PADI4 polymorphisms are not associated with rheumatoid arthritis in the Spanish population
A. Martinez,
A. Valdivia,
D. Pascual-Salcedo2,
J. Ramón Lamas1,
M. Fernández-Arquero,
A. Balsa3,
B. Fernández-Gutiérrez1,
E. G. de la Concha and
E. Urcelay
Immunology and 1 Rheumatology Department, Hospital Clínico San Carlos, 2 Immunology and 3 Rheumatology Department, Hospital Universitario La Paz, Madrid, Spain.
Correspondence to: A Martinez, Immunology Department, Hospital Clinico San Carlos, C/Martín Lagos, s/n, 28040 Madrid, Spain. E-mail: alfmdoncel{at}terra.es
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Abstract
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Objectives. The presence of anti-citrullinated peptide antibodies is the most specific serological marker known of rheumatoid arthritis (RA). The PADI4 gene, encoding a haematopoietic isoform of the peptidylarginine deiminase citrullinating enzyme, has recently been associated with susceptibility to RA in the Japanese population. A subsequent UK report could not confirm this association, and a later French study also yielded a negative result. Given this discrepancy and the importance of antibodies against citrullinated peptides in the early course of the disease, we performed a replication study.
Methods. Three hundred and fifty-four Spanish RA patients and 498 Spanish controls were recruited from two Madrid hospitals. The padi4_104 and padi4_94 single-nucleotide polymorphisms (SNP) were analysed by TaqMan assays.
Results. Similarly to what was described in the British and French population, the less frequent allele of this SNP was not associated with the disease (genotype TT, 16.1% in RA patients vs 14.3% in controls; P = 0.46, odds ratio 1.15, 95% confidence interval 0.781.71). A confirmatory negative result was obtained on analysing another SNP in the same gene, padi4_94, in 248 RA patients and 394 controls.
Conclusions. The results of our group and from the British and French studies strongly suggest that polymorphisms of the PADI4 gene do not play a role in susceptibility to RA in European populations.
KEY WORDS: Rheumatoid arthritis, Citrulline, Polymorphism, Peptidylarginine deiminase, Single-nucleotide polymorphism
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Introduction
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Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases, affecting about 1% of the adult population in Western countries. Similarly to what has been postulated for the majority of common diseases, a polygenic mode of inheritance has been proposed for RA [1]. The predictable small predispositional effect of each of the genes involved has made their identification a daunting task. Linkage analysis, a method with no a priori hypothesis, has been a valuable tool for identifying genes responsible for monogenic diseases, but its results for RA have been difficult to replicate [2]. The association study, although not free from shortcomings and from suspicion due to occasional lack of replication, remains the gold standard in the genetics of complex diseases, because of its accepted higher power to detect small effects. In this kind of study, a candidate gene suggested by its physiological role is investigated to ascertain whether any of its alleles is differently distributed in patients compared with controls, i.e. whether it is associated with the disease. Advances in molecular and clinical studies [3] have already suggested several candidate genes possibly implicated in the aetiology or in the pathology of RA.
One of the most recent advances in the clinical serology of the disease has been the description in RA patients of antibodies directed against citrullinated peptides [4]. These antibodies are fairly specific to RA and they precede the onset of the disease [5], suggesting a causal relationship. The antibodies are directed against a pool of proteins in which some arginine residues have been post-translationally modified by deimination to the amino acid citrulline in the presence of a calcium ion [6]. The role played by the citrullination in a number of proteins, most characteristically filaggrin and fibrin, is not known at present, but it has been related to some apoptosis function [7]. From the immunological point of view, it can be argued that post-translationally modified self-antigens are more likely to break tolerance towards self. In the Spanish population, anti-citrullinated peptide antibodies have a specificity of 94% and a sensitivity of 60% as RA markers (M. C. Martin, A. Valdivia, E. Loza, D. Pascual-Salcedo, C. Ruiz de Alegria, S. López, A. Fernández and M. A. Figueredo, unpublished communication).
There are four citrullinating enzymes in humans. One of these isoforms, expressed specifically in haematological cells and pathological synovial tissues, is encoded by the gene PADI4 (peptidyl arginine deiminase), located on chromosome 1p36. This locus has been linked to RA in an ongoing Japanese linkage project, and it was found to be not very far from one of the markers (D1S228) suggested in the European linkage study [8]. Therefore, because of both its function and its location, the PADI4 gene was a novel candidate gene to be analysed for its association with the disease. A Japanese study [9, 10] has convincingly demonstrated that, in their population, the less frequent variant of the gene is indeed associated with the disease, and that it confers an appreciable (rather than microscopic) relative risk [odds ratio (OR) = 2] on homozygous carriers. A functional rationale for this association was provided by the observation that the less frequent haplotypes yielded more stable mRNAs. Subsequently, however, a British group has been unable to replicate this finding in a large sample from Caucasian origin [11]. Similarly, a recent French study also yielded negative results [12]. We address in this report the question of whether the association described in Japanese patients can be detected in a casecontrol study in another European population, this time from southern Europe.
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Methods
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This study included 354 white Spanish RA patients consecutively recruited at two Madrid Hospitals. For the transmission test, 53 Spanish families of the European Consortium on RA families (ECRAF) were analysed. The diagnosis was established based on the 1987 ACR (formerly ARA) criteria [13]. Their main clinical characteristics are indicated in Table 1. Most of these patients have been included in previous studies from our group [14, 15]. The subjects' written consent was obtained according to the Declaration of Helsinki, and the research was approved by the ethical committees of the Clinico and La Paz hospitals.
The control group included 498 samples: white Spanish blood donors and healthy laboratory staff and students. Although the blood donors were not specifically asked about the presence of RA, the 1% frequency of this disease in our population precludes this from being a major concern.
Due to the concordant structure of haplotypes found in the Japanese [9] and British [11] studies, and to the high linkage disequilibrium between individual nucleotide positions within the PAD14 locus, we studied only one of the SNPs included in the UK study. The padi4_104 polymorphism, located at codon 117 in the fourth exon of the PADI4 gene, was analysed by using the C_7541083_1 Assay on Demand (Applied Biosystems, Foster City, CA, USA), under the conditions recommended by the manufacturer. Allele discrimination was performed in an ABI 7700 Sequence Detector (Applied Biosystems). Another SNP, padi4_94, located 23 nucleotides before padi4_104 and not included in the British study, was also analysed (Assay on Demand C_16176717_10) in most of the samples.
The presence of the shared epitope, a common stretch of amino acids present in HLA-DRB1 alleles associated with the disease, was ascertained as described previously [14]. The shared epitope is the strongest RA susceptibility factor identified [16].
Genotype frequencies, carriage rates and allelic frequencies were compared with the
2 test. A P value under 0.05 was considered significant, if any of our comparisons attained that level of significance. The P values were calculated with a standard free software package (Epi Info v2000; CDC, Atlanta, GA, USA). Allele transmission from heterozygous parents to affected offspring was studied using the transmission disequilibrium test (TDT) [17]. The distribution of transmitted and non-transmitted alleles was compared using a Monte Carlo computer simulation with 20 000 iterations under the null hypothesis of a probability of transmission of 0.5. Haplotypic frequencies were estimated using the expectation maximization algorithm implemented in the Arlequin v. 2.000 software, with number of iterations set at 5000 and initial conditions at 50, with an
value of 107.
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Results
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In control subjects, each genotype frequency of PADI4 polymorphisms conformed to HardyWeinberg expectation. Table 2 shows the genotypic distribution of the padi4_104 polymorphism in patients and controls. As can be observed, the two groups were very similar and no statistically significant difference was observed. When allelic frequencies (38.3% in patients vs 37.6% in controls for the mutant allele; P = 0.76, OR = 1.03, 95% CI 0.841.26) and carriage rates (60.5% in patients vs 60.8% in controls for the mutant allele; P = 0.91, OR = 0.98, 95% CI 0.741.31) were examined, again no difference was apparent. No difference was observed when patients were subgrouped by sex or shared epitope status (data not shown).
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TABLE 2. Comparison of padi4_104 and padi4_94 genotype, and padi4_94/padi4_104 haplotype frequencies in RA patients and controls
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Fifty-three families were studied to verify absence of distorted transmission from heterozygous progenitors by the TDT. The TDT design requires probands and their parents and is based on the Mendelian principle that, for any polymorphism, each parent contributes one allele to an offspring. In summary, the TDT consists of a casecontrol study in which cases the parental alleles are transmitted to the patient and controls are those that were not transmitted. In our family sample, 48 heterozygous progenitors were found. Allele C was transmitted 23 times and allele T 25 times. Therefore, no difference in transmission was detected by Monte Carlo analysis (P = 0.44), which is consistent with the lack of association found previously in the casecontrol study.
We then decided to study another polymorphism located in the vicinity, padi4_94. It was selected because its relationship with RA has not been studied previously in any European population, and because in the Applied Biosystems database (https://myscience.appliedbiosystems.com) its reported allelic frequencies differ between oriental and European populations. It could be, therefore, a key position explaining the discordant predisposition results found in diverse populations. As can be observed in Table 2, no differences were apparent. Of note, the frequencies found in our population were similar to those found in the Japanese study. Finally, Table 2 shows the padi4_94/padi4_104 haplotypic frequencies estimated by the expectation maximization algorithm. These data were also very similar to those observed in Japan. One of the haplotypes (CT) was almost non-existent in our population, and the same absence is observed in the Japanese population.
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Discussion
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In this study we were unable to confirm the association reported in the Japanese population between the less frequent allele of the padi4_104 polymorphism and susceptibility to RA. Our results are concordant with those of previous British and French studies, and they seem to confirm in another European cohort that this polymorphism is not aetiological in white populations. This is the first replication study focused on a southern European population. The British authors already discussed in their paper the most likely explanations of this clearly discordant finding. One point we would like to address further is the genetic heterogeneity of populations. We think that aetiological mutations per se are likely to be molecularly implicated in susceptibility to common diseases in different human populations. The human physiology is similar all around the world. If the increased susceptibility to RA is due to a more stable mRNA of the citrullinating enzyme, this effect in our opinion should be more or less apparent in different populations. However, what may be quite variable among populations is the frequency of a particular mutation. And what is also probably different is the association between mutations, the way they are arranged in haplotypes. Although the haplotypes described in the British and Japanese studies are the same, it has to be taken into account that the Japanese study was more exhaustive, including a higher number of polymorphic positions. The British study focused mainly on exonic polymorphisms, but the functional effect described in the Japanese study (differential metabolism of PADI4 mRNA) is not necessarily due to missense protein mutations. On the contrary, silent mutations in exons or even mutations inside introns may affect mRNA stability, export or processing, resulting in different intracellular concentrations depending on genotype [18]. The stability of the PADI4 mRNA has not been studied in a white population, but a recent study showed no correlation between the PADI4 haplotypes and the presence of anti-citrullinated peptide antibodies [19]. This suggests that the differential mRNA stability of the haplotypes may be irrelevant to the pathophysiology of the disease, and the functional source of the association may reside in another molecular or cellular subtlety. The possibility exists that the aetiological mutation present in the Japanese risk haplotype is not present (at an appreciable frequency) in European populations and that the polymorphisms studied in Europe have no primary effect on susceptibility. It may be that an intronic polymorphism present in the Japanese susceptibility haplotype is not present in our population. It would be interesting in this regard to study the whole set of PADI4 polymorphisms in European populations, not to make a new casecontrol study but to check if the haplotypes found are wholly identical to the haplotypes described in the Japanese report. If this is indeed the case, the explanation for the discordant results should be sought elsewhere, perhaps in different genomic or environmental interactions.
Our study shows how even very well-designed association studies can fail to be confirmed due to population differences. Pessimists may think that variable population association studies are futile if no general conclusions can be drawn. But the positive confirmation of an association even in only one population can lead to a novel insight into the pathological mechanisms or the aetiology of the disease. The therapeutic benefits of such progress are probably mainly restricted at first to the carriers of mutations identified on a one-population-only basis. However, it could be that carriers of these mutations are present world-wide, although at smaller frequencies in other countries, and they could also benefit from these advances.
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Acknowledgments
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We are most grateful to Carmen Martínez Cuervo for her expert technical assistance. A.M. is an employee with support from the Fondo de Investigaciones Sanitarias (04/CP00175) and E.U. is the recipient of a Ramon y Cajal contract from the Spanish Science and Technology Ministry. This work was supported by project 01/0348 from the Fondo de Investigaciones Sanitarias (Spain).
The authors have declared no conflicts of interest.
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Submitted 4 April 2005;
revised version accepted 24 May 2005.