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Original Article |
Pennington Biomedical Research Center (O.U., T.R., C.B.), Baton Rouge, Louisiana 70808; Department of Internal Medicine and Biocenter Oulu (O.U.), University of Oulu, FIN-90220, Oulu, Finland; Laboratory of Molecular Endocrinology (J.G.), Centre Hospitalier de l’Université Laval Research Center, Laval University, Québec GIK 7P4, Canada; School of Kinesiology and Leisure Studies (A.S.L.), University of Minnesota, Minneapolis, Minnesota 55455; Department of Kinesiology (J.S.S.), Indiana University, Bloomington, Indiana 46405; Department of Health and Kinesiology (J.H.W.), Texas A&M University, College Station, Texas 77843-4243; and Division of Biostatistics (D.C.R.), Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Claude Bouchard, Ph.D., Executive Director, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, Louisiana 70808-4124. E-mail: . bouchac{at}pbrc.edu
Abstract
To identify loci-harboring genes affecting steroid hormone and SHBG plasma levels, a genomic-wide scan was performed in the HERITAGE Family Study at baseline. The following steroid hormones were assayed: androstane-3
, 17ß-diol glucuronide, androsterone glucuronide, cortisol, dihydrotestosterone, estradiol, 17-hydroxyprogesterone (OH-PROG), progesterone (PROG), pregnenolone ester, and testosterone. A total of 509 markers on the 22 autosomes were genotyped, and a maximum of 357 pairs of siblings from white families and 103 from black families were available for the study. Significant linkages with LOD scores over 3.6 (P < 2.2 x 10-5) for SHBG were observed in blacks on 1q44 (D1S321), 5p13.3 (D5S1986), 10q24.1 (D10S1239), and 12q12 (D12S1653) in both singlepoint and multipoint analyses. Promising evidence of linkage (1.75 < LOD < 3.6; 2.2 x 10-5 < P < 0.0023) for SHBG was observed on 1q44 in singlepoint analysis in whites. In addition, several other loci in blacks exhibited promising evidence of linkage, suggesting that many genes can potentially regulate SHBG levels. In the case of C21 steroids, promising linkages were found on 1q43 (D1S517) for PROG, 2p25.1 (D2S1400) for pregnenolone ester, and 18q21.32 (D18S38) for OH-PROG in whites and on 3q25.33 (D3S1763) for OH-PROG in blacks, both singlepoint and multipoint analyses (P < 0.0023). The strongest signals for C19 steroids were found on 22q12.3 for testosterone in whites (P = 0.0024 in multipoint) and on 8q22.1 for dihydrotestosterone in blacks. In blacks, the strongest evidence of linkage for estradiol (C18 steroid) was provided by marker D1S1588 on 1p21.3 and in whites by markers D2S2374 and D2S2347 on 2p21, and D6S465 on 6p12.3. Several genes encoding enzymes of the steroid biosynthesis pathways but also other potential candidate genes were located in the vicinity of the genomic regions showing evidence of linkage in this genomic scan.
THE MAIN STEROID hormones, C21 steroids (mineralo- and glucocorticoids), C19 steroids (androgens), and C18 steroids (estrogens), are synthesized in the adrenals and peripheral tissue. The steroid hormones directly or indirectly affect many physiological processes (1). Previous studies have suggested that variations in plasma levels of several steroid hormones include significant genetic effects. For instance, heritability estimates vary from about 50% for total cortisol (2) to 76% for estrogens (3). Heritability of androgens is usually lower (3, 4), although their production rates have been suggested to be under strong genetic control (5). The reported heritability levels for SHBG, the major serum carrier of androgens, ranged from below 5% to 62% (3, 4, 6, 7, 8), with the results influenced by the study design and technical issues. The structural gene for SHBG is on the short arm of chromosome 17 (9). However, a recent segregation analysis study based on the HERITAGE Family Study data suggested that SHBG levels were influenced predominantly by a multifactorial component (heritability, 29%) and potentially several interacting loci (10). The genes that influence quantitative variation in serum steroid levels are largely unknown. A genome-wide scan makes it possible to identify chromosomal regions harboring novel genes influencing quantitative phenotypes. In baboons, a quantitative trait locus (QTL) for estrogen levels has been found on a region homologous to human chromosome 20 (11). Significant linkages for dehydroepiandrosterone-fatty acid esters on chromosomes 1, 4, 7, and 12 were observed in the HERITAGE cohort (12). Here, we report the results of genomic scans for other steroids and SHBG using the data of the HERITAGE Family Study at baseline.
Subjects and Methods
Subjects and phenotypes
Subjects. The HERITAGE Family Study is a multicenter clinical trial conducted at five institutions, and it includes both black and white subjects. The specific aims, design, inclusion, and exclusion criteria and methodology of the study have been described in detail elsewhere (13). The age range of the subjects was from 17–65 yr in both races and sexes. Subjects were required to be healthy (i.e. free of diabetes, cardiovascular diseases, or other chronic diseases, although subjects with mild hypertension were allowed in the study) and sedentary at baseline (defined as no regular strenuous physical activity or exercise over the past 6 months). The study protocol has been approved by each of the Institutional Review Boards of the HERITAGE Family Study research consortium. Written informed consent was obtained from each participant. There were 461 whites (225 men and 236 women) and 281 blacks (107 men and 174 women). The maximum number of sib-pairs available was 357 and 103 in whites and blacks, respectively.
Steroid hormone assays.
The list of steroid hormones examined in the present study is shown in Table 1
. The steroid hormone levels by sex and race have been described earlier (14). Blood samples were obtained in the morning from an antecubital vein of participants in a semirecumbent position after a 12-h fast and put into vacutainer tubes with no anticoagulant. Blood was drawn twice at least 24 h apart. The present study is based on mean values from these two samples. For eumenorrheic women, all samples were obtained in the early follicular phase of the menstrual cycle. None of the women in the reproductive age had dramatically irregular menstrual cycles. Fasting serum was prepared according to a standard protocol. After centrifugation of blood at 2000 x g for 15 min at 4 C, two aliquots of 2 ml in cryogenic tubes were frozen at -80 C until shipment within a month. Frozen serum samples from the three U.S. HERITAGE Clinical Centers were shipped to the HERITAGE Steroid Core Laboratory in the Molecular Endocrinology Laboratory at the Laval University Medical Center in Ste-Foy, Quebec.
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DIOL-G)] and pregnenolone ester (PREG-E)-conjugated steroids, ethanol extraction was performed and was followed by C18 column chromatography (15). Glucuronide conjugates were submitted to hydrolysis with ß-glucuronidase (Sigma, St. Louis, MO). Levels of the steroids were measured by RIA (16). SHBG was determined with a solid phase immunoradiometric assay using I125 (Diagnostic Systems Laboratories Inc.). PCR conditions and genotyping methods have been outlined in Chagnon et al. (17). Automatic genotyping was performed using the computer software SAGA (Rick McIndoe, Roger Bumgarner, and Russ Welti, University of Washington, Seattle, WA; LICOR). SAGA allows for the sample and standard lanes to be automatically found, the different markers to be located on the gel, bands for each sample to be identified, and genotyping to be performed for a given marker on all subjects of the study. Microsatellite markers were selected mainly from the Marshfield panel version 8a. Map locations were taken from the Location Data Base (Southampton, UK; http://cedar.genetics.soton.ac.uk).
Data adjustment. Steroids were adjusted for the effects of sex, generation, age, and body mass index (BMI) using stepwise multiple regressions (18). Steroid phenotypes were regressed on BMI and up to a third-degree polynomial in age (separately within race-by-sex-by-generation subgroups). Only significant terms (5% level) were retained. The residuals from this regression (or the raw score if no BMI or age terms were significant) were then standardized to zero mean and unit variance within each subgroup and were used for the linkage studies.
Linkage analysis. Both singlepoint and multipoint linkage analyses were performed with the sib-pair linkage procedure (19, 20) as implemented in the SIBPAL2 program of S.A.G.E. (4.0 Beta 7) (21). Briefly, if there is a linkage between the marker locus and a putative gene influencing the phenotype, sibs sharing a greater proportion of alleles identical-by-descent (IBD) at the marker locus show also a greater resemblance for the phenotype. Phenotypic resemblance of the sibs, modeled as the mean-corrected trait product of the sibs’ trait values, is linearly regressed on the estimated proportion of alleles that the sib-pair shares IBD at each marker locus. Both singlepoint and multipoint estimates of allele sharing IBD were generated using the GENIBD program of S.A.G.E. (4.0 Beta 7). All analyses were conducted separately for blacks and whites. A total of 509 markers with an average spacing of 6.0 Mb on the 22 autosomes were genotyped. We followed the recommendation of Rao and Province (22) and used an LOD score of 1.75 (P < 0.0023) to identify promising linkages assuming that, on average, we may have one false positive per scan (based on discrete marker density).
Results
The linkage results are reported separately for C21, C19, and C18 steroids and for SHBG. Baseline data of the HERITAGE Family Study is used.
C21 steroids
A summary of promising genome scan results for C21, C19, and C18 steroids is depicted in Table 2. Figures 1–5 depict the regions where interesting signals for C21, C19, and C18 steroids were detected. Promising linkages (1.75 < LOD < 3.6; 2.2 x 10-5 < P < 0.0023) were found on 1q43 (marker D1S517) for PROG, 2p25.1 (D2S1400) for PREG-E, and 18q21.32 (D18S38) for OH-PROG in whites and on 3q25.33 (D3S1763) for OH-PROG in blacks, both in singlepoint and multipoint analyses. On chromosome 1, marker D1S180 at 1q44, about 6 Mb from D1S517, also showed evidence of promising linkage with PROG in singlepoint and suggestive (1.18 < LOD < 1.75; 0.01 < P < 0.0023) evidence of linkage in multipoint analyses. Other areas with promising linkages in singlepoint and suggestive evidence in multipoint with C21 steroids included 14q23.3 (D14S592; PREG-E), 17p13.3 (D17S1298; PREG-E), 10p13 (D10S191; PROG), and 14q11.2 (D14S283; PROG) in whites. Finally, D6S1027 at 6q27 had promising results in multipoint and suggestive in singlepoint for PREG-E in whites. On chromosome 3, within a region of 14 Mb containing D3S1763, two neighboring markers (AT1R1166 and D3S1744) generated evidence of promising linkage in multipoint analyses with OH-PROG in blacks.
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The strongest signals for C19 steroids were found on 22q12.3 (D22S304) for testosterone in whites (P = 0.00236 in multipoint) and on 8q22.1 (D8S1119) for DHT in blacks (P < 0.0023 for both singlepoint and multipoint analyses). Regions with promising (1.75 < LOD < 3.6; 2.2 x 10-5 < P < 0.0023) linkages in singlepoint and suggestive (1.18 < LOD < 1.75; 0.01 < P < 0.0023) evidence in multipoint for C19 steroids included 3q25.33 (testosterone; D3S1763), 7q22.1 (testosterone; D7S821), and 17q23.2 [ADT-G, angiotensin-converting enzyme deletion/insertion (ACEDI)] in blacks, and 19p13.3 (testosterone; D19S1034), 1q22 (DHT; D1S1653), 17q24.1 (DHT; D17S1351), 22q12.3 (DHT; D22S304), and 9q21.11 (ADT-G; D9S175) in whites. Promising linkages in multipoint and suggestive linkages in singlepoint were observed in whites at the following regions: 2p24.3 (DHT; D2S131), 2q37.3 (DHT; D2S427), 10q25.1–10q26.11 (ADT-G; D10S187, D10S190 and D10S1230), 13q12.2 (ADT-G; D13S260), and 2q33.2 (3DIOL-G; D2S1384). In blacks, 3DIOL-G exhibited a similar pattern of linkage at 7q11.1 (D7S3046).
C18 steroids
In blacks, the strongest evidence of linkage for E2 was provided by marker D1S1588 on 1p21.3 and in whites by markers D2S2347 and D2S2374 on 2p21 and D6S465 on 6p12.3 (Table 2
).
SHBG
Table 3 shows a summary of promising linkage results for SHBG. Figure 6 highlights the regions with interesting results for SHBG in blacks. Significant linkages (LOD > 3.6; P < 2.2 x 10-5) both in singlepoint and multipoint analyses were observed at 1q44 (D1S321), 5p13.3 (D5S1986), 10q24.1 (D10S1239, and 12q12 (D12S1653) in blacks. D1S321 at 1q44 showed also promising linkage in singlepoint analysis in whites. Additional promising linkages in both singlepoint and multipoint analyses were revealed in blacks on 1p22.3 (leptin receptor locus), 2p25.1 (D2S1400; in whites also), 2p24.1 (D2S1360), 2p23.3 (D2S305), 5p12 (D5S1470), 7q21.3 (PON2DDEI and PON1ALWI), 10q23.2 (D10S2470), 10q23.31 (D10S677), 10q25.2 (ADRA2MSP and D10S190), 12q21.1 (D12S375), 16p13.13 (D16S764), 18q12.2 (D18S463), 20q13.12 (D20S107, D20S850, D20S43, and D20S880), and 20q13.2 (D20S839 and D20S840).
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Several genes coding for steroid regulatory enzymes were mapped in the vicinity of the genomic regions showing evidence of linkage in the present genomic scans (Table 4). For instance, the 5-reductase gene (2p22.1; 45.7 Mb) was located near the linkage peak for DHT (2p22.1; 43 Mb) in whites. 5-reductase is an enzyme responsible for conversion of testosterone to DHT (1). Additionally, a side-chain cleavage enzyme gene (15q24.1; 80.7 Mb), a rate-limiting enzyme in the biosynthesis of all the steroids, was mapped in the vicinity of the OH-PROG peak (86.4 Mb); and the 17ß-OH-steroid dehydrogenase I gene (17q12; 38.9 Mb), an important enzyme in the peripheral androgen metabolism, was near the 3-DIOL-G peak (42.1 Mb) in blacks. Moreover, the FSH-receptor (2p15; 63.3 Mb) and LH-ß (19q13.3; 56.1 Mb) genes were mapped near the linkage peaks of E2 (2p21; 48.4 Mb) and DHT (19q13.2; 49.5 Mb) in whites, respectively.
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On chromosome 11p15.5 (0.3 Mb), a linkage with cortisol in blacks was observed in the present study. Moreover, an IGF-2 Apa I polymorphism (11p15.5; 0.7 Mb) was linked to cortisol in multipoint analysis in blacks. It is interesting to note that the insulin gene (11p15.5; 0.7 Mb) and a diabetes susceptibility locus (11p15.5; 2.2 Mb) (25) have been mapped to the same region. Cortisol counteracts insulin action, and cortisol excess leads to systemic insulin resistance (26).
On chromosome 2, within a region of about 30 Mb, promising linkages were observed for several steroids in whites: PREG-E (14.9 Mb), DHT (18.9 Mb), and E2 (46.3–48.4 Mb). Interestingly, the POMC locus (18.8 Mb) is precisely in this region. In addition, the strongest peak for SHBG in both blacks and whites occurred in the same region (14.9 Mb). SHBG is a major carrier of androgens and estrogens and regulates the blood levels of free hormones. A QTL for SHBG can therefore have implications for steroid hormone concentrations. Leptin and fat mass have been shown to be linked in Mexican-Americans (27) with markers around the QTL for E2 (48.9 Mb) in whites of the present study. It is important to note that estrogen regulates the LEP gene expression and leptin production in vivo (28). Although an earlier genomic scan identified a QTL influencing estrogen levels in baboons with a synthetic relation to human chromosome 20q13.11 (11), we did not find any significant linkages for estrogen in this region. However, in blacks the strongest evidence of linkage for E2 was provided by marker D1S1588 on 1p21.1 (102.1 Mb) which is near the locus of an interesting candidate gene, TGF-ß receptor type III, on 1p21.3 (102.9 Mb). It has been shown that TGF-ß receptor type III is able to bind with high-affinity inhibin (29), a gonadal protein regulating FSH secretion (30), and functions as an inhibin co-receptor.
Two main androgens, testosterone and DHT, had coinciding linkage peaks: on 8q22.1 (105.1 Mb) among blacks, and on 17q23.3 (ACEDI-marker; 64.6 Mb) and 22q12.3 (34.5 Mb) among whites. One of the QTLs for ADT-G in blacks was also located on 17q23.3. The growth hormone gene (17q21.33) maps in the broad region encompassing these peaks. The interactions and synergistic effects between growth hormone and sex steroids are well known (31). Furthermore, on 22q13.1 (36.6 Mb), near the peak linkages for testosterone and DHT in whites of this study, is the somatostatin receptor 3 gene. Somatostatin inhibits the release of growth hormone and many other pituitary hormones including gonadotropins (32).
Sex steroid hormones have been closely related to the regulation of adiposity (33). Interestingly, there were some regions in the present study in which steroid hormone linkage peaks coincided with peaks observed earlier for body composition phenotypes in the same cohort (34). For instance, in whites on chromosome 19p13.3 (5.8 Mb), there were peaks for testosterone and E2 near the peaks reported for leptin and percentage body fat (19p13.2; 10.9 Mb; Ref. 34). Leptin has negative actions on steroidogenesis, being a major signal linking excess adipose tissue to altered steroid hormone synthesis (35). Another common region (in the HERITAGE cohort) for BMI, fat mass, and steroid hormone (PROG) was on chromosome 14q11.2 (D14S283).
Human SHBG gene has been localized to the p12-p13 bands of chromosome 17 (9). In the present study, chromosome 17 did not generate any evidence of linkage with SHBG levels. It has been suggested that several interacting loci may be involved in determining SHBG levels (10). The latter concept is supported by the findings of the present study with QTLs in blacks on 1q44, 5p13.3, 10q24.1, and 12q12 in both singlepoint and multipoint analyses for SHBG. Several other loci were also suggested in the present study, thus providing indirect evidence that many genes could regulate SHBG levels. Among these loci in whites, one marker (D6S1027) is of particular interest because it is mapped under a peak observed for PREG-E in the present study and under a peak for dehydroepiandrosterone fatty acid ester in an earlier study also based on the HERITAGE cohort (12).
In conclusion, significant QTLs with LOD scores over 3.6 (P < 2.2 x 10-5) were observed in blacks at 1q44, 5p13.3, 10q24.1, and 12q12 for SHBG. In addition, QTLs were found on 1q43 for PROG, 2p25.1 for PREG-E, and 18q21.32 for OH-PROG in whites and on 3q25.33 for OH-PROG in blacks. The strongest signals for testosterone were found on 22q12.3 in whites and for DHT on 8q22.1 in blacks. In blacks, the strongest evidence of linkage for E2 was located at 1p21.3 and, in whites, at 2p21, 6p12.3, and 6q11.1. Several key enzymes of steroidogenesis and other potential candidate genes are located in the vicinity of the genomic regions, showing evidence of linkage in this HERITAGE genomic scan. Further studies are needed to define more precisely these QTLs and narrow down the most important chromosomal regions. A replication in another family study would be highly desirable.
Acknowledgments
We thank Dr. Alain Belanger and his collaborators of the Molecular Endocrinology Laboratory, Centre Hospitalier de l’Université Laval, Laval University (Ste-Foy, Quebec) for performing the steroid assays.
Footnotes
The HERITAGE Family Study is supported by the National Heart, Lung, and Blood Institute through Grants HL-45670 (to C.B.), HL-47323 (to A.S.L.), HL-47317 (to D.C.R.), HL-47327 (to J.S.S.), and HL-47321 (to J.H.W.). A.S.L. is partially supported by the Henry L. Taylor endowed Professorship in Exercise Science and Health Enhancement. C.B. is supported in part by the George A. Bray Chair in Nutrition. The results of this paper were obtained with the program package S.A.G.E., which is supported by a U. S. Public Health Service Resource Grant (1 P41 RR03655) from the National Center for Research Resources.
Abbreviations: ACEDI, Angiotensin-converting enzyme deletion/insertion; ADT-G, androsterone glucuronide; BMI, body mass index; DHT, dihydrotestosterone; 3-DIOL-G, androstane-3 17ß-diol glucuronide; E2, estradiol; IBD, identical-by-descent; OH-PROG, 17-hydroxyprogesterone; PREG-E, pregnenolone ester; PROG, progesterone; QTL, quantitative trait locus.
Received September 20, 2001.
Accepted April 23, 2002.
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