A Common Promoter Variant in the Cytochrome P450c17α (CYP17) Gene Is Associated with Bioavailable Testosterone Levels and Bone Size in Men
Cytochrome P450c17α (CYP17) encodes an enzyme with 17α-hydroxylase and 17,20-lyase activities, which is essential for the normal production of adrenal and gonadal androgens. Because androgens have powerful effects on bone growth and metabolism, we determined whether a single base pair (bp) substitution (T → C) in the promoter region (−34 bp) of CYP17 is associated with sex hormone levels, stature, and femoral mass and size in 333 white men aged 51-84 years (mean ± SD; 66 ± 7 years). Femoral neck bone mineral content (BMC), cross-sectional area (CSA), and bone mineral density (BMD) were measured using dual-energy X-ray absorptiometry (DXA). Genotype frequencies did not deviate from Hardy-Weinberg expectations. Serum bioavailable testosterone levels were 20% or 0.5 SDs higher in men with the C/C compared with the T/T genotype, whereas heterozygous men had intermediate hormone levels (p = 0.019). Men with the C/C genotype also were nearly 3 cm taller and had 0.6 SD greater femoral neck CSA than men with the T/T genotype (p ≤ 0.01 for both). The association with CSA persisted after adjusting for age, height, and body weight. In contrast, CYP17 genotype was not associated with femoral neck BMC, areal BMD (g/cm2), or estimated volumetric BMD (g/cm3). These results suggest that allelic variation at the CYP17 locus may contribute to the genetic influence on stature and femoral size in men.
MALE OSTEOPOROSIS is an increasingly important health problem(1) for which there are few well-established risk factors.(2, 3) A genetic basis is suggested by the stronger concordance of bone mass, size, and geometry in monozygotic compared with dizygotic twins,(4–7) reduced bone density in sons of osteopenic men,(8–10) and increased risk of vertebral fracture among men with a positive family history of fracture.(11, 12) Allele sharing(13, 14) and candidate-gene association(15) studies are beginning to define the genetic contributions to skeletal size and mass in women, but considerably less is known about the genetic determinants of bone-related traits in men.
Androgens have dramatic effects on the development and maintenance of skeletal size and mass.(16) The genes regulating androgen production, metabolism, and action thus are strong candidate genes for skeletal traits. Cytochrome P450c17α (CYP17) encodes an enzyme with both 17α-hydroxylase and 17,20-lyase activities, which is essential for the normal production of gonadal and adrenal androgens.(17) Patients with loss of function mutations in CYP17 fail to undergo a normal pubertal growth spurt(18, 19) and have reduced statural growth(19) and diffuse osteoporosis,(20, 21) but such mutations have been described only rarely.(22) The 5′ untranslated region of CYP17 also contains a common single nucleotide substitution (T → C) located 34 base pairs (bp) upstream from the translation initiation site.(23) This polymorphism creates a potential Sp1-like (CCACC box) binding site, which is thought to increase CYP17 gene expression and androgen biosynthesis.(23) In the present study, we tested the hypothesis that this T → C substitution in the CYP17 promoter region is associated with circulating testosterone levels and femoral size and mass in a well-defined cohort of community-dwelling men.
MATERIALS AND METHODS
From February 1991 to February 1992, a total of 523 community-dwelling white men attended a clinic examination for the Study of Osteoporotic Risk in Men (STORM), a study of the determinants of bone density in men aged 50 years and older.(24) Participants were recruited primarily from population-based lists of age-eligible voters in the Monongahela Valley (30 miles southeast of Pittsburgh, PA, USA). We excluded black men, because of their high bone density and low incidence of fractures, and men who were unable to walk without assistance of another person, had undergone a bilateral hip replacement, or were being treated for osteoporosis. Of the 523 men who participated in the baseline examination, 333 provided peripheral blood leukocytes for DNA extraction and genotyping at subsequent clinic visits and form the basis of the present analyses. Written informed consent was obtained from all participants and the University of Pittsburgh Institutional Review Board approved the protocol.
High molecular weight genomic DNA was isolated from peripheral lymphocytes harvested from the EDTA anticoagulated whole blood using the salting-out procedure.(25) The presence of the T → C substitution at −34 bp in CYP17 creates a recognition site for the restriction enzyme MspAI.(23) Genotyping was facilitated by polymerase chain reaction (PCR) amplification of a region spanning the MspAI site with oligonucleotide primers (forward, 5′-CATTCGCACTCTGGAGTC-3′; reverse, 5′-AGGCTCTTGGGGTACTTG-3′) in 50 μl of reaction volumes containing 2 μl of DNA, 0.30 μM of each primer, 200 μM of deoxynucleoside triphosphate (dNTP), 1.5 of mM MgCl2, 5 μl of 10× PCR buffer (500 mM of KCl and 200 mM of Tris-HCL), 0.8 U of Taq DNA polymerase, and water to a total volume of 50 μl. After an initial 5-minute denaturation at 94°C, 30 cycles of denaturation (94°C for 30 s), annealing (57°C for 40 s), and elongation (72°C for 90 s) were followed by a final elongation step at 72°C for 5 minutes. The PCR products were digested overnight with 5 U of MspAI (New England Biolabs, Beverly, MA, USA), separated on a 1% agarose gel containing ethidium bromide, and the fragments were visualized by UV illumination. Fragment sizes were estimated by comparison to a 1-kilobase (kb) ladder run on the same gel, and ambiguous genotypes were retyped.
The bone mineral content (BMC; g), cross-sectional area (CSA; cm2), and areal bone mineral density (BMD; g/cm2) of the femoral neck were measured with dual-energy X-ray absorptiometry (DXA) using a Hologic QDR-1000 or QDR-2000 densitometer (Hologic, Bedford, MA, USA). The correlation coefficient for BMD measurements in 10 men scanned on the two densitometers was r = 0.98. Bone mineral apparent density (BMAD) was calculated as an estimate of volumetric bone density as previously described: BMAD (g/cm3) = BMC/area2.(26)
Sex steroid hormones
Blood samples were collected in the morning after an overnight fast and stored at −70°C until first thawed for hormone assays. Samples for sex steroid hormone analysis were available on 313 men and were sent directly from storage to the analytical laboratory (Endocrine Sciences, Calabasas Hills, CA, USA) without thawing. Total testosterone was measured by radioimmunoassay after extraction and purification by column chromatography.(27) Estradiol was measured by radioimmunoassay after extraction and purification by column chromatography.(28) Bioavailable testosterone and bioavailable estradiol were determined by separation of the sex hormone-binding globulin (SHBG) bound steroid from albumin bound and free steroid with ammonium sulfate as described by Mayes and Nugent.(29) Aliquots of serum samples were incubated with either H3-testosterone or H3-estradiol. SHBG was precipitated by the addition of ammonium sulfate and the samples were centrifuged. Aliquots of the supernatant containing the “non-SHBG-bound” steroids were removed for scintillation counting. The bioavailable steroid concentration was then derived from the product of the total serum steroid and the percent non-SHBG-bound steroid determined from the separation procedure. Intra- and interassay CVs, respectively, range from 4.9% to 6.6% for total testosterone, 3.3% to 12.4% for bioavailable testosterone, 5.2% to 7.5% for total estradiol, 4.9% to 6.0% for bioavailable estradiol, and 2.0% to 3.2% for SHBG.
Body weight was measured to the nearest 0.1 kg after removal of shoes and heavy outer clothing on a calibrated balance beam scale. Height was measured to the nearest 0.1 cm after removal of shoes using a wall-mounted Harpenden stadiometer (Holtain, Dyved, UK). The average of two height measurements was used in statistical analysis. Body mass index (BMI; kg/m2) was calculated from height and weight measurements.
Participants also completed a self-administered questionnaire, which was reviewed with the participant in the clinic by a trained interviewer. We asked participants if they engaged in rigorous exercise at least once per week, currently smoked cigarettes, drank alcohol and how much, used calcium supplements, and to report their health status (excellent/good/fair/poor/very poor) and height at the age of 25 years.
Allele frequencies were estimated by gene counting. Hardy-Weinberg equilibrium was tested by a χ2 goodness of fit statistic. Analysis of variance (ANOVA) was used to test for differences in continuous variables by CYP17 genotype. We adjusted the BMC, CSA, and BMD of the femoral neck for age, body weight, and height using analysis of covariance. χ2 statistic was used to test for differences in dichotomous variables. Differences in sex steroid hormone levels by CYP17 genotype were tested for statistical significance using a Kruskal-Wallis test because of their skewed distribution. We also performed multiple regression analysis with backward elimination to determine if CYP17 genotype was independently associated with sex steroid hormone levels. Partial coefficients of determination (r2) were used to estimate the independent contribution of the CYP17 polymorphism to the total variance in transformed (square root) levels of sex steroid hormones. We included age, body weight, height, smoking status, regular exercise, alcohol intake, and self-reported health status in the model to control for their potential confounding effects. Men taking gonadotropin-releasing hormone agonists (n = 1), antiandrogens (n = 2), antiestrogens (n = 1), and 5α-reductase inhibitors (n = 15) were excluded from analyses of sex steroid hormones. Statistical analyses were performed with the Statistical Analysis System (SAS) software (version 6.12; SAS Institute, Inc., Cary, NC, USA).
CYP17T and C allele frequencies were 60.5% and 39.5%, respectively. There were 120 men with the T/T genotype (36.0%), 163 men with the T/C genotype (48.9%), and 50 men with the C/C genotype (15.0%). The observed genotype distribution did not differ significantly from Hardy-Weinberg equilibrium (χ2 = 0.20). The average age and weight of the participants were 66.2 ± 7.3 kg (range, 51-84 years) and 83.5 ± 13.2 kg, respectively, and did not differ significantly by CYP17 genotype (Table 1). In contrast, CYP17 genotype was significantly associated with current height measured in the clinic. We found that men with the CYP17C/C genotype were 2.9 cm or 0.5 SDs taller than men with the T/T genotype, whereas heterozygous men had intermediate stature (p = 0.019; Table 1). We found a similar significant association between CYP17 genotype and self-reported height at the age of 25 years. None of the other characteristics of the participants including their exercise habits, smoking status, use of calcium supplements, alcohol intake, or health status differed significantly by CYP17 genotype.
Table Table 1.. Characteristics of Men by Cytochrome P450c17α (CYP17) Genotype
The mean BMC (4.51 ± 0.76 g), CSA (5.84 ± 0.46 cm2), and areal BMD (0.77 ± 0.13 g/cm2) of the femoral neck in our cohort of older men were comparable with values for older U.S. men in the Third National Health and Nutrition Examination Survey (NHANES III).(30) Men with the CYP17C/C genotype had 4.7% or 0.6 SD greater CSA of the femoral neck compared with men with the T/T genotype, whereas heterozygous men had intermediate values (p = 0.002; Table 1). This association persisted after adjusting for age, body weight, and height (p = 0.014; data not shown). In contrast, femoral neck BMC, areal BMD (g/cm2) and BMAD (g/cm3) were not significantly associated with CYP17 genotype.
Serum bioavailable testosterone (128 ± 46 ng/dl) and bioavailable estradiol (13.9 ± 5.1 pg/ml) levels in our cohort were similar to values reported in other large population studies of older men.(31) Median bioavailable testosterone but not bioavailable estradiol concentrations were significantly associated with CYP17 genotype. We found that men with the CYP17C/C genotype had 20% or 0.5 SD higher bioavailable testosterone levels compared with men with the T/T genotype, whereas heterozygous men had intermediate hormone concentrations (p = 0.019; Table 2). The CYP17 polymorphism explained 1.4% of the total variance in bioavailable testosterone levels in multiple regression analysis (p < 0.05).
Table Table 2.. Serum Hormone Levels (Median, Range) in Older Men According to Cytochrome P450c17α (CYP17) Genotype
Correlations among serum hormone levels and measures of stature and femoral size and mass are shown in Table 3. Associations between serum testosterone and estradiol and current height measured in the clinic and self-reported height at the age of 25 years generally were weak and not statistically significant. Total estradiol was correlated inversely with femoral neck CSA (r = −0.13; p < 0.05), but there was no significant association between serum total or bioavailable estradiol concentrations and the other skeletal measurements. Likewise, serum levels of total and bioavailable testosterone were only weakly and nonsignificantly related to femoral neck mass and size.
Table Table 3.. Correlations Between Serum Hormones and Measures of Stature and Femoral Size and Mass in Older Men
A strong genetic influence on the attainment of peak adult skeletal size is well established,(32) but the genes and allelic variants regulating normal skeletal growth are not well defined. Because androgens have powerful effects on bone growth,(33) we tested whether a single nucleotide polymorphism in the promoter region of cytochrome P450c17α (CYP17), an enzyme essential for androgen production, is associated with hormone levels, stature, and femoral mass and size in men. We found that men who were homozygous for a T → C substitution in the CYP17 promoter have higher endogenous testosterone concentrations and greater adult stature and femoral size compared with men who were homozygous for the wild-type allele. These results raise the possibility that common allelic variation at the CYP17 locus may mediate some of the genetic influence on normal skeletal growth.
Our findings are consistent with the growth-promoting effects of androgens. The most dramatic effects of androgens during growth may be on bone size.(16) The importance of androgens for linear and periosteal appositional bone growth is supported by the presence of androgen receptors in epiphyseal chondrocytes(34, 35) and osteoblasts,(36–38) and by the observations that femoral length and width(39) and periosteal bone formation and apposition rates(40) are significantly decreased in orchiectomized growing male rats. Moreover, testosterone replacement increases epiphyseal growth plate width,(41) long bone growth rates,(42) and periosteal bone formation and appositional growth(43) in these animals. The importance of androgens for longitudinal bone growth is illustrated further by the delayed and reduced growth rate(44) and decreased height(45–47) in boys with constitutional delay of growth and adolescence, a variant of pubertal maturation characterized by an extension of the prepubertal hypogonadal state.(44) Administration of nonaromatizable androgens increases growth velocity in these patients,(48) showing that androgens can influence bone growth independent of their conversion to estradiol.
Most candidate-gene association studies to date have focused on the possible genetic influences on BMD.(15) However, BMD is not the only determinant of skeletal fragility and genetic influences on fracture risk may be mediated through factors other than or in addition to BMD.(49) The overall size and geometry of bone also determine its mechanical strength,(50) and such measures predict the risk of fracture independent of BMD.(51–54) As much as 60-80% of the variance in measures of proximal femur geometry, such as femoral neck CSA(5) and hip axis length,(6, 7) may be caused by genetic differences among individuals. Our results indicate that CYP17 genotype may contribute to the genetic regulation of femoral neck CSA, but not BMC, or BMD. These observations are consistent with the hypothesis that different genes may control the size and BMC of bone.(55) Moreover, these results suggest that the BMC, CSA, and BMD of bone should be considered separately in statistical analyses, otherwise important genetic effects might be overlooked.
Comparisons of monozygotic and dizygotic twin pairs suggest that 85% of the variance in testosterone production rates(56) and 60% of the variance in circulating testosterone levels(57) may be under genetic control, but the allelic variants that mediate these effects are not well defined. Cytochrome P450c17α (CYP17) encodes a bifunctional enzyme essential for the biosynthesis of adrenal and gonadal androgens,(17) and allelic variants of CYP17 may contribute to interindividual differences in androgen production. Indeed, we found that men who were homozygous for the CYP17C allele had 20% higher bioavailable testosterone levels compared with men who were homozygous for the CYP17T allele. We did not measure other androgenic hormones in our study, but other recent reports have found increased levels of the adrenal androgens, androstenedione and dehydroepiandrosterone, among postmenopausal women who were homozygous for CYP17C allele.(58) The CYP17 genotype explained <2% of the total variance in bioavailable testosterone levels in our study, indicating that additional allelic variants at the CYP17 locus or other key steroidogenic loci contribute to endogenous testosterone levels in men.
Bone contains androgen receptors(36–38) and is highly sensitive to androgens.(33) However, population studies have not shown consistently a strong positive association between endogenous androgen concentrations and measures of skeletal size or mass.(33) Interpretation of these studies is difficult because a single hormone measurement may not accurately reflect a man's lifetime exposure to androgenic hormones. For instance, we have found previously only a modest correlation (r = 0.44) in serum testosterone concentrations measured 13 years apart in middle-aged men.(59) In the present study, CYP17 genotype was significantly associated with stature and femoral size, whereas total and bioavailable testosterone levels were only weakly and nonsignificantly associated with skeletal measures. These findings suggest that CYP17 genotype may be a useful marker of long-term androgen exposure. Additional support for this possibility comes from recent studies showing that CYP17 genotype is associated with the risk of prostate cancer.(60, 61)
The 5′ promoter region of CYP17 contains several binding sites for the transcription factor Sp1,(62) which mediates the constitutive and cyclic adenosine monophosphate (cAMP)-dependent expression of CYP17.(63) The T → C substitution at 34 bp upstream from the initiation of translation creates an additional Sp1-like binding motif that initially was thought to increase CYP17 expression.(23) Indeed, deletion or mutation of a single Sp1 site in the CYP17 promoter region decreases constitutive and cAMP-dependent expression of CYP17 in bovine ovary cells.(63) However, recent in vitro experiments failed to show differential binding of recombinant Sp1 protein to DNA sequences containing the two allelic variants of the CYP17 promoter region.(64) Thus, it is unclear if the T → C substitution directly alters CYP17 expression in vivo or is in linkage disequilibrium with other functional sequence variation in the regulatory or coding regions of CYP17. However, to our knowledge, other candidate functional variation in CYP17 has not been reported.
The present study has several potential limitations that should be acknowledged. First, our study sample was restricted to older white men, and results will need to be confirmed in and extended to other populations, including women, children, and other ethnic groups. Second, DXA provides a two-dimensional approximation of a three-dimensional structure of bone, and our findings should be confirmed with three-dimensional imaging techniques such as volumetric quantitative computed tomography.(65) Finally, the clinical relevance of our findings is uncertain. Although CYP17 genotype was associated with differences in femoral dimensions, it is unclear if these differences confer a biomechanical advantage that translates into lower fracture risk. Conclusions on this possibility must await the completion of studies examining the relationship between CYP17 genotype and the incidence of fracture.
In conclusion, the present data suggest that the common T → C substitution in the promoter region of CYP17, or a closely linked allelic variant, may contribute to the genetic influence on stature and femoral size in men.
This study was supported in part by grants from the United States Public Health Service grants AR 35582, DK 46204, and 1P60 AR44811.