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Circulating Testosterone and SHBG Concentrations

Are Heritable in Women: The Framingham Heart

Study

A. D. Coviello, W. V. Zhuang, K. L. Lunetta, S. Bhasin, J. Ulloor, A. Zhang,D. Karasik, D. P. Kiel, R. S. Vasan, and J. M. Murabito

Sections of Preventive Medicine and Epidemiology (A.D.C., R.S.V.), Endocrinology, Diabetes, and

Nutrition (A.D.C., S.B., J.U., A.Z.), and General Internal Medicine (J.M.M.), Department of Medicine,

Boston University School of Medicine, and Department of Biostatistics (W.V.Z., K.L.L.), Boston University

School of Public Health, Boston, Massachusetts 02118; Institute for Aging Research (D.K., D.P.K.),

Hebrew SeniorLife, Harvard Medical School, Boston, Massachusetts 02131; and Framingham Heart

Study (K.L.L., R.S.V., J.M.M.), Framingham, Massachusetts 01702

Context: Many factors influence the concentration of circulating testosterone and its primary

bindingprotein, SHBG. However, little is known about the genetic contributionto their circulating

concentrations in women, and their heritability in women is not well established.

Objective: Our objective was to estimate the heritability of circulating total testosterone (TT), free

testosterone (FT), and SHBG in women in families from the Framingham Heart Study.

Methods:Women in the Framingham Heart Study who were not pregnant, had not undergone

bilateral oophorectomy, and were not using exogenous hormones were eligible for this investigation.

TT was measured using liquid chromatography tandem mass spectrometry and SHBG using an immu-

nofluorometric assay (Delfia-Wallac), and FT was calculated. Heritability estimates were calculated

using variance-components methods in Sequential Oligogenic Linkage Analysis Routines (SOLAR) and

were adjusted for age, age2, body mass index (BMI), BMI2, diabetes, smoking, and menopausal status.

Bivariate analyses were done to assess genetic correlation between TT, FT, and SHBG.

Results:A total of 2685 women were studied including 868 sister pairs and 688 mother-daughter

pairs. Multivariable adjusted heritability estimates were 0.26 0.05 for FT, 0.26 0.05 for TT, and

0.56 0.05 for SHBG (P 1.0 107 for all). TT was genetically correlated with SHBG [genetic

correlation coefficient (G) 0.31 0.10] and FT (G 0.54 0.09), whereas SHBG was inversely

correlated with FT (G 0.60 0.08).

Conclusion: Circulating TT, FT, and SHBG concentrations in women are significantly heritable,

underscoring the importance of further work to identify the specific genes that contribute sig-

nificantly to variation in sex steroid concentrations in women. The strong shared genetic compo-

nent among pairs of TT, FT, and SHBG concentrations suggests potential pleiotropic effects for

some of the underlying genes.(J Clin Endocrinol Metab96: E1491E1495, 2011)

Sex steroids areessential for normal sexual differentiationandreproductive healthacrossthelifespan.Sexsteroidsinfluence many age-related chronic diseases in women in-

cluding osteoporosis (1), metabolic syndrome (MetS) (2, 3),

type 2 diabetes (T2DM) (2, 4, 5),and cardiovascular disease

(CVD) (6, 7). The relationship between sex steroids and

chronic disease is affected by changes in hormone profiles in

women, particularly with menopause.

ISSN Print 0021-972X ISSN Online 1945-7197

Printed in U.S.A.

Copyright 2011 by The Endocrine Society

doi: 10.1210/jc.2011-0050 Received January 10, 2011. Accepted June 17, 2011.

First Published Online July 13, 2011

Abbreviations:BMI,Body mass index; CVD,cardiovascular disease;FHS,FraminghamHeart

Study; MetS, metabolic syndrome; T2DM, type 2 diabetes.

J C E M O N L I N E

B r i e f R e p o r t E n d o c r i n e R e s e a r c h

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Testosterone and its primary binding protein, SHBG,

mediate sex-hormone-sensitive phenotypes in women.

Higher circulating testosterone has been associated with

incident T2DM (4) and CVD (8). Lower SHBG has been

associated with MetS and T2DM (4, 9, 10), whereas

higher SHBG has been associated with greater risk for hip

fracture (1).Although circulating testosterone and SHBG are af-

fected by many environmental factors including obesity,

smoking, insulin resistance, and T2DM (11), they are be-

lieved to be at least partially heritable. In men, heritability

estimates range from 2575% for testosterone and 30

50% for SHBG (1214). In contrast, little is known about

the genetic influences of circulating sex hormones in

women. One study estimated the heritability of total tes-

tosterone at 39% and SHBG at 56% in postmenopausal

sisters and twins (15). The purpose of this investigation

was to estimate theheritabilityof circulating total and freetestosterone and SHBG in adult women in families from

the Framingham Heart Study (FHS), a multigenerational

population-based study.

Subjects and Methods

Study populationTheFHS designand participants have been describedin detail

previously (16). In brief, the FHS recruited a population-basedsampleof menand women residingin Framingham, MA,in 1948

(n 5209) with the purpose of prospectively studying CVD. In1971, children of the original cohort and their spouses wererecruited as a second generation (the Offspring Study). In 2002,children of the Offspring cohort were recruited as a third gen-eration. Women from Generation 2 and Generation 3 aged 19and above with sex steroid hormone measurements were eligiblefor this study. Women taking estrogens, progestins, and andro-gens or who were pregnant or had undergone bilateral oopho-rectomy wereexcluded.All participants signed informed consentbefore participation in the FHS. The study was approved by theinstitutional review board at Boston University Medical Center.

All blood samples were collected in the morning, usually be-tween 0730 and 0930 h after participants fasted approximately

10 h overnight. Samples were aliquoted, frozen, and stored at80 C. Samples used for testosterone and SHBG measurementswere not thawed previously.

Assay measurementsTotal testosterone was measured by liquid chromatography

tandem mass-spectrometry (17). Mass spectrometry was per-formed using TSQ Thermo-Finnigan Quantum Ultra (ThermoFisher Scientific, Waltham, MA). The functional limit of detec-tion, defined as the lowest concentration detected with less than20% coefficient of variation, was 2 ng/dl. Cross-reactivity withother steroids including dehydroepiandrosterone/dehydroepi-androsterone sulfate, androstenedione, and estradiol was negli-

gible. SHBG was measured with a two-site directed immuno-fluorometric assay with sensitivity of 0.5 nM and less than 0.1%

cross-reactivity with other circulating proteins (Delfia-Wallac,Inc., Turku, Finland). Free testosterone was calculated fromtotal testosterone and SHBG using the law of mass actionequations (18).

Statistical analysisSerum hormones were transformed by rank normalization to

minimize skew. Covariates considered in the analysis includedage, age2, body mass index (BMI), BMI2, T2DM, smoking, andmenopausal status. Covariates were defined as follows: T2DM,fasting blood sugar of at least 126 mg/dl or use of diabetes med-ications; current smoking, yes/no; andpostmenopause, at least 1yr without menses. SAS version 9.1 (SAS Inc., Cary, NC) wasused. SOLAR (Sequential Oligogenic Linkage Analysis Rou-tines) statistical software was used to estimate heritability (19).All family relationships including first-degree relatives (mother-daughter, sister-sister pairs) and extended family relationships(aunt-niece, cousins) were analyzed. Heritability estimates wereadjusted for the same covariates across all models. Heritabilityestimates for free and total testosterone were also adjusted for

SHBG to assess for genetic influences independent of SHBG.Bivariate analyses were used to examine the genetic correlationbetween circulating testosterone and SHBG and were adjustedfor the above covariates.

Results

The 2685 women studied included first-degree relatives

(868 sister-sister, 688 mother-daughter pairs) and ex-

tended family relationships (Table 1). The older, predom-

inantly postmenopausal Generation 2 women had higher

BMI (28 6vs. 26 6 kg/m2) and more T2DM (10vs.2%) than the younger, predominantly premenopausal

Generation 3. SHBG was higher in Generation 3, whereas

free testosterone was slightly higher in Generation 2.

Heritability estimates (Tables 2 and 3)

Total and free testosterone as well as SHBG showed

strong heritability after adjusting for age, BMI, T2DM,

current smoking, and menopausal status (Table 2). Both

totaland free testosterone showedheritability estimates of

0.26 (SE 0.05). SHBG heritability was estimated at 0.56

TABLE 1. Characteristics of women from the Offspringand Generation 3 cohorts of the FHS

OffspringGeneration 2 Generation 3

Sample (n) 1071 1614Age (yr) 62 10 41 8BMI (kg/m2) 28 6 26 6Postmenopause (%) 80 10Smoker (%) 12 16Diabetes (%) 10 2Total testosterone (ng/dl) 32.1 21.8 27.9 15.3

Free testosterone (pg/ml) 3.6 2.4 2.9 1.9SHBG (nmol/liter) 74.2 38.8 87.2 46.6

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(SE 0.05). Heritability estimates of circulating free and

total testosterone remained significant after adjustment

for SHBG: free testosterone heritability was 0.20 (SE

0.05) and total testosterone heritability was 0.25 (SE

0.05).

Bivariate analyses (Table 3) showed significant genetic

correlation between total testosterone and SHBG [genetic

correlation coefficient (G) 0.31; SE 0.10;P 3.32

103] after adjustment for age, BMI, T2DM, current

smoking, and menopausal status. Total and free testos-

terone were also highly genetically correlated (G 0.54;

SE 0.09;P 1.69 103) as would be expected becausefreetestosterone, a small fraction of total testosterone con-

centrations, is calculated from total testosterone and

SHBG. SHBG was inversely genetically correlated with

free testosterone (G 0.60;P 1.93 108), sug-

gesting that specific genes may have opposite effects on

theircirculating concentrations.Estimates of environmen-

tal influences on circulating SHBG and free testosterone

were inversely correlated as well (environmental correla-

tion, E 0.37; SE 0.05;P 2.23 108), suggesting

that environmental influences may affect SHBG and free

testosterone in opposite directions.

The derived phenotypiccorrelation, P,betweenSHBG

and free testosterone is 0.44 (Table 3), which is consis-

tent with the correlation of the two hormones circulating

concentrations (Pearson correlation 0.41, P

0.0001).

Discussion

We have shown that total and free testosterone and SHBG

are moderately to highly heritable (2656%) in white

women of European ancestry in the FHS. Furthermore,circulating total testosterone and SHBG are genetically

correlated, suggesting that the two are influenced by com-

mon genes or that genetic influences on SHBG may indi-

rectly influence total testosterone given that SHBG is tes-

tosteronesprimary binding protein. Free testosterone and

SHBG are inversely genetically correlated, suggesting that

genes may influence them in opposite directions, which is

consistent with the inverse correlation of their circulating

concentrations. Given the importance of testosterone and

SHBG for both reproductive and nonreproductive health

in women, further analyses of the genetic influences ofcirculating testosterone and SHBG are needed to elucidate

risk factors for the diseases influenced by these hormones,

particularly osteoporosis, MetS, T2DM, and CVD.

Women experience a rapid decline in estrogen produc-

tion at the time of menopause. In contrast, testosterone

declines at a relatively stable rate across the menopause

(20). The menopausal transition is associated with

changes in metabolism and body fat distribution consis-

tent with the MetS. Furthermore, there is an increase in

osteoporosis (1) andCVD (21) after menopause tradition-

ally thought to be due to estrogen deficiency. However,estrogen and estrogen/progestin treatment in clinical trials

have not shown a benefit in CVD risk or mortality and

have suggested possible increased risk for CVD and stroke

(7). The role of testosterone independent of estrogens in

osteoporosis, CVD, and metabolic disorders is not clear.

Some studies (including the FHS) (22) have shown that

higher testosterone levels are associated with increased

prevalence of CVD and T2DM (2, 4). Prospective studies

have found higher testosterone to be associated with in-

creased risk of T2DM (4) and CVD (8). Postmenopausal

women in the WISE (Womens Ischemia Syndrome Eval-uation) study being evaluated for ischemia with a history

TABLE 3. Heritability of total and free testosterone andSHBG in adult women from the FHS Generations 2 and3: bivariate analysis

Totaltestosterone

and SHBG

Totaltestosteroneand free

testosterone

SHBGand free

testosterone

n 2677 2671 2677G (SE) 0.31 (0.10) 0.54 (0.09) 0.60 (0.08)

PvalueHo: G 0 3.32 103 1.69 103 1.93 108

Ho: G 1 3.00 107 1.54 109 1.29 105

E (SE) 0.10 (0.06) 0.85 (0.017) 0.37 (0.053)Pvalue

Ho: E 0 0.14 2.23 108 9.78 1044

P 0.171 0.769 0.440

Covariates included age, age2, BMI, BMI2, menopausal status,

diabetes, and current smoking. E, Environmental correlation;G,genetic correlation; P phenotypic correlation; Ho, null hypothesis.

TABLE 2. Heritability of total and free testosterone andSHBG in adult women from the FHS Generations 2 and3: univariate analysis

Totaltestosterone SHBG

Freetestosterone

n 2671 2677 2671h2 (SE) 0.263 (0.053) 0.556 (0.052) 0.259 (0.052)Pvalue 1.00 107 7.29 1030 3.25 108

SHBG-adjusted

analysisa

n 2671 2671h2 (SE) 0.246 (0.053) 0.203 (0.052)Pvalue 3.00 107 1.24 105

Covariates included age, age2, BMI, BMI2, menopausal status,

diabetes, and current smoking. h2, Heritability estimate.a Total and free testosterone heritability estimates adjusted for

circulating SHBG in addition to age, age2, BMI, BMI2, menopausal

status, diabetes, and current smoking.

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of irregular menses and elevated free testosterone had a

higher burden of angiographic coronary artery disease

and were more likely to have a myocardial infarction or

cardiovascular-related death (23). There are very limited

data on thegenetic influencesof circulating testosteronein

otherwise healthy women.SHBG tightly binds testosterone in women. SHBG pro-

duction and clearance are affected by many factors in-

cluding age, adiposity, smoking, hormone use, liver dis-

ease, insulin resistance, and T2DM, resulting in variable

circulating SHBG levels (11). SHBG was thought to func-

tion only as a carrier protein for sex hormones, but it has

been proposed that SHBG binds to its own receptor and

exerts biological effects as well (24, 25). Lower SHBG has

recently been shown to predict incident T2DM in women

(910). SHBG concentrations are higher in premeno-

pausal women compared with postmenopausal womendue to the stimulation of SHBG production by the liver in

response to higher estrogen levels. Thus, the lower SHBG

levels coincident with lower estrogen levels after meno-

pause may be related to the greater metabolic and cardio-

vascular risk observed after the menopausal transition.

Our adjusted heritability estimate for SHBG of 56%

suggests a significant genetic component and is in the

range estimated in the San Antonio Heart Study in men

and women and extends the observation in the Australian

studyof postmenopausal sisters to premenopausal womenas well (12, 15). Additionally, SHBG had a strong inverse

genetic correlation with free testosterone, suggesting that

the two share common genes that influence their circulat-

ing levels. The candidate gene approach has been used to

identify regions of interest that may influence circulating

hormone levels in conjunction with disease states. A

SHBG single-nucleotide polymorphism, rs179994, has

beenassociated with T2DM (odds ratio 0.94; 95% con-

fidence interval 0.910.97;P 2 105) (10). Further

study of the genetic factors influencing circulating SHBG

levels are warranted to determine whether these genes in-

fluence susceptibility to chronic diseases, particularly

T2DM and CVD.

The use of mass spectrometry, a state of the art method

for measuringtestosteronein thelow concentrations prev-

alent in women, is a significant strength of this study. The

multigenerational design of the FHS is also a significant

strength. Our findings are based on the FHS population,

which is Caucasian of European ancestry. Future studies

of the genetic influences of circulating sex steroid profiles

in women should be conducted in populations of variedracial and ethnic backgrounds.

Summary

Circulating testosterone and SHBG levels are highly

heritable in women of white, European ancestry, suggest-

ing strong genetic influences. Further study to elucidate

the genetic loci that contribute to the determination of

circulating hormone profiles is important given that cir-

culating sex hormone profiles are associated with in-

creased risk for significant chronic disease in women in-

cluding osteoporosis, T2DM, and CVD. The candidate

gene approach is limited by the assumptions underlying

the choice of the genes to be analyzed. Genome-wide as-

sociation scans may be a more fruitful approach for iden-

tifying genetic regions that influence sex hormone and

SHBGlevelsthatarenotencumberedbythebiasofapriori

assumptions about which genes will be important in medi-

ating their synthesis or action. There is evidence of potential

pleiotropic genetic effects with single genes affecting both

testosterone and SHBG. Understanding pleiotropic geneticeffects on circulating hormone concentrations may have im-

portant implications for the development of hormone-tar-

geted therapy for chronic diseases.

Acknowledgments

Address all correspondence and requests for reprints to: AndreaCoviello, M.D., M.Sc., Assistant Professor of Medicine, Sectionof Preventive Medicine and Epidemiology, Section of Endocri-nology and Metabolism, Boston University School of Medicine,

760 Harrison Avenue, Harrison Court B91, Boston, Massachu-setts 02118. E-mail: coviello@bu.edu.

This work was supported by National Institutes of HealthGrants RO1 HL094755 (to A.D.C., S.B., and R.S.V.), RO1AG31206 (to R.S.V. and S.B.), R21 AG032598 (to W.V.Z.,

J.M.M., K.L.L., D.P.K., and D.K.), and RO1 AR/AG41398 (toD.P.K.), andby a Foundation Grant from theCenters forDiseaseControl (to S.B.).

Disclosure Summary: The authors have nothing to disclose.

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