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
Thomas G. Travison
Andr e´ G. Uitterlinden 3 16
Joseph M. Zmuda
Winfried M a¨rz
Eric S. Orwoll
Liesbeth Vandenput 15
Frederick C. W. Wu
Frank H. de Jong 3
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.
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,
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 (
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% (
). 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 (
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 (
). 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) (
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)
). 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 (
Both E2 and testosterone regulate bone mass (
of men with nonfunctional estrogen receptor alpha (
inactivating mutations of the CYP19A1 gene (
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 (
Some studies also show a smaller contribution of
testosterone to BMD in men (
). 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 (
). 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 (
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.
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
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 (
). 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% (
). 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 (
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) (
). 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/ (
Associations with testosterone
Associations with serum testosterone concentrations were
retrieved from the discovery dataset of our previously published
GWAS of testosterone levels (
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 (
). 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.
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
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% (
). 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
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
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.
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
). 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).
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
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).
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 (
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].
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
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 (
). 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 (
). 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
postmenopausal women (
). 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 (
). 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 (
); 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
), 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
), a finding that was later replicated by Jin and
colleagues in a smaller GWAS in men (n = 3225) (
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
). 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
), 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
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 (
). 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 (
The E1 signal rs17277546 in the TRIM4 gene has also
been shown to be associated in our previous GWAS of
dehydroepiandrosterone sulfate (DHEAS)
). Serum levels of DHEAS and
dehydroepiandrosterone are highly collinear (
). 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 (
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 (
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 (
). 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
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 (
). 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 (
). 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
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.
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
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.
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