Single-nucleotide polymorphisms in the lysyl oxidase-like protein 4 and complement component 3 genes are associated with increased risk for endometriosis and endometriosis-associated infertility
2011; Elsevier BV; Volume: 96; Issue: 2 Linguagem: Inglês
10.1016/j.fertnstert.2011.06.001
ISSN1556-5653
AutoresLynnette A. Ruiz, Julie Dutil, Abigail Ruiz, Jessica Fourquet, Sonia Abac, Joaquín Laboy, Idhaliz Flores,
Tópico(s)Reproductive System and Pregnancy
ResumoThis study was conducted to assess genetic associations with endometriosis in a Puerto Rican population. Statistically significant differences in the allelic frequencies and genotype distribution of genetic variants in lysyl oxidase-like protein 4 (LOXL4) and complement component 3 (C3) were documented in patients with endometriosis-associated infertility versus controls, and in patients with endometriosis versus controls, respectively. In women who have the risk genotype at both single-nucleotide polymorphisms, the estimated risk for endometriosis nearly doubled. This study was conducted to assess genetic associations with endometriosis in a Puerto Rican population. Statistically significant differences in the allelic frequencies and genotype distribution of genetic variants in lysyl oxidase-like protein 4 (LOXL4) and complement component 3 (C3) were documented in patients with endometriosis-associated infertility versus controls, and in patients with endometriosis versus controls, respectively. In women who have the risk genotype at both single-nucleotide polymorphisms, the estimated risk for endometriosis nearly doubled. Endometriosis is characterized by chronic painful symptoms and infertility (1Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Google Scholar, 2Paris K. Aris A. Endometriosis-associated infertility: a decade's trend study of women from the Estrie Region of Quebec, Canada.Gynecol Endocrinol. 2010; 26: 838-842Google Scholar, 3Vercellini P. Fedele L. Aimi G. Pietropaolo G. Consonni D. Crosignani P.G. Association between endometriosis stage, lesion type, patient characteristics and severity of pelvic pain symptoms: a multivariate analysis of over 1000 patients.Hum Reprod. 2007; 22: 266-271Google Scholar, 4Giudice L.C. Kao L.C. Endometriosis.Lancet. 2004; 364: 1789-1799Google Scholar). There is strong evidence suggesting that endometriosis is a multifactorial disease resulting from interactions of multiple susceptibility genes and environmental factors (5Montgomery G.W. Nyholt D.R. Zhao Z.Z. Treloar S.A. Painter J.N. Missmer S.A. et al.The search for genes contributing to endometriosis risk.Hum Reprod Update. 2008; 14: 447-457Google Scholar, 6Child T.J. Tan S.L. Endometriosis: aetiology, pathogenesis and treatment.Drugs. 2001; 61: 1735-1750Google Scholar, 7Seli E. Arici A. Endometriosis: interaction of immune and endocrine systems.Semin Reprod Med. 2003; 21: 135-144Google Scholar, 8Tempfer C.B. Simoni M. Destenaves B. Fauser B.C. Functional genetic polymorphisms and female reproductive disorders: part II—endometriosis.Hum Reprod Update. 2009; 15: 97-118Google Scholar, 9Guo S.W. Epigenetics of endometriosis.Mol Hum Reprod. 2009; 15: 587-607Google Scholar, 10Vigano P. Somigliana E. Vignali M. Busacca M. Blasio A.M. Genetics of endometriosis: current status and prospects.Front Biosci. 2007; 12: 3247-3255Google Scholar, 11Kennedy S. Genetics of endometriosis: a review of the positional cloning approaches.Semin Reprod Med. 2003; 21: 111-118Google Scholar, 12Simpson J.L. Where are the genes that cause endometriosis?.J Soc Gynecol Investig. 2005; 12: 143-144Google Scholar). After recent reports of genetic associations with endometriosis, there has been a surge in the interest in uncovering the role of genetic variants in this disease (13Treloar S.A. Wicks J. Nyholt D.R. Montgomery G.W. Bahlo M. Smith V. et al.Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.Am J Hum Genet. 2005; 77: 365-376Google Scholar, 14Painter J.N. Anderson C.A. Nyholt D.R. Macgregor S. Lin J. Lee S.H. et al.Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.Nat Genet. 2011; 43: 51-54Google Scholar). Significant associations with endometriosis have been documented in dozens of genes thus far (15Falconer H. D'Hooghe T. Fried G. Endometriosis and genetic polymorphisms.Obstet Gynecol Surv. 2007; 62: 616-628Google Scholar, 16Guo S.W. Glutathione S-transferases M1/T1 gene polymorphisms and endometriosis: a meta-analysis of genetic association studies.Mol Hum Reprod. 2005; 11: 729-743Google Scholar), but for most of these genes the association was highly dependent on ethnicity (17Garcia-Martin E. Interethnic and intraethnic variability of NAT2 single nucleotide polymorphisms.Curr Drug Metab. 2008; 9: 487-497Google Scholar). None of these studies have been conducted in a Hispanic population.We have previously identified genes that were up-regulated in human and experimental endometriosis, including lysyl oxidases (LOXs) and complement factor 3 (C3) (18Flores I. Rivera E. Mousses S. Chen Y. Rozenblum E. Identification of molecular markers for endometriosis in blood lymphocytes by using deoxyribonucleic acid microarrays.Fertil Steril. 2006; 85: 1676-1683Google Scholar, 19Flores I. Rivera E. Ruiz L.A. Santiago O.I. Vernon M.W. Appleyard C.B. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease.Fertil Steril. 2007; 87: 1180-1199Google Scholar). LOXs are a family of copper-dependent amine oxidases that are involved in collagen and elastin crosslinking at the extracellular matrix (20Molnar J. Fong K.S. He Q.P. Hayashi K. Kim Y. Fong S.F. et al.Structural and functional diversity of lysyl oxidase and the LOX-like proteins.Biochim Biophys Acta. 2003; 1647: 220-224Google Scholar), and they play roles in inflammation, cell migration, and invasion (21Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family.Prog Nucleic Acid Res Mol Biol. 2001; 70: 1-32Google Scholar). LOX and LOXL1 expression is up-regulated in endometriotic lesions (18Flores I. Rivera E. Mousses S. Chen Y. Rozenblum E. Identification of molecular markers for endometriosis in blood lymphocytes by using deoxyribonucleic acid microarrays.Fertil Steril. 2006; 85: 1676-1683Google Scholar, 19Flores I. Rivera E. Ruiz L.A. Santiago O.I. Vernon M.W. Appleyard C.B. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease.Fertil Steril. 2007; 87: 1180-1199Google Scholar), and LOXL1 is down-regulated during the secretory phase in endometrium from healthy women (22Talbi S. Hamilton A.E. Vo K.C. Tulac S. Overgaard M.T. Dosiou C. et al.Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.Endocrinology. 2006; 147: 1097-1121Google Scholar). It is interesting that the LOXL4 gene is located near regions shown to be linked to endometriosis (13Treloar S.A. Wicks J. Nyholt D.R. Montgomery G.W. Bahlo M. Smith V. et al.Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.Am J Hum Genet. 2005; 77: 365-376Google Scholar, 23Ledet E.M. Flores I. Bailey-Wilson J.E. Mandal D.M. Genetic analysis of hereditary endometriosis families in Puerto Rico.Genet Epidemiol. 2008; 32: A112Google Scholar, 24Painter J.N. Nyholt D.R. Morris A. Zhao Z.Z. Henders A.K. Lambert A. et al.High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19.Fertil Steril. 2011; 95: 2236-2240Google Scholar). The important role of LOXs in extracellular matrix stability makes these proteins potential candidates to be studied in the context of infertility (25Levental K.R. Yu H. Kass L. Lakins J.N. Egeblad M. Erler J.T. et al.Matrix crosslinking forces tumor progression by enhancing integrin signaling.Cell. 2009; 139: 891-906Google Scholar, 26Ng M.R. Brugge J.S. A stiff blow from the stroma: collagen crosslinking drives tumor progression.Cancer Cell. 2009; 16: 455-457Google Scholar).In women and animal models of endometriosis, C3 is also dysregulated (19Flores I. Rivera E. Ruiz L.A. Santiago O.I. Vernon M.W. Appleyard C.B. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease.Fertil Steril. 2007; 87: 1180-1199Google Scholar, 27Tao X.J. Sayegh R.A. Isaacson K.B. Increased expression of complement component 3 in human ectopic endometrium compared with the matched eutopic endometrium.Fertil Steril. 1997; 68: 460-467Google Scholar, 28Sherwin J.R. Hastings J.M. Jackson K.S. Mavrogianis P.A. Sharkey A.M. Fazleabas A.T. The endometrial response to chorionic gonadotropin is blunted in a baboon model of endometriosis.Endocrinology. 2010; 151: 4982-4993Google Scholar). Infertile patients with endometriosis expressed high C3 levels in peritoneal fluid (29Kabut J. Kondera-Anasz Z. Sikora J. Mielczarek-Palacz A. Levels of complement components iC3b, C3c, C4, and SC5b-9 in peritoneal fluid and serum of infertile women with endometriosis.Fertil Steril. 2007; 88: 1298-1303Abstract Full Text Full Text PDF Scopus (27) Google Scholar). In normal endometrium, high expression of C3 was observed during the secretory phases of the menstrual cycle (30Sayegh R.A. Tao X.J. Awwad J.T. Isaacson K.B. Localization of the expression of complement component 3 in the human endometrium by in situ hybridization.J Clin Endocrinol Metab. 1996; 81: 1641-1649Google Scholar). Moreover, C3 expression is up-regulated in endometrium of baboons treated with human chorionic gonadotropin (hCG) compared with controls, and C3 levels in eutopic endometrium predict pregnancy success, which indicates that regulation in C3 expression is critical for implantation (31Sherwin J.R. Sharkey A.M. Cameo P. Mavrogianis P.M. Catalano R.D. Edassery S. et al.Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation.Endocrinology. 2007; 148: 618-626Google Scholar, 32Isaacson K.B. Galman M. Coutifaris C. Lyttle C.R. Endometrial synthesis and secretion of complement component-3 by patients with and without endometriosis.Fertil Steril. 1990; 53: 836-841Abstract Full Text PDF Google Scholar, 33Bartosik D. Damjanov I. Viscarello R.R. Riley J.A. Immunoproteins in the endometrium: clinical correlates of the presence of complement fractions C3 and C4.Am J Obstet Gynecol. 1987; 156: 11-15Google Scholar). Because C3 is marker of chronic inflammation, a hallmark of endometriosis, we hypothesized that single-nucleotide polymorphisms (SNPs) in C3 could play a role in the pathogenesis of endometriosis and infertility (34Barrington R. Zhang M. Fischer M. Carroll M.C. The role of complement in inflammation and adaptive immunity.Immunol Rev. 2001; 180: 5-15Google Scholar).Study participants were recruited by the Endometriosis Research Program at the Ponce School of Medicine and Health Sciences (PSMHS). The patients were premenopausal women with surgically confirmed endometriosis. The controls were women without endometriosis or known infertility who had surgery for benign gynecologic conditions. Documentation of the revised American Fertility Society criteria severity (rAFS) was obtained during surgery, and demographic, gynecologic, obstetric, and clinical data were collected (35Revised American Fertility Society classification of endometriosis: 1985.Fertil Steril. 1985; 43: 351-352Crossref Google Scholar). The participants (n = 384) were categorized as follows: [1] control group: women without endometriosis or infertility (n = 147); [2] endometriosis group: women with endometriosis, with or without infertility (n = 214); [3] endo-only: women with endometriosis without known infertility problems (n = 72); [4] endo-associated infertility: women with endometriosis-associated infertility (n = 73). Women were considered "infertile" if they answered positively to "Have you experienced problems getting pregnant?" Most of the infertile women were nulliparous (73.4%), although some had had children (n = 26). These women were surveyed for the specific cause of their perceived infertility. The majority of them (19 of 26) reported either miscarriages and/or a history of infertility (average: 4.9 years; range: 1 to 10 years). One reported being infertile due to endometriosis, and six did not report a specific reason or problem. All participants read and signed an informed consent form before enrollment. The protocols were approved by the PSMHS institutional review board.Isolation of genomic DNA from lymphocytes was conducted using standard procedures (QIAamp DNA Blood kit; Qiagen), and genotyping was conducted on 12 SNPs in C3 and LOXL4 on a custom-made Golden Gate chip from Illumina, Inc., as previously described elsewhere (36Sellers T.A. Huang Y. Cunningham J. Goode E.L. Sutphen R. Vierkant R.A. et al.Association of single nucleotide polymorphisms in glycosylation genes with risk of epithelial ovarian cancer.Cancer Epidemiol Biomarkers Prev. 2008; 17: 397-404Google Scholar). Genotyping was conducted at the Southern California Genotyping Consortium of the University of California, Los Angeles.All statistical analyses were performed using Systat 13.0 (Cranes Software Int. Ltd.). Demographic, gynecologic, and clinical parameters are expressed as mean ± standard error for quantitative variables or as percentages for frequency data. Differences in clinical characteristics between groups were compared using a chi-square for frequencies or Student's t-test for quantitative variables. The SNP genotypes of controls were assessed for departures from Hardy-Weinberg equilibrium (HWE) using a chi-square test. Associations between SNP and risk of endometriosis and/or endometriosis-associated infertility were performed by chi-square test. Odds ratios (OR) and 95% confidence interval (CI) were calculated using a logistic regression model adjusted for confounding factors.Gene–gene interaction was modeled using the multifactor dimensionality reduction (MDR) approach. The MDR is a nonparametric genetic model-free approach for detecting epistatic interactions between SNPs that combines a cross-validation and permutation procedure to assess the statistical significance of the detected interactions. Data were first divided into a training set (9/10 of the data) and a testing set (1/10) (10-fold cross-validation). Performance was estimated by cross-validation consistency and the model's prediction accuracy. Subsequently, permutation analysis was performed in which case-control status was randomized 1,000 times to assess the statistical significance of the modeled interaction.Group characteristics, allelic frequencies, and genotype distribution of statistically significant SNPs associated with endometriosis or endometriosis-associated infertility are shown in Table 1. The prevalence of symptoms such as pain and infertility was statistically significantly higher in patients than controls (37Sinaii N. Plumb K. Cotton L. Lambert A. Kennedy S. Zondervan K. et al.Differences in characteristics among 1,000 women with endometriosis based on extent of disease.Fertil Steril. 2008; 89: 538-545Google Scholar). Thirty percent of patients with endometriosis reported having fertility problems, further demonstrating that infertility commonly coexists with endometriosis (2Paris K. Aris A. Endometriosis-associated infertility: a decade's trend study of women from the Estrie Region of Quebec, Canada.Gynecol Endocrinol. 2010; 26: 838-842Google Scholar, 38Holoch K.J. Lessey B.A. Endometriosis and infertility.Clin Obstet Gynecol. 2010; 53: 429-438Google Scholar). Several studies have suggested a possible link between endometriosis and miscarriages, but there is insufficient evidence for this association (39Vercammen E.E. D'Hooghe T.M. Endometriosis and recurrent pregnancy loss.Semin Reprod Med. 2000; 18: 363-368Google Scholar, 40Tomassetti C. Meuleman C. Pexsters A. Mihalyi A. Kyama C. Simsa P. et al.Endometriosis, recurrent miscarriage and implantation failure: is there an immunological link?.Reprod Biomed Online. 2006; 13: 58-64Google Scholar). In our population, 40% of patients with endometriosis reported having had at least one miscarriage, which was statistically significantly different from the controls. These data add to the current state of knowledge in this controversial issue by providing support for a possible link between pregnancy loss and endometriosis in a Hispanic population.Table 1A. Characteristics of patients with endometriosis versus women without endometriosis (controls).CharacteristicsEndometriosis (n = 214)Controls (n = 147)P valueAge (y)31.9 ± 0.537.8 ± 0.7<.001aP was calculated by Student's t-test.Menstrual cycle characteristics Age at menarche (y)11.9 ± 0.112.1 ± 0.1.198aP was calculated by Student's t-test. Regular cycles (%)73.478.8.242bP was calculated by Pearson Chi-square test. Cycle length (d)36.4 ± 3.730.2 ± 1.0.180aP was calculated by Student's t-test. Menstrual flow length (d)6.6 ± 0.45.8 ± 0.2.164aP was calculated by Student's t-test.Obstetric history Tried to be pregnant (%)75.187.0.007bP was calculated by Pearson Chi-square test. Gestation, average1.7 ± 0.13.0 ± 0.1<.001aP was calculated by Student's t-test. Miscarriages, average1.2 ± 0.70.4 ± 0.1.241aP was calculated by Student's t-test. Miscarriages (%)40.324.8.008bP was calculated by Pearson Chi-square test. Age at first childbirth24.0 ± 1.121.1 ± 0.4.005aP was calculated by Student's t-test.Endometriosis-related symptoms Dysmenorrhea (%)84.664.1<.001bP was calculated by Pearson Chi-square test. Incapacitating pain (%)68.931.3<.001bP was calculated by Pearson Chi-square test. Dyspareunia (%)56.332.1<.001bP was calculated by Pearson Chi-square test. Infertility (%)34.1—NAEndometriosis family history (%)46.32.0<.001bP was calculated by Pearson Chi-square test.B. Allelic frequencies and genotype distribution of statistically significant single-nucleotide polymorphisms (SNPs) associated to endometriosis or endometriosis-associated infertility.SNPMAF count (%)Genotype count (%)ORaP was calculated by Student's t-test.95% CIP valuers737657 (LOXL4)AAAAGGGModel GG vs. AA + AG Controls124 (42.5)24 (16.4)76 (52.1)46 (31.5)Ref—— Endometriosis148 (34.7)25 (11.7)98 (46.0)90 (42.3)1.6831.049–2.700.031 Endo-only49 (34.0)8 (11.1)33 (45.8)31 (43.1)1.6300.908–2.927.102 Endo-infertility40 (27.4)5 (6.8)30 (41.1)38 (52.1)2.3361.273–4.286.006rs17524355 (LOXL4)AAAAGGGModel AA vs. AG + GG Controls52 (17.8)4 (2.7)44 (30.1)98 (67.1)Ref—— Endometriosis84 (19.9)11 (5.2)62 (29.4)138 (65.4)2.3720.702–8.012.164 Endo-only27 (18.8)2 (2.8)23 (31.9)47 (65.3)0.9440.190–6.033.939 Endo-infertility33 (22.9)8 (11.1)17 (23.6)47 (65.3)5.2301.434–19.072.012Rs2213392 (C3)GCCCGGGModel GG vs. CC + CG Controls136 (46.3)39 (26.5)80 (54.4)28 (19.0)Ref—— Endometriosis172 (40.2)72 (33.6)112 (52.3)30 (14.0)0.6310.344–1.159.138 Endo-only54 (37.0)25 (34.2)42 (57.5)6 (8.2)0.3690.144–0.942.037 Endo-infertility66 (45.2)23 (31.5)34 (46.6)16 (21.9)1.0110.485–2.107.978Note: Values are mean ± SD or percentage. CI = confidence interval; MAF = minor allele frequency; NA = not applicable; OR = odds ratio; Ref = reference value.a P was calculated by Student's t-test.b P was calculated by Pearson Chi-square test. Open table in a new tab Genotypes of 12 SNPs were tested for associations using allelic, recessive, dominant, and additive models. Genotype distributions were in HWE in the control group for all SNPs tested. The GG genotype at rs737657 in LOXL4 was more frequent in patients compared with controls, indicating an increased risk for endometriosis in individuals homozygous for the G allele (OR = 1.68; 95% CI, 1.049–2.700; P=.031). This risk increased when patients with endometriosis-associated infertility were compared with controls (OR = 2.34; 95% CI, 1.273–4.286; P=.006). Also, statistically significant differences were observed in the frequency of the AA genotype at rs17524355 in LOXL4 when patients with endometriosis-associated infertility were compared with controls (OR = 5.23; 95% CI, 1.434–19.072; P=.012). In conclusion, these two polymorphisms in the LOXL4 gene (GG genotype at rs737657 and AA genotype at rs17524355) are associated with a higher risk for infertility in the context of endometriosis. In this study, SNP rs737657 was also shown to be associated with a diagnosis of endometriosis, suggesting that some of the risk factors or molecular mechanisms underlying endometriosis and infertility may be linked. Because our population was recruited for an endometriosis case-control study, and we did not focus on recruiting infertility cases, our study design would not allow distinguishing between infertility alone and infertility in the context of endometriosis. Further studies would be needed to assess whether the observed associations remain statistically significant in patients suffering from infertility only.It is interesting that the LOXL4 gene is mapped to 10q24.2, close to two regions of interest in endometriosis: 10q23.3 which had statistically significant linkage to endometriosis in families from Puerto Rico, and 10q26 identified by the Endogene study of 1,176 families as associated with endometriosis (13Treloar S.A. Wicks J. Nyholt D.R. Montgomery G.W. Bahlo M. Smith V. et al.Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.Am J Hum Genet. 2005; 77: 365-376Google Scholar, 23Ledet E.M. Flores I. Bailey-Wilson J.E. Mandal D.M. Genetic analysis of hereditary endometriosis families in Puerto Rico.Genet Epidemiol. 2008; 32: A112Google Scholar). Of note in a recent report, linkage analysis on families grouped by fertility status not only validated the previously observed linkage peak at 10q26 but also detected an association to a SNP in that genetic region (24Painter J.N. Nyholt D.R. Morris A. Zhao Z.Z. Henders A.K. Lambert A. et al.High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19.Fertil Steril. 2011; 95: 2236-2240Google Scholar). Our study thus confirmed that there is a region of interest in chromosome 10 that is associated with endometriosis-associated infertility and, moreover, independently identified a genetic polymorphism in this region associated with an increased risk for this disease. More studies are necessary to identify a possible functional effect of these SNPs in gene expression or protein levels/activity, in particular during the window of implantation; alternatively, these SNPs may be in linkage disequilibrium with other SNP(s) that would prove to be causative of this disease.These studies also showed that the frequency of the GG genotype at rs2241392 in C3 was statistically significantly different between patients with endometriosis and controls (OR = 0.369; 95% CI, 0.144–0.942), indicating a decreased risk for endometriosis associated with this genotype in this population. Statistical significance for this particular SNP was lost when all endometriosis patients (including those with infertility) or when patients with endometriosis-associated infertility were compared with controls; therefore, we speculate that the association is driven by the endometriosis phenotype and not by infertility. It is known that C3 levels are dysregulated in patients with the condition, and aberrant expression of this inflammatory molecule could explain some of the symptoms of endometriosis.To determine whether more than one SNP can interact to increase the risk, we compared the prevalence of endometriosis in the study population grouped by the number of risk genotypes at LOXL4 and/or C3 loci. The prevalence of endometriosis was of 44.7% in women carrying neither of the risk genotype at the C3 and LOXL4 loci. In a group of individuals bearing a risk genotype at either locus, C3 or LOXL4, the prevalence of endometriosis increased to 52.7%, (OR = 1.361; 95% CI, 0.674–2.748; P=.39) in comparison with women not carrying any risk genotype. It is interesting that in the group of women having the risk genotype at both SNPs the prevalence of endometriosis reached 66.1% and the risk for endometriosis nearly doubled (OR = 2.247; 95% CI, 1.106–4.785; P=.0362). These results suggest that, in combination, SNPs in different candidate genes may improve the probability of correctly predicting genetic susceptibility to endometriosis.Some of the P values we report here did not reach statistical significance after applying Bonferroni correction. Also, the SNPs analyzed did not cover all of the potential linkage disequilibrium (LD) blocks in the genes. We also performed a multifactor dimensionality reduction (MDR) analysis to search for possible SNP-SNP interactions between rs737657 and rs2241392. The testing accuracy was 0.5134 with a cross-validation consistency of 10/10. However, when submitted to a permutation analysis, this interaction did not reach statistical significance (P=.5). For those reasons, it would be important to validate these findings in a larger sample size and in other populations. In addition, future studies should incorporate a tag-SNP strategy to provide a better coverage of the whole genes.Our results suggest that polymorphisms in LOXL4 and C3 can be associated with a risk for endometriosis and for infertility in the context of endometriosis. To our knowledge, this is the first report of a genetic association between SNPs in these genes and the risk of endometriosis or endometriosis-associated infertility, and between any SNP and these two diagnoses in a Hispanic population. Because the prevalence of SNPs may vary among ethnic groups, it is important to ascertain effects related to ethnic differences and to validate these findings in other populations (41Hinds D.A. Stuve L.L. Nilsen G.B. Halperin E. Eskin E. Ballinger D.G. et al.Whole-genome patterns of common DNA variation in three human populations.Science. 2005; 307: 1072-1079Google Scholar, 42Spielman R.S. Bastone L.A. Burdick J.T. Morley M. Ewens W.J. Cheung V.G. Common genetic variants account for differences in gene expression among ethnic groups.Nat Genet. 2007; 39: 226-231Google Scholar). Endometriosis is characterized by chronic painful symptoms and infertility (1Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Google Scholar, 2Paris K. Aris A. Endometriosis-associated infertility: a decade's trend study of women from the Estrie Region of Quebec, Canada.Gynecol Endocrinol. 2010; 26: 838-842Google Scholar, 3Vercellini P. Fedele L. Aimi G. Pietropaolo G. Consonni D. Crosignani P.G. Association between endometriosis stage, lesion type, patient characteristics and severity of pelvic pain symptoms: a multivariate analysis of over 1000 patients.Hum Reprod. 2007; 22: 266-271Google Scholar, 4Giudice L.C. Kao L.C. Endometriosis.Lancet. 2004; 364: 1789-1799Google Scholar). There is strong evidence suggesting that endometriosis is a multifactorial disease resulting from interactions of multiple susceptibility genes and environmental factors (5Montgomery G.W. Nyholt D.R. Zhao Z.Z. Treloar S.A. Painter J.N. Missmer S.A. et al.The search for genes contributing to endometriosis risk.Hum Reprod Update. 2008; 14: 447-457Google Scholar, 6Child T.J. Tan S.L. Endometriosis: aetiology, pathogenesis and treatment.Drugs. 2001; 61: 1735-1750Google Scholar, 7Seli E. Arici A. Endometriosis: interaction of immune and endocrine systems.Semin Reprod Med. 2003; 21: 135-144Google Scholar, 8Tempfer C.B. Simoni M. Destenaves B. Fauser B.C. Functional genetic polymorphisms and female reproductive disorders: part II—endometriosis.Hum Reprod Update. 2009; 15: 97-118Google Scholar, 9Guo S.W. Epigenetics of endometriosis.Mol Hum Reprod. 2009; 15: 587-607Google Scholar, 10Vigano P. Somigliana E. Vignali M. Busacca M. Blasio A.M. Genetics of endometriosis: current status and prospects.Front Biosci. 2007; 12: 3247-3255Google Scholar, 11Kennedy S. Genetics of endometriosis: a review of the positional cloning approaches.Semin Reprod Med. 2003; 21: 111-118Google Scholar, 12Simpson J.L. Where are the genes that cause endometriosis?.J Soc Gynecol Investig. 2005; 12: 143-144Google Scholar). After recent reports of genetic associations with endometriosis, there has been a surge in the interest in uncovering the role of genetic variants in this disease (13Treloar S.A. Wicks J. Nyholt D.R. Montgomery G.W. Bahlo M. Smith V. et al.Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.Am J Hum Genet. 2005; 77: 365-376Google Scholar, 14Painter J.N. Anderson C.A. Nyholt D.R. Macgregor S. Lin J. Lee S.H. et al.Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.Nat Genet. 2011; 43: 51-54Google Scholar). Significant associations with endometriosis have been documented in dozens of genes thus far (15Falconer H. D'Hooghe T. Fried G. Endometriosis and genetic polymorphisms.Obstet Gynecol Surv. 2007; 62: 616-628Google Scholar, 16Guo S.W. Glutathione S-transferases M1/T1 gene polymorphisms and endometriosis: a meta-analysis of genetic association studies.Mol Hum Reprod. 2005; 11: 729-743Google Scholar), but for most of these genes the association was highly dependent on ethnicity (17Garcia-Martin E. Interethnic and intraethnic variability of NAT2 single nucleotide polymorphisms.Curr Drug Metab. 2008; 9: 487-497Google Scholar). None of these studies have been conducted in a Hispanic population. We have previously identified genes that were up-regulated in human and experimental endometriosis, including lysyl oxidases (LOXs) and complement factor 3 (C3) (18Flores I. Rivera E. Mousses S. Chen Y. Rozenblum E. Identification of molecular markers for endometriosis in blood lymphocytes by using deoxyribonucleic acid microarrays.Fertil Steril. 2006; 85: 1676-1683Google Scholar, 19Flores I. Rivera E. Ruiz L.A. Santiago O.I. Vernon M.W. Appleyard C.B. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease.Fertil Steril. 2007; 87: 1180-1199Google Scholar). LOXs are a family of copper-dependent amine oxidases that are involved in collagen and elastin crosslinking at the extracellular matrix (20Molnar J. Fong K.S. He Q.P. Hayashi K. Kim Y. Fong S.F. et al.Structural and functional diversity of lysyl oxidase and the LOX-like proteins.Biochim Biophys Acta. 2003; 1647: 220-224Google Scholar), and they play roles in inflammation, cell migration, and invasion (21Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family.Prog Nucleic Acid Res Mol Biol. 2001; 70: 1-32Google Scholar). LOX and LOXL1 expression is up-regulated in endometriotic lesions (18Flores I. Rivera E. Mousses S. Chen Y. Rozenblum E. Identification of molecular markers for endometriosis in blood lymphocytes by using deoxyribonucleic acid microarrays.Fertil Steril. 2006; 85: 1676-1683Google Scholar, 19Flores I. Rivera E. Ruiz L.A. Santiago O.I. Vernon M.W. Appleyard C.B. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease.Fertil Steril. 2007; 87: 1180-1199Google Scholar), and LOXL1 is down-regulated during the secretory phase in endometrium from healthy women (22Talbi S. Hamilton A.E. Vo K.C. Tulac S. Overgaard M.T. Dosiou C. et al.Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.Endocrinology. 2006; 147: 1097-1121Google Scholar). It is interesting that the LOXL4 gene is located near regions shown to be linked to endometriosis (13Treloar S.A. Wicks J. Nyholt D.R. Montgomery G.W. Bahlo M. Smith V. et al.Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.Am J Hum Genet. 2005; 77: 365-376Google Scholar, 23Ledet E.M. Flores I. Bailey-Wilson J.E. Mandal D.M. Genetic analysis of hereditary endometriosis families in Puerto Rico.Genet Epidemiol. 2008; 32: A112Google Scholar, 24Painter J.N. Nyholt D.R. Morris A. Zhao Z.Z. Henders A.K. Lambert A. et al.High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19.Fertil Steril. 2011; 95: 2236-2240Google Scholar). The important role of LOXs in extracellular matrix stability makes these proteins potential candidates to be studied in the context of infertility (25Levental K.R. Yu H. Kass L. Lakins J.N. Egeblad M. Erler J.T. et al.Matrix crosslinking forces tumor progression by enhancing integrin signaling.Cell. 2009; 139: 891-906Google Scholar, 26Ng M.R. Brugge J.S. A stiff blow from the stroma: collagen crosslinking drives tumor progression.Cancer Cell. 2009; 16: 455-457Google Scholar). In women and animal models of endometriosis, C3 is also dysregulated (19Flores I. Rivera E. Ruiz L.A. Santiago O.I. Vernon M.W. Appleyard C.B. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease.Fertil Steril. 2007; 87: 1180-1199Google Scholar, 27Tao X.J. Sayegh R.A. Isaacson K.B. Increased expression of complement component 3 in human ectopic endometrium compared with the matched eutopic endometrium.Fertil Steril. 1997; 68: 460-467Google Scholar, 28Sherwin J.R. Hastings J.M. Jackson K.S. Mavrogianis P.A. Sharkey A.M. Fazleabas A.T. The endometrial response to chorionic gonadotropin is blunted in a baboon model of endometriosis.Endocrinology. 2010; 151: 4982-4993Google Scholar). Infertile patients with endometriosis expressed high C3 levels in peritoneal fluid (29Kabut J. Kondera-Anasz Z. Sikora J. Mielczarek-Palacz A. Levels of complement components iC3b, C3c, C4, and SC5b-9 in peritoneal fluid and serum of infertile women with endometriosis.Fertil Steril. 2007; 88: 1298-1303Abstract Full Text Full Text PDF Scopus (27) Google Scholar). In normal endometrium, high expression of C3 was observed during the secretory phases of the menstrual cycle (30Sayegh R.A. Tao X.J. Awwad J.T. Isaacson K.B. Localization of the expression of complement component 3 in the human endometrium by in situ hybridization.J Clin Endocrinol Metab. 1996; 81: 1641-1649Google Scholar). Moreover, C3 expression is up-regulated in endometrium of baboons treated with human chorionic gonadotropin (hCG) compared with controls, and C3 levels in eutopic endometrium predict pregnancy success, which indicates that regulation in C3 expression is critical for implantation (31Sherwin J.R. Sharkey A.M. Cameo P. Mavrogianis P.M. Catalano R.D. Edassery S. et al.Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation.Endocrinology. 2007; 148: 618-626Google Scholar, 32Isaacson K.B. Galman M. Coutifaris C. Lyttle C.R. Endometrial synthesis and secretion of complement component-3 by patients with and without endometriosis.Fertil Steril. 1990; 53: 836-841Abstract Full Text PDF Google Scholar, 33Bartosik D. Damjanov I. Viscarello R.R. Riley J.A. Immunoproteins in the endometrium: clinical correlates of the presence of complement fractions C3 and C4.Am J Obstet Gynecol. 1987; 156: 11-15Google Scholar). Because C3 is marker of chronic inflammation, a hallmark of endometriosis, we hypothesized that single-nucleotide polymorphisms (SNPs) in C3 could play a role in the pathogenesis of endometriosis and infertility (34Barrington R. Zhang M. Fischer M. Carroll M.C. The role of complement in inflammation and adaptive immunity.Immunol Rev. 2001; 180: 5-15Google Scholar). Study participants were recruited by the Endometriosis Research Program at the Ponce School of Medicine and Health Sciences (PSMHS). The patients were premenopausal women with surgically confirmed endometriosis. The controls were women without endometriosis or known infertility who had surgery for benign gynecologic conditions. Documentation of the revised American Fertility Society criteria severity (rAFS) was obtained during surgery, and demographic, gynecologic, obstetric, and clinical data were collected (35Revised American Fertility Society classification of endometriosis: 1985.Fertil Steril. 1985; 43: 351-352Crossref Google Scholar). The participants (n = 384) were categorized as follows: [1] control group: women without endometriosis or infertility (n = 147); [2] endometriosis group: women with endometriosis, with or without infertility (n = 214); [3] endo-only: women with endometriosis without known infertility problems (n = 72); [4] endo-associated infertility: women with endometriosis-associated infertility (n = 73). Women were considered "infertile" if they answered positively to "Have you experienced problems getting pregnant?" Most of the infertile women were nulliparous (73.4%), although some had had children (n = 26). These women were surveyed for the specific cause of their perceived infertility. The majority of them (19 of 26) reported either miscarriages and/or a history of infertility (average: 4.9 years; range: 1 to 10 years). One reported being infertile due to endometriosis, and six did not report a specific reason or problem. All participants read and signed an informed consent form before enrollment. The protocols were approved by the PSMHS institutional review board. Isolation of genomic DNA from lymphocytes was conducted using standard procedures (QIAamp DNA Blood kit; Qiagen), and genotyping was conducted on 12 SNPs in C3 and LOXL4 on a custom-made Golden Gate chip from Illumina, Inc., as previously described elsewhere (36Sellers T.A. Huang Y. Cunningham J. Goode E.L. Sutphen R. Vierkant R.A. et al.Association of single nucleotide polymorphisms in glycosylation genes with risk of epithelial ovarian cancer.Cancer Epidemiol Biomarkers Prev. 2008; 17: 397-404Google Scholar). Genotyping was conducted at the Southern California Genotyping Consortium of the University of California, Los Angeles. All statistical analyses were performed using Systat 13.0 (Cranes Software Int. Ltd.). Demographic, gynecologic, and clinical parameters are expressed as mean ± standard error for quantitative variables or as percentages for frequency data. Differences in clinical characteristics between groups were compared using a chi-square for frequencies or Student's t-test for quantitative variables. The SNP genotypes of controls were assessed for departures from Hardy-Weinberg equilibrium (HWE) using a chi-square test. Associations between SNP and risk of endometriosis and/or endometriosis-associated infertility were performed by chi-square test. Odds ratios (OR) and 95% confidence interval (CI) were calculated using a logistic regression model adjusted for confounding factors. Gene–gene interaction was modeled using the multifactor dimensionality reduction (MDR) approach. The MDR is a nonparametric genetic model-free approach for detecting epistatic interactions between SNPs that combines a cross-validation and permutation procedure to assess the statistical significance of the detected interactions. Data were first divided into a training set (9/10 of the data) and a testing set (1/10) (10-fold cross-validation). Performance was estimated by cross-validation consistency and the model's prediction accuracy. Subsequently, permutation analysis was performed in which case-control status was randomized 1,000 times to assess the statistical significance of the modeled interaction. Group characteristics, allelic frequencies, and genotype distribution of statistically significant SNPs associated with endometriosis or endometriosis-associated infertility are shown in Table 1. The prevalence of symptoms such as pain and infertility was statistically significantly higher in patients than controls (37Sinaii N. Plumb K. Cotton L. Lambert A. Kennedy S. Zondervan K. et al.Differences in characteristics among 1,000 women with endometriosis based on extent of disease.Fertil Steril. 2008; 89: 538-545Google Scholar). Thirty percent of patients with endometriosis reported having fertility problems, further demonstrating that infertility commonly coexists with endometriosis (2Paris K. Aris A. Endometriosis-associated infertility: a decade's trend study of women from the Estrie Region of Quebec, Canada.Gynecol Endocrinol. 2010; 26: 838-842Google Scholar, 38Holoch K.J. Lessey B.A. Endometriosis and infertility.Clin Obstet Gynecol. 2010; 53: 429-438Google Scholar). Several studies have suggested a possible link between endometriosis and miscarriages, but there is insufficient evidence for this association (39Vercammen E.E. D'Hooghe T.M. Endometriosis and recurrent pregnancy loss.Semin Reprod Med. 2000; 18: 363-368Google Scholar, 40Tomassetti C. Meuleman C. Pexsters A. Mihalyi A. Kyama C. Simsa P. et al.Endometriosis, recurrent miscarriage and implantation failure: is there an immunological link?.Reprod Biomed Online. 2006; 13: 58-64Google Scholar). In our population, 40% of patients with endometriosis reported having had at least one miscarriage, which was statistically significantly different from the controls. These data add to the current state of knowledge in this controversial issue by providing support for a possible link between pregnancy loss and endometriosis in a Hispanic population. Note: Values are mean ± SD or percentage. CI = confidence interval; MAF = minor allele frequency; NA = not applicable; OR = odds ratio; Ref = reference value. Genotypes of 12 SNPs were tested for associations using allelic, recessive, dominant, and additive models. Genotype distributions were in HWE in the control group for all SNPs tested. The GG genotype at rs737657 in LOXL4 was more frequent in patients compared with controls, indicating an increased risk for endometriosis in individuals homozygous for the G allele (OR = 1.68; 95% CI, 1.049–2.700; P=.031). This risk increased when patients with endometriosis-associated infertility were compared with controls (OR = 2.34; 95% CI, 1.273–4.286; P=.006). Also, statistically significant differences were observed in the frequency of the AA genotype at rs17524355 in LOXL4 when patients with endometriosis-associated infertility were compared with controls (OR = 5.23; 95% CI, 1.434–19.072; P=.012). In conclusion, these two polymorphisms in the LOXL4 gene (GG genotype at rs737657 and AA genotype at rs17524355) are associated with a higher risk for infertility in the context of endometriosis. In this study, SNP rs737657 was also shown to be associated with a diagnosis of endometriosis, suggesting that some of the risk factors or molecular mechanisms underlying endometriosis and infertility may be linked. Because our population was recruited for an endometriosis case-control study, and we did not focus on recruiting infertility cases, our study design would not allow distinguishing between infertility alone and infertility in the context of endometriosis. Further studies would be needed to assess whether the observed associations remain statistically significant in patients suffering from infertility only. It is interesting that the LOXL4 gene is mapped to 10q24.2, close to two regions of interest in endometriosis: 10q23.3 which had statistically significant linkage to endometriosis in families from Puerto Rico, and 10q26 identified by the Endogene study of 1,176 families as associated with endometriosis (13Treloar S.A. Wicks J. Nyholt D.R. Montgomery G.W. Bahlo M. Smith V. et al.Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.Am J Hum Genet. 2005; 77: 365-376Google Scholar, 23Ledet E.M. Flores I. Bailey-Wilson J.E. Mandal D.M. Genetic analysis of hereditary endometriosis families in Puerto Rico.Genet Epidemiol. 2008; 32: A112Google Scholar). Of note in a recent report, linkage analysis on families grouped by fertility status not only validated the previously observed linkage peak at 10q26 but also detected an association to a SNP in that genetic region (24Painter J.N. Nyholt D.R. Morris A. Zhao Z.Z. Henders A.K. Lambert A. et al.High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19.Fertil Steril. 2011; 95: 2236-2240Google Scholar). Our study thus confirmed that there is a region of interest in chromosome 10 that is associated with endometriosis-associated infertility and, moreover, independently identified a genetic polymorphism in this region associated with an increased risk for this disease. More studies are necessary to identify a possible functional effect of these SNPs in gene expression or protein levels/activity, in particular during the window of implantation; alternatively, these SNPs may be in linkage disequilibrium with other SNP(s) that would prove to be causative of this disease. These studies also showed that the frequency of the GG genotype at rs2241392 in C3 was statistically significantly different between patients with endometriosis and controls (OR = 0.369; 95% CI, 0.144–0.942), indicating a decreased risk for endometriosis associated with this genotype in this population. Statistical significance for this particular SNP was lost when all endometriosis patients (including those with infertility) or when patients with endometriosis-associated infertility were compared with controls; therefore, we speculate that the association is driven by the endometriosis phenotype and not by infertility. It is known that C3 levels are dysregulated in patients with the condition, and aberrant expression of this inflammatory molecule could explain some of the symptoms of endometriosis. To determine whether more than one SNP can interact to increase the risk, we compared the prevalence of endometriosis in the study population grouped by the number of risk genotypes at LOXL4 and/or C3 loci. The prevalence of endometriosis was of 44.7% in women carrying neither of the risk genotype at the C3 and LOXL4 loci. In a group of individuals bearing a risk genotype at either locus, C3 or LOXL4, the prevalence of endometriosis increased to 52.7%, (OR = 1.361; 95% CI, 0.674–2.748; P=.39) in comparison with women not carrying any risk genotype. It is interesting that in the group of women having the risk genotype at both SNPs the prevalence of endometriosis reached 66.1% and the risk for endometriosis nearly doubled (OR = 2.247; 95% CI, 1.106–4.785; P=.0362). These results suggest that, in combination, SNPs in different candidate genes may improve the probability of correctly predicting genetic susceptibility to endometriosis. Some of the P values we report here did not reach statistical significance after applying Bonferroni correction. Also, the SNPs analyzed did not cover all of the potential linkage disequilibrium (LD) blocks in the genes. We also performed a multifactor dimensionality reduction (MDR) analysis to search for possible SNP-SNP interactions between rs737657 and rs2241392. The testing accuracy was 0.5134 with a cross-validation consistency of 10/10. However, when submitted to a permutation analysis, this interaction did not reach statistical significance (P=.5). For those reasons, it would be important to validate these findings in a larger sample size and in other populations. In addition, future studies should incorporate a tag-SNP strategy to provide a better coverage of the whole genes. Our results suggest that polymorphisms in LOXL4 and C3 can be associated with a risk for endometriosis and for infertility in the context of endometriosis. To our knowledge, this is the first report of a genetic association between SNPs in these genes and the risk of endometriosis or endometriosis-associated infertility, and between any SNP and these two diagnoses in a Hispanic population. Because the prevalence of SNPs may vary among ethnic groups, it is important to ascertain effects related to ethnic differences and to validate these findings in other populations (41Hinds D.A. Stuve L.L. Nilsen G.B. Halperin E. Eskin E. Ballinger D.G. et al.Whole-genome patterns of common DNA variation in three human populations.Science. 2005; 307: 1072-1079Google Scholar, 42Spielman R.S. Bastone L.A. Burdick J.T. Morley M. Ewens W.J. Cheung V.G. Common genetic variants account for differences in gene expression among ethnic groups.Nat Genet. 2007; 39: 226-231Google Scholar).
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