Mol. Hum. Reprod. Advance Access originally published online on December 22, 2005
Molecular Human Reproduction 2005 11(10):745-749; doi:10.1093/molehr/gah225
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Variants of the CTLA4 gene that segregate with autoimmune diseases are not associated with endometriosis
1Department of Obstetrics, Gynaecology and Neonatology, ‘Fondazione Policlinico-Mangiagalli-Regina Elena’ Hospital, University of Milano and 2Molecular Biology Laboratory Istituto Auxologico Italiano, Milan, Italy
3 To whom correspondence should be addressed at: Molecular Biology Laboratory, Istituto Auxologico Italiano, Via Zucchi 18, Cusano Milanino, Milan, Italy. E-mail: a.diblasio{at}auxologico.it
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Abstract |
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An autoimmune etiology has been suggested for endometriosis mostly on the basis of an increased prevalence of autoimmune diseases in affected women. Cytotoxic T lymphocyte antigen (CTLA) 4 gene is recognized as a primary determinant for autoimmunity since specific polymorphisms have been associated with predisposition to most autoimmune disorders. This study was aimed to evaluate whether two variants of CTLA4 gene might be associated with endometriosis in an Italian population. We examined the +49A/G polymorphism and the CT60A/G dimorphism in n = 146 endometriosis subjects classified according to Holt and Weiss criteria. Controls were represented by n = 165 women without laparoscopic evidence of the disease. We found no statistically significant difference in the genotype frequencies between women with and without endometriosis. The proportion of the mutant G allele of the +49A/G polymorphism in the former and in the latter group resulted 34 and 30%, respectively. The proportion of the susceptible G allele of the CT60 A/G dimorphism resulted 51% in both groups. No association was demonstrated between the polymorphisms and the clinical forms of the disease and no susceptibility haplotypes were found. These findings suggest that endometriosis aetiology is not primarily associated with the development of CTLA4-linked autoimmunity.
Key words: autoimmune/CTLA4/endometriosis/genetics/polymorphism
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Introduction |
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Endometriosis is a multifactorial inflammatory disorder characterized by implantation and growth of endometrial cells outside the uterine cavity. Mechanisms underlying the disease are still poorly elucidated but familiar aggregation and a higher rate of concordance in monozygotic twins provide support for the role of genetic factors in the pathogenesis of this disorder (Simpson et al., 2003
; Barlow and Kennedy, 2005). A postulated autoimmune etiology has been also recently suggested based on observations of increased polyclonal B-cell activity, abnormalities in function of B and T cells, familiar inheritance, high B-cell and T-cell counts and reduced natural killer (NK) cell activity in patients affected (Matarese et al., 2003; Giudice and Kao, 2004). Also, high serum concentrations of antiphospholipid immunoglobulin (Ig) G, IgA and IgM antibodies (Ulcova-Gallova et al., 2002) and antibodies to endometrium have been reported (Matarese et al., 2003). More importantly, high rates of autoimmune disorders (fibromyalgia, chronic fatigue syndrome, systemic lupus erythematous, rheumatoid arthritis, hypothyroidism, multiple sclerosis and Sjogren’s syndrome) and atopic diseases have been found in women with endometriosis based on a survey by the US Endometriosis Association and the US National Institutes of Health (Sinaii et al., 2002). This latter study, however, suggests but does not demonstrate that endometriosis is an autoimmune disease. The presence of immune system abnormalities linked to potential autoimmune phenomena can indeed be interpreted as a secondary manifestation of the disease itself. Thus, the possible autoimmune nature of endometriosis remains controversial.
Cytotoxic T lymphocyte antigen (CTLA) 4, whose gene is localized in 2q34, is recognized as a primary determinant of susceptibility to autoimmune diseases (Pociot, 2002; Gough et al., 2005). The CTLA4 molecule is a homologue for CD28 and both molecules and their common ligands (B7-1 and B7-2) constitute the costimulatory pathway for T-cell activation. The CTLA4 molecule, in particular, is an essential negative regulator of T-cell activation either by preventing the activation of self-reactive T cells or attenuating T cell response post-activation and, therefore, is known to be involved in maintenance of immune tolerance (Diamond, 2005). Common autoimmune diseases are thought to be caused in part by inherited changes in CTLA4 expression that increase T cell self-reactivity. Infact, reports of allelic association between specific polymorphisms of CTLA4 gene and an ever increasing number of autoimmune diseases including autoimmune hypothyroidism (Chistiakov and Turakulov, 2003), systemic lupus erythematous (Torres et al., 2004), rheumatoid arthritis (Lei et al., 2005) and primary progressive multiple sclerosis (Dyment et al., 2004) in several different ethnic groups support the idea that the CTLA4 region is an important locus for autoimmune disease in general (GoughWalker and Sansom, 2005). Moreover, studies based on positional genetics approach have demonstrated that there is a common autoimmune disease locus within a 6.1-kb 3' region of CTLA4 and other potential etiological mutations in proximity (Ueda et al., 2003).
To gain insights into the potential autoimmune nature of endometriosis, the aim of this study was to evaluate whether two genetic variants of CTLA4 gene might be associated with endometriosis in a population of Caucasian Italian women. Specifically, genotype and allele frequencies of the +49A/G polymorphism in exon 1 (Vaidya et al., 1999) and a recently described CT60A/G dimorphism within the 3'-UTR (Ueda et al., 2003) have been compared in subjects with and without laparoscopic-proven endometriosis.
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Materials and methods |
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Subjects
The A-G exchange polymorphism at nucleotide position +49 in exon 1 and the CT60A/G variant in the 3'-UTR of CTLA4 gene were evaluated in women who attended the endoscopic surgical service of the Department of Obstetrics and Gynecology of the ‘Fondazione Policlinico-Mangiagalli-Regina Elena’ Hospital of Milan to undergo gynaecological laparoscopy. All studied individuals were of Caucasian Italian origin. From January 2001 to June 2003, 318 women were enrolled in the study. All the women underwent complete pre-surgery clinical examination before the diagnostic-operative laparoscopy. Indications to laparoscopy included chronic pelvic pain, infertility, ovarian cysts and myomas. None of the women were taking hormonal medications. Patients were excluded from the study if they were affected by an autoimmune disorder or had had a previous autoimmune or neoplastic disease. Endometriosis was always diagnosed during the laparoscopic intervention, and criteria for inclusion in the study were those proposed by Holt and Weiss (2000)
. Specifically, these criteria include patients with ovarian endometriomas of any size or with pelvic endometriotic implants of any size >5 mm deep or with pelvic endometriotic implants of any size with adhesions not attributable to another cause. Diagnosis of ovarian endometriotic cysts and deep peritoneal lesions has always been confirmed histologically. Diagnosis of superficial peritoneal lesions was based on direct visualization when endometriotic implants presented as typical lesions. Lesions whose aspect was dubious were always removed to confirm their endometriotic nature by histologic examination. Three physicians active in the evaluation and treatment of endometriosis staged patients according to the revised American Society for Reproductive Medicine (rASRM) classification (1997). Endometriosis was documented in 150 women (47%). Stage of the disease was found to be minimal or mild (stage I–II) in 25 cases (16.6%), moderate (Stage III) in 71 cases (47.4%) and severe (Stage IV) in 54 cases (36%). Other gynaecological benign pathologies were present in five of these 150 women with endometriosis (four cases of uterine myomas and one case of adenomyosis). The presence of endometriotic cysts (endometriomas) was observed in 117 (78%) of these 150 women. Deep endometriosis, defined as lesions infiltrating to a depth of at least 5 mm beneath the peritoneal surface, was observed in 34 (22.7%) women. One hundred and sixty-eight women who underwent laparoscopy and in whom no endometriosis was found served as control group. Specifically, they included 31 cases with a regular pelvis, 46 cases of serous or mucinous cysts, 22 cases of dermoid cysts, 24 cases of uterine myomas, 19 cases of pelvic inflammatory disease, two cases of functional cysts, one case of ovarian fibroid, 14 cases of uterine malformations, eight cases of paraovarian cysts, and one case of appendicitis. The mean age ± SD of patients with endometriosis and controls was 32.7 ± 5.8 and 33.7 ± 6.0 years, respectively. Patients were informed that blood would be used for research purposes and gave written consent. Approval for this study was granted by the local Human Institutional Investigation Committee.
Molecular analysis of the CTLA4 CT60A/G polymorphism
The single-nucleotide CT60A/G polymorphism at position +6230 at the end of the CTLA4 transcript was amplified by PCR from genomic DNA, using the following primers: forward 5'- GAGGTGAAGAACCTGTGTTAAA-3'; reverse 5'-ATAATGCTTCATGAGTCAGCTT-3'. PCR products were then subjected to digestion with the restriction enzyme HpyCH4 IV (New England BioLabs) according to the procedure described by Ban et al. (2004). PCR was performed in a total volume of 25 µl containing genomic DNA, 5 pmoles of each primer, 1x Taq polymerase buffer and 0.5 units of Red Taq (Sigma). Cycling conditions for CT60 was set as follows: one cycle at 94°C for 5 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and one final cycle of extension at 72°C for 5 min. The restriction enzyme HpyCH4 IV cuts the 178 base pair (bp) PCR product only if the A allele is present at this site, resulting in fragments of 107 and 71 bp, which were resolved on a 2.5% agarose gel.
Molecular analysis of the CTLA4 + 49A/G exon 1 polymorphism
Genomic DNA was isolated as previously described (Vaidya et al., 1999). The CTLA4 +49A/G polymorphism, which encodes a threonine (GCC) to alanine (ACC) substitution at codon 17 in exon 1 of the CTLA4 gene, was amplified by PCR from genomic DNA, using the following primers: forward 5'-CCACGGCTTCCTTTCTCGTA-3'; reverse 5'-AGTCTCACTCACCTTTGCAG-3'. PCR products were then subjected to digestion with the restriction enzyme Bbv I (New England BioLabs) according to the procedure described by Vaidya et al. (1999). PCR was performed in a total volume of 25 µl containing genomic DNA, 5 pmoles of each primer, 1x Taq polymerase buffer and 0.5 units of Red Taq (Sigma). Cycling conditions for CTLA4 49A/G was set as follows: one cycle at 94°C for 5 min, 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s, and one final cycle of extension at 72°C for 5 min. The restriction enzyme BbvI cuts the 328bp PCR product only if the G allele is present at this site, resulting in fragments of 244 and 84 bp, which were resolved on a 2.5% agarose gel.
Statistical analysis
Considering the reported 46–53% prevalence of the mutated G allele of the CT60A/G polymorphism (Ueda et al., 2003; Torres et al., 2004) and the 28–38% prevalence of the mutated G allele of the +49A/G polymorphism (Vaidya et al., 1999; Malferrari et al., 2005), the sample size used in this study allowed us to detect at the usual level of study power (80% statistical power of avoiding a type-II error and 5% level of significance) at least a one and half-fold increase in the risk of developing endometriosis. Statistical analysis was done using the Statistics Package for the Social Sciences (SPSS) (SPSS, Chicago, IL, USA). Alleles and genotypes frequencies were compared between groups using the 
2 test or Fisher’s exact test, as appropriate. The odds ratio was used to measure the strength of the association between allele frequencies and endometriosis.
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Results |
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In this prospective study, patients and controls were examined before the results of genetic analysis were known, and laparoscopic surgeons were therefore masked to the CTLA4 genotypes of the individuals. Since diagnosis of endometriosis can be definitively proven by laparoscopy and the disease is often asymptomatic, controls consisted of women in which the disease was laparoscopically ruled out. In any case, the frequencies of the G alleles of the CT60A/G dimorphism and the +49A/G polymorphism in our control population resulted perfectly in line with those reported by Ueda et al. (2003)
and Vaidya et al. (1999) for a group of healthy white UK controls (53.2 and 31%, respectively). Some individuals were not successfully genotyped for both mutations tested since amplifications of the correspondent DNA fragments did not result in satisfactory products, and thus restriction enzyme analysis was not performed. For this reason, the number of cases analysed for the two polymorphisms did not coincide. For the single-nucleotide polymorphism CT60A/G, two alleles were present (i.e. allele A and allele G) and three different genotypes (i.e. homozygotes AA and GG or heterozygote AG). Table I summarizes the genotype frequencies of this gene polymorphism in 143 cases of endometriosis and 165 controls. In both populations, we observed a similar prevalence of allele A (49%) and G (51%). The distribution of the three different genotypes did not differ between controls and endometriosis patients. We then analysed the endometriosis group according to the presence or absence of the different forms and features of the disease (presence of deep endometriosis, peritoneal surface implants, adhesions and endometriotic cysts). As summarized in Table II, the frequency of the G allele does not appear to be associated with any of the parameters indicative of a specific manifestation of the disease or a more severe disease.
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For the single-nucleotide polymorphism +49A/G located in the first exon of the gene, two alleles were present (i.e. allele A and allele G) and three different genotypes (i.e. homozygotes AA and GG or heterozygote AG). Table I summarizes the genotype frequencies of this polymorphism in 146 cases of endometriosis and 153 controls. In both groups, there was a strong predominance of allele A, whereas allele G was present less frequently (33.5% in the endometriosis group and 30% in controls). The distribution of the different genotypes resulted similar between women with endometriosis and controls. Moreover, as summarized in Table II, the frequency of the G allele does not appear to be associated with any of the parameters indicative of a specific manifestation of the disease. Therefore, evidence does not indicate an association between endometriosis and the CTLA4 polymorphic loci evaluated.
Based on the haplotype study, we found that distribution of the different haplotypes of the two polymorphisms were not independent (P < 0.001). When the haplotype frequencies were compared between endometriosis patients and controls, we did not find any significant association with any of the haplotype regarded (Table III).
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Discussion |
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This is the first study that has evaluated the potential association between two polymorphisms of the CTLA4 gene, the well-known variant +49A/G in exon 1 (Vaidya et al., 1999
) and the recently described CT60A/G dimorphism (Ueda et al., 2003), and the susceptibility to endometriosis. No evidence of association was found either considering the single polymorphisms separately or combining the data of the two different variants. These findings may have certain relevance in relation to the aetiologic bases of the disease.
Recently, an autoimmune aetiology has been proposed for endometriosis since the disease fulfills most of the classification criteria for such disorders (Giudice and Kao, 2004). Specifically, it presents with tissue damage, production of autoantibodies (against endometrium, ovary, phospholipids) and association with other autoimmune diseases. An increased prevalence of hypothyroidism, autoimmune inflammatory diseases, fibromyalgia and chronic fatigue syndrome has been found in US members of the Endometriosis Association compared with the general US female population. Women with endometriosis also resulted more likely to have allergies, asthma and eczema. Moreover, family members of these women also had autoimmune inflammatory diseases and chronic fatigue states (Sinaii et al., 2002).
Autoimmune diseases, which affect about 5% of the population and disproportionately affect women, comprise a heterogeneous group of disorders (Diamond, 2005). The recent recognition that these diseases need to be considered, at a genetic and mechanistic level, as a related group of diseases has helped the field forward. Recent genetic analyses have supported the existence of common pathways to autoimmunity and have identified susceptibility loci shared by several autoimmune diseases (Knight, 2005). These observations suggest that there are common pathways of intracellular signalling or cellular interactions that are dysregulated in more than one autoimmune disease. Moreover, they provide an explanation for the evidence that these diseases cluster in families with different individuals having different diseases.
In general, one of the most important locus for the susceptibility to autoimmune diseases is represented by the CTLA4 region (Gough et al., 2005). CTLA4 controls common immunity traits such as the amplitude of the immune response and peripheral tolerance, disruption of which can cause autoimmune disease. On the other hand, CTLA4 gene cannot account for determining the organ specificity of autoimmunity. Apparently, CTLA4 splice variants regulate immunoregulatory pathways that are essential (but not sufficient) for autoimmunity even if, as for most genes that contribute to complex diseases, they typically exert small effect on disease risk (Chistiakov and Turakulov, 2003; Gough et al., 2005).
Until recently, the exact polymorphisms responsible for the association of CTLA4 with autoimmune diseases remained unclear. One of the most reported significant associations was the polymorphism +49A/G in exon 1, although it has not been ascertain whether its effect might be due to a linkage disequilibrium with other variants (Ueda et al., 2003). Ueda et al. (2003) have recently studied many single-nucleotide polymorphisms across the CTLA4 region and have detected a maximum pick of association at CT60. They also presented evidence from functional studies that the CT60 genotypes determine the ratio of two alternative RNA splice forms: a full-length form and a soluble form (sCTLA4). Individuals with the protective CT60A/A genotype showed elevated levels of sCTLA4 that participates in the B7–CTLA4–CD28 signalling pathway of T-cell regulation protecting against the development of autoimmune disease.
In line with these observations, both polymorphisms are associated with many autoimmune diseases including those with a high prevalence in endometriosis patients as indicated in Table IV. The results of this study, in contrast, do not support an association of these CTLA4 variants with endometriosis. Failure to observe any significant difference in the distribution of CTLA4 alleles between affected women and controls is unlikely to be due to a type II error (false negative results), since our study had sufficient power to disclose effects similar in magnitude to those observed in most autoimmune diseases.
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As a consequence, three pathogenetic hypotheses can be put forward:
- endometriosis may have an autoimmune etiology without a CTLA4-dependent predisposition. Some autoimmune diseases, for instance Crohn disease, do not seem to be associated with CTLA4 variants (Rueda et al., 2005) but such CTLA4-independent diseases are in general not associated with CTLA4-linked affections as endometriosis is. In any case, we cannot exclude that other gene variants implicated in general autoimmunity (i.e. CD28 or inducible co-stimulator) (Ueda et al., 2003) might be involved.
- endometriosis may not have an autoimmune etiology but may predispose to the development of autoimmune reactions. This idea is also supported by the recent demonstration that ovarian endometriosis is also not associated with particular human leukocyte antigen class II alleles that confer susceptibility to many autoimmune disorders (Roszkowski et al., 2005). The reduced NK cell activity characteristic of endometriosis has been claimed to explain at least in part the increased autoimmune reactivity associated with the disease. NK cells play a key role in the delicate balance of immune self-tolerance by eliminating potentially dangerous cells presenting self-antigens. In the peritoneal cavity, NK cells might be less effective in killing autologous dendritic cells loaded with endometrial self antigens, facilitating their presentation to autoreactive T cells and the production of autoantibodies (Matarese et al., 2003).
- finally, patients included in this study are affected by different forms of the disease. Thus, even if we did not observe any association to any clinical manifestations, the sample size might be insufficient to assess an association with specific entities.
At present, the postulated autoimmune aetiology of endometriosis is not well substantiated. The results presented herein are in line with evidence suggesting that autoimmune reactions do not represent the primum movens of the disease.
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Submitted on July 5, 2005; revised on July 29, 2005; accepted on August 15, 2005
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