Rheumatology Advance Access originally published online on June 14, 2007
Rheumatology 2007 46(8):1274-1276; doi:10.1093/rheumatology/kem093
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APRIL polymorphism and systemic lupus erythematosus (SLE) susceptibility
1Division of Rheumatology, Department of Internal Medicine, Korea University Medical Center, College of Medicine, Korea University, Seoul, Korea, 2Arthritis and Immunology Research Program and 3Genetic Epidemiology Unit, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
Correspondence to: S. K. Nath, Arthritis and Immunology Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, USA E-mail: swapan-nath{at}omrf.ouhsc.edu
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Abstract |
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Objective. Two novel non-synonymous polymorphisms of the APRIL gene, codon 67 (rs11552708) and 96 (rs3803800), were recently identified and tested for disease association. The 67G allele was reported to be associated with systemic lupus erythematosus (SLE) in a Japanese population. The aim of the study is to investigate whether the APRIL polymorphism associated with susceptibility to SLE in a Japanese population is associated with the susceptibility to SLE in other ethnic groups.
Methods. Three hundred and forty-eight SLE patients (204 European-American, 103 African-American and 41 Hispanic) and 345 ethnicity-matched controls (201 European-American, 104 African-American and 40 Hispanic) were included from the Lupus Multiplex Registry and Repository (LMRR) and evaluated for genetic association. The APRIL codon 67 and codon 96 were genotyped by a 3-base extension method. Statistical evaluations were performed using both chi-square and logistic regression analysis.
Results. Both the single-nucleotide polymorphisms (SNPs) were in Hardy–Weinberg equilibrium in cases and controls within each ethnic group. The APRIL codon 67 was significantly associated with SLE risk under the dominant model adjusted by ethnicity (odds ratio, 95% confidence interval and P-values were 1.45 and 1.02–2.06 and 0.036, respectively). Race-specific analysis also showed a trend for association in African-American and Hispanic SLE subjects.
Conclusion. The APRIL codon G67R polymorphism associated with SLE in a Japanese population may also be associated with SLE in other populations.
KEY WORDS: Systemic lupus erythematosus, APRIL, Polymorphisms
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Introduction |
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Systemic lupus erythematosus (SLE) is the prototype of human autoimmune diseases and a disorder of generalized autoimmunity. While the aetiology of SLE remains unclear, a strong genetic background is known to be important in the pathogenesis of SLE.
APRIL (also known as a proliferation-inducing ligand, TNFSF13, TRDL-1, CD256 and TALL2) is a member of the tumour necrosis factor (TNF) superfamily and plays a role in immune responses and tumour cell growth [1]. APRIL plays a regulatory role in B-cell proliferation by binding to B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) on B cells [2]. Serum APRIL levels are inversely associated with anti-dsDNA titre in SLE [3]. B-lymphocyte stimulator (BlyS, BAFF) is a member of the TNF superfamily and is a potent B-cell co-stimulator and promotes B-cell differentiation, proliferation and survival [4]. BLyS, like APRIL, binds to BCMA and TACI; and both belong to the TNF receptor super family and are mainly expressed in B-cells. Transgenic mice over expressing BlyS develop symptoms characteristic of SLE [5]. It has been reported that APRIL affects the T-cell response [6] and the soluble decoy receptors for APRIL prolong the survival of lupus-prone NZBWF1 mice [7]. These findings suggest that APRIL may be one of candidate genes involved in SLE.
Interestingly, the APRIL gene is located on chromosome 17p13.1, a genomic location that has been known to be the linkage area in European-American families with vitiligo-related SLE and the hypothetical putative gene is known as SLEV1 [8]. This linkage was supported by analysis in a separate population of European-American vitiligo multiplex families [9].
Recently, Koyama et al. [10] identified two novel polymorphisms at the APRIL codons 67 in exon 1 (rs11552708) and 96 in exon 2 (rs3803800) [10]. At amino acid residue 67, the first nucleotide G of the codon GGG for Gly was replaced by A, which resulted in an amino acid change from Gly to Arg (G67R). At codon 96, the second nucleotide A was replaced by G, which resulted in an amino acid change from Asn (AAT) to Ser (AGT) (N96S). They demonstrated that the 67G allele was significantly increased in patients with SLE in a Japanese population.
The APRIL G67R polymorphism was found to be associated with SLE in a Japanese population but it is unknown if this finding can be extrapolated to other populations. The aim of the study is to investigate whether the APRIL polymorphism associated with susceptibility to SLE in a Japanese population is also associated with the susceptibility to SLE in other ethnic groups.
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Subjects and methods |
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Patients and controls
Genomic DNA samples from SLE patients and control subjects were collected. There are 348 unrelated SLE patients (204 European-American, 103 African-American and 41 Hispanic) and 345 ethnicity-matched unrelated controls (201 European-American, 104 African-American and 40 Hispanic) were obtained from the Lupus Multiplex Registry and Repository (LMRR) (http://omrf.ouhsce.edu/lupus). Whenever possible we tried to match the age and gender between cases and controls. All of the patients in the LMRR were diagnosed according to the revised criteria of the American College of Rheumatology (ACR) for SLE [11].
Upon obtaining informed consent, blood samples, buccal swabs and/or mouthwash samples were collected from each participant. The study was approved by the Institutional Review Boards at the Oklahoma Medical Research Foundation and University of Oklahoma where the subjects were recruited.
Polymorphism typing at APRIL
A novel single-nucleotide polymorphism (SNP) assay using no proprietary chemicals, which is relatively inexpensive and has been adapted into a moderate throughput genotyping facility, was used to determine the genotypes for these SNPs [12]. Primers flanking the polymorphism (5'-TAACCATCCTCTCCCAGACA-3' and 5'-AACAGAGCTGCAGAGCCTCA-3' for G67R, 5'-GGAAGCCTCACTCTTCTGTT-3' and 5'-TTTCCATGAGCAGAGTTCCG-3' for N96S) were used to amplify the genomic DNA using standard PCR conditions (95°C for 5 min, followed by 44 cycles of 94°C for 30 s, 57°C for 90 s and 72°C for 90 s, after the final cycle the reactions were incubated at 72°C for 10 min, then held at 10°C) in a 10 µl reaction (20 ng genomic DNA, 1.6X PCR Buffer, 2 mM dNTP, 1.9 mM MgCl2, 5 pmol each primer 1 unit Taq polymerase). Two microlitres of the PCR product was treated with 2 units of Shrimp alkaline phosphatase to destroy the initial PCR primers and dNTPs. An extension reaction was then performed on the treated PCR product using 5'-IR700 (Li-Cor, Lincoln, NE, USA) labelled primers located immediately upstream of the polymorphic site (A 5'-TTCTGGGAGGGGCCTCCTGT-3' for G67R and B 5'-TCTCCTTTTCCGGGATCTCT-3' for N96S). The extension reaction was performed in 7.4 ul with 2 µl of the treated PCR product, 1 U Taq polymerase, 1X PCR buffer, 1.5 mM MgCl2, 1 pmol labelled primer and nucleotides dTTP, dATP, dCTP (Primer A) or dTTP, dCTP, dGTP (Primer B) at 0.2 mM. The extension reactions were then incubated at 92°C for 3 min, followed by 30 cycles of 92°C for 30 s, 45°C for 15 s and 70°C for 15 s, after the final cycle the reactions were incubated at 10°C. The extension reactions were analysed on a 8% denaturing Long Ranger gel on a Li-Cor 4200 DNA sequencer. All samples were assayed with both primers, providing an independent assessment of the genotype at this locus for each subject. This was used as a quality control measure to minimize errors. The few genotyping discrepancies were resolved.
Statistical analysis
A test for departure from Hardy–Weinberg equilibrium (HWE) in cases and controls was tested for both SNPs with a Pearson 
2 test. The genotype and allele frequencies in SLE cases were compared with those in control subjects using the 2 test. Statistical evaluations for testing genetic effects were performed using logistic regression analysis. Fixed effect meta-analysis was also performed to combine three ethnic populations. Haplotypes were estimated using an expectation–maximization (EM) algorithm (SNP-EM). Case-control comparisons were analysed by an omnibus test [SNP-EM omnibus likelihood ratio (LR) test] [13]. A P-value <0.05 was considered to be significant.
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Results |
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The genotype distributions of the APRIL codon 67 and 96 are shown in Table 1. Out of 693 individuals (348 cases and 345 controls), two to three individual's genotypes were unscored for both SNPs, even after third round repeats. We treated these individual's genotypes as missing genotypes. These two SNPs are only 414 basepair apart from each other. The linkage disequilibrium (LD) is significant between SNPs in each ethnic group (D' > 0.7 and P < 0.01). The genotype frequencies of the codon 67 and codon 96 were under Hardy-Weinberg Equilibrium (HWE) within each population samples. Although the distribution of genotypes of the APRIL codon 67 and codon 96 did not differ significantly between SLE patients and control subjects, but the alleles at codon 67 were significantly different [odds ratio (OR), 95% confidence intervals (CIs) and P-values are 1.38, 1.009–1.902 and 0.043, respectively]. This result is also supported by ethnicity adjusted logistic regression test under the dominant model (95% CI and P-values were 1.45 and 1.02–2.06 and 0.036, respectively). Moreover, a fixed effect meta-analysis of three different cohorts for the APRIL 67G vs 67R allele also showed nearly significant association (OR = 1.360, 95% CI = 0.987–1.873, P = 0.06). Stratification by ethnicity did show some degree of allelic association in Hispanic samples (P = 0.056) (Table 1). In Hispanics, under the dominant model codon 67 was nearly significant (OR, 95% CI and P-values were 2.54, 0.98–6.54 and 0.05, respectively)
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Generally, haplotype analysis yields more power to detect differences between cases and controls. However, none of the observed haplotypes were significantly different between cases and controls. The omnibus LR test in each ethnic group is non-significant (P > 0.05) (Table 2). However, a trend is observed in African-American samples (P = 0.07)
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Discussion |
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A previous study identified two novel polymorphisms at codons G67R and N96S. Both created single nucleotide polymorphisms with amino acid substitution [10] and both polymorphisms were located in the stalk region of the extracellular domain. Of these, the 67G allele was associated with susceptibility of SLE in 148 Japanese SLE patients. The amino acid substitution from Gly to Arg may change the structure of the whole molecule because it is a substitution from a non-polar to a charged polar amino acid.
We investigated two novel polymorphisms of APRIL in other ethnic groups to assess whether the APRIL polymorphism associated in a Japanese population is associated in other populations. Although not overwhelming evidence, our study showed that the APRIL codon G67R polymorphism associated with SLE in a Japanese population may also be associated with SLE, especially in African-Americans and Hispanics. Moreover, despite the substantially lower allele frequency of 67R in the tested populations compared with that in the Japanese, the same tendency of increase of 67G/G genotype in SLE is observed in all tested populations. This tendency is the same as in the Japanese study.
There are possible explanations as to why the association was not overwhelmingly replicated. First, there may be ethnic difference of the APRIL polymorphism among different populations. We compared the genotype distribution of the APRIL codon G67R in controls between Japanese and non-Japanese population. There was a significant difference of the G67R genotype distribution between ethnic groups, suggesting evidence of ethnic difference of the polymorphism. Second, the findings may have arisen due to a type 2 error (false negative). This is unlikely because genotype frequencies did not differ from HWE expectations in the control population and the study had more than 80% power to detect the relative risk for individual SNPs reported in the Japanese study (OR = 2.8). However, we could not rule out the possibility of association of the APRIL G67R polymorphism in Hispanics and African-Americans. Third, the border-line significance in association within Hispanics and Africans may be due to the small sample size. We need more samples to assess this association. Fourth, it is possible that G67R is in LD with ‘unknown’ susceptibility gene or polymorphism and the LD vary among ethnic populations.
In conclusion, while the APRIL codon G67R polymorphism is associated with SLE in a Japanese population it was not associated with SLE in European-American population. However, there was a possibility of the association of the APRIL polymorphism with SLE in Hispanic and African-American populations. Further studies are needed to examine whether the APRIL polymorphism contributes to pathogenesis of SLE.
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Acknowledgements |
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The authors are grateful to Dr John B. Harley for encouraging them to perform this study, and to two anonymous referees for their thoughtful and helpful comments. This study was supported by the National Institutes of Health (AR048928, AI063622, RR020143, AR049084 and AR48940).
The authors have declared no conflicts of interest.
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References |
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