Genetic Determinants of Circulating Estrogen Levels and Evidence of a Causal Effect of Estradiol on Bone Density in Men

The Journal of Clinical Endocrinology & Metabolism, Mar 2018

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Genetic Determinants of Circulating Estrogen Levels and Evidence of a Causal Effect of Estradiol on Bone Density in Men

J Clin Endocrinol Metab, March Genetic Determinants of Circulating Estrogen Levels and Evidence of a Causal Effect of Estradiol on Bone Density in Men Anna L. Eriksson 15 John R. B. Perry 12 14 Andrea D. Coviello 20 Graciela E. Delgado 19 Luigi Ferrucci 18 Andrew R. Hoffman 17 Ilpo T. Huhtaniemi 21 22 M. Arfan Ikram 16 Magnus K. Karlsson 9 Marcus E. Kleber 19 Gail A. Laughlin 10 Yongmei Liu 7 Mattias Lorentzon 8 15 Kathryn L. Lunetta 5 6 Dan Mellstro¨ m 8 15 Joanne M. Murabito 2 Anna Murray 12 Maria Nethander 15 Carrie M. Nielson 4 Inga Prokopenko 0 13 Stephen R. Pye 11 Leslie J. Raffel 1 Fernando Rivadeneira 3 16 Priya Srikanth 4 Lisette Stolk 3 Alexander Teumer Thomas G. Travison Andr e´ G. Uitterlinden 3 16 Dhananjay Vaidya Dirk Vanderschueren Joseph M. Zmuda Winfried M a¨rz Eric S. Orwoll Pamela Ouyang Liesbeth Vandenput 15 Frederick C. W. Wu Frank H. de Jong 3 Shalender Bhasin Douglas P. Kiel 6 Claes Ohlsson 15 0 Department of Genomics of Common Disease, School of Public Health, Imperial College London , London W12 0NN , United Kingdom 1 Division of Genetic and Genomic Medicine, Department of Pediatrics, University of California , Irvine, California 92868 , USA 2 Department of Medicine, Section of General Internal Medicine, Boston University School of Medicine , Boston, Massachusetts 02118 , USA 3 Department of Internal Medicine, Erasmus MC , Rotterdam 3000 CA , USA 4 School of Public Health, Oregon Health & Science University , Portland, Oregon 97239 , USA 5 Boston University School of Public Health , Boston, Massachusetts 02118 , USA 6 Framingham Heart Study , Framingham, Massachusetts 07012 , USA 7 Department of Epidemiology and Prevention, Division of Public Health Sciences, Wake Forest School of Medicine , Winston-Salem, North Carolina 27157 , USA 8 Geriatric Medicine, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, University of Gothenburg and Geriatric Medicine, Sahlgrenska University Hospital , 43180 M o ̈lndal , Sweden 9 Department of Orthopaedics and Clinical Sciences, Sk a ̊ne University Hospital, Lund University , 217 74 Malmo ̈ , Sweden 10 Family Medicine and Public Health, University of California-San Diego , San Diego, California 92093 , USA 11 Arthritis Research UK Centre for Epidemiology, Centre for Musculoskeletal Research, The University of Manchester, Manchester Academic Health Science Centre , Manchester M13 9PT , United Kingdom 12 University of Exeter Medical School, University of Exeter , Exeter EX1 2LU , United Kingdom 13 Hammersmith Hospital , London W12 0NN , United Kingdom 14 Medical Research Council Epidemiology Unit, University of Cambridge School of Clinical Medicine, Institute of Metabolic Science , Cambridge Biomedical Campus, Cambridge CB20QQ , United Kingdom 15 Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska University Hospital , 413 45 Gothenburg , Sweden 16 Department of Epidemiology, Erasmus MC , Rotterdam 3000 CA , The Netherlands 17 Division of Endocrinology, Stanford University School of Medicine , Stanford, California 94305 , USA 18 Longitudinal Studies Section, Clinical Research Branch, Gerontology Research Center, National Institute on Aging , Baltimore, Maryland 21224 , USA 19 Vth Department of Medicine, Medical Faculty Mannheim, Heidelberg University , Mannheim 68167 , Germany 20 Duke University School of Medicine , Durham, North Carolina 27710 , USA 21 Department of Physiology, Institute of Biomedicine, University of Turku , Turku 20100 , Finland 22 Department of Surgery and Cancer, Imperial College London , Hammersmith Campus, London W12 0NN , United Kingdom - *These authors contributed equally to this work †These authors were joint senior authors on this work. Abbreviations: BMD, bone mineral density; BMI, body mass index; DHEAS, dehydroepiandrosterone sulfate; E1, estrone; E2, estradiol; EMAS, European Male Ageing Study; FAM9B, FAMily with sequence similarity 9, member B; FHS, Framingham Heart Study; FN, femoral neck; GC-MS, gas chromatography-mass spectrometry; GEFOS, Genetic Factors in Osteoporosis Consortium; GOOD, Gothenburg Osteoporosis and Obesity Determinants; GWAS, genome-wide association study; KAL1, Kallman syndrome 1; LD, linkage disequilibrium; LS, lumbar spine; LURIC, Ludwigshafen Risk and Cardiovascular Health; MrOS, Osteoporotic Fractures in Men; RS1, Rotterdam 1 study; SD, standard deviation; SE, standard error; SHBG, sex hormone binding globulin; SNP, single nucleotide polymorphism; TRIM4, Tripartite motif containing 4. https://academic.oup.com/jcem The Netherlands; 24Institute for Community Medicine, University Medicine Greifswald, 17475 Greifswald, Germany; 25Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, 17475 Greifswald, Germany; 26Institute for Aging Research, Hebrew Senior Life and Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02131; 27Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; 28Department of Clinical and Experimental Medicine, Katholieke Universiteit Leuven, Laboratory of Clinical and Experimental Endocrinology, Leuven, B-3000, Belgium; 29Department of Epidemiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; 30Synlab Academy, Synlab Holding Deutschland GmbH, Mannheim 68161, Germany; 31Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, 8036 Graz, Austria; 32Bone & Mineral Unit, Oregon Health & Science University, Portland, Oregon 97239; 33Andrology Research Unit, Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical and Human Sciences, The University of Manchester, Central Manchester University Hospitals National Health Service Foundation Trust, Manchester M13 9WL, United Kingdom; and 34Research Program in Men’s Health: Aging and Metabolism, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 Context: Serum estradiol (E2) and estrone (E1) levels exhibit substantial heritability. Objective: To investigate the genetic regulation of serum E2 and E1 in men. Design, Setting, and Participants: Genome-wide association study in 11,097 men of European origin from nine epidemiological cohorts. Main Outcome Measures: Genetic determinants of serum E2 and E1 levels. Results: Variants in/near CYP19A1 demonstrated the strongest evidence for association with E2, resolving to three independent signals. Two additional independent signals were found on the X chromosome; FAMily with sequence similarity 9, member B (FAM9B), rs5934505 (P = 3.4 3 1028) and Xq27.3, rs5951794 (P = 3.1 3 10210). E1 signals were found in CYP19A1 (rs2899472, P = 5.5 3 10223), in Tripartite motif containing 4 (TRIM4; rs17277546, P = 5.8 3 10214), and CYP11B1/B2 (rs10093796, P = 1.2 3 1028). E2 signals in CYP19A1 and FAM9B were associated with bone mineral density (BMD). Mendelian randomization analysis suggested a causal effect of serum E2 on BMD in men. A 1 pg/mL genetically increased E2 was associated with a 0.048 standard deviation increase in lumbar spine BMD (P = 2.8 3 10212). In men and women combined, CYP19A1 alleles associated with higher E2 levels were associated with lower degrees of insulin resistance. Conclusions: Our findings confirm that CYP19A1 is an important genetic regulator of E2 and E1 levels and strengthen the causal importance of E2 for bone health in men. We also report two independent loci on the X-chromosome for E2, and one locus each in TRIM4 and CYP11B1/B2, for E1. (J Clin Endocrinol Metab 103: 991–1004, 2018) Emajor biologically active estrogens in men. E2 is more strogens 17 b-estradiol (E2) and estrone (E1) are the potent than E1. Aromatase, encoded by the CYP19A1 gene, is the key enzyme responsible for the final step in the synthesis of both E2 and E1. E2 is formed from aromatization of testosterone, and E1 is formed from aromatization of androstenedione. E2 can also be formed from conversion of E1 by 17b-hydroxysteroid dehydrogenase ( 1 ). In men, the circulating levels of E2 and E1 are determined by both genetic and environmental factors. The heritability for E2 in men has been estimated to be ;30% to 45% and for E1 ;40% ( 2, 3 ). Early studies of the genetic regulation of circulating E2 and E1 levels were hampered by their small size and the use of immunoassays with poor specificity, precision, and accuracy at lower concentrations. However, in 2010, Orwoll and colleagues performed a large study of 5000 elderly men of European, Asian, and African origin in Sweden, the United States, Hong Kong, and Tobago (4). Serum sex steroid levels were measured using gas chromatographymass spectrometry (GC-MS), thereby avoiding the previously mentioned problems with immunoassays. In addition to geographical differences in E2 and E1 levels, suggestive of environmental influences, they also found racial differences. Both E2 and E1 levels, as well as the E2 to testosterone and E1 to androstenedione ratios, were higher in black than in Asian and Caucasian men ( 4 ). These data suggested that genetically determined differences in aromatase activity among black, Asian, and Caucasian men might be responsible for the observed racial differences in E2 and E1 levels. We made a first attempt to find genetic loci involved in the determination of estrogen levels in men by analyzing 604 single nucleotide polymorphisms (SNPs) in 50 candidate sex steroid-related genes ( 5 ). In a screening cohort, the CYP19A1 SNP rs2470152 showed the most significant association with E2 levels measured by GC-MS. This was confirmed in two replication cohorts. Rs2470152 was also significantly associated with E1 levels in all three cohorts (n = 5531) ( 5 ). Meta-analyses of genome-wide association studies (GWASs) enable a comprehensive analysis of the whole genome in a large number of subjects. Chen and colleagues performed a GWAS in 3495 Chinese men in which E2 concentrations were determined using an immunoassay. They found two independent SNPs in the CYP19A1 gene to be associated with E2 levels (rs2414095 and rs2445762) ( 6 ). These findings further strengthened the evidence for a major role of CYP19A1 in the regulation of serum E2 levels in men, but because of the relatively small sample size and low power, genetic loci in other regions of the genome could have been missed. To date, no GWAS has been performed in men of European origin. In women, a smaller GWAS meta-analysis of 1583 postmenopausal women found no genome-wide significant SNPs. Among variants that were suggestively associated with E2, several were located at the CYP19A1 locus ( 7 ). Both E2 and testosterone regulate bone mass ( 8 ). Studies of men with nonfunctional estrogen receptor alpha ( 9 ), and inactivating mutations of the CYP19A1 gene ( 10 ), have demonstrated that estrogens are important for peak bone mass acquisition in men. Population-based studies have shown that in men, low serum levels of E2 are associated with a lower bone mineral density (BMD), higher rates of bone loss, and an increased risk of fractures ( 8, 11–14 ). Some studies also show a smaller contribution of testosterone to BMD in men ( 8, 11 ). The relative contribution of androgens vs estrogens in the regulation of bone mass in men remains incompletely understood, and studies showing evidence of a causal effect of serum E2 on BMD in men are still sparse (15). Mendelian randomization is a method used to strengthen or refute the causality of a biomarker, such as E2, and an outcome measure of interest, such as BMD, when a randomized controlled trial is not possible. Mendelian randomization uses genetic data and relies on the principle that, because of the random assortment of genetic variants at conception, these genetic variants are independent of many factors that bias observational studies, such as confounding and reverse causation. Therefore, if a biomarker is etiologically involved in an outcome measure, the genetic factors that influence the biomarker will also influence the outcome measure ( 16 ). To date, no Mendelian randomization has been performed to investigate causality between E2 levels and BMD in men. Case reports of men with aromatase deficiency from an inactivating mutation of the CYP19A1 gene, mechanistic animal studies and clinical studies also suggest that estrogen signaling through estrogen receptor alpha is important for insulin sensitivity in men ( 17–23 ). Thus, genetic factors regulating estrogen levels may also be of relevance for the regulation of insulin sensitivity in men. Here, we present the results of a GWAS of estrogen levels combining several population-based cohorts of men of European origin. We also present results of our analyses of the association of resultant genome-wide significant associations with two major estrogen related traits: BMD and insulin sensitivity. Methods Study samples The discovery stage of the E2 GWAS included 11,097 men of European origin drawn from nine epidemiological cohorts: the Framingham Heart Study (FHS), the Gothenburg Osteoporosis and Obesity Determinants (GOOD) study, the Invecchiare in Chianti study, the Ludwigshafen Risk and Cardiovascular Health (LURIC) study, the Multi-Ethnic Study of Atherosclerosis study, the Osteoporotic Fractures in Men (MrOS) Sweden Gothenburg study, the MrOS Sweden Malm o¨ study, the MrOS US study, and the Rotterdam 1 study (RS1). Replication of one SNP displaying considerable heterogeneity in genome-wide significant fixed-effect models but nominal significance only in random effects models, was performed in the European Male Ageing Study (EMAS; n = 1641). EMAS is a cohort of men predominantly of European origin, with only 0.62% (n = 21) of the sample used here being of non-European descent. The discovery stage of the E1 GWAS included 7570 men of European origin drawn from six of the previously mentioned cohorts: FHS, GOOD, MrOS Sweden Gothenburg, MrOS Sweden Malm o¨, MrOS United States, and RS1. Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5-alpha reductase inhibitors and sex hormone antagonists. All studies were approved by local ethics committees and all participants provided written informed consent. Characteristics of the study samples and detailed descriptions of the participating cohorts, genotyping, quality control, and imputation procedures are provided in the Supplemental Appendix and Supplemental Tables 1, 2, and 3. Sex hormone measurements In six discovery cohorts (FHS, GOOD, MrOS Sweden Gothenburg, MrOS Sweden Malm o¨, and MrOs United States), measurements of E1 and E2 were performed using either GC-MS or liquid chromatography tandem mass spectrometry. In the remaining discovery cohorts (LURIC, Invecchiare in Chianti, Multi-Ethnic Study of Atherosclerosis, and RS1) measurements were performed using immunoassays. In the replication cohort (EMAS), E2 was measured using the GC-MS technique. Methods for all measurements are given in the Supplemental Appendix. Genotyping and statistical analyses Nine discovery and one replication study populations were genotyped using a variety of genotyping platforms including Illumina (HumanHap 550k, 610k, 1M-Duo, Omni1-Quad, Omni express) and Affymetrix (500K Dual GeneChip + 50K genecentered MIP set, Array 6.0) (Supplemental Table 2). To increase genomic coverage and allow the evaluation of the same SNPs across as many study populations as possible, each study imputed genotype data based on the HapMap CEU Build 36. Algorithms were used to infer unobserved genotypes in a probabilistic manner using either MACH (http://www.sph.umich.edu/ csg/abecasis/MACH) or IMPUTE2 ( 24 ). We analyzed only those SNPs (genotyped or imputed) that had a minor allele frequency of .0.01 and an imputation quality of $0.3. The X chromosome was available for analysis in six cohorts (FHS, GOOD, LURIC, MrOS Sweden Gothenburg, MrOS Sweden Malmo¨ , and MrOS United States) in this study. Imputations of the X chromosome were performed in all of these cohorts except MrOS United States. Altogether, ;2.5 million SNPs were tested for association with serum E2 and E1 in the discovery stage. GWAS analyses were performed using an additive genetic linear regression model adjusted for: 1) age and body mass index (BMI; E2 and E1) or 2) age, BMI, testosterone, and sex hormone binding globulin (SHBG; E2 only), in each of the discovery cohorts. In FHS, a linear mixed-effect model with a random effect to account for relationships was used. Imputed genotypes were analyzed in all cohorts, taking the genotype uncertainties into account. The meta-analyses were performed in the METAL software (https:// www.sph.umich.edu/csg/abecasis/MACH), using an inversevariance weighted fixed effect model. Random effects models were used when fixed effect models displayed heterogeneity defined as an I2 value .50% ( 25 ). These models were calculated using the R package (http://www.r-project.org). A threshold of P , 531028 was established a priori as the level for genome-wide significance in the discovery analyses ( 26 ). Approximate conditional analyses for E2 and E1 were performed using the Genome-wide Complex Trait Analysis (GCTA) software (27), and the genotypes of the European Prospective Investigation of Cancer Norfolk study cohort used as a reference panel to estimate patterns of linkage disequilibrium (LD) ( 28 ). The gas chromatography–corrected and quality control–filtered meta-analysis results and a condition list containing the lead SNPs of the final loci were used as input for the conditional analysis. An additional association was declared when the conditional P value was below the genome-wide significance threshold. Subsequently, this SNP was added to the list of conditional analysis SNPs and the conditional analysis was performed again in a stepwise fashion until no additional substantial independent associations were found. Gene expression analyses We analyzed associations between identified SNPs associated with serum estrogen levels and gene expression in the eQTL dataset generated by the GTEx Consortium (version 6p), which was obtained from http://www.gtexportal.org/ ( 29 ). Associations with testosterone Associations with serum testosterone concentrations were retrieved from the discovery dataset of our previously published GWAS of testosterone levels ( 30 ). Associations with other traits We hypothesized, based on data in the literature, that our genome-wide significant SNPs and secondary signals from conditional analyses could be associated with BMD and/or insulin sensitivity. To test these hypotheses, we searched publicly available databases for associations with lumbar spine (LS) and femoral neck (FN) BMD in men [Genetic Factors in Osteoporosis Consortium (GEFOS); www.gefos.org] (31). Data on glycemic traits in men and women combined were downloaded from http://www.magicinvestigators.org/downloads/ (32, 33). Data on glycemic traits in men and women separately were contributed by Meta-Analysis of Glucose and Insulin-Related Traits Consortium (MAGIC) investigators (32, 33). Homeostatic model assessment-estimated insulin resistance was calculated as (fasting insulin 3 fasting glucose)/22.5. Mendelian randomization of serum E2 on BMD To investigate if E2 has a causal effect on BMD, we performed a summary statistic two sample inverse varianceweighted Mendelian randomization ( 34 ). We selected the five top loci from our E2 meta-analysis and extracted summary statistics [b and standard error (SE)] from the corresponding SNPs in both our E2 study and the GEFOS study on LS and FN BMD. The variant specific associations were used to create an inverse variance weighted estimate of the causal effect size and its SE. Results We performed a GWAS of serum E2 and E1 concentrations, investigating ;2.5 million SNPs in up to 11,097 men. In analyses of autosomal chromosomes, all nine discovery cohorts (n = 11,097) were included in the discovery analyses of E2; six cohorts (n = 7570) were included in the discovery analyses of E1. In analyses of the X chromosome, six cohorts (n = 8953) were included in the discovery analyses of E2 and five cohorts (n = 6917) were included in the discovery analyses of E1. E2 In the model adjusted for age and BMI (model 1), two loci were associated with E2 concentrations at the genome-wide significance threshold of P , 5 3 1028 in the discovery analyses [Supplemental Fig. 1(A)]. The strongest association was found within the CYP19A1 locus on chromosome 15q21.1 (rs727479, effect size 1.39 pg/mL per effect allele; SE, 0.12; P = 8.2 3 10230) [Table 1; Fig. 1(a); Supplemental Figs. 2(A) and 3(A)]. This SNP, which is located in the second intron of the gene, showed heterogeneity of effect size across studies as indicated by an I2 value of 57% ( 25 ). To take this heterogeneity into account, we additionally calculated a random effects model, which was also genome-wide significant (effect size = 1.35 pg/mL; SE, 0.19; P = 2.0 3 10212). The second locus was found on the X chromosome where one SNP, rs5934505, reached genome-wide significance (P = 3.4 3 1028). This SNP is located 79 kb downstream of the FAMily with sequence similarity 9, member B (FAM9B) gene (Xp22.31) [Table 1; Fig. 1(a); SNPs Associated With Serum E2 and E1 Concentrations: Genome-Wide Results of Meta-Analysis Effect size is given per effect allele as picogram per milliliter. Location is given according to human GRCh38/hg38. All cohorts (n = 11,097) were included in the E2 GWAS of chromosomes 1 through 22. The E1 GWAS of chromosomes 1 through 22 included FHS, GOOD, MrOS Sweden Gothenburg, MrOS Sweden Malm o¨, MrOS United States, and RS1. X chromosome data were available for FHS, GOOD, LURIC, MrOS Sweden Gothenburg, MrOS Sweden Malmo¨ , and MrOS United States. Model 1 is adjusted for age and BMI; model 2 is adjusted for age, BMI, testosterone, and SHBG. Abbreviations: Chr, chromosome; EA, effect allele (i.e., the allele associated with increased serum E2); freq., frequency of effect allele. aSecondary signal from GCTA analysis. bConditional P value from GCTA analysis. Fixed-effect meta-analysis P values for secondary signals from GCTA analysis: rs2899472 (E2, model 2): P = 4.3 3 10221; rs16964258 (E2, model 2): P = 2.3 3 10217; rs727479 (E1, model 1): P = 2.1 3 10222. Supplemental Figs. 2(B) and 3(B)]. There was heterogeneity of effect size across studies for this SNP (I2 = 72%). A random effects model displayed nominal, but not genome-wide, significance in the same direction as the result from the fixed-effect meta-analysis [C-allele associated with higher E2 levels, effect size 0.74 pg/mL per effect allele (SE, 0.24), P = 0.002]. Therefore, we attempted replication for rs5934505 in the EMAS cohort (n = 1641). In this cohort, the C-allele was also associated with higher E2 levels; effect size of 1.59 pg/mL per effect allele (SE, 0.39), P = 5.2 3 1025. In the model that was adjusted for testosterone and SHBG levels, in addition to age and BMI [model 2; Supplemental Fig. 1(B)], the associations between E2 and the CYP19A1 locus remained significant [rs727479: P = 3.1 3 10243; Table 1; Fig. 1(b); Supplemental Figs. 2(C) and 3(C)]. In this analysis, the I2 value was 69%, but the random effects model was genome-wide significant [effect size, 1.42 pg/mL per effect allele (SE, 0.20), P = 3.5 3 10213]. A genome-wide significant locus on the X chromosome also appeared in this analysis. rs5951794 (P = 3.1 3 10210, I2 = 6%) is located in the distal part of the long arm on chromosome X (Xq27.3), ;137 Mb from the FAM9B SNP rs5934505 [Table 1; Fig. 1(b); Supplemental Figs. 2(D) and 3(D)]. To identify multiple statistically independent SNPs within the same genomic region, we performed stepwise approximate conditional analyses (GCTA) for each of the genome-wide significant loci. In the model adjusted for testosterone and SHBG, the analysis revealed two additional genome-wide significant SNPs in the CYP19A1 locus: rs2899472 in intron 4 (conditional P = 1.1 3 1028) and rs16964258 in intron 1 (conditional P = 8.2 3 10215) [Table 1; Fig. 1(b); Supplemental Figs. 2(C), 3(E), and 3(F)]. In the model adjusted for age and BMI only, no additional independent associations were found. In model 1, rs727479 explained 0.9% of the overall variance of E2 levels. When the other identified SNP from model 1, rs5934505 (FAM9B), was added, 1.1% of the overall variance in E2 levels was explained. In model 2, independent CYP19A1 SNPs explained 1.3% of the overall variance in E2 levels. When the other genomewide significant SNP from model 2, rs5951794 (Chr X), was added, 1.4% of the overall variance in E2 levels was explained. E1 Three genome-wide significant loci, located on chro mosomes 7, 8, and 15, respectively, were associated with E1 levels [Supplemental Fig. 1(C)]. The strongest association was found for the CYP19A1 locus on chromosome 15. The lead SNP was rs2899472 (P = 5.5 3 10223) [Table 1; Fig. 1(c); Supplemental Figs. 2(E) and 3(G)]. Because of heterogeneity in effect size at this variant (I2 = 59%), a random effects model was run, which was genome-wide significant (effect size, 2.55 pg/mL per effect allele; SE, 0.41, P = 4.6 3 10210). In conditional analyses of this locus, the SNP with the most significant association with E2, rs727479, was also genome-wide significantly associated with E1 (conditional P = 3.5 3 10210) [Table 1; Fig. 1(c); Supplemental Figs. 2(E) and 3(H)]. On chromosome 7, the SNP most significantly associated with E1 levels was rs17277546 (P = 5.8 3 10214), located in the 30 UTR of the Tripartite motif containing 4 (TRIM4) gene [Table 1; Fig. 1(c); Supplemental Figs. 2(F) and 3(I)]. On chromosome 8, the SNP most significantly associated with E1 levels was rs10093796 (P = 1.2 3 1028). This SNP is located between the CYP11B1 and the CYP11B2 genes [Table 1; Fig. 1(c); Supplemental Figs. 2(G) and 3(J)]. E1 is not derived from testosterone and not bound to SHBG in the circulation; therefore no analyses of E1 adjusted for these parameters were performed. Independent CYP19A1 SNPs explained 1.5% of the overall variance in E1 levels. Rs17277546 (TRIM4) and rs10093796 (CYP11B1/B2) explained 0.5% and 0.1%, respectively, of the variance. In total, 2.1% of the overall variance in E1 levels was explained by these genome-wide significant SNPs. The alleles associated with higher E2 levels were associated with higher gene expression levels [rs727479: b = 0.23, P = 1.9 3 1025 (skin); rs2899472: b = 0.20, P = 9 3 1028 (whole blood)]. Rs727479 was also associated with the expression level of signal peptide peptidase like 2A [b = 0.18, P = 1.3 3 1024 (transformed fibroblasts)], which is located 442 kB upstream of CYP19A1. The E1-associated SNP on chromosome 8, rs10093796, was associated with the expression levels of two adjacent genes in several tissues (Lys6/neurotoxin1); pancreas: b = 0.68, P = 5.6 3 1029; lymphocyte antigen 6 complex, locus K skin b 0.32, P = 2.3 3 1027. Both are located 95 and 168 kB, respectively, upstream of CYP11B1. The other SNPs in our study were not associated with expression levels in the GTEx database. Gene expression analyses In the GTEx database, two of the CYP19A1 SNPs were robustly associated with the expression level of CYP19A1. Associations with estrogen-related traits To further investigate the physiological relevance of our E2 GWAS findings, we performed look-up analyses of other GWAS that had data on phenotypes known or suspected to be related to E2 levels. Testosterone To better understand the mechanism underlying the association between our E2-related SNPs and E2 levels, we studied the association between these SNPs and serum testosterone levels. If the effect of the SNPs on E2 levels was exerted upstream of the aromatase enzyme, one would expect that those SNPs would be associated with higher testosterone as well as higher E2 levels. On the other hand, if the effect of the SNPs on E2 levels were exerted through alteration in either the amount or the activity of the aromatase enzyme, only E2 levels would be expected to be increased, with no increase in testosterone levels. The C-allele of the E2 X chromosome SNP rs5934505 (FAM9B) was positively associated with levels of both testosterone and E2, suggesting that the effect of rs5934505 is exerted upstream of aromatase (Table 2; Fig. 2). Indeed, we have previously reported that the X chromosome SNP rs5934505 (FAM9B) is associated with circulating testosterone levels in men (P = 1.6 3 1028) ( 30 ). None of the other E2 SNPs were associated with increased levels of testosterone, suggesting that these SNPs are affecting either the amount or the activity of aromatase or E2 clearance. In fact, the G-allele of the other E2 X chromosome SNP, rs5951794, was associated with increased E2 levels and slightly decreased testosterone levels [effect size, 27.68 ng/dL per effect allele (SE, 3.05), P = 0.01] (Table 2; Fig. 2). Additionally, for CYP19A1 SNPs, there were indications of associations with testosterone in the opposite direction compared with E2, but these associations did not reach statistical significance (rs727479 P = 0.05 and rs16964258: P = 0.26) (Table 2; Fig. 2). BMD The primary SNP in CYP19A1, rs727479, and the secondary signals rs2899472 and rs16964258, were all significantly associated with LS BMD in men (P # 0.01; Table 3). Rs727479 and rs2899472 were also associated with FN BMD in men (P , 0.01). The direction of the effect was the same for all markers (i.e. alleles associated with higher levels of E2) were associated with a higher BMD. Moreover, rs5934505 (FAM9B) was associated with both FN (P = 0.01) and LS (P = 7 3 1026) BMD. As in the case of CYP19A1 SNPs, the allele associated with higher E2 levels was associated with a higher BMD (Table 3). Mendelian randomization E2 and BMD The data from the GEFOS database show associations between individual SNPs and BMD, but do not provide information on possible causality between the E2 levels resulting from these SNPs and BMD. To overcome this, we performed a summary statistic Mendelian randomization analysis, which suggested that there is a causal effect of serum E2 on BMD. A 1 pg/mL genetically increased E2 was associated with a 0.048 standard deviation (SD) (SE, 0.008), P = 2.8 3 10212 increase in LS BMD. For the femoral neck, the increase was 0.037 SD (SE, 0.007, P = 4.4 3 1028) (Fig. 3). Insulin sensitivity The publicly available GWAS results for measures of insulin sensitivity included only autosomal chromosomes, and did not include results for men and women separately. Thus the following results apply for men and women combined. Insulin resistance expressed as homeostatic model assessment-estimated insulin resistance was negatively associated with the E2 increasing A alleles of rs727479 (P = 0.004) and rs2899472 (P = 0.003) in CYP19A1. This was due to a negative association of these alleles with fasting insulin (P = 0.003 for rs727479; P = 0.017 for rs2899472) (Supplemental Table 4). Adjustments for BMI had no effect on the results (BMI-adjusted fasting insulin, P = 0.002 for rs727479 and P = 0.031 for rs2899472). There were no associations with fasting glucose for these SNPs. The MAGIC investigators also provided us with data not publicly available on fasting Effect size is given per effect allele as nanogram per deciliter. Numbers in bold represent statistical significance. Testosterone levels were retrieved from our previous GWAS of testosterone levels ( 30 ). aTotal number of study participants, information for individual SNPs not available. insulin and fasting glucose for men and women separately (fasting insulin: men, n 26,000; women, n 32,000; fasting glucose: men, n 36,000; women, n 43,000). In this dataset, the association between rs727479 and fasting insulin was significant in women [b = 20.014 (SE, 0.004), P = 0.002]. In men, the direction of the association was the same as in women, but was not statistically significant [rs727479: b = 20.006 (SE, 0.005), P = 0.19]. Discussion In this GWAS, SNPs in the CYP19A1 gene showed the strongest associations with both E1 and E2 levels. This confirms data from previous studies ( 5, 6, 35 ) and establishes CYP19A1 as an important genetic regulator of estrogen levels in men. We found three independent signals in CYP19A1, which extends the results from previous studies. We also identified two additional signals for E2 on chromosome X and two additional signals for E1 on chromosomes 7 and 8, respectively. Moreover, SNPs found to be associated with E2 levels in this study were also associated with known or suspected estrogenrelated traits including BMD and insulin sensitivity. Mendelian randomization analysis using the independent E2 SNPs suggests a causal effect of E2 on BMD in men. The finding of several independent signals for both E1 and E2 in CYP19A1 is consistent with the findings in the previously reported GWAS in Chinese men, where two independent SNPs were found. This strengthens the conception that the regulation of estrogen levels is governed by more than one signal in the gene. The organization of CYP19A1 is rather complex. The gene consists Effect size for BMD is given as standardized values per copy of the SNP allele from fixed-effects meta-analysis. Numbers in bold represent statistical significance after Bonferroni correction for two phenotypes (LS BMD and FN BMD). aTotal number of study participants, information for individual SNPs not available. of a 30-kb coding region and a 93-kb regulatory region including 10 tissue-specific promoters ( 36 ). There are four blocks of LD in the gene. Rs727479, which displayed the most important association with E2 levels in our study, is located in intron 2 in LD block 4, which covers 50 kB, including the entire coding region, exons/ promoters I.6, I.3, and PII, through 5.8 kb downstream of exon 10 ( 37 ). Rs727479 has been associated with E2 levels in previous candidate gene studies investigating haplotype-tagging SNPs in CYP19A1 as well as in more comprehensive studies investigating larger numbers of SNPs in many genes in both men ( 35, 38, 39 ) and postmenopausal women ( 40 ). Moreover, rs727479 was the second-most significant SNP in the GWAS of E2 levels in postmenopausal women performed by Prescott and colleagues, although it did not reach genome-wide significance (P = 5 3 1027), perhaps because of the relatively low number of study participants ( 7 ). In all of these studies, the direction of the effect was the same as in our study: the A allele was associated with higher E2 levels. The most significant SNP in the male Chinese GWAS performed by Chen and colleagues, rs2414095, is in very strong linkage (r2 = 0.96) with rs727479 ( 6 ); it is also located in intron 3 in LD block 4. The findings from our gene expression analyses that rs727479 is associated with the expression of CYP19A1 in two tissues further support the relevance of this SNP in the regulation of E2 levels. To our knowledge, the CYP19A1 loci rs2899472 and rs16964258 have not been linked to E2 levels in previous studies. Rs2899472 is located in intron 4, in LD block 4. Rs16964258 is located in a different region of the gene; intron 1, between LD blocks 1 and 2. Interestingly, the SNP most significantly associated with estrogen levels in our previous extended candidate gene study, rs2470152 ( 5 ), is also located in this region, 10 kb downstream of rs16964258. The D0 for rs2470152 and rs16964258 is 1.0 but the r2 is 0.062, indicating that the SNPs are probably linked but, because of different allele frequencies, they are not proxy SNPs of one another. The signal in the FAM9B region on the X chromosome, rs5934505, has not been associated with E2 levels before, but associations of this locus with testosterone levels are known from our earlier testosterone GWAS ( 30 ), a finding that was later replicated by Jin and colleagues in a smaller GWAS in men (n = 3225) ( 41 ). Because testosterone is the precursor of E2, it is likely that the association of rs5934505 in the FAM9B region with E2 levels is mediated through the regulation of testosterone production and not through the conversion of testosterone to E2 per se. Rs5934505 is located in a CNVinsertion area (Xp22), 145 kb upstream of FAM9A and 79 kb downstream of FAM9B genes. Both genes are expressed exclusively in the testes and share 46% amino acid identity. Very little is known about their functions ( 42 ). The Kallman syndrome 1 (KAL1) gene is located 214 kb downstream of rs5934505. KAL1 encodes the extracellular matrix glycoprotein anosmin-1 implicated in the embryonic migration of gonadotropin-releasing hormone and olfactory neurons. Deleterious mutations in KAL1 cause X-linked Kallmann syndrome, characterized by hypogonadotropic hypogonadism and anosmia ( 43 ), but there are no previous data supporting that minor alterations in the function of KAL1 are associated with sex steroid levels. Moreover, rs5934505 is correlated (r2 = 0.35) with another SNP, rs5978985, in this region, which was associated with male puberty in a recent GWAS ( 44 ). The other signal on the X chromosome, rs5951794, has not previously been associated with sex steroid levels, and the mechanism underlying the association in our study is not known. In contrast to rs5934505 (FAM9B), rs5951794 was not associated with higher testosterone levels. Therefore, the effect of this SNP would be expected to be exerted through alteration in the amount or activity of the aromatase enzyme or through regulation of E2 clearance. In fact, rs5951794 was associated with slightly lower levels of testosterone. This might be the result of E2-mediated suppression of luteinizing hormone, which in turn would result in decreased testosterone levels. Rs5951794 is located approximately 65 kb downstream of a region rich in micro-RNAs (506 through 510, 513, and 514), expressed mainly in the testes ( 45 ). Aside from the micro-RNA cluster, Fragile X mental retardation 1 is the closest gene located approximately 700 kb downstream of rs5951794. Keeping the distance in mind, one could speculate that rs5951794 could affect the regulation of Fragile X mental retardation 1, a gene that, in addition to its crucial role in the pathogenesis of fragile X syndrome– associated mental retardation, is also the leading molecular cause of premature ovarian failure ( 46 ). The E1 signal rs17277546 in the TRIM4 gene has also been shown to be associated in our previous GWAS of dehydroepiandrosterone sulfate (DHEAS) concentrations ( 47 ). Serum levels of DHEAS and dehydroepiandrosterone are highly collinear ( 48 ). Serum levels of DHEAS could therefore be a marker of serum levels of DHEA. In our earlier GWAS, the G allele was associated with higher levels of DHEAS; in the current study, the G allele was associated with higher levels of E1. Thus, an increased amount of adrenal-derived precursors for estrogen synthesis is a possible explanation for the present findings. TRIM4 is a member of the TRIM family. Members of this family have been implicated in many biological processes, including cell differentiation, apoptosis, and transcriptional regulation ( 49 ). The mechanism relating rs17277546 to DHEAS levels is not known, but in our previous GWAS, we found that rs17277546 is strongly associated with expression levels of TRIM4 in cell lines from liver and adipose tissue in publicly available databases. This indicates that rs17277546 is a functional SNP or is linked to such an SNP ( 47 ). The chromosome 8 signal, rs10093618, is located 1.5 kb upstream of the CYP11B1 gene. The product of CYP11B1, the steroid 11b-hydroxylase enzyme, catalyzes the conversion of 11-deoxycortisol to cortisol, representing the final step in cortisol biosynthesis, and 11-deoxycorticosterone to corticosterone. Deficiency of this enzyme leads to congenital adrenal hyperplasia. Hyperandrogenism is a hallmark of this condition because accumulated precursors are shunted into the androgen synthesis pathway ( 50 ). One could thus speculate that rs10093618, or an unknown variant linked with it, affects the production or efficiency of the steroid 11b-hydroxylase enzyme, and thereby regulates the level of adrenal precursors for the sex steroid synthesis pathway, notably androstenedione, which is a direct precursor in E1 biosynthesis. Because serum E2 levels in men are positively associated with BMD, the SNPs associated with higher E2 levels would be expected to be associated with higher BMD. In fact, in our previous extended candidate gene study, there was such an association between the lead CYP19A1 SNP, rs2470152, and BMD ( 5 ). Thus, the association in the current study between E2-associated SNPs in CYP19A1 as well as FAM9B and BMD is a plausible finding. In fact, rs5934505 is in complete linkage (r2 = 1.0) with rs5934507, which was identified as the only male-specific signal in our previous GWAS of BMD (31). Because of the known association of rs5934505 with testosterone, the BMD signal was thought to be mediated via testosterone levels in the BMD GWAS. Given the findings in the current study of an association between rs5934505 and E2, it seems more likely that the association with BMD is mediated at least in part via E2 levels rather than solely via a direct effect of testosterone (Fig. 3). Although an association between serum E2 levels and BMD in men has been shown in earlier association studies, a causal relation has not been demonstrated. In this study, using Mendelian randomization analysis, we provide evidence that there is a causal effect of E2 on BMD. For instance, in the RS1 cohort, where the E2 levels were 12.7 pg/mL (SD, 6.6), 1 SD of genetically instrumented decrease in E2 would result in a 6.6 3 0.048 = 0.32 SD decrease in LS BMD and 6.6 3 0.037 = 0.24 SD decrease in FN BMD. According to Johnell and coworkers, the relative risk for hip fracture in men aged 65 years was 2.94 (95% confidence interval, 2.02 to 4.27) for each SD decrease in FN BMD ( 51 ). Using this information of the association between FN-BMD and hip fracture risk together with the causal effect of serum E2 on FN-BMD as estimated in the present Mendelian randomization analysis, 1 SD (using the SD of serum E2 from the RS1 cohort) decrease in genetically instrumented E2 level could increase the relative risk for hip fracture by 47%. In this study, SNPs in CYP19A1 that were associated with higher E2 levels were also associated with improved insulin sensitivity and lower fasting insulin in men and women combined. In men, the role of estrogens in the regulation of insulin sensitivity is not fully understood. However, mechanistic studies and clinical trials suggest that estrogen signaling is important in the regulation of insulin sensitivity in men ( 18, 20, 22, 23 ). Furthermore, men with aromatase deficiency resulting from an inactivating mutation of the CYP19A1 gene are overweight or obese, and display and insulin resistance, which often improves with estrogen replacement therapy (17). The strengths of our study include the large sample size, with 11,097 men in the discovery analysis of E2 levels, and the large proportion of serum samples analyzed using the MS technique. This enabled us to find multiple signals in the CYP19A1 locus and signals on other chromosomes for both E1 and E2. A potential weakness of our study is that not all samples were analyzed by MS. As a result of the lower specificity of the immunoassays, weaker genetic signals might have been missed. It is likely that future studies with even larger numbers of samples analyzed by MS could uncover signals not found in this study. Nevertheless, we believe that, because of the large proportion of samples analyzed by MS, our findings are robust and the risk for falsepositive signals is low. We also found SNP associations with BMD and measures of insulin sensitivity. Additionally, the Mendelian randomization analysis provides evidence of a causal effect of E2 on BMD in men. The mechanisms underlying some of the associations in our study should be further investigated to expand our understanding of the regulation of sex steroid levels. Acknowledgments The authors thank the Meta-Analysis of Glucose and InsulinRelated Traits Consortium investigators for providing us with data on fasting insulin and fasting glucose for men and women separately. Financial Support: This work was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, the ALF/LUA (Avtal om L a¨karutbildning och Forskning/L a¨karutbildningsavtalet) research grant in Gothenburg, the Lundberg Foundation, Knut and Alice Wallenberg Foundation, the Torsten and Ragnar S o¨derberg’s Foundation, and the Novo Nordisk Foundation. Correspondence and Reprint Requests: Claes Ohlsson, MD, PhD, Centre for Bone and Arthritis Research, Klin Farm Laboratory, Vita Str a˚ket 11, Department of Internal Medicine and Clinical Nutrition, Sahlgrenska University Hospital, SE41345 Gothenburg, Sweden. E-mail: . Disclosure Summary: D.V. is a consultant for Consumable Science Inc. I.T.H. received grants from Ferring Pharmaceutical and did consultation for Novartis and Takeda. W.M. is employed by Synlab Holding Deutschland GmbH. The other authors have nothing to disclose. Eriksson et al 1. Payne AH , Hales DB . 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Eriksson, Anna L, Perry, John R B, Coviello, Andrea D, Delgado, Graciela E, Ferrucci, Luigi, Hoffman, Andrew R, Huhtaniemi, Ilpo T, Ikram, M Arfan, Karlsson, Magnus K, Kleber, Marcus E, Laughlin, Gail A, Liu, Yongmei, Lorentzon, Mattias, Lunetta, Kathryn L, Mellström, Dan, Murabito, Joanne M, Murray, Anna, Nethander, Maria, Nielson, Carrie M, Prokopenko, Inga, Pye, Stephen R, Raffel, Leslie J, Rivadeneira, Fernando, Srikanth, Priya, Stolk, Lisette, Teumer, Alexander, Travison, Thomas G, Uitterlinden, André G, Vaidya, Dhananjay, Vanderschueren, Dirk, Zmuda, Joseph M, März, Winfried, Orwoll, Eric S, Ouyang, Pamela, Vandenput, Liesbeth, Wu, Frederick C W, de Jong, Frank H, Bhasin, Shalender, Kiel, Douglas P, Ohlsson, Claes. Genetic Determinants of Circulating Estrogen Levels and Evidence of a Causal Effect of Estradiol on Bone Density in Men, The Journal of Clinical Endocrinology & Metabolism, 2018, 991-1004, DOI: 10.1210/jc.2017-02060