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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1417
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2198-2204
Copyright © 2005 by The Endocrine Society

Common Genetic Variation in the Sex Steroid Hormone-Binding Globulin (SHBG) Gene and Circulating SHBG Levels among Postmenopausal Women: The Multiethnic Cohort

Christopher A. Haiman, Stephanie E. Riley, Matthew L. Freedman, Veronica W. Setiawan, David V. Conti and Loïc Le Marchand

Department of Preventive Medicine, Keck School of Medicine, University of Southern California (C.A.H., S.E.R., V.W.S., D.V.C.), Los Angeles, California 90089; Broad Institute of Massachusetts Institute of Technology and Harvard (M.L.F.), Cambridge, Massachusetts 02139; Departments of Medicine, Molecular Biology, and Hematology/Oncology, Massachusetts General Hospital (M.L.F.), Boston, Massachusetts 02114; and Etiology Program, Cancer Research Center of Hawaii, University of Hawaii (L.L.M.), Honolulu, Hawaii 96813

Address all correspondence and requests for reprints to: Dr. Christopher Haiman, University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Los Angeles, California 90089. E-mail: haiman{at}usc.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
SHBG transports sex steroid hormones in the blood, and levels in humans are thought to partially be genetically determined. Recently, studies have found a pentanucleotide (TAAAA)n repeat polymorphism in the promoter of the SHBG gene and a missense polymorphism in exon 6 (Asp327Asn) to predict circulating SHBG levels. Based on the potential role of common genetic variation in SHBG to serve as a marker of SHBG levels in the general population, we evaluated the association between the (TAAAA)n repeat polymorphism, Asp327Asn polymorphism, and SHBG levels in a population of African-American, Native Hawaiian, Japanese, Latina, and white healthy postmenopausal women from the Multiethnic Cohort Study (n = 372). Mean SHBG levels were not significantly different between carriers and noncarriers of the Asn327 allele [minor allele frequency range across ethnic groups, 0.02–0.14; Asp/Asn and Asn/Asn genotypes, 33.6 mol/liter; 95% confidence interval (CI), 28.2–40.0; n = 49; Asp/Asp genotype, 30.8 mol/liter (95% CI, 28.7–33.1; n = 296); P = 0.37]. For the repeat polymorphism, we observed six different SHBG repeat alleles segregating in the population (TAAAA6–11), and the distribution of these alleles varied widely across populations. We found suggestive evidence of linkage disequilibrium between the Asn327 allele and the eight-repeat allele in all populations except African-Americans (P ≥ 0.08). In analysis of the repeat polymorphism, SHBG levels among carriers of two short alleles (seven or fewer repeats; 31.2 nmol/liter; 95% CI, 27.3–35.6; n = 82) were not statistically different from those of carriers of two long alleles (more than seven repeats; 32.7 nmol/liter; 95% CI, 29.4–36.3; n = 124; P = 0.59). We did, however, observe individual genotypic classes (n = 16) to contribute modestly to the overall prediction of SHBG levels (by analysis of covariance, P = 0.03). Carriers of the six-repeat allele (27.9 nmol/liter; 95% CI, 25.2–30.8; n = 147) were found to have nominally significantly lower SHBG levels than noncarriers (32.4 nmol/liter; 95% CI, 29.7–35.2; n = 202; P = 0.03). This effect was stronger among the subset of women who also carried the Asn327 allele (interaction, P = 0.006). In summary, these results suggest that genetic variation at the SHBG locus may contribute to modest differences in SHBG levels among healthy postmenopausal women, and that much larger studies will be needed to better comprehend the effects of common variations at this locus in predicting circulating SHBG levels.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SHBG BINDS ANDROGENS and estradiol in the blood and influences the bioavailability of these hormone fractions to their target tissues (1). In humans, SHBG is produced mainly by the liver (2), although gene expression has been noted in other extrahepatic sites, including testis, ovary, placenta, and endometrium and in breast cancer tissue (3, 4, 5, 6, 7). Circulating levels of SHBG are inversely associated with body weight (8, 9) and serve as a marker of androgenicity and insulin resistance (10, 11). Physiologically low levels of SHBG are commonly observed in women with hyperandrogenic conditions such as polycystic ovarian syndrome (PCOS) (12) and, independent of body weight, have been associated with higher insulin levels (13, 14) and increased risk of type II diabetes (15). Lower SHBG levels are also associated with increased risk of postmenopausal breast cancer (16) and are an indication of a higher proportion of circulating estradiol that is free (non-SHBG bound) and biologically active.

Functional genetic variation at the SHBG gene locus may contribute to our understanding of these SHBG-linked disorders by serving as surrogate markers of circulating SHBG levels and long-term exposure to biologically important steroid hormones, estradiol and testosterone. Recently, a pentanucleotide repeat polymorphism (TAAAA)n in the 5' promoter region of the SHBG gene has been characterized (17, 18, 19), with alleles varying from six to 11 TAAAA repeats. Hogeveen et al. (17) reported the six-TAAAA allele to be associated with decreased transcriptional activity in an in vitro assay tested in hepatoblastoma cells (17). The relationship between this polymorphism and circulating SHBG levels has been examined in three separate studies of premenopausal women with PCOS (18) and hirsutism, (19) and of young men (20). In contrast to the in vitro results, all of these studies found that individuals with shorter (TAAAA)n repeat genotypes had significantly higher circulating SHBG levels. Whether this polymorphic repeat contributes to interindividual differences in SHBG levels among postmenopausal women without common endocrine disorders has yet to be determined.

Additional variants in SHBG have also been detected and evaluated as predictors of SHBG levels (19, 21, 22). A common missense single nucleotide polymorphism in exon 8 (Asp327Asn; frequency, 5–15%) (22, 23) is thought to introduce an N-glycosylation site and result in a decreased rate of clearance of SHBG (24). This variant (Asn327 allele) has been found to be in strong linkage disequilibrium (LD) with the eight-TAAAA allele, and two studies that have investigated this single nucleotide polymorphism have found carriers of the less common Asn327 allele to have lower circulating SHBG levels (19, 22).

In the present study we extend these previous observations by evaluating the frequency of the pentanucleotide repeat and Asp327Asn missense polymorphisms in the SHBG gene among African-American, Native Hawaiian, Japanese, Latina, and white postmenopausal women in the Multiethnic Cohort Study (MEC) and investigated whether specific alleles at this locus predict circulating SHBG levels in these healthy women.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Women in this study are participants in the MEC (25), a large prospective cohort consisting of over 215,000 men and women from Hawaii and California (mainly Los Angeles). In brief, the cohort is comprised predominantly of Native Hawaiians, Japanese, and whites in Hawaii and African-Americans, Japanese, and Latinos in Los Angeles. Between 1993 and 1996, participants entered the MEC by completing a 26-page, self-administered questionnaire that asked detailed information about dietary habits, demographic factors (ethnicity, education, and migrant status), personal behaviors (smoking, sun exposure, and physical activity), history of prior medical conditions (e.g. heart attack, diabetes, and cancer), and, for women, reproductive history and exogenous hormone use. No personal information was collected regarding specific endocrine conditions, such as hirsutism or PCOS. Potential cohort members were identified through the Department of Motor Vehicles drivers’ license files and, additionally for African-Americans, Health Care Financing Administration data files. The participants were between the ages of 45 and 75 yr when they entered the cohort.

Beginning in 1994, blood samples were collected from a random sample of MEC participants to serve as a control pool for genetic association studies in the cohort. Blood samples were collected at the participants’ homes after an overnight fast, processed within 8 h, and stored at –80 C. Four hundred and fifty-four postmenopausal women were originally selected for hormone analysis (100 African-Americans, 78 Native Hawaiians, 92 Japanese, 97 Latinas, and 87 whites). Participants provided their informed consent, and the protocol was approved by the institutional review boards at the University of Southern California and the University of Hawaii.

Hormone assay

SHBG levels were measured by an immunoradiometric assay (Cis-Bio, Gif-sur-Yvette, France) in 14 separate batches. The number of women from each ethnic group was evenly distributed across the batches. Analyses were performed in the Hormone and Cancer Group laboratory at the International Agency for Research on Cancer (Lyon, France). The intra- and interbatch laboratory coefficients of variation calculated from blind duplicate samples were 2.9% and 13.6%, respectively.

Genotyping of the (TAAAA)n and Asp327Asn polymorphisms

DNA was extracted from the buffy coat fraction using the QIAGEN 96 DNA Spin Kit (QIAGEN, Valencia, CA). Genotyping of the (TAAAA)n was performed as follows. PCR amplification of the fragment was generated using primers 5'-GATCGCTTGAACTCGAGAGG-3' (forward) and 5'-GCCTGTCTCCCAAGAAGGTA-3' (reverse). This resulted in a PCR product of 275 bp (eight TAAAA repeats). Two forward primers were used in the reaction: one 5'-labeled with a fluorescent dye, and one without. Thirty nanograms of genomic DNA were used per 20 µl reaction with 200 nmol of each forward and reverse primer (180 nmol unlabeled and 20 nmol 5'-labeled forward primers), 25 mM MgCl2, 10 mM deoxy-NTPs, 10x QIAGEN PCR buffer, and 0.5 U QIAGEN HotStarTaq (QIAGEN). Amplification conditions were 5 min of initial denaturation at 95 C, followed by 40 cycles of 1 min at 95 C, 1 min at 60 C, and 1 min at 72 C, and a final extension of 10 min at 72 C. Fragment length was determined using the ABI 3700 DNA analyzer (Applied Biosystems, Foster City, CA). Amplified products were evaluated relative to Genescan-500 [Rox] size standard using Genescan and Genotyper software (Applied Biosystems). Twenty-three samples did not amplify and were excluded from the analysis of the TAAAA polymorphism.

Genotyping of the Asp327Asn polymorphism was performed by the 5'-nuclease TaqMan allelic discrimination assay (Applied Biosystems) as follows. PCR amplification of the fragment was generated using primers 5'-AATGCTCTAATGCCACCTTTGC-3' (forward) and 5'-TGCCCAAAGGCCATTCAG-3' (reverse). Five nanograms of genomic DNA were used per 5 µl reaction, which included 0.0625 µl 80x custom primer/probe mix (Applied Biosystems), which contained the primers and 6-carboxyfluorescein- and VIC-labeled probes (6-carboxyfluorescein, 5'-TCTAGGAGAAAACTCT-3'; VIC, 5'-CTCTAGGAGAAGACTC-3'), 2.5 µl TaqMan Universal PCR Master Mix (Applied Biosystems), and 2.44 µl water. Amplification conditions were 10 min of initial denaturation at 95 C, followed by 50 cycles of 15 sec at 95 C and 1 min at 60 C. Ten replicate samples were inserted to assess quality control, and the concordance of genotyping calls for both the TAAAA and Asp327Asn polymorphisms was 100%.

Statistical analysis

Because SHBG levels have been found to be lower in women with breast cancer, to decrease the variability in the SHBG values we excluded women with a history of breast cancer (n = 59). In addition, one woman missing body mass index (BMI) measurement, one woman who self-reported taking hormone replacement therapy at the time of blood sampling, and 22 women who had estradiol values above 100 pg/ml at the time of blood sampling, indicating hormone replacement therapy use or premenopausal status, were also excluded. The natural logarithm of the plasma SHBG values was used to reduce the skewness. After this transformation, only two subjects had values more than 1.5 times the interquartile range (both had values <7.0 nmol/liter). The removal of these women from the analysis had no bearing on the final results. ANOVA and analysis of covariance (ANCOVA) were used to test for differences between the ethnic groups with regard to demographic factors and SHBG levels. For demographic factors, results are provided as the mean ± SD. Least-squares geometric mean SHBG levels were calculated for each ethnic group, and by SHBG genotype [(TAAAA)n and Asp327Asn], using multiple regression methods while adjusting for age (continuous), BMI (continuous), and laboratory batch; adjustment for these factors as categorical variables produced similar results. Other potential predictors of SHBG levels (age at menarche, alcohol intake and smoking status) did not alter the results and thus were not included in the final models. Ninety-five percent confidence intervals (95% CIs) were calculated for mean SHBG values to allow comparison between ethnic groups and genotype classes.

To assess the relationship between the repeat polymorphism and SHBG levels, we performed analyses similar to those described by Xita et al. (18) and Cousin et al. (19). First, we performed a cut-point sensitivity analysis and evaluated SHBG levels between individuals with two short-repeat vs. two long-repeat alleles (e.g. both alleles with seven or fewer repeats vs. both alleles with more than seven repeats). Second, we evaluated SHBG levels by specific SHBG repeat genotypes; we observed 16 of the 36 possible combinations of alleles. Lastly, to assess whether a specific repeat allele may be functional or a marker in LD with a functional variant located elsewhere in the gene, we evaluated differences in SHBG levels between carriers and noncarriers of each allele [e.g. noncarriers of the six-repeat allele (no 6) vs. heterozygotes (6/X) and homozygotes (6/6)]. A similar analysis was conducted for the Asp327Asn polymorphism. For both polymorphisms, gene dosage was also evaluated by modeling genotype as an ordinal variable. LD between the (TAAAA)n and Asp327Asn alleles was evaluated for each ethnic group using the {chi}2 test. We explored the potential interaction between TAAAA repeat alleles and the Asp327Asn polymorphism by including interaction terms between carriers and noncarriers of specific alleles in multivariate linear regression models. Two-sided tests were conducted, and P < 0.05 was considered statistically significant. For all analyses, we used SAS statistical package version 8.2 (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Three hundred seventy-two women between the ages of 56 and 82 yr were included in this analysis; of these, 81 were African-American, 65 were Native Hawaiian, 70 were Japanese, 89 were Latina, and 67 were white. Descriptive characteristics by ethnic group are presented in Table 1Go. The mean age was similar across groups, whereas Japanese women were shorter and weighed the least (by ANCOVA, P < 0.0001). In adjusted analyses, Native Hawaiians had significantly lower SHBG levels than African-Americans (–24.1%; P = 0.007) and whites (–25.9%; P = 0.006), whereas the differences between the other ethnic groups were not statistically significant (P ≥ 0.12). As expected, heavier women (BMI, ≥25 kg/m2) in all groups had lower SHBG levels (Table 1Go).


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  		TABLE 1. Descriptive data among all women (n = 372) by ethnicity

 
The frequency of the Asn327 allele varied across ethnic groups (African-Americans, 0.02; Native Hawaiians, 0.08; Japanese, 0.14; Latinas, 0.04; whites, 0.10; Table 2Go); the genotype frequencies were in Hardy-Weinberg equilibrium in each group (P > 0.33). Among all ethnic groups combined, carriers of the Asn327 allele (Asp/Asn plus Asn/Asn genotypes, 33.6 mol/liter; 95% CI, 28.2–40.0; n = 49) had nonsignificantly higher SHBG levels than noncarriers (Asp/Asp genotype; 30.8 mol/liter; 95% CI, 28.7–33.1; n = 296; P = 0.37). This relationship, however, was not observed consistently across ethnic groups, and the power was limited to assess ethnic-specific effects because the Asp327 allele was rare in some populations [i.e. African-Americans (0.02) and Latinas (0.04)].


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  		TABLE 2. Geometric least-squares mean SHBG levels by Asp327Asn genotype classification (n = 345)

 
In these five populations, we observed (TAAAA)n pentanucleotide alleles of six, seven, eight, nine, 10, and 11 repeats. The frequency of these alleles varied widely across populations, and distribution of the repeat alleles by ethnic group is presented in Fig. 1Go. Among whites, the repeat allele frequencies were comparable to those previously reported by Cousin et al. (19) (six-repeat allele, 0.34; seven-repeat allele, 0.06; eight-repeat allele, 0.32; nine-repeat allele, 0.20; 10-repeat allele, 0.05; 11-repeat allele, 0.03). Although the power to detect differences in Hardy-Weinberg equilibrium in each ethnic group was limited because of the small sample size, the expected genotype distributions based on the allele frequencies within each ethnic group were similar to the observed distributions (for Hardy-Weinberg equilibrium in each ethnic group, P > 0.67). The six-, eight-, and nine-repeat alleles were common to all groups (frequencies, ≥0.15). The eight-repeat allele was the most common allele among African-Americans (0.47), whereas the nine-repeat allele was the most common among Japanese (0.39). The seven-repeat allele was the most common allele among Native Hawaiians (0.38) and Latinas (0.29) and was relatively rare among African-Americans and whites (0.06). The 10-repeat allele was of low frequency in all populations (frequencies, <0.09). The 11-repeat allele was extremely rare; we observed only three white women to be carriers.



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  		FIG. 1. The distribution of SHBG (TAAAA)n repeat alleles (n = 6, 7, 8, 9, 10, and 11) by ethnic group in the MEC. AA, African-Americans; NA, Native Hawaiians; JA, Japanese; LA, Latinas; WH, white.

 
We observed no consistent pattern of association between SHBG levels and number of (TAAAA)n repeats when comparing women with two short vs. two long alleles (Table 3Go). We did, however, observe individual genotypic classes (n = 16) to contribute modestly to the overall prediction of SHBG levels (by ANCOVA, P = 0.03; Table 4Go). The genotypic classes with outlying values [genotypes 8/11 (n = 1) and 9/11 (n = 2)] had no effect on this global test; the significance level for the global test remained P < 0.05 when one or both of these genotypic classes were removed from the analysis. The major contribution to the significance of this test was the six-repeat allele heterozygote carriers (genotypes 6/7, 6/8, 6/9, and 6/10; we designate this group 6/X). When these genotypic classes were removed from the analysis, the global test was no longer statistically significant (P = 0.26).


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  		TABLE 3. Cut-point analysis of SHBG TAAAA genotype (n = 349)

 

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  		TABLE 4. Geometric least-squares mean SHBG levels by TAAAA genotype

 
Based on this observation, we next evaluated associations by repeat allele carrier status to assess whether specific alleles predict SHBG levels. Compared with noncarriers, carriers of the six-repeat allele had significantly lower plasma levels of SHBG (–13.9%; P = 0.03; Table 5Go). This association was influenced by the six-repeat allele heterozygotes (6/X), who had significantly lower mean SHBG levels than noncarriers of the six-repeat allele (–17.9; P = 0.006). The direction of this association was also observed to be consistent in four of the five ethnic groups (all but Native Hawaiians; Table 5Go). Women who were homozygous for the six-repeat allele had nonsignificantly higher mean levels than noncarriers (+9.3; P = 0.51); however, only 24 women were homozygous for the six-repeat allele, and the width of the CI does not exclude the possibility that homozygous carriers of the six-repeat allele do have levels comparable to six-repeat allele heterozygotes. It is also interesting to note that in three of the five ethnic groups (African-Americans, Latinas, and whites), those groups with a higher prevalence of the six-repeat allele, mean SHBG levels were nonsignificantly lower among women with the 6/6 genotype compared with noncarriers of the six-repeat allele. In the entire sample, the association with the six-repeat allele was observed among both heavy and lean women; no significant difference was observed in the effect of the six-repeat allele in strata of BMI (defined by the median in each ethnic group; P = 0.50 for interaction).


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  		TABLE 5. Geometric least-squares mean SHBG levels by TAAAA genotype classification (n = 349)

 
Women homozygous for the eight- and nine-repeat alleles were also observed to have higher levels of SHBG (vs. noncarriers: 8/8 genotype, +20.9%, P = 0.09; 9/9 genotype, +21.1%, P = 0.18), although these associations failed to reach statistical significance. No significant associations were observed with the 10- and 11-repeat alleles, although our ability to make inferences regarding the effects of these alleles was limited due to the small number of allele carriers in these populations.

As observed in other white populations (19), we found the eight-repeat allele and the Asn327 allele to be in LD. Evidence of this relationship was noted in all populations (whites, P = 0.005; Latinas, P < 0.0001; Native Hawaiians, P = 0.03; Japanese, P = 0.08), except African-Americans (P = 0.49). Next, we explored the combined effects of the six-repeat allele and the Asn327 allele in predicting SHBG levels (Table 6Go). The test for interaction between carriers and noncarriers of the six-repeat allele and the Asn327 allele was statistically significant (P = 0.006). Compared with noncarriers of the six-repeat allele and the Asn327 allele (31.8 mol/liter; 95% CI, 29.0–35.0; n = 159), carriers of both of these alleles had significantly lower SHBG levels (20.6 mol/liter; 95% CI, 14.7–28.7.0; n = 13; P = 0.01). Although preliminary, these data suggest that the association observed with the six-repeat allele may be stronger among, and perhaps limited to, those who also carry the Asn327 allele.


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  		TABLE 6. Geometric least-squares mean SHBG levels by Asn327 and six TAAAA allele carriers (n = 326)

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This is the first study to characterize the frequency distribution of the SHBG (TAAAA)n repeat polymorphism in a multiethnic sample and to assess its role in predicting plasma SHBG levels in a population of healthy postmenopausal women. We found the distribution of repeat alleles to vary widely across populations; four of the six-repeat alleles were observed to be the most prevalent allele in four of the five ethnic populations, and five of the six-repeat alleles were found in all populations studied. The frequency of the Asn327 allele was also observed to vary widely across populations. Overall, in this multiethnic sample, we observed carriers of the (TAAAA)6 allele to have lower mean circulating plasma SHBG levels than noncarriers, and suggestive evidence that this association may be more pronounced among a relatively small subset of individuals who also carry the Asn327 allele.

Previous studies have suggested that interindividual differences in circulating SHBG levels may in part be genetically determined (18, 19, 20, 21, 22, 26). Three previous studies have examined the SHBG (TAAAA)n repeat polymorphism in relation to SHBG levels, and in all three of these studies, individuals with longer repeat alleles were reported to have significantly lower circulating SHBG levels than those with shorter repeat alleles (18, 19, 20). Xita et al. (18) examined the association between the (TAAAA)n repeat polymorphism and PCOS and its relation to SHBG levels among Greek women with PCOS (n = 185) and ethnically matched controls (n = 324). They observed that women with PCOS were more likely to be carriers of longer (TAAAA)n repeat alleles (more than eight repeats) compared with controls (P = 0.03). Among women with PCOS, they also found that carriers of two long-repeat alleles (eight or more repeats; n = 92) had significantly lower SHBG levels (–22.5%; P = 0.02) compared with women homozygous for two short alleles (fewer than eight repeats; n = 47). This difference, however, was not observed in the premenopausal control women (n = 324). Cousin et al. (19) also examined whether the (TAAAA)n repeat polymorphism influences SHBG levels among hirsute women (n = 303). In this sample, comprised mainly of white French women, they observed women with the 9/9 repeat genotype (n = 21) to have significantly lower SHBG levels than women with the 6/6 genotype (n = 8; –38.4%; P = 0.04). In a more recent study among 993 young men, Wood et al. (20) reported carriers of longer repeat alleles to have lower serum SHBG levels. These findings support those previously reported among women (18, 19). However, these previous studies are contrary to expectation based on the in vitro results, where the six-allele was the only allele with lower transcriptional activity (17). In addition, a direct comparison of the results across these three previous studies is difficult, because each study grouped SHBG repeat genotype classes differently in their analyses. Based on the previous clinical studies among premenopausal women as well as the present report, one may infer that SHBG production through this (TAAAA)n repeat polymorphism may be differentially regulated between women depending on their androgenic status. Hogeveen et al. (17) identified a nuclear factor that was found to bind to the six-TAAAA allele and silence SHBG transcription in hepatoma cells. This in vitro result supports our findings that women with the six-repeat allele may have lower SHBG levels. Additional work will be required to characterize this factor and to understand under what conditions (i.e. high androgen levels) it interacts with this repeat polymorphism and is regulated.

Two recent studies have reported the Asn327 allele of the Asp327Asn polymorphism to be associated with higher circulating SHBG levels. Among hirsute women, Cousin et al. (19) reported both heterozygous (n = 52) and homozygous (n = 2) carriers of the Asn327 allele to have higher SHBG levels than women with the Asp/Asp genotype (n = 249; P = 0.01 for trend). In a more recent study by Dunning et al. (22), among 1975 postmenopausal women from the United Kingdom, the Asn327 allele was found to explain only 0.6% of the variance in SHBG levels in the population (P = 0.005). We also found weak evidence that carriers of the Asn327 allele may have higher circulating SHBG levels, which is consistent with these previous studies as well as a laboratory study that found the human variant SHBG protein to have a lower clearance rate in rabbits (24). Although preliminary, we also found suggestive evidence that the association we observed with the six-repeat allele may depend on Asn327 carrier status, because the inverse relationship between the six-repeat allele and SHBG levels was more apparent among Asn327 allele carriers. One possible explanation may be that down-regulation of SHBG production via the six-repeat allele may be enhanced among women with modestly higher circulating SHBG levels (i.e. Asn327 carriers). Based on our limited sample size, however, it is also possible that the associations we observed with the six-repeat allele and the suggestive interaction with the Asn327 allele may be false positives.

Additional variants in SHBG have been detected and evaluated as predictors of SHBG levels (19, 21, 22). Dunning et al. (22) reported a second polymorphism in the 5'-untranslated region to be highly statistically significantly associated with SHBG levels (P < 10–6) and account for as much as 2.4% of the variance. This 5'-untranslated region polymorphism does not appear to be inherited on the same haplotype as the Asn327 allele (22), and it is presently not known whether this polymorphism is linked with a specific repeat allele. LD-based studies in multiple populations that include the TAAAA repeat polymorphism as well as other known common variants will be required to better understand the independent and joint effects of putative functional variation in the SHBG gene. Rare missense variants in SHBG have also been identified, one in exon 4 (P156L) and two in exon 1 (R25H, R25C; www.genome.wi.mit.edu/cvar_snps) (21). Hogeveen et al. (21) found the rare L156 variant in five patients with hyperandrogenism and ovarian dysfunction, whereas the influence of the two extremely rare missense variants in exon 1 on SHBG levels has not been studied.

In summary, our data suggest that the (TAAAA)6 repeat allele of the SHBG gene may be associated with modestly lower circulating SHBG levels in healthy postmenopausal women. Our sample size within each ethnic group, however, is not large enough to definitively evaluate modest ethnic-specific effects, and larger studies evaluating the combined effects of TAAAA repeat alleles and the Asp327Asn polymorphisms are needed. A comprehensive LD-based approach will be required to clarify the contribution of genetic variation in SHBG to lifetime exposure to androgens and estrogens and risk of SHBG-linked disorders.


   Acknowledgments
 
We thank Drs. Sabina Rinaldi and Rudolf Kaaks for the hormone analysis, and David Van Den Berg for his assistance with genotyping. We are most indebted to the participants of the MEC for their participation and commitment.


   Footnotes
 
This work was supported by NCI Grants CA-63464 and CA-54281 and a General Motors Cancer Research Scholar’s Grant (to C.A.H.).

First Published Online January 5, 2005

Abbreviations: ANCOVA, Analysis of covariance; BMI, body mass index; CI, confidence interval; PCOS, polycystic ovarian syndrome.

Received July 19, 2004.

Accepted December 28, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Siiteri PK, Murai JT, Hammond GL, Nisker JA, Raymoure WJ, Kuhn RW 1982 The serum transport of steroid hormones. Recent Prog Horm Res 38:457–510
  2. Rosner W, Aden DP, Khan MS 1984 Hormonal influences on the secretion of steroid-binding proteins by a human hepatoma-derived cell line. J Clin Endocrinol Metab 59:806–808[Abstract]
  3. Hammond GL, Underhill DA, Rykse HM, Smith CL 1989 The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular message RNAs. Mol Endocrinol 3:1869–1876[Abstract]
  4. Forges T, Gerard A, Hess K, Monnier-Barbarino P, Gerard H 2004 Expression of sex hormone-binding globulin (SHBG) in human granulosa-lutein cells. Mol Cell Endrocrinol 219:61–68[CrossRef][Medline]
  5. Larrea F, Diaz L, Carino C, Larriva-Sahd J, Carrillo L, Orozco H, Ulloa-Aguirre A 1993 Evidence that human placenta is a site of sex hormone-binding globulin gene expression. J Steroid Biochem Mol Biol 46:497–505[CrossRef][Medline]
  6. Misao R, Nakanishi Y, Fujimoto J, Tamaya T 1995 Expression of sex hormone-binding globulin mRNA in uterine leiomyoma, myometrium and endometrium of human subjects. Gynecol Endocrinol 9:317–323[Medline]
  7. Moore KH, Bertram KA, Gomez RR, Styner MJ, Matej LA 1996 Sex hormone binding globulin mRNA in human breast cancer: detection in cell lines and tumor samples. J Steroid Biochem Mol Biol 59:297–304[CrossRef][Medline]
  8. Haffner SM, Katz MS, Dunn JF 1991 Increased upper body and overall adiposity is associated with decreased sex hormone binding-globulin in postmenopausal women. Int J Obes 15:471–478[Medline]
  9. Wu F, Ames R, Evans MC, France JT, Reid IR 2001 Determinants of sex hormone-binding globulin in normal postmenopausal women. Clin Endocrinol (Oxf) 54:81–87[CrossRef][Medline]
  10. Nestler JE 1993 Editorial. Sex hormone-binding globulin: a marker for hyperinsulinaemia and/or insulin resistance? J Clin Endocrinol Metab 76:273–274[CrossRef][Medline]
  11. Lee CC, Kasa-Vubu JZ, Supiano MA 2004 Androgenicity and obesity are independently associated with insulin sensitivity in postmenopausal women. Metabolism 53:507–512[CrossRef][Medline]
  12. Holte J, Bergh T, Gennarelli G, Wide L 1994 The independent effects of polycystic ovary syndrome and obesity on serum concentrations of gonadotrophins and sex steroids in premenopausal women. Clin Endocrinol (Oxf) 41:473–481[Medline]
  13. Haffner SM, Dunn JF, Katz MS 1992 Relationship of sex hormone-binding globulin to lipid, lipoprotein, glucose, and insulin concentrations in postmenopausal women. Metabolism 41:278–284[CrossRef][Medline]
  14. Preziosi P, Barrett-Conner E, Papoz L, Roger M, Saint-Paul M, Nahoul K, Simon D 1993 Interrelation between plasma sex hormone-binding globulin and plasma insulin in healthy adult women: the telecom study. J Clin Endocrinol Metab 76:283–287[Abstract]
  15. Lindstedt G, Lundberg PA, Lapidus L, Lundgren H, Bengtsson C, Bjorntorp P 1991 Low sex-hormone-binding globulin concentration as independent risk factor for development of NIDDM. 12-yr follow-up of population study of women in Gothenburg, Sweden. Diabetes 40:123–128[Abstract]
  16. The Endogenous Hormones and Breast Cancer Collaborative Group 2002 Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 94:606–616[Abstract/Free Full Text]
  17. Hogeveen KN, Talikka M, Hammond GL 2001 Human sex hormone-binding globulin promoter activity is influenced by a (TAAAA)n repeat element within Alu sequence. J Biol Chem 276:36383–36390[Abstract/Free Full Text]
  18. Xita N, Tsatsoulis A, Chatzikyriakidou, Georgiou I 2003 Association of the (TAAAA)n repeat polymorphism in the sex hormone-binding globulin (SHBG) gene with polycystic ovary syndrome and relation to SHBG serum levels. J Clin Endocrinol Metab 88:5976–5980[Abstract/Free Full Text]
  19. Cousin P, Calemard-Michel L, Lejeune H, Raverot G, Yessaad N, Emptoz-Bonneton A, Morel Y, Pugeat M 2004 Influence of SHBG gene pentanucleotide TAAAA repeat and D327N polymorphism on serum sex hormone-binding globulin concentrations in hirsute women. J Clin Endocrinol Metab 89:917–924[Abstract/Free Full Text]
  20. Wood K, Kopp P, Colangelo LA, Liu K, Gapstur SM A cross-sectional analysis of the association between serum sex hormone-binding globulin (SHBG) levels and the SHBG gene pentanucleotide TAAA repeat: the CARDIA Male Hormone Study. Program of the 86th Annual Meeting of The Endocrine Society, New Orleans, LA, 2004, pp 539–540 (Abstract P3–311)
  21. Hogeveen KN, Cousin P, Pugeat M, Dewailly D, Soudan B, Hammond GL 2002 Human sex hormone-binding globulin variants associated with hyperandrogenism and ovarian dysfunction. J Clin Invest 109:973–981[CrossRef][Medline]
  22. Dunning AM, Dowsett M, Healey CS, Tee L, Luben RN, Folkerd E, Novik KL, Kelemen L, Ogata S, Pharoah PDP, Easton DF, Day NE, Ponder BAJ 2004 Polymorphisms associated with circulating sex hormone levels in postmenopausal women. J Natl Cancer Inst 96:936–945[Abstract/Free Full Text]
  23. Försti A, Jin Q, Grzybowska E, Söderberg M, Zientek H, Sieminska M, Rogozinska-Szczepka J, Chmielik E, Utracka-Hutka B, Hemminki K 2002 Sex hormone-binding globulin polymorphisms in familial and sporadic breast cancer. Carcinogenesis 23:1315–1320[Abstract/Free Full Text]
  24. Cousin P, Déchaud H, Grenot C, Lejeune H, Pugeat M 1998 Human variant sex hormone-binding globulin (SHBG) with an additional carbohydrate chain has a reduced clearance rate in rabbit. J Clin Endocrinol Metab 83:235–240[Abstract/Free Full Text]
  25. Kolonel LN, Henderson BE, Hankin JH, Nomura AM, Wilkens LR, Pike MC, Stram DO, Monroe KR, Earle ME, Nagamine FS 2000 A multiethnic cohort in Hawaii and Los Angeles: baseline characteristics. Am J Epidemiol 151:346–357[Abstract/Free Full Text]
  26. Ukkola O, Rankinen T, Gagnon J, Leon AS, Skinner JS, Wilmore JH, Rao DC, Bouchard C 2002 A genome-wide linkage scan for steroids and SHBG levels in black and white families: the HERITAGE Family Study. J Clin Endocrinol Metab 87:3708–3720[Abstract/Free Full Text]



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