcriptional activity, and as a result, a higher expression of IL12p40.10 In this sense, there are increasing data to suggest that NFκB plays a pivotal role in many nephropathies.18,19 Thus further investigation into the role of SUMO4 polymorphisms in the development of renal involvement should be of interest, using SLE patients from different populations.
Finally, it is possible that the genes evaluated do not play a major role in the development of SLE, although they may be associated with the development of other related autoimmune diseases.
Acknowledgements
This work was supported by Plan Nacional de I+D (grant SAF03‐3460), Fondo de Investigaciones Sanitarias (PI 04 0067), and in part by Junta de Andalucía, grupo CTS‐180. We thank Ma Paz Ruiz and Sonia Morales for their excellent technical assistance.
Supported also by grants from the Alliance for Lupus Research, the Swedish Research Council (grant 12673) and the Torsten and Ragnar Söderbergs Foundation to MEAR.
Abbreviations
SLE - systemic lupus erythematosus
SNP - single nucleotide polymorphism
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