Mol. Hum. Reprod. Advance Access originally published online on August 26, 2005
Molecular Human Reproduction 2005 11(9):641-647; doi:10.1093/molehr/gah221
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Association between polymorphisms in the progesterone receptor gene and endometriosis
Genetic Epidemiology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia
1 To whom correspondence should be addressed at: Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia. E-mail: sue.treloar{at}qimr.edu.au
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Abstract |
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The progesterone receptor (PR) is a candidate gene for the development of endometriosis, a complex disease with strong hormonal features, common in women of reproductive age. We typed the 306 base pair Alu insertion (AluIns) polymorphism in intron G of PR in 101 individuals, estimated linkage disequilibrium (LD) between five single-nucleotide polymorphisms (SNPs) across the PR locus in 980 Australian triads (endometriosis case and two parents) and used transmission disequilibrium testing (TDT) for association with endometriosis. The five SNPs showed strong pairwise LD, and the AluIns was highly correlated with proximal SNPs rs1042839 (
2 = 0.877, D9 = 1.00, P < 0.0001) and rs500760 (2 = 0.438, D9 = 0.942, P < 0.0001). TDT showed weak evidence of allelic association between endometriosis and rs500760 (P = 0.027) but not in the expected direction. We identified a common susceptibility haplotype GGGCA across the five SNPs (P = 0.0167) in the whole sample, but likelihood ratio testing of haplotype transmission and non-transmission of the AluIns and flanking SNPs showed no significant pattern. Further, analysis of our results pooled with those from two previous studies suggested that neither the T2 allele of the AluIns nor the T1/T2 genotype was associated with endometriosis. Key words: endometriosis/linkage disequilibrium/polymorphism/progesterone receptor/transmission disequilibrium test
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Introduction |
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Endometriosis is a common and complex gynaecological disorder. Estimates of its true prevalence vary (Zondervan et al., 2002
), but given a proportion of affected women are asymptomatic, the reported rate of 7% in our large Australian twin study (Treloar et al., 1999) suggests a lifetime prevalence of around 10%. Endometriosis is influenced by both environmental and genetic factors (Treloar et al., 1999). There is strong evidence for familial aggregation in population based samples (Stefansson et al., 2002), hospital samples (Kennedy et al., 1995; Simpson and Bischoff, 2002), twin studies (Moen, 1994; Hadfield et al., 1997; Treloar et al., 1999) and in non-human primates (Zondervan et al., 2004). Finding genes predisposing to endometriosis will help to define mechanisms of disease, and efforts are being directed towards association studies of plausible candidate genes, and more recently towards positional cloning approaches (Kennedy, 2003).
In endometriosis, ectopic endometrium-like tissue continues to proliferate and invade in response to the monthly cycles of ovarian steroid hormones (Meresman et al., 2002). Endometriosis has characteristics of invasion and metastasis, although pathologically it resembles a benign tumour (Koninckx et al., 1999). The complex nature of the disease means variation in many genes may contribute to disease prevalence. Possible candidates include genes involved in endometrial tissue proliferation and invasion including those from steroid hormone pathways. Estrogen is a potent mitogen in the endometrium and endometriosis (Gurates and Bulun, 2003), whereas progesterone counteracts the estrogen-dependent proliferation of endometrial tissue and promotes differentiation (Gurates and Bulun, 2003). Combined oral contraceptives have been shown to down-regulate cell proliferation and to enhance apoptosis in eutopic endometrium from women with endometriosis (Meresman et al., 2002). Endometriosis appears less responsive to the antiproliferative and differentiating effects of progesterone (Gurates and Bulun, 2003), but progesterone receptor (PR) modulators are included in the range of promising new medical treatments for endometriosis (D’Hooghe, 2003).
Steroid receptor gene polymorphisms and protein changes have been correlated with endometriosis (Moutsatsou and Sekeris, 2003). The chromosomal region on 11q22-23 encompassing the PR gene is a frequent site for loss of heterozygosity in women with endometriosis (Jiang et al., 1996). Response to progestins may differ in women with endometriosis and differential PR expression has been reported in endometriotic tissue compared with eutopic endometrium (Jiang et al., 2002). Variants in PR include rs1042838 in exon 4, rs1042839 in exon 5 and an Alu insertion (AluIns) polymorphism in intron 7 (Rowe et al., 1995). There is strong but not complete linkage disequilibrium (LD) between these variants, and the combination of the T allele at rs1042838, T allele at rs1042839 and the AluIns has been referred to as PROGINS (Agoulnik et al., 1997; Kieback et al., 1998). Evidence for the direct involvement of the PR gene in endometriosis comes from two small case-control studies that reported a significant association between the T2 AluIns allele of the intron G (PROGINS) of the PR gene (Wieser et al., 2002; Lattuada et al., 2004). A two-fold increase in risk of endometriosis in women carrying the T2 allele was found from analysis of the combined data from both studies (Lattuada et al., 2004). The same pathophysiology may contribute to progression of endometriosis and transformation to endometrioid or clear cell ovarian neoplasias (Ness, 2003). The PROGINS AluIns element (T2 allele) has been associated with sporadic ovarian carcinoma (Rowe et al., 1995) and the PROGINS complex has been associated with the prevalence of ovarian cancer in women carrying BRCA1 and BRCA2 mutations (Runnebaum et al., 2001).
The human PR gene encodes several transcripts with two major isoforms, PR-A and PR-B, regulated by distinct promoter regions (Kastner et al., 1990). Transcription from separate promoters and translational start sites give rise to the major isoforms PR-A and PR-B, which are identical except for an additional 165 amino acids present only in the N terminus of PR-B (Conneely et al., 1987, 1989; Kastner et al., 1990). Although PR-A and PR-B share several structural domains, they are distinct transcription factors that mediate their own response genes and physiologic effects with little overlap (Conneely et al., 2003). A SNP in the promoter of PR (+331G/A; rs10895068) creates a unique start site which increases transcription of the PR-B isoform and has been associated with increased risk for developing endometrial cancer (De Vivo et al., 2002). A second polymorphism in the promoter region (+44C/T; rs518162), identified also by De Vivo et al. (2002) was not associated with endometrial cancer. Expression of the major isoforms may be altered in endometriotic lesions, but results are inconsistent. In one study a higher ratio of PR-B to PR-A mRNA was found in ectopic compared with eutopic endometrial tissue (Misao et al., 1999), whereas in another, PR-B mRNA was absent from endometriotic tissue (Attia et al., 2000).
In summary, variation in the PR may contribute to the incidence of endometriosis and related ovarian cancers (Del Carmen et al., 2003). Therefore we tested association between endometriosis and genetic polymorphism in the PR by analysing selected single-nucleotide polymorphisms (SNPs) across the locus in a sample of 980 triads (affected individual and both parents) from our Australian study of endometriosis (Treloar et al., 2002). Because typing the AluIns polymorphism is labour intensive, we typed it in only a subsample and used strong LD with one of the flanking SNPs to infer presence/absence of the insertion.
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Materials and methods |
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Participants and sample collection
Samples and genotype data were from Australian participants in the International Endogene Study, recruited between 1996 and 2002 (Treloar et al., 2002
). The median age at participation of affected daughters was 31 years, with a range of 16–54 years. Median age at diagnosis reported by daughters was 24 years, with a range of 12–50 years. Participants were mostly of European ancestry (Treloar et al., 2002). The Australian study recruited 1055 triads of affected women plus two parents, of which 980 families provided adequate DNA. Triads were independent of 931 recruited affected sister-pair families used in our parallel linkage study. All women had been diagnosed by surgical means, with visualisation a minimum requirement for inclusion. Revised American Fertility Society (rAFS) criteria (American Fertility Society, 1985) were used for staging of disease severity (Treloar et al., 2002). The project was approved by the Human Research Ethics Committee of the Queensland Institute of Medical Research and all DNA samples were collected with written, informed consent.
Triads included daughters with all rAFS stages of endometriosis. We used two broad categories: stage A (minimal to mild, N = 673, 69%) and stage B (moderate to severe, N = 307, 31%) for stratified analyses, given that stage was in some cases assigned retrospectively (Treloar et al., 2002). Subfertility was measured by reported problems conceiving. In this sample, 388 daughters who had tested their fertility (40%) reported problems conceiving and 215 (22%) reported no problems; 377 cases (38%) had never tried to conceive. In those who had tested their fertility there was a strong positive association (
12= 24.45, P < 0.0001) between more severe stage and subfertility in this sample, with minimal–mild endometriosis having a relatively protective effect (OR = 0.38, 95% CI = 0.25–0.56).
DNA was extracted from peripheral blood lymphocytes by the salt precipitation method (Miller et al., 1988) and was then stored at 4°C at Queensland Institute of Medical Research (QIMR, Brisbane, Australia). Two participants provided a buccal swab sample and DNA was extracted from buccal swabs using Microcon Centrifugal Filter Devices (Amicons) and stored at 4°C. Aliquots of sample genomic DNA were adjusted to final concentrations of 3 ng/ul and 10 ng/ul for SequenomTM MassARRAY and AluIns PCR, respectively.
SNP selection
We selected four polymorphisms in the PR gene on the basis of published reports: the 306 base pair AluIns in intron G, part of the PROGINS complex, which had been reported to be associated with endometriosis (Wieser et al., 2002), a functional polymorphism +331G/A (rs10895068) in the promoter region associated with risk of endometrial cancer and two further SNPs, rs518162 and rs1042838 (Val660Leu), identified by De Vivo et al. (2002). Five additional SNPs–rs208112, rs3740754 (Ser344Thr), rs1042839 (His770His), rs2020880 (Leu865Ser) and rs500760 (Gln886Gln), the latter two flanking the AluIns–were selected from the NCBI database (NCBI GenBank Database, entry x51730, human mRNA and promoter DNA for the PR). Three SNPs, rs2008112; rs3740754 and rs2020880 were monomorphic in our sample and were excluded from data analyses. Figure 1 shows the positions of the SNPs genotyped in the PR gene.
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Genotyping
Forward and reverse PCR primers and a primer extension probe were designed using SpectroDESIGNER software (SequenomTM, San Diego, CA, USA) and assembled into a multiplex set. SNPs were typed using the SequenomTM matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MassARRAY protocol. The PCR volume of 5 µl contained 15 ng DNA, 10x PCR buffer, 25 mM MgCl2, 25 mM dNTP, forward and reverse primers to a final concentration of 50 nM for all SNPs (excluding rs10895068 which was 75 nM) and 5 U/µl of Taq polymerase. PCR cycling conditions were an initial denaturation step of 15 min at 95°C, followed by 45 cycles of 20 s at 95°C, 30 s at 56°C, 1 min at 72°C and a final extension step of 3 min at 72°C. To neutralize and remove residual unincorporated dNTPs from the PCR, shrimp alkaline phosphatase (1 U/µl) was added and the reaction incubated at 37°C for 20 min and then 85°C for 5 min.
The MassEXTEND PCR mix contained allele-specific extension primers at 300nM each, h-ME (ACT or ACG) extension mix and 32 U/µl of ThermoSequenase. PCR cycling conditions were a denaturation step of 2 min at 94°C, followed by 55 cycles of 5 s at 94°C, 5 s at 52°C and 5 s at 72°C. Purification of h-ME reaction products was performed by the addition of resin. Primer products were spotted onto 384-well chips, precoated with a 3-hydroxypicolinic acid matrix, to help bind and stabilize DNA during ionisation by the MassARRAY. Loading of the products was performed using a nanolitre pipetting system (SpectroCHIP, SpectroJet; Sequenom) and reaction products analysed through MALDI-TOF MassARRAY (Sequenom).
We used a modified protocol to examine the presence/absence of the AluIns polymorphism in a subsample (Spurdle et al., 2002). A subset of DNA samples (n = 101) was typed successfully by PCR using forward and reverse primers 5'-ATACGGTATCCATGACATGAG-3' and 5'-AAGTATTTTCTTGCTAAATGTCTG-3', respectively. The reaction volume of 10 ul contained 15 ng of DNA, primer concentrations of 10 pmol each, dNTPs (200 nM), 1x Perkin-Elmer Taq polymerase buffer, 1 unit of Taq polymerase and 1.5 mM MgCl2. Amplification conditions were an initial denaturation step of 10 min at 94°C, 35 cycles of 94°C for 20 s, 51°C for 20 s, 72°C for 60 s and a final extension step of 72°C for 10 min. PCR products of 65 bp for wild type and 385 bp with AluIns were resolved on a 2.5% agarose gel (Agarose 1000 UltraPure, Invitrogen, Carlsbad, CA, USA).
Statistical analysis
To avoid spurious associations arising from population admixture we used a family-based design to study parental alleles/haplotypes transmitted and not transmitted to affected offspring. Our strategy for data analysis was based on (i) typing selected SNPs in the triad set as outlined, (ii) typing the AluIns in a subset of individuals, (iii) estimating LD between the AluIns and flanking SNPs, and between all the SNPs typed and (iv) using the transmission disequilibrium test (TDT) for detecting association between endometriosis and individual SNP and haplotypes. Because inference about association between the AluIns and endometriosis depends on the extent of LD with its proximal SNPs, it was important to estimate LD before proceeding.
Data handling was performed using SAS (SAS Institute Inc., 2001) and Mendelian error checking using SIB-PAIR software (Duffy, 2001). For estimating LD between pairs of variants in the PR gene, in addition to Lewontin’s D¢ linkage disequilibrium statistic, we also estimated 2, effectively providing an estimate of inter-SNP correlation (r = 
2). LD between the SNPs across the gene was estimated using SNPSpD (Nyholt, 2004). LD calculations involving the AluIns were based on data from up to 101 individuals, and for other SNPs using data based on individuals in the 980 triads, numbers ranged between 2889 and 2913.
The single-locus TDT (Spielman et al., 1993), a robust test for allelic association in the presence of population stratification, which uses information from heterozygous parents, was performed on SNP data from 980 cases and both parents (2940 individuals) using SIB-PAIR software (Duffy, 2001). This TDT statistic is the Pearson goodness-of-fit based test of symmetry in the square table of transmitted versus non-transmitted alleles to each affected child (Haberman, 1979). The genotypic TDT P-value is estimated through gene-dropping based on the genotypes of both typed parents of the proband. The test statistic compares the observed number of each genotype transmitted with the number expected based on the parental genotypes. Multi-locus TDT, using a sliding window approach across the 5 SNP genotypes, was performed using GENEHUNTER 2.0 (Kruglyak et al., 1996) to compare the frequencies of transmitted and untransmitted SNP haplotypes, excluding the AluIns. These analyses were also performed for phenotypic strata of endometriosis families, to assess whether any effects were restricted to certain phenotypes. We stratified triad families according to three important phenotypes of cases: age at diagnosis, subfertility and disease stage.
We first estimated haplotype frequencies between AluIns and proximal SNPS rs1042839 and rs500760 in 87 affected probands where complete data were available, using ARLEQUIN version 2.000 (Schneider et al., 2000). Because we did not type the full PROGINS complex and typed the AluIns component in a relatively small subsample, we conducted a final check of haplotype transmission. We used TDTPHASE (Dudbridge, 2004) to imput the missing genotypes, enabling estimation of haplotypes for the AluIns and its two flanking SNPs for a much larger sample, and to conduct a full likelihood ratio test in a log-linear model using the expectation maximization (EM) algorithm to obtain maximum-likelihood estimates for haplotype transmission disequilibrium.
Finally, given the strong LD between the AluIns and rs1042839, we used the latter alleles and genotypes to draw comparisons and analyse pooled results from our own and the Austrian (Wieser et al., 2002) and Italian (Lattuada et al., 2004) studies. Because of the different designs, we used transmitted and untransmitted alleles of rs1042839, which was in strong LD with the AluIns in the Australian data, to combine with case and control data, respectively, from other studies. Given the significant heterogeneity between the results of the three studies, a pooled allelic odds ratio was calculated, under a random effects model, from genotype counts using the methods of DerSimonian and Laird (1986) and Whitehead and Whitehead (1991).
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Results |
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All SNPs were in Hardy–Weinberg equilibrium (probabilities 0.37–0.84, restricted to founders). Minor allele frequencies for the five SNPs in founders ranged from 0.061 to 0.241 (Table I), and for the minor (T2) allele of the AluIns was 0.124 (N = 101).
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There was evidence for significant allelic association with endometriosis for SNP rs500760 in exon 8 (P = 0.0270), with the ratio of transmission to non-transmission for the A allele of 386:326 (Table II). Genotypic transmission disequilibrium was not significant (empirical P = 0.075). Results were consistent when triads involving identically heterozygous parents were removed from analysis (P = 0.026). There was no evidence for association with SNPs in the promoter region of PR.
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There was strong LD across the PR locus (Table III). Five common haplotypes accounted for more than 99% of chromosomes in the sample (Table IV). There was significant over-transmission of the GGGCA haplotype (378:315, P = 0.017), but no significant under-transmission or ‘protective’ haplotype. This result stood with the more stringent test after eliminating triads with double-heterozygous parents, although the significance level for the susceptibility haplotype decreased to P = 0.041 (Table IV).
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) measures of association between pairs of variants in the progesterone receptor (PR) gene
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We stratified triad families according to phenotype of cases: age at diagnosis, subfertility and disease stage. There were no effects of age at diagnosis. The association with rs500760 was very strong in the subgroup reporting subfertility (problems conceiving), with overtransmission of the A allele and consequent undertransmission of G allele (P = 0.007, Table V). No association was shown in either those who reported no problems conceiving or in those who had never tried conceiving. Interestingly, the evidence for allelic association between endometriosis and rs500760 was also increased in triads with stage A (minimal to mild) disease (P = 0.006), despite the negative phenotypic association between subfertility and disease stage. In triads with stage B (moderate to severe) disease, there was no association with rs500760 and no significant association with any other SNP. Over the five SNPs, there was both a significant susceptibility and a protective haplotype in stage A probands (Table V). When triads with double heterozygote parents were removed from analysis, the evidence for significance of the GGGCA susceptibility haplotype decreased to P = 0.049, and that of the protective GGTTG haplotype increased to P = 0.0280. In the subset of families with both subfertility and minimal–mild disease overtransmission of the GGGCA haplotype failed to reach significance. The GGGCG haplotype appeared protective, although numbers were small.
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Inference about association between the AluIns and endometriosis depends on the extent of LD with its proximal SNPs. We estimated 2, effectively providing an estimate of inter-SNP correlation r = 2. There was very strong LD between rs1042839 and the AluIns (2 = 0.877, D' = 1.000, P = 0.000). LD between the AluIns and rs500760 was also strong but not complete (2 = 0.438, D' = 0.942, P = 0.000), and similarly between the two flanking SNPs themselves (2 = 0.568, D' = 0.987, P = 0.000). Analysis of LD between the AluIns and the two flanking markers in 99 individuals typed for all three markers showed that the C T1 A haplotype accounted for 74% of PR chromosomes in these families, and the T T2 G haplotype accounted for only 13% of the 87 affected probands typed for the AluIns (Table VI). Further, the low frequency of the T T1 G haplotype suggests a misclassification rate of no more than 2% if the T G haplotype for rs1042839 and rs500760 were used to infer the AluIns genotype.
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Because we did not type the full PROGINS complex and typed the AluIns component in a subsample, we assessed haplotype transmission of the AluIns and its flanking SNPs, imputing missing genotypes using TDTPHASE (Dudbridge, 2004). The non-significant likelihood ratio statistic of 6.259, df = 5 (P = 0.2818), excluding rare haplotypes, suggested no haplotype transmission disequilibrium and provided no reason to modify our conclusion that our data suggested no convincing evidence for association between the AluIns and endometriosis.
Given the strong LD between the AluIns and rs1042839, we used the latter alleles and genotypes to draw comparisons and analyse pooled results from our own and the Austrian (Wieser et al., 2002) and Italian (Lattuada et al., 2004) studies. Because of the different designs, we used transmitted and untransmitted alleles of rs1042839, which was in strong LD with the AluIns in the Australian data, to combine with case and control data, respectively, from other studies. Combined analysis suggests that a significant risk effect of the T2 allele or of the T1/T2 genotype for endometriosis is unlikely (Table VII).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The AluIns polymorphism in intron G of the PR gene was reported to be associated with susceptibility to endometriosis (Wieser et al., 2002
; Lattuada et al., 2004). There is strong LD between two SNP variants (rs1042838 in exon 4, rs1042839 in exon 5) and the AluIns polymorphism (Agoulnik et al., 1997; Kieback et al., 1998). We therefore investigated five SNPs in the PR gene for association with endometriosis in a large set of Australian families with clinically confirmed endometriosis. We typed the two SNPs reported in LD with the AluIns polymorphism (rs1042838 and rs1042839), one SNP in exon 8 flanking the AluIns polymorphism (rs500760) and two SNPs in the promoter region (rs518162 and rs10895068) including +331G/A (rs10895068) reported to be associated with endometrial cancer (De Vivo et al., 2002). We found no association between rs1042838 or rs1042839 and endometriosis in our families, and unconvincing evidence of association between endometriosis and the exon 8 SNP rs500760. The confidence bounds of the association were broad considering multiple testing, so we consider the association to be marginal. If multiple comparison adjustment is made using a false discovery rate (Benjamini and Hochnerg, 1995) of 0.05, neither the result in Table II (P = 0.027) nor in Table IV (P = 0.017) is significant. Further, the rs500760 SNP is located in the third codon position (Gln886Gln) and may not have functional significance.
Our results cannot be interpreted as supporting association between variation in PR and susceptibility to endometriosis and do not replicate previous findings. The AluIns has been reported in complete LD with the two exonic SNPs, rs1042838 and 3764C/T (Spurdle et al., 2001; De Vivo et al., 2002). We genotyped the AluIns allele in a subset of samples. Our data showed very strong, but not complete LD. The frequency of the AluIns mutant T2 allele in our 87 typed endometriosis probands was 0.128, approximately mid-way between its frequency in cases (0.17) and controls (0.08) in the Austrian study (Wieser et al., 2002) and the Italian study (0.17 for cases and 0.11 for controls) (Lattuada et al., 2004). Our T2 frequency was also lower than the frequency of 0.16% reported for an Australian control sample (Spurdle et al., 2002). The homozygote AluIns was rare in our sample (n = 2, 1.9%), slightly less common than the 3% and 2.52% observed in ovarian cancer cases and controls, respectively (Spurdle et al., 2001), although our standard error would be very high. Our heterozygote T1/T2 genotype was 23%, compared to the 22% and 21% reported for ovarian cases and controls (Spurdle et al., 2001).
Unlike Lattuada et al.’s study (2004), there was no trend for increased prevalence of the mutant T2 allele in cases of more severe disease stage. In fact, the T1/T1 genotype was much more prevalent in cases of stage B disease (87%) compared with stage A disease (66%), and the heterozygous T1/T2 genotype was more prevalent in stage A than stage B cases (32% compared with 11%) (P = 0.055). Given that we found a stronger association with rs500760 and endometriosis in women with minimal to mild disease, and given the very strong LD between the PR AluIns polymorphism and proximal SNPs, no association could be inferred for endometriosis with the T2 allele in our data; it was not evident particularly in cases of moderate–severe disease. Therefore we did not replicate findings from the earlier two case control studies. Results of pooling of our data, using a flanking SNP (rs1042839) in strong LD with the AluIns, with the published data from the Austrian (Wieser et al., 2002) and Italian (Lattuada et al., 2004) studies suggested that association with endometriosis is unlikely.
There was no evidence for association between endometriosis and SNPs in the promoter region of the PR gene, which included a functional polymorphism associated with increased endometrial cancer risk (De Vivo et al., 2002). We found very strong LD across the PR locus. Serum prolactin levels have been implicated as a cause for infertility in patients with minimal to mild endometriosis (Gregoriou et al., 1999; Martinez et al., 2002). The promoter SNP rs10895068 was associated with prolactin levels in a population sample (Westberg et al., 2004); although this SNP did not show individual association with endometriosis in our families, or in the subset with minimal–mild endometriosis and subfertility, it may warrant further investigation.
Like De Vivo et al. (2002), we estimated eight haplotypes with a frequency greater than 1%, and five common haplotypes. We typed SNPs in the PR promoter (De Vivo et al., 2002) and SNPs in LD with the insertion/deletion polymorphism (Wieser et al., 2002) implicated in endometriosis. Recent data from large scale genotyping across the human genome typed 44 SNPs across the PR locus (http://www.hapmap.org). The data show strong LD in general agreement with results of our study, but greater numbers of SNPs that identify all common alleles would need to be typed to completely exclude association between common variants in PR and endometriosis. An in-depth sequence approach to the PR gene would be necessary to detect allelic variants that were large in number but individually rare. Nevertheless, caution is again required as transcriptional differences as unmasked by microarray studies may well be the result of epigenetic factors, which are being shown to be increasingly important in influencing gene expression over the lifespan (Fraga et al., 2005).
The fact that our results did not support the association between (more severe) endometriosis and the AluIns T2 allele reported in earlier studies may be a function of the poor predictive value of early studies reporting ‘positive’ findings (Trikalinos et al., 2004); indeed studies are seldom replicated when the initial sample size is low (Ioannidis et al., 2001). A more directed approach to targeting potential candidate genes for future association studies may come from different approaches. Using microarray analysis, Arimoto et al. (2003) analysed the expression patterns of 23 040 genes of endometrial tissue in various phases throughout the menstrual cycle. Borthwick et al. (2003) examined transcript abundance and changes in transcript level for 60 000 gene targets between normal endometrium in the proliferative and secretory phases of the menstrual cycle. These studies have shown altered levels of expression of genes not previously thought to cycle during the different endometrial phases.
In a recent genome-wide scan of microsatellite markers, we found linkage between various chromosomal regions and endometriosis (Treloar et al., 2000; Wicks et al., 2002). Obviously, these regions warrant further investigation. The genome-wide scan data showed no evidence for a locus for endometriosis on chromosome 11q (Treloar et al., 2005). Our findings fail to provide support for association between variants in PR and endometriosis and demonstrate the complexity of genotype–phenotype associations in this disease.
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Acknowledgements |
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We thank women with endometriosis and their families for participation in the study, Barbara Haddon for coordinating recruitment, Anjali Henders and Renée Mayne for sample management and genotyping, Lien Le for bioinformatics, David Smyth and Olivia Zheng for data management, Michael James for advice on SNP genotyping methods, and Chris Collet (Queensland University of Technology) for helpful discussions. This study was supported in part by grants from the Cooperative Research Centre for the Discovery of Genes for Common Human Diseases established and supported by the Australian Government’s Cooperative Research Centre’s Program and National Health and Medical Research Council of Australia (339430). This study was supported by the Cooperative Research Centre for Discovery of Genes for Common Human Diseases, Australia (1997–2004) and the National Health and Medical Research Council of Australia (339430).
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Submitted on May 9, 2005; accepted on July 27, 2005.
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