Lack of an association human dioxin detoxification gene polymorphisms with endometriosis in Japanese women: results of a pilot study

Objectives Endometriosis is a chronic disease caused by the presence of endometrial tissue in ectopic locations outside the uterus. Chronic exposure to the environmental pollutant dioxin has been correlated with an increased incidence in the development of endometriosis in non-human primates. We have therefore examined whether there is an association between the polymorphisms of ten dioxin detoxification genes and endometriosis in Japanese women.

This was a pilot study in which 100 patients with endometriosis and 143 controls were enrolled. The prevalence of five microsatellite and 28 single nucleotide polymorphism markers within ten dioxin detoxification genes ( AhR, AHRR, ARNT, CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, GSTT1, NAT2) was examined.

Taking into account that this analysis was a preliminary study due to its small sample size and genetic power, the results did not show any statistically significant difference between the cases and controls for any of the allele and genotype frequency distributions examined. In addition, no significant associations between the allele/ genotype of all polymorphisms and the stage (I–II or III– IV) of endometriosis were observed.

Based on the findings of this pilot study, we conclude the polymorphisms of the ten dioxin detoxifica- tion genes analyzed did not contribute to the etiology of endometriosis among our patients.

Endometriosis /C1Dioxin detoxification genes /C1 Single nucleotide polymorphisms /C1Microsatellite /C1 Association study

Endometriosis (MIM131200) is an estrogen-dependent inflammatory disease that is characterized histologically by the presence of endometrial glands and stroma outside the uterine cavity. Approximately 6–10 % of women of repro- ductive age have been reported to suffer from this condition [1, 2]. In a study of the genetic influence on the risk of endo- metriosis among twins in Australia, the risk ratio of affected versus population prevalence was 3.58 for monozygotic twins and 2.32 for dizygotic twins [3]. A strong familial occurrence, Electronic supplementary material The online version of this article (doi:10.1007/s12199-012-0281-y) contains supplementary material, which is available to authorized users. Y. Matsuzaka ( &) /C1Y. Y. Kikuti /C1A. Oka /C1H. Inoko /C1 J. K. Kulski /C1M. Kimura ( &) Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan e-mail: [email protected] M. Kimura e-mail: [email protected] Present Address: Y. Matsuzaka Institute of Experimental Genetics, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Neuherberg, Germany K. Goya /C1T. Suzuki /C1L. Cai /C1S. Izumi ( &) Department of Obstetrics and Gynecology, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan e-mail: [email protected] J. K. Kulski Centre for Forensic Science, The University of Western Australia, Nedlands, WA 6009, Australia 123 Environ Health Prev Med (2012) 17:512–517 DOI 10.1007/s12199-012-0281-y in which the incidence of the disease is approximately sev- enfold higher in relatives of women with endometriosis than in those without family history, supports a genetic role [4, 5]. Ligand-bound AhR translocates to the nucleus, where it heterodimerizes with ARNT.T h e AhR–ARNT heterodimer binds to xenobiotic response element sequences and indu- ces the activation of the target genes that encode the cytochrome P450 enzymes CYP1A1 and CYP2E1 [ 6]. In this heterodimer formation, the AhRR competes with AhR to repress the expression of AhR regulatory genes [ 7]. Crosstalk between dioxin-activated AhR and the functional estrogen receptor (ER) has been reported [ 8]. Dioxin has also been shown to induce a dose-dependent increase in the severity of endometriosis in rhesus monkeys [ 9]. In order to gain a better understanding of the association between detoxification genes and endometriosis in Japanese women, we performed a case–control study of endometriosis as pilot study involving 100 cases and 143 controls.

Subjects All patients and controls were female and had given their written informed consent (IC), following an explanation of the laparoscopy and histological examination, to participate in this study, which was performed at the Department of Obstetrics and Gynecology, Tokai University School of Medicine. The rate of approval was almost 100 % after a reflection period between the explanation and admittance to the hospital. The patients with endometriosis were diagnosed by laparoscopy and histological examination, and the severity of disease was staged according to the revised classifica- tion of the American Society of Reproductive Medicine (r-ASRM) [10]. Of the 100 endometriosis cases (mean age of patient 38 ± 4.8 years), 27, 10, 31, 32 cases were classified as stage I, II, III, and IV levels of disease, respectively. The 143 healthy controls (mean age 43 ± 12.3 years) were patients of unexplained infertility who had undergone a diagnostic laparoscopy that proved the absence of endome- triosis (Table 1). Patients with apparent disease(s) were excluded from the control group. Ethics approval for this study was obtained from the Institutional Review Board at Tokai University School of Medicine. DNA extraction Genomic DNA samples were isolated from peripheral blood leukocytes that were drawn from each subject into tubes containing heparin by using the Qiagen DNA Extraction kit (Qiagen, Tokyo, Japan) after lysis with proteinase K and 0.5 % sodium dodecyl sulfate at 37 /C176C for 1 h. Analysis of microsatellite polymorphisms in the AhR and ARNT gene regions To determine the number of repeat units of the microsat- ellite loci exhibiting polymorphisms in the AhR and ARNT genes, we synthesized unilateral primers by labeling the 5 0 end with the fluorescent reagent 6-FAM (PE Biosystems, Foster City, CA). Two and four PCR primer sets were used for the amplification of two and four microsatellite loci in AhR and ARNT gene regions, respectively [Electronic Supplementary Material (ESM) Table 1]. PCR amplifica- tion and GenScan (Applied Biosystems Japan, Tokyo, Japan) detection of these microsatellites were carried out as previously described [ 11]. Genotyping To examine the distribution of the alleles and genotype frequency of each single nucleotide polymorphism (SNP) and deletion, we performed direct sequencing analysis of ten endometriosis candidate genes using 25 pairs of oli- gonucleotide PCR primers (ESM Table 2). The reaction mixture (20 ll) contained 2 ml of dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), genomic DNA (5 ml; 2 ng/ ll), 2 ml of 10 9 buffer (100 mM Tris–HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2), 20 pmol of forward and reverse primers, and 0.5 U of r Taq (TaKaRa Bio, Shiga, Japan). PCR amplification was performed in a GeneAmp PCR system 9700 automated thermal cycler (Applied Biosystems Japan) under the following PCR reaction conditions: an initial denaturation for 5 min at 96 /C176C, followed by 30 amplification cycles of 45 s at 96 /C176C, 45 s at various temperatures depending on the Table 1 Characteristics of cases of endometriosis and controls Study cohort Cases Controls Number of patients 100 143 Age (years)a 38 ± 4.8 43 ± 12.3 Endometriosis stageb Stage I 27 (27.0) Stage II 10 (10.0) Stage III 31 (31.0) Stage IV 32 (32.0) Smoking (%) c 14.7 14.7 Body mass index d 20.6 ± 3 21.5 ± 3 a Mean and standard deviation (SD) of 100 cases and 143 controls b Number of patients, with the percentage (of 100 cases) given in parenthesis c Smoking rates for 34 cases and 34 controls d Mean and standard deviation for 32 cases and 30 controls Environ Health Prev Med (2012) 17:512–517 513 123 primers used (ESM Table 2) for annealing, and 2 min at 72 /C176C, with a final extension of 7 min at 72 /C176C. Each PCR product was purified by exonuclease I and then sequenced using an ABI 3100 automated sequencer (Applied Bio- systems Japan). Statistical analyses Allele and genotype frequencies were determined by direct counting. The significance of differences in the distribution of alleles and genotypes between the patients and controls was tested using a case–control design by the Fisher’s exact probability test ( P value test). A P c of\0.05, which indicates significant corrections for multiple testing, such as the Bonferroni method [ P 9 L 9 (n - 1)], was con- sidered to indicate statistical significance for all allelic and genotypic frequencies where P, L, and n indicate the P value, number of loci examined, and the number of alleles examined, respectively. The odds ratio (OR) and 95 % confidence interval (CI) were also calculated for all SNPs. The program Haploview (MIT/Harvard Broad Institute, Cambridge, MA) was used to estimate pairwise linkage disequilibrium (LD) and haplotype frequency [ 12]. The Genetic Power Calculator was used to calculate a genetic power for association study ( http://pngu.mgh. harvard.edu/*purcell/gpc/).

Association of microsatellite markers around the AhR and ARNT genes with endometriosis A total of 100 Japanese patients with endometriosis and 143 healthy controls were enrolled in our analysis aimed at investigating the association between genetic variations in the AhR and ARNT genes and endometriosis. To this end, we used six microsatellites found in close proximity and within the region of the AhR and ARNT genes (Table 2). The two microsatellites with the dinucleotide repeat (AC)n and (CA) n were positioned 1,206 and 882 bp upstream of the exons 2 and 3 in the AhR gene region, respectively. The other four microsatellites, with di- to pentanucleotide repeats (TG) n, (TAA), (ATTT) n, and (TTTTG)n, were positioned 9,907 and 10,880 bp upstream of the first methionine of exon, 16,306 bp upstream of exon 2, and 2,642 bp upstream of exon 14, respectively, in the ARNT gene. As shown in Table 2, the microsatellite with the CA repeats, at intron 2 of the AhR gene and the four microsatellites in the ARNT gene region had no statistically significant association with endometriosis. The other microsatellite with the dinucleotide repeat (AC) n in the AhR gene region was not polymorphic (data not shown). Association of endometriosis candidate gene polymorphisms with endometriosis Twenty-eight SNPs in the AhR, AHRR, ARNT, CYP1A1 , CYP2E1, EPHX1, GSTP1, and NAT2 genes were geno- typed in all 100 patients with endometriosis and 143 con- trols. The allele frequencies of each polymorphism are shown in Table 3. There was no significant association found between the gene polymorphisms and the presence of endometriosis. However, the TT genotypes in intron 10 (rs2106728) of the AhR gene showed a prominent—but not significant—association with the risk of developing endo- metriosis (Table 4, P c = 0.087). To also test whether there was an association between endometriosis and the gluta- thione S-transferase (GST) M1 and T1 null mutation, we genotyped these genotype frequencies in 97 patients with endometriosis and 143 controls. No significant differences were found between the cases and controls for the fre- quencies of the GSTM1 and GSTT1 null mutations (Table 4). We evaluated the LD extension of approximately 42 kb of the AhR genomic region with 12 SNPs. For the LD block evaluation, we included SNPs with a minor allele fre- quency of [0.2, a genotype success rate of [0.8, and P[ 0.001 in the Hardy–Weinberg equilibrium test. All SNPs analyzed met the criteria, and the pairwise LD index Table 2 Results of the association tests for five microsatellite markers with endometriosis Gene Location Position a Repeat unit Number of alleles Patients b Controlsb Odds ratio (90 % CI) P value AhR Intron 2 22,322 (CA) n 8 39 (24.1) 49 (20.9) 1.20 (0.74–1.93) 0.461 ARNT Promoter -10,880 (TAA) n 3 65 (50.8) 79 (44.9) 1.27 (0.80–2.00) 0.309 ARNT Promoter -9,907 (TG) n 14 20 (17.2) 20 (12.2) 1.50 (0.77–2.93) 0.235 ARNT Intron 1 11,774 (ATTT) n 2 20 (17.5) 21 (15.4) 1.17 (0.60–2.28) 0.655 ARNT Intron 13 55,880 (TTTTG) n 3 107 (75.9) 128 (68.8) 1.43 (0.87–2.34) 0.159 CI, Confidence interval a Position is the number of nucleotides from the first nucleotide (A) of the start codon b Data are presented as the number with the percentage in parenthesis 514 Environ Health Prev Med (2012) 17:512–517 123 Table 3 Nucleotide variations within the human candidate genes and the significance of their association with endometriosis Gene dbSNP accession no. Location Allele a Amino acid substitution Allele frequencyb OR (90 % CI) P value Pc value Patients Controls AhR rs713150 Intron 1 C/g – 79 (40.7) 87 (30.4) 1.57 (1.07–2.30) 0.020 0.540 rs2282886 Intron 1 A/g – 109 (69.9) 157 (66.5) 1.17 (0.75–1.80) 0.487 1.000 rs2237299 Intron 1 T/c – 139 (73.2) 192 (68.1) 1.28 (0.85–1.92) 0.238 1.000 rs2237298 Intron 1 T/c – 139 (73.2) 192 (68.1) 1.28 (0.85–1.92) 0.238 1.000 rs3802083 Intron 1 T/c – 131 (68.2) 186 (65.5) 1.13 (0.77–1.67) 0.535 1.000 rs2282883 Intron 2 T/c – 122 (69.3) 170 (65.9) 1.17 (0.78–1.76) 0.455 1.000 rs1476080 Intron 2 T/g – 131 (68.2) 183 (66.3) 1.09 (0.74–1.62) 0.663 1.000 rs2237297 Intron 2 G/a – 86 (45.7) 127 (45.4) 1.02 (0.70–1.02) 0.934 1.000 rs3802082 Intron 5 A/t – 100 (52.6) 131 (52.0) 1.03 (0.70–1.50) 0.893 1.000 rs2066853 Exon 10 G/a Arg/Lys 91 (47.9) 155 (45.8) 1.09 (0.75–1.57) 0.654 1.000 rs2040623 Intron 10 C/a – 96 (49.5) 137 (47.9) 1.07 (0.74–1.53) 0.734 1.000 rs2106728 Intron 10 T/c – 169 (87.1) 219 (76.6) 2.07 (1.26–3.39) 0.004 0.108 AHRR rs2292596 Exon 6 C/g Pro/Ala 69 (37.5) 103 (36.8) 1.03 (0.70–1.51) 0.876 1.000 ARNT rs11204735 Intron 1 A/g – 84 (46.7) 112 (43.8) 1.13 (0.77–1.65) 0.547 1.000 rs3768015 Intron 5 C/t – 127 (66.8) 164 (61.2) 1.28 (0.87–1.89) 0.216 1.000 rs10305711 Intron 9 A/g – 127 (67.6) 177 (62.8) 1.24 (0.84–1.82) 0.287 1.000 CYP1A1 rs3826042 Promoter G/a – 157 (89.2) 220 (84.6) 1.50 (0.84–2.68) 0.169 1.000 rs3826041 Promoter T/g – 73 (41.5) 104 (40.0) 1.06 (0.72–1.57) 0.758 1.000 rs4646421 Intron 1 C/t – 71 (37.0) 90 (31.9) 1.25 (0.85–1.84) 0.253 1.000 rs4646422 Exon 2 G/a Gly/Asp 29 (14.9) 32 (11.3) 1.37 (0.80–2.35) 0.248 1.000 rs1048943 Exon 7 A/g Ile/Val 46 (24.2) 61 (22.3) 1.12 (0.72–1.73) 0.624 1.000 rs5030838 3 0 flanking region T/c – 72 (37.1) 92 (32.4) 1.23 (0.84–1.81) 0.286 1.000 CYP2E1 rs2070673 Promoter T/a – 95 (55.9) 133 (54.5) 1.06 (0.71–1.06) 0.782 1.000 EPHX1 rs1051740 Exon 3 T/c Tyr/His 113 (58.2) 145 (51.1) 1.34 (0.93–1.93) 0.121 1.000 rs2292566 Exon 3 G/a synonymous 57 (29.4) 76 (26.8) 1.14 (0.76–1.71) 0.530 1.000 GSTP1 rs1695 Exon 5 A/g Ile/Val 29 (14.9) 40 (14.1) 1.07 (0.64–1.80) 0.792 1.000 rs4891 Exon 7 T/c synonymous 165 (85.1) 239 (84.2) 1.07 (0.65–1.78) 0.790 1.000 NAT2 rs1799930 Exon 2 G/a Arg/Gln 131 (74.4) 183 (73.2) 1.07 (0.69–1.65) 0.776 1.000 dbSNP, Single Nucleotide Polymorphism Database; OR, odds ratio; CI, confidence interval a In the allele column, a nucleotide on the left-hand side of the slash is a more frequent allele in the controls; each allele is represented by the nucleotide sequence of the sense strand of each gene b Allele frequency is presented as the number, with the percentage in parenthesis; frequencies listed are higher in the patients than in the controls Table 4 Genotype frequencies of the human AhR, GSTM1, and GSTT1 genes and the significance of their association with endometriosis Gene dbSNP accession no. Genotype a Genotype frequency OR (90 % CI) P value Pc value Patients (%) Controls (%) AhR rs2106728 CC 1 (1.0) 5 (3.5) – CT 23 (23.7) 57 (39.9) – TT 73 (75.3) 81 (56.6) 2.33 (1.33–4.08) 0.003 0.087 GSTM1 – Deletion 43 (44.3) 67 (46.9) – Present 54 (55.7) 76 (53.1) 1.11 (0.66–1.86) 0.700 1.000 GSTT1 – Deletion 38 (39.2) 56 (39.2) – Present 59 (60.1) 87 (60.1) 1.00 (0.59–1.69) 0.998 1.000 a Each genotype is represented by the nucleotide sequence of the sense strand of each gene Environ Health Prev Med (2012) 17:512–517 515 123 (D) was calculated and plotted (ESM Fig. 1). We further analyzed the haplotype constitution with six SNPs, namely, rs1476080, rs2237297, rs3802082, rs2066853, rs2040623, and rs2106728. Three haplotypes were estimated to have a frequency of [0.1 using Haploview. Haplotype 2 (TGA- GAT) was more frequently observed in patients, but not significantly so, with a difference in frequency between the case and the control group of P c = 0.072 (Table 5). Although we evaluated whether all polymorphisms were also associated with any of the four stage of endometriosis, we found that all SNPs were not significantly associated with stages I/II and III/IV of endometriosis. However, the frequencies of the allele and the TT genotype in intron 10 (rs2106728) of the AhR gene showed a significant associ- ation with developing the severe stage (stage III/IV) of endometriosis without corrections for multiple testing (ESM Table 3a, P = 0.012 and 0.008, respectively). To assess the relationship between smoking and rs2106728, we examined the allele and genotype frequencies of five smoking patients and five smoking controls, or 29 non- smoking patients and 29 non-smoking controls. Although no significant differences were found between the cases and controls in the smoking group, the frequencies of the allele and the TT genotype for rs2106728 showed a sig- nificant association in the non-smoking group without corrections for multiple testing (ESM Table 3b, P = 0.016 and 0.017, respectively).

The aim of this pilot study, in which 100 cases and 143 controls were enrolled, was to evaluate whether the poly- morphisms and null mutations in the dioxin receptor, the regulatory factor, and the phase I/II drug-metabolizing genes AhR, AHRR, ARNT, CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, GSTT1, and NAT2 are associated with the susceptibility of endometriosis. Our results indicate a fail- ure to detect a significant association between polymor- phisms of dioxin detoxification genes and endometriosis in this small number of subjects. However, the TT genotypes in intron 10 (rs2106728) of the AhR gene showed a prom- inent—but not significant—association with the risk of developing endometriosis (Table 4, P c = 0.087), leading us to calculate the genetic power in this association study (ESM Table 4). The genetic power for significance at P = 0.05 is 0.505 and the number of cases required for 80 % power is 192; moreover, the genetic power at P = 0.001 is 0.096 and number of cases required for 80 % power is 392. However, if genetic power is increased simply by increasing the number of subjects, the effect sizes in the risk loci for complex diseases identified using the associa- tion study of genetic polymorphisms with a disease are typically small. The problem could be overcome using a classification by some risk factors, such as serum cytokines and growth factors and exposure of patients to dioxins, because some association of genetic polymorphisms of immunological factors, cytokines and growth factors with endometriosis has been reported, in addition to higher levels of dioxin in peritoneal fluid [13, 14]. In addition, there have been two recent genome-wide association studies which have demonstrated strong associations betweenCDKN2BAS (antisense non-coding RNA in the cyclin-dependent kinase inhibitor 2A locus), Wnt4, and an intergenic region upstream of the NFE2L3 and HOXA10 genes and endo- metriosis [15, 16]. The CDKN2BAS and HOXA10 genes are involved in the regulation of cell growth and Wnt4 plays a role in the development of the female genital tract. Although the ethnic populations in these two studies com- prised Japanese, Australia and UK populations, the asso- ciation of Wnt4 with endometriosis in both studies was shown. This finding may suggest that the etiology of endometriosis is common between populations but that risk variants are often population-specific. In conclusion, we have genotyped five microsatellites, 23 SNPs and two deletion markers in human dioxin detoxification genes using 100 Japanese patients with endometriosis and 143 controls as pilot study. However, there were no statistically significant differences in the genotype or allele frequency distributions between the cases and controls. Overall, these results suggest that these genetic variations analyzed in this study do not appear to contribute to the development of endometriosis. However, additional studies on different female populations are required to further confirm its role in the pathogenesis of endometriosis. Table 5 Association between AhR gene haplotypes and endometriosis Haplotype no. Haplotype Patients (%) Controls (%) P value Pc value Hap1 TATACT 0.409 0.348 0.169 0.506 Hap2 TGAGAT 0.217 0.139 0.024 0.072 Hap3 GGAGAT 0.151 0.121 0.926 1.000 Haplotypes with a frequency of \0.1 were not included in the table. The P c value was corrected for three tests 516 Environ Health Prev Med (2012) 17:512–517 123 Acknowledgments This study was partly supported by Health Sciences Research grants from the Ministry of Health, Labor and Welfare of Japan. We thank the members of Teaching and Research Support Center, Tokai University for DNA sequencing. Conflict of interest None.

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