Association of the CTLA4 3′ untranslated region polymorphism with the susceptibility to rheumatoid arthritis

Association of the CTLA4 3ⴕ Untranslated Region Polymorphism With the Susceptibility to Rheumatoid Arthritis M. R. Rodrı´guez, A. Nu´n˜ez-Rolda´n, F. Aguilar, A. Valenzuela, A. Garcı´a, and M. F. Gonza´lez-Escribano ABSTRACT: Cytotoxic T-lymphocyte antigen 4 (CTLA4) gene polymorphism located in the 3⬘ untranslated region (UTR) was investigated in 141 Spanish patients (38 men and 103 women) with rheumatoid arthritis (RA) and in 194 ethnically-matched healthy controls. Twenty alleles having different numbers of (AT) repeats (from 7 to 32) were found in this population. (AT)7 and (AT)16 were the most frequent alleles, and accounted for almost two-thirds of the allelic frequency in the control population. Consequently, alleles were assigned as L (large: 16 or more AT repeats) or S (short: less than 16 AT repeats). When the L/S distribution in patients and controls were compared, an increase of L alleles was observed among patients (49.9% vs. 39.7%; p ⫽ 0.02; pc ⫽ 0.04, odds ratio [OR] ⫽ 1.46; 95% confidence interval [CI], 1.06 –2.01). Hence, the frequency of S alleles was decreased among patients (51.1% vs. 60.3%; p ⫽ 0.02; pc ⫽ 0.04; OR ⫽ 0.69; 95%CI, 0.50 – 0.95). Moreover, a sta-

INTRODUCTION Rheumatoid arthritis (RA) is a frequent, chronic, and progressive joint disease characterized by leukocyte invasion of the synovial lining and hyperplasia of the resident synoviocytes. Its etiology and pathogenesis remain controversial, although twin and family studies have already indicated that both major genetic and environmental factors contribute to its etiopathology [1, 2]. The extent of the genetic contribution has been estimated as being approximately 50%. Of this, onethird has been accounted for by genes in the HLA complex, and for the remaining the contribution of many

From the Servico de Inmunologı´a, Servicio Andaluz de Salud, Hospital Universitario Vergen del Rocı´o, Sevilla, Spain. Address reprint requests to: Dr. Antonio Nu´n˜ez-Rolda´n, Servicio de Inmunologı´a, Hospital Universitario Vergen del Rocı´o, Avda. Manuel Siurot s/n. 41013, Sevilla, Spain; Tel: ⫹34 (95) 501 3228; Fax: ⫹34 (95) 501 3221; E-mail: [email protected] Received January 29, 2001; revised August 28, 2001; accepted September 25, 2001. Human Immunology 63, 76 – 81 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.

tistically significant decrease in the frequency of S/S individuals was observed among RA patients (27.7% versus 40.7%; p ⫽ 0.01; pc ⫽ 0.03; OR ⫽ 0.56; 95%CI, 0.34 – 0.91). These differences were irrespective of the HLA “shared epitope” (SE) status, and were observed similarly among SE⫹ as well as among SE⫺ patients. After combining these data with other reported previously by us, from studies of CTLA4 49 (A/G) and ⫺318 (C/T) polymorphisms, we conclude that the strongest association between CTLA4 gene polymorphisms and RA susceptibility occurs with the 3⬘ UTR polymorphism. Human Immunology 63, 76 – 81 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: CTLA4; rheumatoid arthritis; genetic susceptibility; genetic polymorphism

other non-HLA genes has been suggested [3–5]. The cytotoxic T-lymphocyte antigen 4 (CTLA4) molecule is a homologue of CD28, and both molecules and their common ligands (B7-1 and B7-2) constitute the B7/ CD28-CTLA4 costimulatory pathway of T-cell activation. Whereas interaction of CD28 with its ligands plays a critical role in increasing and maintaining a T-cell response initiated through T-cell antigen receptor (TCR) engagement, interaction of CTLA4 with the same ligands has an inhibitory effect on T-cell activation and might contribute to peripheral tolerance [6, 7]. Three different polymorphisms have been described in the CTLA4 gene: one in the promoter region at the ⫺318 position from the ATG start codon consisting of a C/T change [8], a second in position 49 of the exon 1, which lies in a A/G transition resulting in a threonine (Thr) or alanine (Ala) dimorphism [9], and a third in the 3⬘ untranslated region (3⬘ UTR) with variant lengths of a dinucleotide (AT)n repeat [10]. The CTLA4 49 G (Ala) allele 0198-8859/02/$–see front matter PII S0198-8859(01)00358-5

CTLA4 3⬘ UTR Polymorphism and RA

has been found to be associated with insulin-dependent diabetes mellitus (IDDM) [11–14], Graves’ disease [11, 15], and other autoimmune disease [16 –19], whereas studies in RA patients have provide inconsistent results [20 –24]. In addition, CTLA4 3⬘ UTR polymorphism has been also found associated to several autoimmune disease [13, 19, 25–28]. The aim of this work was to further investigate the influence of the CTLA4 polymorphisms in the susceptibility to RA in Spanish patients.

MATERIAL AND METHODS The present study includes 141 Spanish RA patients (38 men and 103 women) fulfilling the American College of Rheumatology (ACR) 1987 criteria for RA [29] and 194 ethnically matched healthy controls (105 men and 89 women). DNA from patients and controls was obtained from peripheral blood as previously described [30]. The (AT)n microsatellite polymorphism in the 3⬘ UTR of the CTLA4 gene was defined employing a polymerase chain reaction (PCR) carried out in a Perkin-Elmer 9600 Thermal Cycler (Perkin-Elmer, Foster City, CA, USA) with mixes consisting of 0.5 ␮l of genomic DNA, 2 pmoles of each primer, 400 ␮M of each dNTPs, 2.5 mM MgCl2, and 0.2 U Taq DNA polimerase and ddH2O to a final volume of 10 ␮l. Primers used in this study were those previously described [10]; the forward primer was labeled at the 5⬘-end with HEX (Perkin-Elmer). The following thermal profile was run: a 95°C, 5-min initial denaturation followed by 30 cycles of 94°C for 1 min, 52°C for 2 min, 72°C for 2 min, and a final extension at 72°C for 7 min. PCR amplified fragments were electrophoresed in a capillar system (ABI PRISM 310, PerkinElmer) and their lengths analyzed using the Genescan 672 software (Perkin-Elmer) using 500 TAMRA (Perkin Elmer) as size standard marker. Some samples were sequenced in order to know the number of AT repeats for each allele, using unlabeled primers [10] for amplification followed by AmpliTaqFS dye terminator cycle-sequencing protocol (Perkin-Elmer). Sequenced samples were run in an ABI-PRISM 310 and analyzed using the Navigator software (Perkin-Elmer). Statistical comparisons were performed by ␹2 test calculated on 2 ⫻ 3 or 2 ⫻ 2 contingency tables using the Statcalc program (Epi Info version 6.0; Centers for Disease Control and Prevention, Atlanta, GA, USA) Odds ratio (OR) with 95% confidence intervals (95%CI) was calculated using the same software. The p values were corrected by multiplying by the number of comparisons. The strongest association study for different positions in the CTLA4 gene was performed, as recommended by Svejgaard [31].

77

RESULTS Twenty different alleles having from 7 to 32 (AT) repeats in the 3⬘ UTR of the CTLA4 gene were found and their allelic frequencies are listed in Table 1. The most common alleles among patients and controls were (AT)7 and (AT)16, the remainders revealed allelic frequencies lower than 5%. When the allelic frequencies from RA patients and controls were compared, a slight decrease of (AT)7 was found among RA patients (48.6% versus 56.9%; p ⫽ 0.03; pc ⬎ 0.05; OR ⫽ 0.71; 95%CI, 0.52– 0.98). The alleles were grouped into two categories according to the number of AT repeats in the 3⬘ UTR. The group identified as “short” (S) includes alleles presenting ⬍16 AT repeats, and the one called “long” (L) includes alleles with 16 AT repeats or more. A statistically significant increase of L alleles (49.9% versus 39.7%; p ⫽ 0.02; pc ⫽ 0.04; OR ⫽ 1.46; 95%CI, 1.06 –2.01) and a parallel decrease of S alleles (51.1 vs. 60.3; p ⫽ 0.02; pc ⫽ 0.04; OR ⫽ 0.69; 95%CI, 0.50 – 0.95) was observed among RA patients (Table 2). When the frequencies of the different “genotypes,” according to these categories, were compared a statistically significant decrease in the frequency of S/S individuals was observed among RA patients (27.7% versus 40.7%; p ⫽ 0.01; pc ⫽ 0.03; OR ⫽ 0.56; 95%CI, 0.34 – 0.91). After gender stratification, no statistical significant differences between male and female patients were found with respect to the 3⬘ UTR polymorphism. In order to test previous findings [21], which suggest a HLA-DRB1 phenotype dependence of the CTLA4 49 A/G and RA association, patients were stratified according to the DRB1 specificities. An increase in the frequency of L alleles among patients bearing DRB1*03 was observed (90.5% vs. 69.2%, p ⫽ 0.04), although, after correction this difference became nonsignificant. Likewise, no differences were found when L/S polymorphism was studied with respect to the HLA shared epitope status (results not shown). The present results were combined with those previously reported by us concerning studies of ⫺318 (C/T) and 49 (A/G) CTLA4 gene polymorphisms [21]. The analysis of samples homozygous for three or two of these positions allows to assign different combinations as CTLA4 “alleles.” The rest of the samples were not considered in this study except ⫺318T/C 49G/A 3⬘ UTR S/S and ⫺318T/C 49G/A 3⬘ UTR L/L that were assigned as ⫺318T 49A 3⬘ UTR S/⫺318C 49G 3⬘ UTR S and as ⫺318T 49A 3⬘ UTR L/⫺318C 49G 3⬘ UTR L, respectively, because the combination ⫺318T 49G was not observed among homozygous samples nor in family studies. Among RA patients, all the CTLA4 “alleles” bearing 3⬘ UTR L were slightly increased. The analysis performed with data from Table 3 demonstrated that the

78

M. R. Rodrı´guez et al.

TABLE 1 Frequency of the CTLA4 3⬘ UTR alleles among Spanish RA patients and healthy controls Healthy controls (2n ⫽ 388)

RA (2n ⫽ 282)

3⬘ UTR alleles (number of AT repeats)

N

%

N

%

p Value

OR (95%CI)

7 11 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

137 3 4 46 9 8 8 7 1 7 2 12 13 4 9 7 1 2 2 0

48.6 1.1 1.4 16.3 3.2 2.8 2.8 2.5 0.4 2.5 0.8 4.3 4.6 1.4 3.2 2.5 0.4 0.8 0.8 0

221 10 3 61 12 8 7 3 6 7 1 5 12 6 6 11 2 1 3 3

56.9 2.6 0.8 15.7 3.1 2.1 1.8 0.8 1.6 1.8 0.3 1.3 3.1 1.6 1.6 2.8 0.5 0.3 0.8 0.8

0.03 pc(20) ⬎ 0.05

0.71 (0.52–0.98)

pc ⫽ corrected p value; number in paratheses indicates correction factor (number of alleles found). Abbreviations: CTLA 4 ⫽ cytotoxic T-lymphocyte antigen 4; OR ⫽ odds ratio; RA ⫽ rheumatoid arthritis; UTR ⫽ untranslated region.

only significant association between RA susceptibility and CTLA4 polymorphisms occurs with the 3⬘ UTR position (p ⫽ 0.006; pc ⫽ 0.036; OR ⫽ 1.71; 95%CI, 1.14 –2.57), whereas the positions ⫺318 and 49 were not significantly associated (Table 4). Results from secondary comparisons between positions are also included in Table 4, and indicate that, indeed, the 3⬘ UTR position polymorphism seems to be responsible for the association between CTLA4 gene and RA.

DISCUSSION The main conclusion of this study is that polymorphism in the 3⬘ UTR of the CTLA gene influences the susceptibility to RA, and that individuals bearing allelic variants with 16 or more (AT) repeats in this region are overrepresented in the patient population. In our study, the assignation of alleles according to the number of AT repeats was done after sequencing several samples, because

TABLE 2 Genotypic distribution and allelic frequencies of CTLA4 3⬘ UTR polymorphism RA N ⫽ 141

Healthy controls N ⫽ 194

Genotypes

N

%

N

%

p Value

OR (95%CI)

S/S S/L L/L

39 66 36

27.7 46.8 25.5

79 76 39

40.7 39.2 20.1

0.01 pc(3) ⫽ 0.03

0.56 (0.34–0.91)

RA (2n ⫽ 282) Allelic frequency S L

Healthy controls (2n ⫽ 388)

N

%

N

%

p Value

OR (95%CI)

144 138

51.1 49.9

234 154

60.3 39.7

0.02 pc(2) ⫽ 0.04

0.69 (0.50–0.95) 1.46 (1.06–2.01)

Alleles are grouped into two categories according to the number of AT repeats (S ⬍ 16 AT repeats; L ⫽ 16 or more AT repeats). pc ⫽ corrected p value; number in parentheses indicates correction factor (number of genotypes and alleles respectively). Abbreviations: CTLA4 ⫽ cytotoxic T-lymphocyte antigen 4; OR ⫽ odds ratio; RA ⫽ rheumatoid arthritis; UTR ⫽ untranslated region.

CTLA4 3⬘ UTR Polymorphism and RA

79

TABLE 3 Distribution of CTLA4 alleles according to polymorphisms in three different positions CTLA4 “alleles” ⫺318C ⫺318C ⫺318C ⫺318C ⫺318T ⫺318T

49A 3⬘ UTR S 49A 3⬘ UTR L 49G 3⬘ UTR S 49G 3⬘ UTR L 49A 3⬘ UTR S 49A 3⬘ UTR L

RA (2n ⫽ 176)

Percent

Healthy controls (2n ⫽ 272)

Percent

87 26 3 43 1 16

49.4 14.8 1.7 24.4 0.6 9.2

160 33 5 51 11 12

58.8 12.1 1.8 18.8 4.0 4.4

Abbreviations: CTL A4 ⫽ cytotoxic T-lymphocyte antigen 4; RA ⫽ rheumatoid arthritis; UTR ⫽ untranslated region.

assignations based on the length of the DNA fragment are not unequivocal and are influenced by the primers and the method used. In our population, the shortest and most frequent allele found was (AT)7 that corresponds to the allele named 86 bp in most of the reported studies. The majority of alleles depict frequencies too low for statistical analysis, and therefore we grouped the alleles into two categories: ⬍16 AT repeats, and 16 or more AT repeats in this region. This cut off generates two similar groups in terms of the number of healthy individuals in each group, and, also, one common allele [(AT)7 and (AT)16] was present in each group. In order to determine which of the three CTLA4 polymorphisms is responsible for the association, primary and secondary association studies were conducted following the Svejgaard’s method [31]. Both studies

indicate that the association is mainly with the 3⬘ UTR position, although the relatively small number of samples after stratification, and, especially, the strong linkage disequilibrium between alleles in different positions limit the statistical significance of the results obtained; therefore, confirmatory studies carried on other populations would be necessary. Differences in the strength of linkage disequilibrium among diffrent populations could explain the discrepant results obtained in studies on RA [20 –24]. The significant increase of alleles having 16 or more AT repeats among RA patients has been also observed in some organ-specific diseases (Grave’s disease, IDDM, and myasthenia gravis) [13, 25–28] and in a systemic autoimmune disease (SLE) [19]. These finding are very interesting because it is known that AU-rich regions of the

TABLE 4 Analysis for strongest CTLA and rheumatoid arthritis association

Basic comparisons ⫺318 T vs. ⫺318 C 49 G vs. 49 A 3⬘ UTR “L” vs. 3⬘ UTR “S” Secondary comparisons ⫺318 T and 49G ⫹⫹ vs. ⫺⫹ ⫹⫺ vs. ⫺⫺ ⫹⫹ vs. ⫹⫺ ⫺⫹ vs. ⫺⫺ ⫺318 T and 3⬘ UTR “L” ⫹⫹ vs. ⫺⫹ ⫹⫺ vs. ⫺⫺ ⫹⫹ vs. ⫹⫺ ⫺⫹ vs. ⫺⫺ 49G and 3⬘ UTR “L” ⫹⫹ vs. ⫺⫹ ⫹⫺ vs. ⫺⫺ ⫹⫹ vs. ⫹⫺ ⫺⫹ vs. ⫺⫺

a

b

c

d

p Value

OR (95%CI)

Test number

17 46 85

23 56 96

159 130 91

249 216 176

NS NS 0.006 pc(6) ⫽ 0.036

1.71 (1.14–2.57)

0 17 0 46

46 113 17 113

0 23 0 56

56 193 23 193

* NS * NS

3 4 5 6

16 1 16 69

69 90 1 90

12 11 12 84

84 165 11 165

NS NS 0.01 pc(6) ⫽ 0.06 0.05 pc(6) ⫽ 0.30

14.67 (1.61–669.74) 1.51 (0.98–2.32)

3 4 5 6

43 3 43 42

42 88 3 88

51 5 51 45

45 171 5 171

NS NS NS 0.02 pc(6) ⫽ 0.12

1.81 (1.08–3.06)

3 4 5 6

The question in tests 3 and 4 is: Is factor A associated independently of factor B? The question in tests 5 and 6 is: Is factor B associated independently of factor A? * ␹2 could not be evaluated (one or more entry ⫽ 0); pc ⫽ corrected p value; number in parentheses indicate correction factor (number of comparisons). Abbreviations: CI ⫽ confidence interval; OR ⫽ odds ratio; UTR ⫽ untranslated region.

80

3⬘ UTR influence the mRNA stability [32–34] and, indeed, it has been proposed that large mRNAs may be more unstable than the small ones [13, 32]. As CTLA4 plays a downregulator role in T-cell activation [7], mRNA instability may contribute to disrupt the T-cell homeostasis. The fact that analogous findings were obtained in RA as in some organ-specific autoimmune diseases provides additional weight to this hypothesis. Recently, an increase of IL-2 sR␣ serum levels and a higher level of telomerase activity in peripheral blood mononuclear cells has been described in individuals with myasthenia gravis bearing a high number of AT repeats in the 3⬘ UTR of the CTLA4 gene [35]. Our previous study [21] suggested a possible interaction between HLA-DRB1 specificities and CTLA4 A/G polymorphism in the susceptibility to RA. Although results from the present study revealed some differences in the distribution of DRB1 specificities, after correction of the statistical results, differences became nonsignificant. Other groups have also reported similar results involving different DRB1 specificities [20, 22, 23, 36]. Overall, our findings in RA patients, as well as results obtained in certain autoimmune diseases by others, may suggest that mechanisms implying CTLA4 molecule could be involved in the pathogenesis of these conditions, although the exact nature of such mechanism and the coparticipation of the human leukocyte antigen system remain to be clarified. ACKNOWLEDGMENTS

This study was supported by grants from Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain (FIS 99/0255 and 00/0566), Fundacion Reina Mercedes, and from Plan Andaluz de Investigacio´ n (PAI, grupo CTS-0197), Junta de Andalucı´a.

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