Estrogen receptor 2 and progesterone receptor gene polymorphisms and lipid levels in women with different hormonal status S Almeida N Franken M R Zandoná M C Osório-Wender M H Hutz info:doi/10.1038/sj.tpj.6500272 2005-02-00T00:00:00Z 2005-02-00T00:00:00Z (c) 2005 Nature Publishing Group The Pharmacogenomics Journal 5 1 30 34 1470-269X 1473-1150 (?-mix:\s+\W+|\W+\s+|\s+|\/) (?-mix:\.\s+(?=[A-Z])) (?-mix:\s+\W+|\W+\s+|\s+|\/) women levels HRT ESR2 or gene estrogen G polymorphism 1082GA lipid postmenopausal LDL-C association not premenopausal PGR polymorphisms T-chol 1730AG lipoprotein PROGINS results disease genotype associated observed significant study years 2 according analyses cardiovascular changes effect frequencies genetic HDL-C l mmol P respectively TG 11 14 3 age Alu analysis associations bp effects genes genotypes heterozygotes homozygotes least oral other plus receptor sample samples serum status studies users 1 12 15 16 17 20 4 5 allele band CHD cholesterol clinical detected different endogenous ERT ESR1 exogenous exposed factors gels hepatic hormonal hormones important insertion investigated lipoproteins means months mutation n only power receptors restriction risk site statistical steroid therapy three two unexposed well 0.001 0.625 18 19 21 24 32 34 7 8 abundant acetate Adjusted after agarose al Allele amplification anorexia beneficial benefit bias bleeding blood BMI body breast calculated cancer chromosome compared concentrations conditions conjugated consequently considered coronary CVD decline decrease demonstrated described detect determined development differ DNA equine estimated estradiol estrogens et European excluded exon expected expression fasting female following fragment glucose Hardy–Weinberg health heart height hormone incidence included influence influenced interaction interview ladder leiomyoma located low-density metabolism mRNA multiple nervosa nonusers nucleotide out ovarian pathways performed pharmacological population presence previous progesterone progestogen range receiving replacement reported RNA same score silent single sizes SNPs Statistical Study subgroup subgroups Table test trials type under uterine variance variation variations weight (1 (2 0.017 0.024 0.027 0.035 0.66 0.96 1.5 10 100 1080GA 1082 118 11q22–23 13 149 14q21–22 167 1730 187 18–50 2.48±0.64 2.5 2.76±0.86 22 23 25 26 27 28 29 3.25±0.78 3.43±0.75 30 306-bp 30–35 31 328 33 35 36 37 38 38–85 39 39–75 4.0 4.49±0.89 4.57±0.56 4.98±0.84 40 41 42 455 472 5.67±0.86 50 56 58±9.8 6 6.7 6.9 60 62 8.0.0 84 9 9.7 abolished above access According account accounted actions activity addition adjusted administration adolescents adverse After agreement aim alcohol alendronate alterations ALU Alzheimers amplified Analysis analyzed androgenic angiographic answering antiestrogens apolipoprotein aspect Association atherosclerosis atherosclerotic attenuate best bilateral bipolar Blood bromide bulimia calculate carcinoma care carried carriers cause cells centers chain characteristics children chromosomes clearly clothes codon codons cohorts collected combined commercial common complications conclusions confirmed consent consider consisted consistent consumption containing contraceptive contraceptives contribution counting credible criteria cross-sectional CT=94 data declared. decreases definitive deletion denominated denoting density dependent descent determine diabetes differences differently digested Discussion diseases disequilibrium disorder divided dose doses drugs DUALITY E electrophoresis elucidated encoding Endogenous enzymes equilibrium error estimations Estrogen Estrogens ethidium European-derived even evidence Exclusion exert expectations explained extracted favorable Federal folding follow-up formula Frequencies Friedewald Functional functional Genes Genomic Genotype genotype-by-HRT genotyped Grande h harm Health Heart Herrington HERS high-density Howard HS-1 human hyperlipidemia identified idiopathic II implications increase increased increases Independent independently index indicating individual individually Individuals induces Information Informed Initiative intake interact interacting INTEREST INTRODUCTION intron investigations kits knowledge lack Lack larger LDL-C=94 lead leukocytes Levels lifestyle ligand-actived light limitations linkage lipase Lipid lipid-lowering Lipoprotein ln-transformed locus low lower majority manufacturer mass Materials mean Measurements mediated medication medroxyprogesterone menstrual messenger meters methods Methods mineral miscarriage modify more Moreover myeloperoxidase need None nontranslated nonusers—mean noretisterone number obesity obvious occur oligonucleotide one-third one-way oophorectomy osteoporosis Other package pathological pattern PEPI peripheral physical physiological plasma polymerase Polymorphisms populations possibility possible Postmenopausal potency predicted prediction Premenopausal present previously primers procedure product products progestagen Progestin progestin progestogens Program progression prospective protein provide PV ratio rats reaching reaction reasons recommended recorded recurrent reduced region regression regular related relevant remove renal Replacement replication response responses responsiveness result Results reveal Rio risks role Rosenkranz Rowe Rsa RsaI salting secondary seems selected selection separately severity sex Significant similar simultaneous skewness smoking socioeconomic sources specific SPSS squared stability standard statistically stimulated stratification structure Students subfamily subject subjects subsequently suggest Sul synonymous t T1 tables taking target tends tested tests thyroid total transcription transfer transformations translate translation trial triglyceride tRNA Two two-way uncertain Universidade untransformed usage user users—mean vaginal variable variant variants verifying Version vessels virtue warranted wearing whereas whether WHI wild Women womens Womens year (?-mix:\.\s+(?=[A-Z])) A 50 bp ladder was used to score the band sizes in all gels According to Herrington and Howard, 6 the increased risk for CHD observed in clinical trials could be explained by a womens subgroup at risk for adverse cardiovascular effects of HRT by virtue of genetic variation in genes in pathways of female steroid hormones Adjusted means of lipoprotein and lipid levels were compared among genotypes of the three polymorphisms by one-way analysis of variance or Students t -test, to access genotype 1080GA by HRT use interaction, two-way analysis of variance was performed After answering the interview, women wearing light clothes had their body weight and height recorded and their body mass index (BMI) calculated by the ratio of weight (in kg) and height squared (in meters) All other tests and transformations were performed with SPSS 8.0.0 statistical package Although the possibility of a type II error could not be excluded because the power to detect this association in the premenopausal analysis was only about 60%, although the association of ESR2 1082GA genotype with T-chol and LDL-C levels was independently detected in both the premenopausal women and HRT users samples Association of ESR2 and PGR Genes Polymorphisms with Lipid and Lipoprotein Levels in Premenopausal and Postmenopausal Women Adjusted T-chol, HDL-C, LDL-C and TG means according to the genotypes of 1082GA polymorphism in premenopausal and postmenopausal women, are shown in Table 1 At pharmacological estradiol levels, hepatic low-density lipoprotein receptor is stimulated and consequently plasma LDL-C is reduced in rats. 37 These responses to pharmacological doses of estrogen can be abolished by simultaneous administration of antiestrogens, indicating that the effects are mediated by estrogen receptors. 38 Our results showed that the 1082GA polymorphism in ESR2 gene influenced LDL-C and, consequently, T-chol levels only in women with abundant endogenous (premenopausal women) or exogenous estrogen levels (postmenopausal women HRT users), whereas in postmenopausal women unexposed to HRT (lower endogenous estrogen levels) no effect was observed Discussion The allele and genotype frequencies observed for 1082GA and 1730AG polymorphisms of ESR2 gene and PROGINS of PGR gene were similar to those reported in other European or European-derived populations. 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 Several studies demonstrated that the effect of ERT/HRT on lipid levels or atherosclerosis progression in postmenopausal women may differ according to genotypes of genes with important role in lipid metabolism or CVD development, such as apolipoprotein E, 22 , 23 , 24 hepatic lipase 25 and myeloperoxidase. 26 Other studies have also shown associations between ESR1 polymorphisms and lipid levels, CVD disease or some vessels’ angiographic complications. 27 , 28 , 29 , 30 , 31 , 32 The association between ESR1 polymorphisms with lipid levels 33 and atherosclerotic severity, 34 in response to HRT have also been reported DUALITY OF INTEREST None declared. Estrogens exert the majority of their actions on target cells interacting with two specific estrogen receptors (ESR1 and ESR2), 7 , 8 while progestogens interact with the progesterone receptor (PGR), 9 therefore genetic variations in these genes may also be important to prediction of risks associated with HRT Exclusion criteria were secondary hyperlipidemia due to renal, hepatic or thyroid disease, and diabetes or fasting blood glucose levels above 6.9 mmol/l. 39 Individuals that were taking lipid-lowering medication were excluded For association analyses, the lipid and lipoproteins levels were adjusted for age, BMI and oral contraceptive usage (in premenopausal women) by multiple regression analyses Functional studies of 1082GA polymorphism as well as its contribution to cardiovascular disease development are also clearly warranted However, before reaching definitive conclusions, the results of this study need to be confirmed in larger samples and in prospective follow-up investigations However, this association was not observed in postmenopausal women unexposed to HRT In the present study, we investigated the association of two common single nucleotide polymorphisms of the ESR2 gene locus, 1082GA and 1730AG, and PGR gene PROGINS polymorphism with lipid and lipoprotein levels in premenopausal and postmenopausal women exposed or unexposed to HRT, with the aim of verifying whether T-chol, LDL-C, HDL-C, and TG levels are influenced differently by these genetic variations according to hormonal status In this cross-sectional study, women were divided into three subgroups according to hormonal status, so in the sample considered 187 were premenopausal women (mean age= 34 ± 9.7 years, 18–50 years), 118 were postmenopausal women receiving oral HRT with estrogen or estrogen plus progestagen (0.625 mg of conjugated equine estrogen, n =24; 0.625 mg of conjugated equine estrogen plus 2.5 mg of medroxyprogesterone acetate, n =62; 2 mg of estradiol plus noretisterone acetate, n =32) for at least 4 months (HRT users—mean age = 56 ± 6.7 years, range 39–75 years), and 167 were postmenopausal women that were not receiving any HRT (HRT nonusers—mean age = 58±9.8 years, range 38–85 years) for at least 1 year Independent replication of significant associations in different cohorts from the same population is an important aspect of a credible genetic association result Information about health and lifestyle factors (physical activity, smoking status, alcohol consumption and hormonal contraceptives or drugs intake) was obtained from each individual by an interview Informed consent was obtained from each subject included in the sample INTRODUCTION Endogenous and exogenous sex steroid hormones have multiple effects on lipid and lipoprotein metabolism Lack of association between 1730AG polymorphism of ESR2 gene and PROGINS of PGR gene with lipid levels in this study, as well as the consistent lack of association with other diseases may be denoting no functional effect of these variants LDL-C was calculated according to the Friedewald formula. 40 DNA analyses Genomic DNA was extracted from peripheral blood leukocytes by a salting out procedure. 41 The ESR2 SNPs, 1082GA and 1730AG, and fragment containing the PROGINS polymorphism were amplified by polymerase chain reaction, using the same conditions and oligonucleotide primers as previously described. 14 , 15 , 20 The PROGINS polymorphism amplification product was genotyped in 1.5% agarose gels Materials and Methods Study subjects The sample consisted of 472 women of European descent, which were selected from two clinical centers at the Universidade Federal do Rio Grande do Sul Measurements of lipid serum levels Blood samples were collected from women after 12 h fasting Moreover, no significant effect was detected for ESR2 1730AG polymorphism and PGR PROGINS variant on lipid levels (data not shown) Our results suggest that ESR2 1082GA genotype may influence LDL-C levels in women with abundant estrogen levels, due to either endogenous or exogenous sources Results Allele and Genotype Frequencies The allele frequencies for ESR2 gene polymorphisms were 1082 * G (0.96) and 1730 * A (0.66) Significant associations for this polymorphism were detected in postmenopausal women exposed to HRT (LDL-C: 2.48±0.64 and 3.43±0.75 mmol/l, P =0.001 for G/A heterozygotes and G/G homozygotes, respectively, and T-chol: 4.57±0.56 and 5.67±0.86 mmol/l, P =0.001 for G/A heterozygotes and G/G homozygotes, respectively) Statistical analyses Allele frequencies were estimated by gene counting T-chol, HDL-C, TG, and glucose levels were determined by standard methods using commercial kits TG levels were ln-transformed to remove skewness, but untransformed means are shown in the tables The 1082GA polymorphism of ESR2 gene has been associated with anorexia nervosa, 11 , 17 but it did not influence the mineral density and responsiveness to alendronate therapy in postmenopausal osteoporosis. 14 Analysis of 1730AG polymorphism at ESR2 showed no significant association with anorexia, 11 , 17 bulimia nervosa and obesity in children and adolescents, 11 uterine leiomyoma, 16 bipolar disorder 35 and Alzheimers disease when analyzed individually. 15 The PROGINS polymorphism was associated with risk for breast cancer, 19 but was not associated with ovarian carcinoma, 18 uterine leiomyoma, 16 idiopathic recurrent miscarriage 20 and ovarian and breast cancer. 21 The implications of these polymorphisms in ESR2 and PGR expression or in protein structure have not been elucidated The agreement of genotype frequencies with Hardy–Weinberg expectations was tested using the χ 2 -test The amplification products of ESR2 gene were subsequently digested with the following restriction enzymes under conditions recommended by the manufacturer: RsaI (1082GA) and Alu I (1730AG) The characteristics of the women included in the sample are shown in Table 2 The ESR2 1082GA polymorphism is a silent mutation in codon 328, 14 thus different gene expression pattern might occur by the following reasons: (1) transfer RNA (tRNA) concentrations may be variable for different codons; (2) synonymous changes can modify the predicted messenger RNA (mRNA) folding and lead to changes in mRNA stability and translation; 36 (3) this polymorphism might only be in linkage disequilibrium to a relevant ESR2 mutation that is associated with lipoprotein levels The genotype frequencies observed did not reveal statistically significant differences compared to those expected under Hardy–Weinberg equilibrium The genotypes were determined after electrophoresis on agarose gels with ethidium bromide, using a 100 bp ladder to score the band sizes The HRT clinical trials have shown that HRT induces favorable changes in lipid levels; however, these changes do not translate into decrease in cardiovascular disease. 4 , 5 It is estimated that beneficial alterations in lipoproteins account for one-third of decline in coronary heart disease (CHD) incidence. 3 However, these estimations do not consider the possible genetic variation in genes in pathways of female steroid hormones that could determine a subgroup of women that would benefit more from estrogens The human estrogen receptor 2 (ESR2) encoding gene is located on chromosome 14q21–22. 10 Two single nucleotide polymorphisms (SNPs) in this gene were described by Rosenkranz et al , 11 a Rsa I restriction site at 1082GA (a silent mutation in exon 5) and an Alu I restriction site at 1730AG (3’ nontranslated region of the exon 8) The number of ESR2 G/A carriers was low in the subgroups, but the HRT users analysis has statistical power (for CT=94% and LDL-C=94%) to detect the association observed The oral estrogen replacement therapy (ERT) in postmenopausal women decreases serum total cholesterol (T-chol) and low-density lipoprotein cholesterol (LDL-C) concentrations, as well as increases serum high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels. 1 The effects of combined (estrogen plus progestin) hormone replacement therapy (HRT) are uncertain; the addition of a progestogen to estrogens tends to attenuate the increase in serum HDL-C and the decrease in LDL-C, obtained with ERT, although this effect seems to be related to the dose and to androgenic potency of the progestogen. 2 The beneficial changes in these lipoproteins should be associated with at least 30–35% decline in coronary heart disease (CHD) incidence. 3 However, the results from the Heart and Estrogen/Progestin Replacement Study (HERS) 4 and the Womens Health Initiative (WHI) trial 5 demonstrated that oral HRT does not provide cardiovascular benefit or even some evidence of cardiovascular harm The PEPI Statistical Program Version 4.0 42 was used to calculate the statistical power of the samples The PGR wild type allele (T1 — without insertion Alu) accounted for 84% of the chromosomes investigated The premenopausal women had regular menstrual bleeding within the previous three months The premenopausal women showed significant associations between the 1082GA site with LDL-C (2.76±0.86 and 3.25±0.78 mmol/l; P =0.027 for G/A heterozygotes and G/G homozygotes, respectively) and T-chol (4.49±0.89 and 4.98±0.84 mmol/l; P =0.035 for G/A heterozygotes and G/G homozygotes, respectively) The presence of an ALU insertion results in a 455 bp band, while the deletion genotype results in 149 bp fragment The progesterone receptor gene is located on chromosome 11q22–23, 12 and Rowe et al . 13 identified a 306-bp insertion of the PV/HS-1 Alu subfamily in intron 7, denominated PROGINS The study was exposed to selection bias and factors, such as population stratification, socioeconomic status or health care, may differ between HRT user and nonusers and therefore bias our results The women were considered postmenopausal if they had no vaginal bleeding for at least 12 months without other obvious pathological or physiological cause or bilateral oophorectomy There was a significant genotype-by-HRT interaction ( P for T-chol and LDL-C levels was 0.024 and 0.017, respectively) There were some limitations to our study These analyses were carried out for premenopausal, postmenopausal women HRT users and HRT nonusers separately These results were expected since estrogen receptors are ligand-actived transcription factors dependent on the presence of hormone To the best of our knowledge, no previous studies investigated ESR2 and PGR gene polymorphisms with lipid and lipoprotein levels info:doi/10.1016/S0015-0282(01)01699-5 info:doi/10.1016/j.maturitas.2003.09.015 info:doi/10.1161/01.ATV.0000046033.32478.6D info:doi/10.1001/jama.280.7.605 info:doi/10.1001/jama.288.3.321 info:doi/10.1517/phgs.4.3.269.22686 info:doi/10.1038/320134a0 info:doi/10.1210/jc.82.12.4258 info:doi/10.1016/0167-4781(93)90156-8 info:doi/10.1210/jc.83.12.4524 info:doi/10.1016/S0960-0760(02)00061-4 info:doi/10.1016/S0304-3940(01)01806-7 info:doi/10.1038/sj.mp.4001929 info:doi/10.1002/1097-0215(20011120)95%3A6%3C394%3A%3AAID-IJC1070%3E3.0.CO%3B2-X info:doi/10.1016/S1071-5576(01)00123-X info:doi/10.1210/jc.2002-020008 info:doi/10.1016/S0378-5122(01)00237-7 info:doi/10.1016/S0021-9150(01)00632-3 info:doi/10.1016/S0021-9150(01)00675-X info:doi/10.1097/01.LAB.0000077981.49367.46 info:doi/10.1161/01.ATV.0000014424.18209.21 info:doi/10.1016/S0009-8981(03)00080-9 info:doi/10.1097/00006254-200406000-00022 info:doi/10.1161/01.ATV.0000101181.81022.BF info:doi/10.1007/s00109-001-0311-5 info:doi/10.1046/j.1365-2796.2003.01179.x info:doi/10.1056/NEJMoa012952 info:doi/10.1016/S0378-5122(02)00293-1 info:doi/10.1097/00041444-200112000-00008 info:doi/10.1093/hmg/ddg055 12