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

Abstract 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 × 10−8) and Xq27.3, rs5951794 (P = 3.1 × 10−10). E1 signals were found in CYP19A1 (rs2899472, P = 5.5 × 10−23), in Tripartite motif containing 4 (TRIM4; rs17277546, P = 5.8 × 10−14), and CYP11B1/B2 (rs10093796, P = 1.2 × 10−8). 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 × 10−12). 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.

(A) rs727479, E2 adjusted for age and BMI, (B) rs5934505, E2 adjusted for age and BMI, (C) rs727479, rs2899472 and rs16964258, E2 adjusted for age, BMI, testosterone and SHBG, (D) rs5951794, E2 adjusted for age, BMI, testosterone and SHBG, (E) rs2899472 and rs727479, E1 adjusted for age and BMI, (F) rs17277546, E1 adjusted for age and BMI, (G) rs10093796, E1 adjusted for age and BMI. Location is given according to Human NCBI36/hg18. Independent signals are indicated by purple diamond to evaluate linkage with other single-nucleotide polymorphisms in the region. The r 2 is based on the CEU HapMap II samples. The blue line and right hand Y axis represent CEU HapMap II based recombination rates. The location of secondary signals is marked with arrows in figures C (rs2899472 and rs16964258) and E (rs727479). (A) rs727479, E2 adjusted for age and BMI, (B) rs5934505, E2 adjusted for age and BMI, (C) rs727479, E2 adjusted for age, BMI, testosterone and SHBG, (D) rs5951794, E2 adjusted for age, BMI, testosterone and SHBG, (E) rs2899472, E2 adjusted for age, BMI, testosterone and SHBG, (F) rs16964258, E2 adjusted for age, BMI, testosterone and SHBG, (G) rs2899472, E1 adjusted for age and BMI, (H) rs727479, E1 adjusted for age and BMI, (I) rs17277546, E1 adjusted for age and BMI, (J) rs10093796, E1 adjusted for age and BMI. Beta given as pg/ml E2/allele (A-F), or pg/ml E1/allele (G-J). Vertically left, the populations included in the GWAS. The boxes represent precision, and horizontal lines represent the confidence intervals. The diamond represents the pooled effect estimate from the meta-analysis of all cohorts. The horizontal axis shows the scale of the effects.  Supplemental Table 2 Additional genotyping information for the 10 cohorts included in the genome-wide association study meta-analysis  Table 3 Characteristics of 12,774 men from 10 cohorts included in the genome-wide association study meta-analysis

Estrone and Estradiol assays
Blood samples were drawn in the supine position, typically between 07:30 AM and 09:30 AM after an overnight fast. Sera were aliquoted and immediately stored at -80º C, remaining frozen until the time of assay. Serum estradiol and estrone levels were measured simultaneously using LC-MS/MS after derivatization with dansyl chloride. The limit of quantitation for both hormones was 2 pg/mL. Interassay CVs for estrone were 4.5%, 7.7%, and 6.9% at estrone concentrations of 8, 77, and 209 pg/mL, respectively, and for estradiol 6.9%, 7.0%, and 4.8% at estradiol concentrations of 8, 77, and 206 pg/ml, respectively (5, 6).

Testosterone assay
Serum testosterone levels were measured by liquid chromatography tandem mass-spectrometry (LC-MS/MS) as previously described. The functional sensitivity of the testosterone assay was 2 ng/dl and the interassay coefficient of variation was 15.8%, at 12.0 ng/dL, 10.6%, at 23.5 ng/dL, 7.9%, at 48.6 ng/dL, 7.7% at 241 ng/dL, 4.4% at 532 ng/dL, and 3.3% at 1016 ng/dL respectively. As part of the Centers for Disease Control's (CDC) Testosterone Assay Harmonization Initiative, quality control samples provided by the CDC were run every three months; the bias in quality control samples with testosterone concentrations in 100 to 1000 ng/dL range was consistently less than 6% (6, 7) SHBG assay SHBG was measured using a two-site directed immunofluorometric assay that had a sensitivity of 0.5 nM (Delphia-Wallac, Inc., Turku, Finland) (7).

Height and weight
Height was measured without shoes using a stadiometer and was rounded to the nearest one quarter inch. Weight was measured with a calibrated balance beam scale without clothes.

Exclusions
Chemical or surgical castration and/or medications affecting sex hormones such as steroid 5alpha reductase inhibitors, and sex hormone antagonists.

Disclosures
No disclosures

Gothenburg Osteoporosis and Obesity Determinants (GOOD) study
The Gothenburg Osteoporosis and Obesity Determinants (GOOD) study was initiated to determine both environmental and genetic factors involved in the regulation of bone and fat mass. Male study subjects from the greater Gothenburg area in Sweden were randomly selected from national population registers, contacted by telephone, and invited to participate. To be enrolled in the GOOD study, subjects had to be between 18 and 20 years of age. There were no other exclusion criteria, and 49% of the study candidates agreed to participate (n = 1068). The study was approved by the ethics committee at the University of Gothenburg. Written and oral informed consent was obtained from all study participants (8).

Height and weight
Height and weight were measured using standardized equipment. The CV values were <1% for these measurements.

Exclusions
Chemical or surgical castration and/or medications affecting sex hormones such as steroid 5alpha reductase inhibitors, and sex hormone antagonists.

Disclosures
No disclosures.

InCHIANTI study
The InCHIANTI study is a population-based epidemiological study aimed at evaluating factors that influence mobility in the older population living in the Chianti region of Tuscany, Italy. Details of the study have been previously reported (10,11). Briefly, 1616 residents were selected from the population registry of Greve in Chianti (a rural area; 11,709 residents with 19.3% of the population greater than 65 years of age) and Bagno a Ripoli (Antella village near Florence; 4,704 inhabitants, with 20.3% greater than 65 years of age). The participation rate was 90% (n= 1,453) and participants ranged between 21-102 years of age. The study protocol was approved by the Italian National Institute of Research and Care of Aging Institutional Review.

Estradiol assay
Total E2 was measured in the Laboratory of the University of Parma using ultrasensitive RIA (DSL-4800, Chematil, Angri, Italy) with a minimum detectable concentration (MDC) of 2.2 pg/ml and intra-and interassay coefficients of variation (CVs) of 8 and 10%, respectively.

Height and weight
Height and weight were measured using standardized equipment. The CV values were <1% for these measurements.

Exclusions
Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5-alpha reductase inhibitors, and sex hormone antagonists. Nine individuals were excluded due to extreme E2 values (> 4 SDs).

The Ludwigshafen Risk and Cardiovascular Health (LURIC)
The Ludwigshafen Risk and Cardiovascular Health (LURIC) study is a prospective study of more than 3,300 individuals of German ancestry in whom cardiovascular and metabolic phenotypes (CAD, MI, dyslipidemia, hypertension, metabolic syndrome and diabetes mellitus) have been defined or ruled out using standardized methodologies in all study participants. Inclusion criteria for LURIC were: German ancestry (limitation of genetic heterogeneity), clinical stability (except for acute coronary syndromes) and availability of a coronary angiogram. Exclusion criteria were: any acute illness other than acute coronary syndromes, any chronic disease where non-cardiac disease predominated and a history of malignancy within the last five years. Genome-wide analyses using the Affymetrix 6.0 have been completed in all participants. A 10-year clinical follow-up for total and cause specific mortality has been completed. The study was approved by the ethics committee at the "Landesärztekammer Rheinland-Pfalz" and was conducted in accordance with the "Declaration of Helsinki". Informed written consent was obtained from all participants.
Testosterone assay Testosterone was measured in serum using a solid-phase chemoluminescence enzyme immunoassay (Testosterone Immulite; DPC Biermann GmbH, Bad Nauheim, Germany) with an intra-and interassay coefficient of variation (CV) of 7.2 and 9.1%, respectively.
SHBG assay SHBG was measured by a luminescence immunoassay (Roche, Basel, Switzerland) with an intra-and interassay CV of 1.3 and 2.1%, respectively.

Height and weight
Height and weight were measured using standardized equipment.

Exclusions
Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5-alpha reductase inhibitors, and sex hormone antagonists.

Disclosures
Winfried März is employed with Synlab Holding Deutschland GmbH.

Multi-Ethnic Study of Atherosclerosis (MESA)
The Multi-Ethnic Study of Atherosclerosis is a 6-field center population-based study that enrolled 6,814 men and women age 45 to 85 years, without clinical cardiovascular disease from six United States communities (Baltimore, MD; Chicago, IL; Forsyth County, NC; Los Angeles County, CA; northern Manhattan, NY; and St. Paul, MN). The principal aim of MESA is to investigate subclinical cardiovascular disease and its progression. Sampling and recruitment procedures have been previously described in detail (reference below).
Adults with symptoms or history of medical or surgical treatment for cardiovascular disease were excluded. During the recruitment process, potential participants were asked about their race/ethnicity. Self-reported ethnicity was used to classify participants into groups. This analysis included only men of European descent (12).

Estradiol assay
Estradiol was measured using an ultra-sensitive radioimmunoassay kit from Diagnostic System Laboratories (Webster, TX) at the University of Massachusetts Medical Center, Worcester, MA (limit of quantification 2.2 pg/mL, CV 10.5%) (13).

Testosterone assay
Testosterone was measured using a radioimmune assay at the University of Massachusetts Medical Center, Worcester, MA (Limit of Quantification: not provided by the laboratory, CV 12.3%).

SHBG assay
Sex hormone binding globulin (SHBG) was measured by chemiluminescent enzyme immunometric assay using Immulite kits (Diagnostic Products Corporation, Los Angeles, CA) at the University of Massachusetts Medical Center, Worcester, MA (Limit of Quantification: not provided by the laboratory, CV 9%).

Height and weight
Height and weight were measured using standardized equipment.

Exclusions
Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5-alpha reductase inhibitors, and sex hormone antagonists; symptoms or history of medical or surgical treatment for cardiovascular disease; and non-availability of fasting blood sample.

Disclosures
Dhananjay Vaidya is a consultant for Consumable Science Inc.

MrOS Sweden -Gothenburg and Malmö
The Osteoporotic Fractures in Men (MrOS) study is a prospective multicenter study including older men in Sweden (3014), Hong Kong (~2000), and the United States (~6000). This study comprises a subsample of men included in the Gothenburg (n=1010) and Malmö (n= 1005) parts of the Swedish cohort (14,15). At baseline, letters were sent to a randomly selected group of subjects (men aged 69 to 81 years old) identified in national population registers and contacted by telephone. To be eligible for the study, the participants had to be able to walk without aid, sign an informed consent, and complete a questionnaire. The inclusion rate at baseline for the Swedish part of the MrOS study was 45% (16). Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5alpha reductase inhibitors, and sex hormone antagonists. The study was approved by the ethics committee at the University of Gothenburg and Lund. Written and oral informed consent was obtained from all study participants.

Height and weight
Height was measured using a Harpenden stadiometer, and weight was measured by a standard balance beam or an electric scale. Two consecutive measurements of height were performed in the same session, and the average of these measurements was calculated. If there was a difference of ≥5 mm between the first two measurements, a third measurement was performed, and the average of the two values with the least mutual discrepancy was calculated.

Exclusions
Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5-alpha reductase inhibitors, and sex hormone antagonists.

Disclosures
No disclosures.

The Osteoporotic Fractures in Men (MrOS) study/US
The MrOS study enrolled 5994 participants between March 2000 and April 2002. Details of the MrOS study design and recruitment have been published elsewhere (17,18). In brief, recruitment occurred at six US clinical centers (Birmingham, AL; Minneapolis, MN; Palo Alto, CA; Pittsburgh, PA; Portland, OR; and San Diego, CA) and was accomplished primarily through mass mailings targeted to age-eligible men. Eligible participants were communitydwelling men who were at least 65 years of age, able to walk without assistance from another person, and had not had bilateral hip replacements. Written informed consent was obtained from all participants, and the Institutional Review Board at each study site approved the study.

Estrone, Estradiol and Testosterone assays
The assay method used to determine total serum testosterone, estradiol and estrone was a gas chromatograph/mass spectrometry (GCMS) assay (Taylor Technology, Princeton NJ). A combined gas chromatographic negative ionization tandem mass spectrometry (GC/NCI/MS/MS) and liquid chromatographic electrospray tandem mass spectrometry (LC/ESI/MS/MS) bioanalytical method was used to measure steroids in serum. Briefly, the analytes and their deuterated internal standards were extracted from 1.00 mL of human serum using BondElut Certify solid phase cartridges. Estradiol, estrone, and testosterone were eluted from the solid phase cartridges with ethyl acetate. The analytes underwent three separate derivatization steps: (1) reaction with pentafluorobenzoyl chloride, (2) reaction with O (2,3,4,5,6 pentafluorobenzyl) hydroxylamine hydrochloride, and (3) reaction with N-Methyl-N-(trimethylsilyl)trifluoroacetamide. Then the derivatized analytes were separated by gas chromatography using a DB 17 fused silica capillary column and detected by tandem mass spectrometry using negative ion chemical ionization. A 1/(concentration) 2 weighted least squares regression procedure was used to fit a linear function to the calibration data. The limits of detection are: for estradiol, for estrone, and for testosterone.

SHBG assay
The assay method used to determine SHBG concentration used an Immulite Analyzer with chemiluminescent substrate, (Diagnostic Products Corp., Los Angeles CA). Limit of detection was 0.2 nM, intraassay CV 4.6% and interassay CV 5.8%.

Height and weight
Height and weight were measured using standardized equipment.

Exclusions
Subjects were excluded if they reported having had surgical castration or reported antiandrogen or androgen therapy.

Rotterdam Study (RS)
From 1991 to 1995 all inhabitants of Ommoord, a district of Rotterdam, The Netherlands, who were 55 years or older, were invited to participate in the Rotterdam study (RS). Genotyping information was available for 5,974 participants. All of the participants were followed for incident diseases through linkage to the general practitioner data base and record review by trained medical investigators. General practitioners', hospital records as well as death certificates were used for identification of deaths and health events through 01.01.2009 (19).

Hormone measurements
Serum levels of testosterone, E1, E2 and sex hormone-binding globulin (SHBG) were estimated in 12 batches by coated-tube or double-antibody radioimmunoassays, purchased from Diagnostic Systems Laboratories (Webster, TX, USA). For E2 estimations, the ultrasensitive system was used. The results of these assays were compared with the results obtained with other commercial immunoassays, which in turn had been validated by comparison with in-house immunoassays, making use of steroid extraction and purification by column chromatography (testosterone (20), E2 (21) and E1 (22)). The same procedure was used for SHBG, where the in-house method used ammonium sulphate precipitation (23). Correlation coefficients varied from 0.925 to 0.980. The sensitivities of the assays, defined as the value representing the blank plus twice the standard deviation of the blank, were 4.8 pmol/l for E2 (24), 0.28 nmol/l for testosterone and 5 nmol/l for SHBG. Because of the relatively small volumes of serum available, all values reported are single-sample estimations. Intra-assay coefficients of variation, determined on the basis of duplicate results of internal quality control (QC) serum pools with three different levels of each analyte, were below 15% for all assays, with the exception of E2 (18%) and E1 (21%). Since inter-assay variations were relatively large (20-30%, with the exception of testosterone (19%) and SHBG (14%)), the results of all batches were normalized by multiplying all concentrations within a batch by a factor, a method which equalized results for the internal quality-control pools. This was considered justified because the results of these pools and the mean results for male and female sera in each assay batch showed very similar patterns (25).

Height and weight
Height and weight were measured using standardized equipment.

Exclusions
Exclusion criteria included chemical or surgical castration and/or medications affecting sex hormones such as steroid 5-alpha reductase inhibitors, and sex hormone antagonists.

European Male Ageing Study (EMAS)
The EMAS is a prospective, population-based study of ageing in middle aged and elderly European men. Men were recruited from population based sampling frames in 8 centres: Florence (Italy), Leuven (Belgium), Lodz (Poland), Malmo (Sweden) Manchester (UK) Santiago del Compostella (Spain), Szeged (Hungary), Tartu (Estonia). Details regarding recruitment, response rates and assessments have been described previously (26). Stratified random sampling was performed in each centre with the aim of recruiting one hundred men in each of four 10-year age bands: 40-49 years, 50-59 years, 60-69 years, and 70-79 years. Subjects were invited by letter to attend for a clinical assessment. A single fasting morning (before 10 a.m.) venous blood sample was obtained from all subjects. Serum was separated immediately after phlebotomy and stored at -80°C until assay at the end of the baseline study. Ethical approval for the study was obtained in accordance with local institutional requirements in each centre (26).

Estrone and Estradiol assays
Measurement of E2 was carried out by gas chromatography mass spectrometry (GC-MS) as described in Labrie et al (27,28). The lower limit of E2 quantitation was 7.34 pmol/L. The coefficients of variation of E2 measurements were 3.5% within runs and 3.7% between runs.
Testosterone assay Measurement of T was carried out by GC-MS as described in Labrie et al. (27,28). The lower limit of T quantitation was 0.17 nmol/L. The coefficients of variation of T measurements were 2.9% within runs and 3.4% between runs.
SHBG assay SHBG was measured by the Modular E170 platform electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). Within-and between-assay coefficients of variation were 1.70 and 3.18%, respectively. The detection limit was 0.35 nmol/L.

Height and weight
Height and weight were measured in a standardized manner in the standing position.

Exclusions
From a total of 3,369 participants, men with missing genotype or phenotype data were excluded, giving 1,641 men in the analysis sample.

Disclosures
Ilpo T Huhtaniemi received grants from Ferring Pharmaceutical, and did consultation for Novartis and Takeda

GOOD
Financial support was received from the Swedish Research Council, the Swedish Foundation for Strategic Research, the ALF/LUA research grant in Gothenburg, the Lundberg Foundation, the Torsten and Ragnar Söderberg's Foundation, the Novo Nordisk Foundation, and the European Commission grant HEALTH-F2-2008-201865-GEFOS.

LURIC
We extend our appreciation to the participants of the LURIC study and thank the LURIC study team who were either temporarily or permanently involved in patient recruitment as well as sample and data handling, in addition to the laboratory staff at the Ludwigshafen General Hospital and the Universities of Freiburg and Ulm, Germany. LURIC was supported by the 7th Framework Program (AtheroRemo, grant agreement number 201668 and RiskyCAD, grant agreement number 305739) of the EU and by the INTERREG-IV-Oberrhein-Program (Project A28, Genetic mechanisms of cardiovascular diseases) with support from the European Regional Development Fund (ERDF) and the Wissenschaftsoffensive TMO.