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Rheumatology 2001; 40: 1370-1374
© 2001 British Society for Rheumatology

Original Papers

HLA-DQB1 CAR1/CAR2, TNFa IR2/IR4 and CTLA-4 polymorphisms in Tunisian patients with rheumatoid arthritis and Sjögren's syndrome

H. Hadj Kacem, N. Kaddour1, F.-Z. Adyel, Z. Bahloul1 and H. Ayadi

Laboratoire de Génétique Moléculaire Humaine, Faculté de médecine, Sfax and
1 Service de médecine interne CHU Hédi Chaker, Sfax, Tunisia


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives. To evaluate the contribution of HLA class II region and the CTLA-4 gene in genetic susceptibility to rheumatoid arthritis (RA) and Sjögren's syndrome (SS) in the Tunisian population.

Methods. The polymorphisms of a (CA)n microsatellite of HLA-DQB1 CAR1/CAR2, TNFa IR2/IR4 and an (AT)n microsatellite in the 3'-untranslated region of exon 3 of the CTLA-4 gene were analysed after specific polymerase chain reaction (PCR) amplification. Typing of CTLA-4 A/G exon 1 polymorphism was achieved by the PCR–restriction fragment length polymorphism method.

Results. Genomic DNA from 60 patients with RA, 58 patients with SS and 150 healthy individuals was genotyped. The distribution of HLA-DQ CAR1/CAR2 allele frequencies differed between patients and controls in both diseases (RA, P<10-15; SS, P=7.6x10-15; RA+SS, P<10-15). The analysis of TNFa IR2/IR4 and CTLA-4 A/G polymorphisms did not show any differences in allele or genotype frequencies between patients and control subjects in either disease. The distribution of CTLA-4 (AT)n allele frequencies differed between patients with RA and controls (P=10-3), whereas no significant difference was detected between patients with SS and controls.

Conclusion. These data suggest the involvement of HLA-DQ CAR1/CAR2 polymorphisms in genetic susceptibility to RA and SS and the participation of the CTLA-4 gene, or a gene closely associated with it, in the development of RA.

KEY WORDS: HLA-DQ CAR, TNF, CTLA-4, Association, Polymorphism, Rheumatoid arthritis, Sjögren's syndrome.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Rheumatoid arthritis (RA) and Sjögren's syndrome (SS) are chronic autoimmune diseases with worldwide distribution. Susceptibility to RA and SS is determined by environmental, immunological and multigenic factors. Estimates of the relative genetic and environmental contributions to RA, based on covariance analysis of twin data, suggest that the genetic contribution to disease development approaches 50–60% [1]. Human leucocyte antigens (HLA) have been estimated to account for approximately 40% of the genetic component of susceptibility to RA [2].

The association between HLA-DR4 and genetic susceptibility to RA is well documented. It was originally described by Stastny [3] in a white North American population, and has since been reported in many other ethnic groups, including black Americans, Mexicans and Japanese [4]. Moreover, there are some lines of evidence suggesting the genetic association of HLA-DR3 with primary SS in the Caucasian population [5, 6]. These findings were based on the use of standard microlymphocytotoxicity, restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) single specific oligonucleotide methods.

Recently, several studies, using dimorphic and/or polymorphic genetic markers, reported the involvement of the HLA-DQ, TNF and CTLA-4 loci in genetic susceptibility to many autoimmune diseases [7–12]. Indeed, the HLA-DQ CAR1/CAR2 and TNFa IR2/IR4 microsatellites are in linkage disequilibrium with the HLA-DQB1 and TNFB genes respectively [13, 14]. These polymorphic markers are considered reasonably informative because of the number of alleles (more than five) and of the polymorphism information content (PIC) and heterozygosity value, which is above 0.75 in the Caucasian population [15]. The CTLA-4 gene, located on chromosome 2, is polymorphic in untranslated sequences in exon 3, with a dinucleotide (AT)n repeat of varying length [16], and dimorphic with a single-nucleotide polymorphism (SNP) in exon 1 at position 49; this results in a threonine/alanine (Thr/Ala) polymorphism at the protein level [17]. These polymorphisms have not been studied in the Tunisian population.

In this work, we analysed the distribution of allele frequencies of HLA-DQ CAR, TNFa IR2/IR4 and CTLA-4 polymorphisms between Tunisian patients with RA and SS and controls.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Patients and controls
Sixty patients (12 men and 48 women) affected with RA according to the American College of Rheumatology criteria [18] (mean age 45 yr, range 18–80 yr) and 58 patients (six men and 52 women) affected with SS (41 with primary SS and 17 with secondary SS) based on the Kaplan classification criteria [19] (mean age 48 yr, range 26–76 yr), were included prospectively in this study. Data for the patient group were compared with those obtained from a control population of 150 unrelated healthy subjects (70 men and 80 women) originating from the same area, with no clinical evidence or family history of RA, SS or inflammatory joint disease. DNAs from patients and controls were obtained from peripheral blood as described previously [20].

Methods
The polymorphisms of dinucleotide repeats of HLA-DQB1 CAR1/CAR2, TNFa IR2/IR4 and CTLA-4 (AT)n were analysed after specific PCR amplification using appropriate primers [13, 14, 16]. The 5' end of the forward primers was labelled with [{gamma}32P]ATP. Amplified products were resolved on 5–7% sequencing gels and detected by autoradiography. The CTLA-4 exon 1 A/G transition at position 49 was typed by PCR–RFLP. The primers used were 5'-CAAGGCTCAGCTGAACCTGGGT-3' and 5'-TACCTTTAACTTCTGGCTTTG-3'. The amplified products were digested with the restriction enzyme KpnI (Amersham International, Amersham, UK) and analysed on a 4% agarose gel. The G allele corresponds to the presence of two fragments [173 and 22 base pairs (bp)] generated by KpnI digestion, and the A allele corresponds to the 195-bp uncleaved fragment. The distribution of the alleles in patients with RA or SS versus controls were compared by the {chi}2 test. The association of RA and SS with HLA-DQB1, TNF-B and CTLA-4 alleles was evaluated with the relative predispositional effects methods [21]. Statistical significance was reached when P<0.05, and Fisher's exact test was used when necessary. Odds ratios (OR) were calculated according to Woolf's formula [22].


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
The HLA-DQB1 CAR1/CAR2 microsatellite marker revealed 10 alleles in the controls and eight alleles in the patients, with sizes ranging from 97 to 123 bp.

The distribution of allele frequencies (Table 1Go) differed between patients and controls for both diseases and the overall {chi}2 was significant (RA, {chi}2=98.5, 9 degrees of freedom, P<10-15; SS, {chi}2=86.6, 9 degrees of freedom, P=7.6x10-15; RA+SS, {chi}2=123.0, 9 degrees of freedom, P<10-15). The allele frequency of HLA-DQB1 CAR1/CAR2 97-bp in both RA (57.6%) and SS (54.3%) was significantly higher than that in the normal population (13.9%), with OR 8.39 (4.99<OR <14.17; P<10-15) and 7.34 (4.36<OR<12.38; P<10-15) respectively. To reveal the relative effects (predisposing, protective or neutral) of the HLA-DQB1 CAR1/CAR2 alleles, we used the relative predispositional effects (RPE) method [21]. After removing the 97-bp allele from both patient and control data and repeating the comparisons, the significance of overall {chi}2 was preserved (RA, {chi}2=23.762, 8 degrees of freedom, P=0.0025; SS, {chi}2=21.349, 8 degrees of freedom, P=0.0063; RA+SS (pooled data), {chi}2=21.891, 8 degrees of freedom, P=0.0051). However, the strong contribution of the 97-bp allele to susceptibility to RA and SS was obvious when the variation of probabilities (from 10-15 to 10-3) were taken into account. The absence of the 105- and 123-bp alleles in the patients did not show any protective effect against either disease (P<10-15). The absence of the 105-bp allele in our patients has already been observed in Tunisian patients with Graves disease but without any protective effect against the development of disease [23]. The low frequency of the 105-bp allele in the Tunisian population could explain its absence in Graves disease and in RA and SS patients.


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  		TABLE 1. Frequency of HLA-DQB1 CAR1/CAR2 alleles in RA and SS patients

 
Recently, some studies have shown the involvement of HLA-DQ CAR alleles in susceptibility to RA. HLA-DQ CAR 113 and HLA-DQ CAR 115 in combination with HLA-DRB1 alleles were significant markers of susceptibility to RA in a Korean population [24]. In addition, a significant positive association between RA and HLA-DQ CAR 117 was reported in a Canadian population [25], whereas in the present study these alleles were present at modest frequencies in the Tunisian patients and controls, in whom the HLA-DQ115 allele was absent (Table 1Go).

Until now, it has been unclear whether these HLA-DQ alleles play a role by themselves or reflect linkage disequilibrium with other genes located in the same chromosomal region. Although the role of microsatellites in eukaryotic genes is still unknown, an association between microsatellites located in the class II and class III HLA regions and different diseases has been reported [8–10]. Microsatellite polymorphism could reveal non-MHC genes associated with autoimmune diseases in the 6p21 chromosomal segment.

The TNFa IR2/IR4 microsatellite marker revealed 14 alleles in healthy subjects, 13 alleles in RA patients and 11 alleles in SS patients. The allele frequencies in RA and SS patients and controls are shown in Table 2Go. The most frequent allele was TNFa2 (21.19 and 28.57% in RA and SS patients respectively). No significant difference was observed between patients and controls despite a trend to an increased frequency of satellite a2 in patients (19.26% in controls; P>0.05).


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  		TABLE 2. Frequency of TNFa IR2/IR4 alleles in RA and SS patients

 
By the use of the PCR–RFLP method, the CTLA-4 exon 1 A/G transition polymorphism was assigned in 60 RA patients, 56 SS patients and 150 controls (Table 3Go). As in Spanish and UK populations [26], data on allele frequencies in our study showed no difference between patients and controls.


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  		TABLE 3. CTLA-4 exon 1 polymorphism in RA and SS patients and controls

 
Analysis of the genotype distribution of the diallelic polymorphism of CTLA-4 resulted in no significant difference between patients and controls in either disease (P>0.05) (Table 3Go). The phenotypic frequency of Thr A-positive individuals was marginally higher in patients with RA (61.6%) than in controls (54.6%), and frequencies of Ala G-positive individuals were elevated in SS patients (92.8%) and controls (86.6%).

Analyses of the CTLA-4 A/G polymorphism with respect to the HLA-DQB1 CAR1/CAR2 97-bp allele demonstrated no significant increase in G allele frequency in patients (P>0.05) [RA, 56.2% (54/96), SS, 70% (63/90)] compared with controls [60.4% (29/48)]. Comparison of the CTLA-4 exon 1 polymorphism between female RA patients and controls showed the same allelic distribution (P>0.05) and no significant increase in the AG genotype (P>0.05). These results are different from those of a study in the Spanish population, in which a high frequency of heterozygous CTLA-4 49 AG individuals among female patients with RA was observed [27]. We did not analyse the CTLA-4 exon 1 polymorphism in the SS patients with respect to sex because of the small number of male patients in the sample (six out of 58).

The polymorphism of CTLA-4 (AT)n revealed 20 alleles in controls compared with 19 and 14 alleles in patients with RA and SS respectively (Table 4Go). The distribution of allele frequencies differed between patients with RA and controls ({chi}2=43.77, 19 degrees of freedom, P=10-3), and the highest OR was found with the CTLA-4 (AT)18 allele (OR 7.33, 1.73<OR<35.56; P=0.0007; Fisher's exact test, P=0.002). After removing the CTLA-4 (AT)18 allele from both patient and control data, and repeating the comparisons, the significance of the overall {chi}2 was preserved ({chi}2=33.4, 18 degrees of freedom, P=0.015). Analysis of the CTLA-4 (AT)n polymorphism in patients with SS did not show any difference in the distribution of allele frequencies between patients and controls (P>0.05).


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  		TABLE 4. CTLA-4 (AT)n repeat polymorphism in RA and SS patients and controls

 
The molecular basis of the regulated expression of CTLA-4 is poorly understood. Exon 1 A/G polymorphism is an unlikely candidate, because the Thr/Ala substitution is not expected to affect the function of the leader peptide [7]. On the contrary, the CTLA-4 (AT)n repeat seems to be a good candidate because it may be involved in the stability of mRNA [28]. Long runs of A and T occur in the 3'-untranslated regions of numerous lymphokines, cytokines and proto-oncogene mRNAs, most of which are expressed transiently [28]. Furthermore, stable wild-type rabbit ß-globin mRNA becomes highly unstable when a 51-nucleotide AT sequence is introduced into the 3'-untranslated region of this gene [28]. Therefore, we can speculate about a relationship between CTLA-4 (AT)n polymorphisms and CTLA-4 expression on the cell surface. Further understanding of the regulated expression of this gene is central to the elucidation of the regulation of T-cell-mediated immune response, which may affect the pathogenesis of autoimmune diseases. However, lack of agreement between SNP and microsatellite results with respect to the CTLA-4 gene could be explained by a linkage disequilibrium between CTLA-4 (AT)n markers and other genes in the same chromosomal region that are involved in genetic susceptibility to RA. Indeed, the CTLA-4 gene region contains other nearby candidate genes for autoimmune diseases, including CD28, STAT-1 and -4, and caspases 8 and 10 (http://gdbwww.gdb.org/).

Although the level of consanguinity in the general Tunisian population is high [29], the HLA-DQ CAR1/CAR2, TNFa IR2/IR4 and CTLA-4 (AT)n microsatellite markers show almost the same degree of polymorphism in the Tunisian population (present work) and in occidental populations [13–15, 30]. These results are in concordance with our previous studies reporting variable immunoglobulin heavy-chain genes [31, 32] and DFNB1-linked markers [33]. They presumably reflect genetic exchanges and the migration of people around the Mediterranean sea. Indeed, the allele number of a particular genetic marker could be reduced in a large consanguineous family but be similar to that found in one panmictic population in the general population.

In conclusion, the present data show that the HLA-DQB1 CAR 97-bp allele is associated with both RA and SS, and the CTLA-4 gene, or a gene closely associated with it, could be one of the non-HLA-linked susceptibility genes for RA.


   Acknowledgments
 
This work was supported by the Délégation de la Recherche Scientifique et Technique (Tunisia). We would like to thank Dr Hafedh Makni for her helpful review of the manuscript.


   Notes
 
Correspondence to: H. Ayadi, Faculté de Médecine, Laboratoire de Génétique Moléculaire Humaine, Av. Majida Boulila 3029, Sfax, Tunisia Back


   References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Submitted 3 October 2000; Accepted 4 June 2001


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