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Genetic Polymorphisms of DNMT3L Involved in Hypermethylation of Chromosomal Ends Are Associated with Greater Risk of Developing Ovarian Endometriosis

2012; Elsevier BV; Volume: 180; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2012.01.009

1525-2191

Bruno Borghese, Piétro Santulli, Delphine Héquet, Guillaume Pierre, Dominique de Ziegler, Daniel Vaiman, Charles Chapron,

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Endometrioma is a common ovarian cyst associated with pain and infertility, but its pathogenesis remains enigmatic. Demonstration of the subtelomeric location of hypermethylation in endometrioma has been reported by genome-wide profiling of methylated promoters. Recently, rs113593938, a polymorphism in the DNA methyltransferase 3-like (DNMT3L) gene has been associated with subtelomeric hypomethylation. We investigated the association between endometrioma and rs113593938, rs8129776, rs7354779, and rs2276248, which were chosen for thoroughly covering the locus of interest. We enrolled 127 patients with histologically proved endometrioma and no associated deep endometriotic lesions and 317 healthy subjects for a case-control genetic association study. Genotyping was performed after PCR amplification of the region encompassing the polymorphisms, restriction enzyme digestion, and detection of fragments on an agarose gel. Differences in genotype and allele distributions between cases and controls were tested for each polymorphism separately using the χ2 test. The rs8129776 was significantly associated with endometrioma (P = 0.003). Haplotype analysis showed a higher risk for the patients carrying the ACCC+T haplotypes for rs8129776, rs7354779, rs113593938, and rs2276248 (odds ratio, 7.15; 95% CI, 2.63 to 19.44). We report, for the first time to our knowledge, the association of DNMT3L genetic variants and endometrioma; DNMT3L expression itself was not modified. Our study constitutes a first milestone toward a plausible role of DNMT3L in the establishment of specific DNA methylation patterns in endometrioma. Endometrioma is a common ovarian cyst associated with pain and infertility, but its pathogenesis remains enigmatic. Demonstration of the subtelomeric location of hypermethylation in endometrioma has been reported by genome-wide profiling of methylated promoters. Recently, rs113593938, a polymorphism in the DNA methyltransferase 3-like (DNMT3L) gene has been associated with subtelomeric hypomethylation. We investigated the association between endometrioma and rs113593938, rs8129776, rs7354779, and rs2276248, which were chosen for thoroughly covering the locus of interest. We enrolled 127 patients with histologically proved endometrioma and no associated deep endometriotic lesions and 317 healthy subjects for a case-control genetic association study. Genotyping was performed after PCR amplification of the region encompassing the polymorphisms, restriction enzyme digestion, and detection of fragments on an agarose gel. Differences in genotype and allele distributions between cases and controls were tested for each polymorphism separately using the χ2 test. The rs8129776 was significantly associated with endometrioma (P = 0.003). Haplotype analysis showed a higher risk for the patients carrying the ACCC+T haplotypes for rs8129776, rs7354779, rs113593938, and rs2276248 (odds ratio, 7.15; 95% CI, 2.63 to 19.44). We report, for the first time to our knowledge, the association of DNMT3L genetic variants and endometrioma; DNMT3L expression itself was not modified. Our study constitutes a first milestone toward a plausible role of DNMT3L in the establishment of specific DNA methylation patterns in endometrioma. Endometriosis is a worldwide gynecological disease, histologically defined by the presence of endometrial implants growing outside of the uterine cavity. Endometriosis involves complex traits whose presence is favored by the additive effects of genetics and environment. 1Dun E.C. Taylor R.N. Wieser F. Advances in the genetics of endometriosis.Genome Med. 2010; 2: 75Crossref PubMed Scopus (29) Google Scholar This univocal definition encompasses a broad variety of clinical classes: i) superficial endometriosis, in which the lesions lie on the surface of the peritoneum; ii) endometriomas (OMA), which are endometriotic ovarian cysts; and iii) deeply infiltrating endometriosis (DIE), in which endometriotic nodules infiltrate the muscularis propria of the organs surrounding the uterus, such as the bladder, rectum, or ureters. 2Chapron C. Bourret A. Chopin N. Dousset B. Leconte M. Amsellem-Ouazana D. de Ziegler D. Borghese B. Surgery for bladder endometriosis: long-term results and concomitant management of associated posterior deep lesions.Hum Reprod. 2010; 25: 884-889Crossref PubMed Scopus (140) Google Scholar These lesions are frequently combined and multifocal, possibly extending to more than one organ. 3Chapron C. Fauconnier A. Vieira M. Barakat H. Dousset B. Pansini V. Vacher-Lavenu M.C. Dubuisson J.B. Anatomical distribution of deeply infiltrating endometriosis: surgical implications and proposition for a classification.Hum Reprod. 2003; 18: 157-161Crossref PubMed Scopus (427) Google Scholar In addition, endometriosis produces inconstant symptoms, spanning from no symptoms to severe chronic pelvic pain and infertility. 4Stratton P. Berkley K.J. Chronic pelvic pain and endometriosis: translational evidence of the relationship and implications.Hum Reprod Update. 2011; 17: 327-346Crossref PubMed Scopus (253) Google Scholar, 5de Ziegler D. Borghese B. Chapron C. Endometriosis and infertility: pathophysiology and management.Lancet. 2010; 376: 730-738Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar This clinical heterogeneity is a fundamental feature of endometriosis. It likely explains the difficulty at singling out the genes responsible for the disease and the discrepant outcomes of the genetic association studies published. 6Di W. Guo S.W. The search for genetic variants predisposing women to endometriosis.Curr Opin Obstet Gynecol. 2007; 19: 395-401Crossref PubMed Scopus (31) Google Scholar, 7Painter J.N. Anderson C.A. Nyholt D.R. Macgregor S. Lin J. Lee S.H. Lambert A. Zhao Z.Z. Roseman F. Guo Q. Gordon S.D. Wallace L. Henders A.K. Visscher P.M. Kraft P. Martin N.G. Morris A.P. Treloar S.A. Kennedy S.H. Missmer S.A. Montgomery G.W. Zondervan K.T. Genome-wide association study identifies a locus at 7p15.2 associated with endometriosis.Nat Genet. 2011; 43: 51-54Crossref PubMed Scopus (238) Google Scholar, 8Uno S. Zembutsu H. Hirasawa A. Takahashi A. Kubo M. Akahane T. Aoki D. Kamatani N. Hirata K. Nakamura Y. A genome-wide association study identifies genetic variants in the CDKN2BAS locus associated with endometriosis in Japanese.Nat Genet. 2010; 42: 707-710Crossref PubMed Scopus (203) Google Scholar Epigenetic alterations also occur during the development of the disease. 9Guo S.W. Epigenetics of endometriosis.Mol Hum Reprod. 2009; 15: 587-607Crossref PubMed Scopus (231) Google Scholar Methylation appears to be the predominant mechanism of regulation of some key genes in endometriosis, especially by acting at the promoter region of HOX-A10, 10Wu Y. Halverson G. Basir Z. Strawn E. Yan P. Guo S.W. Aberrant methylation at HOXA10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis.Am J Obstet Gynecol. 2005; 193: 371-380Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 11Kim J.J. Taylor H.S. Lu Z. Ladhani O. Hastings J.M. Jackson K.S. Wu Y. Guo S.W. Fazleabas A.T. Altered expression of HOXA10 in endometriosis: potential role in decidualization.Mol Hum Reprod. 2007; 13: 323-332Crossref PubMed Scopus (196) Google Scholar, 12Lee B. Du H. Taylor H.S. Experimental murine endometriosis induces DNA methylation and altered gene expression in eutopic endometrium.Biol Reprod. 2009; 80: 79-85Crossref PubMed Scopus (161) Google Scholar steroid hormone receptors, 13Wu Y. Strawn E. Basir Z. Halverson G. Guo S.W. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in endometriosis.Epigenetics. 2006; 1: 106-111Crossref PubMed Scopus (236) Google Scholar, 14Xue Q. Lin Z. Cheng Y.H. Huang C.C. Marsh E. Yin P. Milad M.P. Confino E. Reierstad S. Innes J. Bulun S.E. Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis.Biol Reprod. 2007; 77: 681-687Crossref PubMed Scopus (242) Google Scholar, 15Wu Y. Starzinski-Powitz A. Guo S.W. Prolonged stimulation with tumor necrosis factor-alpha induced partial methylation at PR-B promoter in immortalized epithelial-like endometriotic cells.Fertil Steril. 2008; 90: 234-237Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar aromatase, 16Izawa M. Harada T. Taniguchi F. Ohama Y. Takenaka Y. Terakawa N. An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells.Fertil Steril. 2008; 89: 1390-1396Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar or DNA methyltransferases (DNMT), 17Wu Y. Strawn E. Basir Z. Halverson G. Guo S.W. Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis.Fertil Steril. 2007; 87: 24-32Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar although methylation alterations occurring in endometriosis do not systematically correlate with the level of gene expression. 18Borghese B. Barbaux S. Mondon F. Santulli P. Pierre G. Vinci G. Chapron C. Vaiman D. Research resource: genome-wide profiling of methylated promoters in endometriosis reveals a subtelomeric location of hypermethylation.Mol Endocrinol. 2010; 24: 1872-1885Crossref PubMed Scopus (84) Google Scholar By genome-wide profiling of methylated promoters, we also reported an unbalanced distribution of methylation all along the chromosomes in endometriosis. We demonstrated a subtelomeric location of hypermethylated regions in all clinical classes of endometriosis (superficial endometriosis, OMA, and DIE), whereas hypomethylated regions were uniformly spotted on the chromosomes. 18Borghese B. Barbaux S. Mondon F. Santulli P. Pierre G. Vinci G. Chapron C. Vaiman D. Research resource: genome-wide profiling of methylated promoters in endometriosis reveals a subtelomeric location of hypermethylation.Mol Endocrinol. 2010; 24: 1872-1885Crossref PubMed Scopus (84) Google Scholar In endometriosis, DNMT1 and DNMT2 are down-regulated, 19Borghese B. Mondon F. Noel J.C. Fayt I. Mignot T.M. Vaiman D. Chapron C. Gene expression profile for ectopic versus eutopic endometrium provides new insights into endometriosis oncogenic potential.Mol Endocrinol. 2008; 22: 2557-2562Crossref PubMed Scopus (119) Google Scholar which could limit their efficiency and induce defective methylation during cell divisions accompanying lesion development. This is in line with random hypomethylation. On the contrary, DNMT3A and DNMT3B, the de novo methyltransferases, are not significantly modified and are, thus, fully active. 19Borghese B. Mondon F. Noel J.C. Fayt I. Mignot T.M. Vaiman D. Chapron C. Gene expression profile for ectopic versus eutopic endometrium provides new insights into endometriosis oncogenic potential.Mol Endocrinol. 2008; 22: 2557-2562Crossref PubMed Scopus (119) Google Scholar However, the mechanisms that target DNA methylation to the chromosome ends are unknown. Recently, a possible mechanism implying genetic variants of DNMT3-like (DNMT3L) has been suggested. 20El-Maarri O. Kareta M.S. Mikeska T. Becker T. Diaz-Lacava A. Junen J. Nusgen N. Behne F. Wienker T. Waha A. Oldenburg J. Chedin F. A systematic search for DNA methyltransferase polymorphisms reveals a rare DNMT3L variant associated with subtelomeric hypomethylation.Hum Mol Genet. 2009; 18: 1755-1768Crossref PubMed Scopus (49) Google Scholar DNMT3L is essential for de novo methylation, but its mode of action primarily involves interaction with DNMT3A and DNMT3B, because it does not have methyltransferase activity itself. In this study, a rare DNMT3L variant (rs113593938), located in exon 10, was associated with significant subtelomeric hypomethylation. 20El-Maarri O. Kareta M.S. Mikeska T. Becker T. Diaz-Lacava A. Junen J. Nusgen N. Behne F. Wienker T. Waha A. Oldenburg J. Chedin F. A systematic search for DNA methyltransferase polymorphisms reveals a rare DNMT3L variant associated with subtelomeric hypomethylation.Hum Mol Genet. 2009; 18: 1755-1768Crossref PubMed Scopus (49) Google Scholar Based on these data, we examined whether rs113593938 and three additional polymorphisms (rs8129776, rs7354779, and rs2276248), chosen for thoroughly covering the locus of interest, could modify the risk of endometriosis in a white population. In this genetic association study, we intentionally limited our case population to only include patients with histologically proved OMA, but no DIE lesion, per preoperative magnetic resonance imaging workup and surgical inspection findings. This investigative choice aimed at having a well-defined phenotype. Indeed, in light of the disease-known heterogeneous character, OMA probably represents the most distinct and clear-cut phenotype of all endometriosis subtypes. The major conclusion of the present work is that DNMT3L polymorphisms, possibly associated with chromosome end hypermethylation, are risk markers of OMA. The data were obtained from 444 unrelated women of reproductive age operated on between March 2008 and January 2010 in the surgery department of our tertiary referral center. Ethnicity was based on self-report and restricted to the white population. A total of 127 consecutive patients with OMA underwent an ovarian cystectomy procedure, allowing systematic histological confirmation of the diagnosis. None of these patients had associated DIE lesions, as checked preoperatively by transvaginal ultrasonography and/or magnetic resonance imaging and confirmed by a thorough inspection of the abdominopelvic cavity during surgery. A total of 317 control subjects without endometriosis were also recruited in the same period of inclusion. They consisted of women undergoing surgery for a benign gynecological disorder (eg, fibroids or ovarian cysts), in whom no lesion suggestive of endometriosis was found. All these patients were operated on by the same team and underwent a thorough inspection of the reproductive organs, appendix, small and large bowel, peritoneum, and diaphragm. All patients provided written informed consent. The local Institutional Review Board (Consultative Committee for the Protection of Persons in Biomedical Research) approved the study protocol. Samples of venous blood were collected for extraction of DNA by using the salting-out procedure. 21Miller S.A. Dykes D.D. Polesky H.F. A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17628) Google Scholar DNA quality control was performed by repetitive measurement with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Genotyping of rs8129776, rs7354779, rs113593938, and rs2276248 was performed using PCR amplification of the region encompassing the polymorphisms, followed by restriction enzyme digestion and detection of fragments on 3% agarose gel. The primers, provided by Eutogentec (Angers, France), were as follows: 5′-GTTGTGCGCTTGAGGGCCTG-3′ and 5′-GATTTTGTTCAGTACGCACGCA-3′ for rs8129776, 5′-CACCTCCAGGAAGCGAGAT-3′ and 5′-AACAGGTACCAGCCTGGAGT-3′ for rs7354779, 5′-TTGTCCACGAACATCCAGAA-3′ and 5′-GAACAGGTACCAGCCTGGAG-3′ for rs113593938, and 5′-TCCATAGCTCAGGGCTCTA-3′ and 5′-TACCCGTCATCGTCGTACAG-3′ for rs2276248. Restriction fragment length polymorphism enzymatic digestions were conducted according to the manufacturer's recommendations, using BglII, MspI-HpaII, BsuRI, and PvuII (Fermentas, Vilnius, Lithuania) for rs8129776, rs7354779, rs113593938, and rs2276248, respectively. For each polymorphism, allele frequencies were calculated from genotype frequencies in cases and controls. Deviation from Hardy-Weinberg equilibrium was assessed by the Pearson's χ2 test with one df. Because no statistically significant differences were found, we assumed the homogeneity of the studied population with respect to the polymorphisms studied. Differences in genotype and allele distributions between cases and controls were tested by the Pearson's χ2 test, and odds ratios (ORs) with 95% CIs were computed to compare the prevalence of endometrioma among the genotypes of each polymorphism. Haplotype analysis was performed using Haploview software version 4.02 (http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview; last accessed October 7, 2011). When applicable, the haplotypic OR and 95% CI were also computed to assess the effect of the corresponding haplotypes on developing an endometrioma. P<0.05 was considered to be significant. We calculated the sample power by PS Power and Sample Size Calculations 3.0 (http://biostat.mc.vanderbilt.edu/PowerSampleSize; last accessed October 7, 2011). With 127 cases (and 2.5 times more control subjects) and considering an OR of controls relative to subjects with endometrioma of 1.5 and a type I error of 0.05, we will be able to reject the null hypothesis with a P (power) of 0.797. Basic clinical characteristics of the cases and controls are shown in Table 1. All patients were white and had a similar age; 60% of the patients with OMA had a revised American Fertility Society stage of III and 40% had a stage of IV.Table 1Clinical Characteristics of the Cases and ControlsCharacteristicsCase patients with endometrioma (n = 127)Control subjects (n = 317)P valueAge (years)32.032.10.413BMI (kg/m2)21.221.50.512Gravidity 095172<0.001 ⁎P < 0.05 was considered significant. 12358 ≥2987Parity 01122310.002 ⁎P < 0.05 was considered significant. 1938 ≥2648Infertility [no. (%)]46 (36.2)113 (35.6)0.455Mean rAFS score Implant24NANA Adhesion20NA Total43NArAFS stage [no. (%)]NA I0NANA II0NA III76 (59.8)NA IV51 (40.2)NAThe Student's t-test and χ2 test were used for comparisons.BMI, body mass index; NA, not applicable; rAFS, revised American Fertility Society. P < 0.05 was considered significant. Open table in a new tab The Student's t-test and χ2 test were used for comparisons. BMI, body mass index; NA, not applicable; rAFS, revised American Fertility Society. The position of each studied polymorphism on the DNMT3L locus is depicted in Figure 1. The rs7354779 is a nonsynonymous polymorphism resulting in the amino acid change G278R; rs113593938, related to a C-to-T change at codon 271, imposes a glycine to arginine substitution. These two single-nucleotide polymorphisms occur in the C-terminal portion of DNMT3L, which interacts with the active catalytic methyltransferase domain of DNMT3A and DNMT3B. 22Chen Z.X. Mann J.R. Hsieh C.L. Riggs A.D. Chedin F. Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family.J Cell Biochem. 2005; 95: 902-917Crossref PubMed Scopus (165) Google Scholar, 23Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation.Nature. 2007; 449: 248-251Crossref PubMed Scopus (599) Google Scholar The rs8129776 and rs2276248 were chosen in close proximity to the others ( Figure 1). PCR amplification and/or restriction enzyme digestion assays failed in some cases. A total of 64.2%, 61.0%, 61.0%, and 44.4% of the samples were informative on 3% agarose gel after amplification of rs8129776, rs7354779, rs113593938, and rs2276248, respectively, and/or enzymatic digestion. However, proportions of defective and informative samples were similar between cases and controls (P = 0.17). Figure 2 shows a scheme of each agarose gel with mention of the size of the fragments that should be obtained after restriction enzyme digestion for the four studied polymorphisms. Interpretation of fragments generated by PCR–restriction fragment length polymorphism was performed independently by two of the authors (B.B. and P.S.). Only incontrovertible concordant results were selected for further statistical analysis. The distribution of genotypes and allele frequencies in cases and controls for each polymorphism is figured in Table 2. The distribution of the genotypes for the four polymorphisms was in agreement with the Hardy-Weinberg proportion in cases and controls. All calculated χ2 values for Hardy-Weinberg proportion deviation were <3.84, which is the 5% significance level for 1 df ( Table 2). Because no statistically significant differences were found, we assumed the homogeneity of the studied population with respect to the polymorphisms studied. The rs8129776 polymorphism was significantly associated with OMA (P = 0.003), with an OR estimated at 1.61 (95% CI, 1.26 to 2.29). The frequency of the A allele was much higher in the OMA group (62.6%) than in the control group (51.0%). Association with OMA for the other variants did not reach statistical significance (P = 0.209, P = 0.585, and P = 0.482 for rs7354779, rs113593938, and rs2276248, respectively).Table 2Distribution of Genotype and Allele Frequencies in Cases and ControlsVariableGenotypesAlleles (%)χ2 Test for HWP deviationOR95% CIP valuers8129776AAAGGGAG Cases35491162.637.40.611 Controls60745651.049.00.0021.611.26–2.290.003rs7354779TTCTCCTC Cases3121671.628.40.701 Controls132711078.621.40.9930.680.43–1.090.209rs113593938CCCTTTCT Cases6200100.00.0NA Controls2081099.80.20.999NANA0.585rs2276248TTCTCCTC Cases578093.86.20.870 Controls12012095.44.60.8610.730.29–1.820.482The testing deviation from the HWPs was performed using Pearson's χ2 test (with a 5% significance level for 1 df at 3.84).HWP, Hardy-Weinberg proportion; NA, not applicable. Open table in a new tab The testing deviation from the HWPs was performed using Pearson's χ2 test (with a 5% significance level for 1 df at 3.84). HWP, Hardy-Weinberg proportion; NA, not applicable. As displayed in Table 3, haplotype analysis showed a higher risk of developing an OMA for the patients carrying the ACCT haplotype for rs8129776, rs7354779, rs113593938, and rs2276248 (OR, 5.99; 95% CI, 2.17 to 16.52). When considering the first three polymorphisms (rs8129776, rs7354779, and rs113593938), irrespective of the rs2276248 allele (C or T), the haplotypic OR reached 7.15 (95% CI, 2.63 to 19.44). On the contrary, the GTCT haplotype appeared protective toward endometrioma (OR, 0.438; 95% CI, 0.215 to 0.893). The global P value for haplotype analysis is T variant alters the amino acid sequence of the C-terminal portion of DNMT3L, which interacts with the active catalytic methyltransferase domain of DNMT3A and DNMT3B. 23Jia D. Jurkowska R.Z. Zhang X. Jeltsch A. Cheng X. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation.Nature. 2007; 449: 248-251Crossref PubMed Scopus (599) Google Scholar A recent study 20El-Maarri O. Kareta M.S. Mikeska T. Becker T. Diaz-Lacava A. Junen J. Nusgen N. Behne F. Wienker T. Waha A. Oldenburg J. Chedin F. A systematic search for DNA methyltransferase polymorphisms reveals a rare DNMT3L variant associated with subtelomeric hypomethylation.Hum Mol Genet. 2009; 18: 1755-1768Crossref PubMed Scopus (49) Google Scholar reported differences in the methylation level of some genes in association with the DNMT3L 11330C>T variant. In addition, El-Maarri et al 20El-Maarri O. Kareta M.S. Mikeska T. Becker T. Diaz-Lacava A. Junen J. Nusgen N. Behne F. Wienker T. Waha A. Oldenburg J. Chedin F. A systematic search for DNA methyltransferase polymorphisms reveals a rare DNMT3L variant associated with subtelomeric hypomethylation.Hum Mol Genet. 2009; 18: 1755-1768Crossref PubMed Scopus (49) Google Scholar performed a systematic search for polymorphisms in all known human DNMT genes in 192 healthy individuals. By differential methylation hybridization, they revealed that a rare change at DNMT3L (rs113593938) was associated with significant DNA hypomethylation. Interestingly, hypomethylation preferentially clustered to subtelomeric genomic regions. 20El-Maarri O. Kareta M.S. Mikeska T. Becker T. Diaz-Lacava A. Junen J. Nusgen N. Behne F. Wienker T. Waha A. Oldenburg J. Chedin F. A systematic search for DNA methyltransferase polymorphisms reveals a rare DNMT3L variant associated with subtelomeric hypomethylation.Hum Mol Genet. 2009; 18: 1755-1768Crossref PubMed Scopus (49) Google Scholar This study provided, for the first time to our knowledge, a relation between DNMT3L and distribution of methylation to the chromosome extremities. By accounting for all these data, the following assumption might be hypothesized in endometriosis: DNMT3L protein could harbor some structural alterations, possibly caused by genetic variants, that could drift methylation activity toward chromosomal ends. However, the precise mechanisms by which this specific DNA methylation pattern is instructed by DNMT3L are unknown. DNMT3L was admitted to focus DNA methylation away from CpG island promoter regions. 28Ooi S.K. Qiu C. Bernstein E. Li K. Jia D. Yang Z. Erdjument-Bromage H. Tempst P. Lin S.P. Allis C.D. Cheng X. Bestor T.H. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA.Nature. 2007; 448: 714-717Crossref PubMed Scopus (1091) Google Scholar Likewise, other mechanisms, such as recruitment of the de novo methylation machinery by direct association with various DNA binding proteins 32Di Croce L. Raker V.A. Corsaro M. Fazi F. Fanelli M. Faretta M. Fuks F. Lo Coco F. Kouzarides T. Nervi C. Minucci S. Pelicci P.G. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor.Science. 2002; 295: 1079-1082Crossref PubMed Scopus (682) Google Scholar or possibly by small noncoding RNAs, 33Aravin A.A. Sachidanandam R. Bourc'his D. Schaefer C. Pezic D. Toth K.F. Bestor T. Hannon G.J. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice.Mol Cell. 2008; 31: 785-799Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar are also likely to operate. To conclude, this first-time report of an association between genetic variation in DNMT3L and OMA represents an initial step toward involving DNMT3L in the establishment of altered methylation patterns in endometriosis. Further work should confirm and replicate this association in other populations. We thank all of the staff in the operating room for having collected and transported the samples used in this study.