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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is a key negative regulator of the T cell immune response, and the CTLA4 gene is highly polymorphic. Many positive associations between CTLA4 single-nucleotide polymorphisms (SNPs) and various autoimmune diseases have been identified. Two CTLA4 SNPs that are important relative to genetic susceptibility in human autoimmune diseases are the +49GA polymorphism in exon 1 and the CT60A/G polymorphism in the 3′-untranslated region. Using these 2 polymorphisms as markers, we investigated possible genetic associations of CTLA4 in Australian patients with primary Sjögren's syndrome.

Methods

One hundred eleven Australian Caucasian patients with primary SS and 156 population-based controls were genotyped for CTLA4 by polymerase chain reaction–restriction fragment length polymorphism methods, using the restriction enzymes BseXI (+49G/A) and HpyCh4 IV (CT60).

Results

The CT60 and +49G/A SNPs were in strong linkage disequilibrium, and only 3 haplotypes were observed. Significant differences in the haplotype frequencies between patients with primary SS and controls (P = 0.032) were observed, with susceptibility to primary SS associated with both the +49A;CT60A haplotype and the +49A;CT60G haplotype, whereas the +49G;CT60G haplotype was protective against primary SS. The +49A;CT60G haplotype association was predominantly with Ro/La autoantibody–positive primary SS, and the dose of this haplotype influenced the severity of daytime sleepiness (P = 0.036). The +49A;CT60A haplotype appeared to be protective against the development of Raynaud's phenomenon in patients with primary SS (odds ratio 0.49, 95% confidence interval 0.27–0.91).

Conclusion

The CTLA4 +49G/A and CT60 haplotypes are associated with susceptibility to primary SS and with some extraglandular manifestations of the disease.

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) protein is a key negative regulator of the T cell immune response and is expressed predominantly in activated and regulatory T cells. Distinct functions of CTLA-4 include the following: setting the threshold for T cell activation, thereby contributing to the maintenance of peripheral tolerance (1); suppression of T cell proliferation and inflammatory cytokine production; and induction of apoptosis in activated T cells (2). Alterations in the expression or function of CTLA-4 are therefore likely to influence susceptibility to autoimmune diseases.

More than 100 single-nucleotide polymorphisms (SNPs) have been identified in the CTLA4 gene region (3), which maps to chromosome 2q33 (Figure 1A). However, only 1 coding-region SNP, the +49A[RIGHTWARDS ARROW]G polymorphism in exon 1 resulting in a threonine-to-alanine conversion at codon 17, has been identified. Many autoimmune disease association studies of individual CTLA4 SNPs have yielded mixed results (4–7), although there is consistent evidence for an association with Graves' disease, autoimmune hypothyroidism, and type 1 diabetes mellitus. A recent study (3) that screened all known SNPs in the CTLA4 region demonstrated that the linkage with Graves' disease, autoimmune hypothyroidism, and type 1 diabetes mellitus was strongest with the G allele of the noncoding CT60A/G polymorphism, which was correlated with lower messenger RNA (mRNA) levels of a soluble, alternatively spliced form of CTLA4.

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Figure 1. A, The CTLA4 locus is located at chromosome 2q33 and lies between the CD28 and inducible costimulator (ICOS) loci. The direction of transcription is from left to right, as shown by the arrow. The solid boxes represent the 4 exons, and the 5′- and 3′-untranslated regions are depicted by open boxes. The relative locations (not to scale) of some of the previously studied CTLA4 single-nucleotide polymorphisms (SNPs) in relation to the +49 and CT60 SNPs are shown. The +49 SNP in exon 1 is the only coding-region SNP identified, and the CT60 SNP is adjacent to the 3′-untranslated region, close to the (AT)n microsatellite repeat. B, Strong linkage disequilibrium across 8 of these SNPs, resulting in 3 common haplotypes, has been identified in Scandinavian Caucasians (13) and European Caucasians (14). These haplotypes can be identified or “tagged” by the +49;CT60 haplotype combinations identified in the present study.

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Primary Sjögren's syndrome (SS) is a common systemic autoimmune inflammatory condition that is associated with exocrine failure of the salivary and lacrimal (tear) glands and predominantly affects women. Characteristic features of primary SS are severe dryness of the eyes and mouth (sicca) and a variety of extraglandular and systemic manifestations. Anti-Ro/SSA and/or anti-La/SSB autoantibodies are present in up to 80% of patient sera and are markers for both disease and disease severity. The etiology of SS is multifactorial, and the genetic and environmental risk factors are far from being elucidated (8). Studies performed to date have failed to identify any confirmed genetic associations of major significance, other than an association with HLA class II genes. Previously, we demonstrated that the degree of diversification of the anti-Ro/La autoantibody response is influenced by distinct HLA haplotypes (9).

The current study was undertaken to investigate genetic associations of the CTLA4 CT60 and +49G/A SNPs in Australian Caucasian patients with primary SS, and to determine CTLA4 haplotypes and their associations with susceptibility to primary SS and/or extraglandular and serologic features of the disease.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Subjects.

The study group comprised 111 Australian Caucasian patients with primary SS meeting the revised 2002 American–European Consensus Group criteria (10) and 156 population-based healthy Caucasian controls. The patients with primary SS were predominantly female (99 of 111 [89%]), and the median age at disease onset was 50 years (interquartile range 38–59 years). The patients were recruited, with written informed consent, from rheumatology clinics at The Queen Elizabeth Hospital and Flinders Medical Centre, Adelaide, South Australia. For each patient, the Ro/La autoantibody status, information concerning the presence or absence of Raynaud's phenomenon, the Epworth Sleepiness Scale (ESS) score, the FACIT-F fatigue score, and the American Urological Symptom Index (AUA-7) score (11) were obtained from hospital clinical records.

CTLA4 genotyping

DNA was extracted, using the salt precipitation method, from whole blood of patients and controls. The polymerase chain reaction (PCR)–restriction fragment length polymorphism method was used to genotype samples. The +49G/A SNP is located at base 84553 (CTLA4 sequence; GenBank accession no. AF411058). A 360-bp fragment spanning bases 84340–84699 was amplified using primers 5′-TTCAAGTGCCTTCTGTGTGTG-3′ and 5′-AATCACTGCCCTTGACTGCT-3′, with an annealing temperature of 58°C. Twenty microliters of PCR product was digested with 2 units of Bse XI (Fermentas, Hanover, MD) in a 1× digestion buffer (supplied by the manufacturer) for 4 hours at 65°C. Digestion products were visualized on 2% agarose gels stained with ethidium bromide.

Samples demonstrating only a single (undigested) 360-bp band were identified as genotype AA, samples demonstrating 2 bands of 162 bp and 198 bp were typed as GG, and samples demonstrating 3 bands of 162 bp, 198 bp, and 360 bp were typed as AG. The CT60 SNP is located at base 90734 (CTLA4 sequence; GenBank accession no. AF411058). A 382-bp fragment spanning bases 90481–90862 was amplified using primers 5′-ATCTGTGGTGGTCGTTTTCC-3′ and 5′-CCATGACAACTGTAATGCCTG T-3′, with an annealing temperature of 58°C.

Similarly, digestion was performed for 4 hours at 37°C, using the restriction enzyme HpyCh4 IV (New England Biolabs, Beverly, MA). Samples demonstrating a single (undigested) band at 382 bp were assigned genotype AA, samples demonstrating 2 cut bands at 130 bp and 252 bp were typed as GG, and samples demonstrating 3 bands at 130 bp, 252 bp, and 382 bp were typed as AG. Appropriate controls (no template and known genotype) were included in each typing run.

Statistical analysis.

CTLA4 SNP and haplotype frequency distributions for patients with primary SS versus controls and primary SS patient subgroups were compared and interpreted using the log-likelihood ratio chi-square test and odds ratios (ORs), both of which were estimated by logistic regression. Analysis of the association of the haplotype dose with the ESS, AUA-7, and FACIT-F symptom scores was performed using the gamma correlation, a nonparametric correlation coefficient most appropriate for multiple tied ranks.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The CTLA4 +49 and CT60 SNPs were in strong linkage disequilibrium, and only 3 haplotypes were identified in both patients with primary SS and controls: +49A;CT60A, +49A;CT60G, and +49G;CT60G. These are identical to the +49;CT60 haplotypes observed in Spanish Caucasians (12) and effectively define the 3 extended haplotypes (Figure 1B) observed in Scandinavian and European Caucasians (13, 14).

The individual +49 and CT60 SNPs and their haplotypes were in Hardy-Weinberg equilibrium in both patients with primary SS and controls. Both allele and haplotype frequencies were therefore analyzed for both SNPs, but the haplotype analysis resulted in a more consistent interpretation of the data. There were significant differences in the frequency of the CTLA4 haplotype between patients and controls (P = 0.032) (Table 1). Relative to the +49G;CT60G haplotype, the frequency of both the +49A;CT60A haplotype (OR 1.53, 95% confidence interval [95% CI] 1.04–2.25) and the +49A;CT60G haplotype (OR 1.78, 95% CI 1.08–2.93) was increased in patients with primary SS compared with controls.

Table 1. CTLA4 +49A/G and CT60 allele and haplotype frequencies in patients with primary Sjögren's syndrome and controls*
 Controls (n = 156)Patients (n = 111)OR (95% CI)
  • *

    For the frequency of the +49 single-nucleotide polymorphism (SNP) in patients versus controls, χ2 (1 degree of freedom [df]) = 6.46, P = 0.011; for the frequency of the CT60 SNP in patients versus controls, χ2 (1df) = 1.70, P = 0.19; for the frequency of the CTLA4 haplotype in patients versus controls, χ2 (2df) = 6.86, P = 0.032. OR = odds ratio; 95% CI = 95% confidence interval.

  • P < 0.05.

+49A0.5870.6941.60 (1.11–2.30)
+49G0.4130.3061
CT60G0.5710.5140.79 (0.56–1.12)
CT60A0.4290.4861
+49A;CT60A0.4290.4861.53 (1.04–2.25)
+49A;CT60G0.1570.2071.78 (1.08–2.93)
+49G;CT60G0.4130.3061

The CTLA4 haplotype frequencies in Ro/La autoantibody–positive versus Ro/La autoantibody–negative patients with primary SS were suggestive of some differences that did not reach statistical significance (P = 0.11), but the power was low because only 17 Ro/La-negative patients were available for analysis (Table 2). The frequency of the +49A;CT60G haplotype was increased in Ro/La-positive patients with primary SS compared with controls (OR 2.00, 95% CI 1.17–3.41) (Table 2), but not in Ro/La-negative patients compared with controls (OR 0.76, 95% CI 0.20–2.97) (Table 2). In contrast, the frequency of the +49A;CT60A haplotype was relatively increased in both autoantibody subgroups, but relatively more so in the Ro/La autoantibody–negative subgroup (Table 2). Therefore, although the frequency of both of these CTLA4 haplotypes was increased in SS, there were differences between their associations in terms of the Ro/La autoantibody response. There was no evidence of interaction between CTLA4 genotypes and HLA–DR2 or DR3 (both of which are associated with Ro/La autoantibodies in primary SS) (9), or any association with increasing diversification of the Ro/La response, IgG levels, rheumatoid factor titers, sex, or age at disease onset.

Table 2. CTLA4 +49G and CT60 allele and haplotype frequencies in Ro/La autoantibody–negative and autoantibody-positive patients with primary Sjögren's syndrome
 Ro/La statusOR (95% CI)*
Negative (n = 17)Positive (n = 94)Ro/La negative vs. controlsRo/La positive vs. controls
  • *

    OR = odds ratio; 95% CI = 95% confidence interval.

  • P < 0.05.

+49A0.7060.6911.66 (0.78–3.55)1.52 (1.05–2.22)
+49G0.2940.30911
CT60G0.3820.5370.49 (0.24–1.00)0.90 (0.63–1.27)
CT60A0.6180.46311
+49A;CT60A0.6180.4631.89 (0.88–4.05)1.40 (0.94–2.08)
+49A;CT60G0.0880.2290.76 (0.20–2.97)2.00 (1.17–3.41)
+49G;CT60G0.2940.30911

There were significant differences in the CTLA4 haplotype frequencies between patients with primary SS with and those without Raynaud's phenomenon (P = 0.046) (Table 3). The +49A;CT60A haplotype was protective against Raynaud's phenomenon (OR 0.49, 95% CI 0.27–0.91) (Table 3). There was, however, no association between Raynaud's phenomenon and Ro/La autoantibody status.

Table 3. Allele and haplotype frequencies showing that the CTLA4 +49A;CT60A haplotype is protective against RP in primary Sjögren's syndrome*
 RP negative (n = 64)RP positive (n = 42)OR (95% CI)
  • *

    For the frequency of the +49 single-nucleotide polymorphism (SNP) in Raynaud's phenomenon (RP)–negative versus RP-positive patients, χ2 (1 degree of freedom [df]) = 3.56, P = 0.059; for the frequency of the CT60 SNP in RP-negative versus RP-positive patients, χ2 (1df) = 6.01, P = 0.01; for the frequency of the CTLA4 haplotype in RP-negative versus RP-positive patients, χ2 (2df) = 6.13, P = 0.046. OR = odds ratio; 95% CI = 95% confidence interval.

  • P < 0.05.

+49A0.7460.6160.58 (0.33–1.03)
+49G0.2540.3841
CT60G0.4440.6281.92 (1.12–3.29)
CT60A0.5550.3721
+49A;CT60A0.5560.3720.49 (0.27–0.91)
+49A;CT60G0.1900.2440.87 (0.41–1.84)
+49G;CT60G0.2540.3841

We recently demonstrated that daytime sleepiness and urologic symptoms are more severe in patients with primary SS (11), and data regarding these symptoms were available for 63 of the 111 patients with primary SS who were typed for CTLA4 SNPs. The ESS measures daytime sleepiness and is a comprehensively validated, simple, self-administered questionnaire that addresses the likelihood of falling asleep in a variety of situations (15). The dose of the 49A;CT60G haplotype, which is associated with Ro/La autoantibody–positive primary SS, influenced the severity of daytime sleepiness in patients with primary SS (P = 0.036) (Figure 2), although there was no overall difference in the ESS score between Ro/La autoantibody–positive and Ro/La autoantibody–negative patients with primary SS. There were no observed associations between CTLA4 haplotypes and urologic symptom or fatigue severity within the primary SS cohort.

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Figure 2. The Epworth Sleepiness Scale (ESS), a measure of daytime sleepiness. ESS scores have previously been shown to be more severe in patients with primary Sjögren's syndrome compared with controls with osteoarthritis (11). In 63 patients for whom data were available, the CTLA4 +49A;CT60G haplotype dose was significantly correlated with the ESS score (P = 0.036 [gamma correlation]). Values are the mean ± SEM.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The CTLA4 region is highly polymorphic, and the majority of previous studies addressing CTLA4 and autoimmune disease have focused on analysis of individual SNPs, with somewhat conflicting results. We and other investigators (12, 14, 16) have identified 3 common CTLA4 haplotypes. In the current study, we were able to reduce the analysis of haplotypes to 2 SNPs, identified by +49A;CT60A, +49A;CT6G, and +49G;CT60G SNP combinations (Figure 1B). Differences in CTLA4 function are most likely to be associated with the haplotype rather than with individual SNPs. Because individual SNPs may occur on multiple haplotypes, we believe that individual SNP analysis may result in failure to detect an association or inconsistent findings between studies, particularly if the haplotype frequencies vary. For example, we observed a decrease in the frequency of the +49G;CT60G haplotype in patients with primary SS compared with controls and a relative increase in the frequency of both the +49A;CT60A haplotype and the +49A;CT60G haplotype (primarily in Ro/La-positive patients). Therefore, a haplotype carrying the CT60G allele, previously associated with autoimmune disease (3), is clearly implicated in primary SS, yet analysis of the individual CT60 SNPs detected no association (Tables 1 and 2).

There is considerable clinical and serologic overlap between primary SS and systemic lupus erythematosus (SLE) (17), and the frequency of the +49A;CT60G haplotype, which was associated with Ro/La-positive primary SS in this study, was also increased in Spanish patients with SLE (12). This same haplotype is also associated with celiac disease (13), for which patients with primary SS are at increased risk (18). An increased prevalence of lymphoma in primary SS is well recognized (19), and, interestingly, both of the primary SS CTLA4 susceptibility haplotypes carry the +49A allele, which is associated with non-Hodgkin's lymphoma (20). Further study of the effect of CTLA4 haplotypes on the risk of lymphoma in patients with primary SS is clearly warranted.

The CTLA4 haplotypes associated with the systemic rheumatic diseases primary SS and SLE are probably different from the haplotypes associated with organ-specific autoimmune diseases. In Graves' disease, autoimmune hypothyroidism, and type 1 diabetes mellitus, associations with both CT60G and +49G SNPs were observed, with the association with CT60G being statistically stronger (3). This implies an association with the +49G;CT60G haplotype, which was protective against primary SS in the present study. Unlike these organ-specific autoimmune diseases, both primary SS and SLE are associated with systemic B cell activation, polyclonal hypergammaglobulinemia, antinuclear autoantibodies, and multisystem involvement.

CTLA4 exerts distinct independent effects during different phases of T cell responses, including setting the threshold for T cell activation, suppression of T cell proliferation, and induction of apoptosis in already-activated T cells (2). It may even contribute directly to the regulation of B cell responses, because B cells express CTLA4 after cell–cell contact with activated T cells (21). A recent study in patients with SLE demonstrated that CTLA4 genotypes influenced immune responsiveness to Epstein-Barr virus (EBV) (22). EBV has long been hypothesized to play a role in the etiology of primary SS and SLE, and there is evidence that the anti-Ro autoantibody response, which is common in these patients, may be a result of molecular mimicry between Epstein-Barr nuclear antigen 1 and the 60-kd Ro antigen (23). In this context, it is interesting that in the present study, individual CTLA4 haplotypes influenced the expression of Ro/La autoantibodies in primary SS.

These demonstrated differential effects of CTLA4 polymorphisms on the risk of organ-specific versus systemic autoimmune diseases imply differing pathways of autoimmune pathogenesis resulting from altered CTLA4 expression and/or regulation. Therefore, an understanding of the functional effects of the CTLA4 haplotypes may provide key insights into the pathogenesis of autoimmunity. Unfortunately, existing functional studies are difficult to interpret, because they have focused on individual SNPs and have not examined the combined functional effects of the haplotypes on which they occur. The +49G SNP may be associated with incomplete CTLA4 glycosylation, lower cell surface expression (24), and reduced control of T cell proliferation in vitro (25). The CT60G SNP is implicated in lower mRNA levels of the soluble alternatively spliced form of CTLA4 (3), although this was not confirmed in a more recent study (26). Binding of soluble CTLA4 to B7 ligands on antigen-presenting cells may be an important mechanism for blocking co-stimulation of T cell activation, and is the rationale behind the therapeutic use of CTLA-4Ig in human autoimmune diseases such as rheumatoid arthritis. CTLA-4Ig has demonstrated efficacy in a variety of animal models of autoimmunity (27); however, this may be an oversimplification, because transient CTLA-4Ig blockade has also been shown to enhance antigen-specific T cell responses against murine tumors (28).

The severity of infiltration of immune cells in primary SS salivary gland biopsy specimens is associated with both increased titers of Ro/La autoantibodies and an increased frequency of extraglandular manifestations (11). Although within-patient subgroup comparisons are hypothesis-generating rather than definitive, we observed some potentially interesting associations. The CTLA4 +49A;CT60A haplotype was protective against the development of Raynaud's phenomenon in patients with primary SS. Raynaud's phenomenon is associated with a variety of autoimmune rheumatic diseases, but the associated risk factors and pathogenesis are not well understood. A previous study linked interleukin-1 (IL-1) haplotypes with susceptibility to Raynaud's phenomenon in primary SS (29). Genetic associations with both CTLA4 and IL-1 thus implicate immune mechanisms in the development of Raynaud's phenomenon, although in our cohort Raynaud's phenomenon was not associated with other indicators of the severity of autoimmune disease, such as Ro/La autoantibodies.

We previously showed that the severity of daytime sleepiness (based on the ESS score) is significantly increased in patients with primary SS (11). In the present study, we observed that the +49A;CT60G haplotype was associated with predominantly Ro/La-positive primary SS, and also correlated with daytime sleepiness in a dose-dependent manner. Our working hypotheses to explain increased daytime sleepiness in patients with primary SS include upper airway collapse due to altered surface tension resulting from dry airways with consequent obstructive sleep apnea, or, alternatively, central nervous system circadian dysfunction mediated by antimuscarinic receptor autoantibodies (30). In addition, evidence is now accumulating for a direct relationship between sleep and the immune system (31), and the CTLA4 haplotype association with more severe daytime sleepiness observed in this study is further evidence of this link. We are currently evaluating the nature of the sleep disturbance in primary SS, as well as the role of CTLA4 haplotypes in patients with sleep apnea in the absence of autoimmune disease.

In conclusion, this study has demonstrated that CTLA4 haplotypes are associated with susceptibility to primary SS, as well as with autoantibody production and some extraglandular features in patients with primary SS. The +49A;CT60G haplotype is associated with susceptibility to primary SS, Ro/La autoantibodies, and increased daytime sleepiness. The +49A;CT60A haplotype is associated with susceptibility to primary SS and protection against Raynaud's phenomenon. This study is consistent with other studies implicating CTLA4 as an important susceptibility locus in autoimmune disorders; however, the associations observed for systemic rheumatic autoimmune diseases such as primary SS and SLE are different from those reported for organ-specific diseases such as Graves' disease, autoimmune hypothyroidism, and type 1 diabetes mellitus, which may imply separate mechanisms for the pathogenesis of autoimmunity.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES