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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 917-924
Copyright © 2004 by The Endocrine Society

Influence of SHBG Gene Pentanucleotide TAAAA Repeat and D327N Polymorphism on Serum Sex Hormone-Binding Globulin Concentration in Hirsute Women

Patrice Cousin, Laurence Calemard-Michel, Hervé Lejeune, Gérald Raverot, Nadia Yessaad, Agnès Emptoz-Bonneton, Yves Morel and Michel Pugeat

Hospices Civils de Lyon (P.C., G.R., N.Y., A.E.-B., M.P.), Fédération d’Endocrinologie du Pôle Est, Hôpital Neurologique Pierre Wertheimer, 69394 Lyon, France; and Laboratoire de Biologie Endocrinienne et Moléculaire (L.C.-M., Y.M.), Institut National de la Santé et de la Recherche Médicale U 418 (H.L.), and Equipes de Recherche et d’Innovations Technologiques ou Méthodologiques 0322 (P.C., G.R., A.E.-B., M.P.), Hôpital Debrousse, 29 rue Soeur Bouvier, 69322, Lyon, France

Address all correspondence and requests for reprints to: Michel Pugeat, Fédération d’Endocrinologie du Pole Est, Hôpital Neurologique Pierre Wertheimer, Aile A1 Bât HPGO, 59 Boulevard Pinel, 69394 Lyon Cedex 03, France. E-mail: michel.pugeat{at}chu-lyon.fr.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
SHBG is the specific plasma transport protein for sex steroid hormones in humans. Plasma SHBG concentration follows a gender dimorphism but varies with nutritional and hormonal status in both sexes. In addition, a genetic influence on SHBG in humans has recently been suggested by family studies. We investigated the relationship between a point mutation (D327N) in SHBG gene exon 8 that delays human SHBG half-life and a pentanucleotide repeat polymorphism [PNRP (TAAAA)n] in the SHBG gene 5' untranslated region that influences transcription in vitro, on the one hand, and SHBG levels on the other, in a population of 303 women referred for hirsutism. Of these patients, 154 (51%) met the criteria for polycystic ovary syndrome (PCOS) and 124 (41%) were overweight [body mass index (BMI) >= 25 kg/m2]. The two SHBG gene alleles for D327N substitution, wild-type (W) and variant (v), were identified by restriction fragment length polymorphism in the whole population, and the GeneScan method was used to identify PNRP alleles in 245 subjects. Six alleles of the pentanucleotide motif with six to 11 repeats were present in our population. Plasma SHBG concentration was related to PCOS status, non-SHBG-bound testosterone, BMI, fasting blood glucose level, fasting insulinemia, and D327N allele v. The v allele was associated with higher SHBG levels [36.9 ± 15.9 nmol/liter for W/v (n = 52) and 43.5 ± 3.5 nmol/liter for v/v (n = 2)] than was the wild-type W allele [31.1 ± 16.1 nmol/liter (n = 249); P = 0.039]. Multivariate analysis showed that BMI, PCOS status, and D327N polymorphism influenced plasma SHBG concentrations, each of these parameters contributing independently of the others.

Investigating the role of each allele of the TAAAA repeat polymorphism on SHBG levels was more complex because of the number of different genotypes (as many as 18 in our population) and the low frequency of some of them. Moreover, a strong disequilibrium linkage was found between D327N allele v and the eight-TAAAA repeat allele (P < 0.0001). This could mask the effect of the TAAAA repeat polymorphism on SHBG concentration in vivo. Nevertheless, SHBG levels in patients who were homozygous for six repeats (34.9 ± 16.2 nmol/liter; n = 21) were significantly (P = 0.043) higher than in nine-repeat homozygous patients (21.5 ± 13.0 nmol/liter; n = 8), and lay between the two for eight-repeat homozygous patients (28.5 ± 15.8 nmol/liter; n = 44). Delineating the precise role of this PNRP polymorphism will need further investigation in a large healthy population.

In summary, although BMI and PCOS status have a major influence on circulating SHBG levels in hirsute women, the present results support the notion that polymorphism(s) within the coding sequence and, potentially, in the regulatory sequence of the SHBG gene are associated with circulating SHBG levels and may represent part of the genetic background of sex steroid hormone activity in humans.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN HUMAN BLOOD, sex steroid hormones circulate mainly bound to SHBG, a 373-amino-acid glycoprotein of hepatic origin (1, 2). By its high specific binding affinity, SHBG influences the bioavailability of androgens and estrogens and thus their access to target cells (3). Considerable variations in serum SHBG concentration exist between individuals, depending on gender (4) and hormonal (5, 6, 7) and nutritional status (8, 9, 10), and can therefore affect sex steroid action. In many women with hirsutism, SHBG blood levels are low and do not correlate with androgen levels but are in negative correlation with body mass index (BMI) and fasting insulin level (11). The inhibitory effect of insulin on SHBG secretion, reported in vitro (12), suggests that increased insulin secretion could decrease SHBG concentration and increase biodisposal of circulating testosterone, with resulting excess hair growth (13). Interestingly, SHBG concentration has been proposed as a marker of hyperinsulinemia and/or insulin resistance (14); and indeed, in postmenopausal women, an increased risk of developing non-insulin-dependent diabetes mellitus (15, 16) and cardiovascular disease (17) has been found to be associated with decreased SHBG levels.

The SHBG concentration level remains relatively stable during adult life, with some variation with age, mainly related to variation in nutritional status (18). Irrespective, however, of these well documented causes of variation, a genetic influence on SHBG levels has been reported (19, 20, 21), suggesting that polymorphisms in the SHBG gene may alter either the production or the metabolism of the protein, thus contributing to individual variability in SHBG concentration in humans. Mutations and polymorphisms of the human SHBG gene have rarely been reported, but a few nucleotide variations have been described both in coding and regulatory sequences. We recently described two different mutations (P156L and {Delta}326), which led to an apparent absence of SHBG in one individual with symptoms of androgen excess (22), but found that these mutations were extremely rare, at least in the French female population. In contrast, a single-nucleotide polymorphism, found worldwide, which introduces an additional consensus site for N-glycosylation (D327N), is present in the SHBG gene exon 8 (23). This substitution leads to an increase in human SHBG half-life (24) and may be associated with higher SHBG levels in variant allele carriers. More recently, a pentanucleotide repeat polymorphism [PNRP (TAAAA)n] has been characterized in the SHBG gene 5'-flanking region, at approximately -800 bp from the transcription start site (25). This sequence is highly polymorphic, with several alleles of variable numbers of repeats. Furthermore, preliminary data have shown that it binds a 46-kDa nuclear factor and influences the transcriptional activity of human SHBG promoter in a hepatoma cell context (25).

The present study focuses on the respective influences of D327N substitution and TAAAA PNRP on circulating SHBG level in a large population of patients with complaint of hirsutism.


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

A population of 303 women (age = 26.8 ± 9.9 yr) referred to the Fédération d’Endocrinologie du Pôle Est, Hospices Civils de Lyon, for hirsutism was studied. At the time of blood sampling, patients were not taking any medication known to influence SHBG level, including birth control pills or antiandrogen treatment. Normal TSH and/or T4 levels allowed thyroid function to be assumed to be normal. Patients with severe oligomenorrhea (six to eight menses during the last year) and increased plasma concentration of at least one androgen level [non-SHBG-bound testosterone (non-SHBG-T) >5.5 ng/dl and/or androstenedione (A) >230 ng/dl] were classified as having polycystic ovary syndrome (PCOS). Patients were classified as lean or overweight when they had a BMI lower or higher than 25 kg/m2, respectively. Informed written consent was obtained from each subject. All patients were genotyped for D327N polymorphism and 245 for PNRP.

Assays

SHBG concentrations were measured in serum, using a specific immunoradiometric assay for human SHBG (SHBG-RIACT, CisBio International, Gif-sur-Yvette, France). Plasma concentrations of non-SHBG-T, T, A, and dehydroepiandrosterone sulfate (DHAS) were determined by specific RIAs as previously described (26, 27). Plasma glucose level (Glc) was determined by the Glc oxidase method and serum insulin (Ins) level by a double-antibody RIA (CisBio).

SHBG gene polymorphism analysis

Genomic DNA was extracted from white blood cells by a standard phenol-chloroform method and stored at 4 C until use. D327N substitution was screened for SHBG gene exon 8 by restriction fragment length polymorphism after PCR amplification, using oligonucleotide primers and reaction conditions, as previously described (24). Amplification products were digested with BbsI (New England Biolabs, Beverly, MA), run on a 2% agarose gel, and stained with ethidium bromide for visualization under UV light. Patients were then assigned to three genotypes: homozygous for the wild-type (W/W) or the variant allele (v/v) or heterozygotic (W/v). Subjects were genotyped for SHBG gene promoter PNRP after PCR amplification of a region encompassing six TAAAA repeats according to the published gene sequence (28). Primers were synthesized by Eurogentec (Seraing, Belgium), and reverse primer was labeled with 6-carboxyfluorescein (6-FAM): forward 5'-GAACTCGAGAGGCAGAGGCAGCAGTGA and reverse 5'-6-FAM-AGAAATCACCCACTCCCTGA. PCR conditions were 35 amplification cycles at 94 C for denaturation, at 63 C for annealing, and at 72 C for elongation for 1 min per step. Amplification products were then run on an ABI PRISM 373A DNA sequencer (Applied Biosystems, Foster City, CA), and alleles were assigned using the GeneScan analysis software (Applied Biosystems). The number of TAAAA repeats was calculated from the size of the PCR products by comparison with size standards (GeneScan-350 ROX, Applied Biosystems) and was also confirmed by automated sequencing of PCR products in four homozygous subjects (data not shown).

Statistical analysis

Frequencies for the various alleles were estimated by direct gene count, and the Hardy-Weinberg law was used to calculate expected genotype frequencies, which were then compared with the observed frequencies by {chi}2 test. The effects on SHBG levels of the following variables were studied: non-SHBG-T, T, A, DHAS, PCOS/non-PCOS, lean/overweight, BMI, fasting glucose and fasting insulin, and Glc/Ins ratio (29). For the D327N polymorphism, the patients were assigned to three groups according to the D327N genotypes W/W, W/v, and v/v. Because of the low frequency of the v allele, only two homozygous v/v patients were found in our population. Thus, for statistical analysis, the patients were classified in two groups according to the presence of the v allele: W (for W/W genotype) or v (for v/W or v/v genotypes). The number of v alleles was used to include D327N polymorphism as a quantitative variable in multivariate linear regression analysis. For the PNRP, six, seven, eight, nine, 10, or 11 TAAAA repeats were observed in our population, which should theoretically have resulted in 21 genotypes; however, no patients with genotypes 7/7, 7/11, or 11/11 were in fact found in our population. Thus, according to their TAAAA repeat genotype, the patients could be assigned to one of the following 18 groups: 6/6, 6/7, 6/8, 6/9, 6/10, 6/11, 7/8, 7/9, 7/10, 8/8, 8/9, 8/10, 8/11, 9/9, 9/10, 9/11, 10/10, or 10/11. The elevated number of TAAAA repeat genotypes and the low frequency of some of them conferred a low statistical power to analyses encompassing all these genotypes. Because no consensual model has been validated to explain the role of the number of pentanucleotide repeats on gene transcription at the molecular level (30, 31, 32), we explored the possible effect on SHBG levels of individual alleles in different ways. First, we studied the effect of the most frequent alleles in a subpopulation composed only of the homozygous subjects observed in our population: 6/6, 8/8, and 9/9. Then, we studied the effect of the homo- or heterozygous presence or the absence of each possible allele, resulting, for example, for the six TAAAA repeats, in three groups of patients: 6/6, 6/x, and x/x (x != 6). For some statistical analyses, the patients were classified in two groups according to the presence of the TAAAA repeats allele: 6 (for 6/6 or 6/x genotypes) or x (for x/x genotype). The number of the given allele (0, 1, or 2) was used as a quantitative variable in multivariate linear regression analysis. The effects of these variables on SHBG levels were first studied with univariate methods: Student’s t test or one-way ANOVA for the qualitative variables and linear regression for the quantitative variables. Because several of the variables might represent the same biological phenomenon, we systematically explored the relationships between all the variables by linear regression between quantitative variables, by Student’s t test or one-way ANOVA between a quantitative and a qualitative variable, and by {chi}2 test between two qualitative variables. The effects of the various variables on SHBG levels were also studied by multifactor ANOVA for the qualitative variables and by multivariate linear regression. The variables were included or excluded according to their interrelationships (colinearity) and to the biological phenomenon or phenomena they represented. Stepwise procedures were used to determine the relevant variables for inclusion in the multivariate linear regression models. Inclusion threshold was P <= 0.05 and exclusion threshold P > 0.10. All the results are presented as mean ± SD. P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Characteristics of the population

Of 303 hirsute women, 154 (51%) were classified as PCOS and 149 (49%) as non-PCOS according to their menstrual status and androgen levels, and 179 (59%) were classified as lean and 124 (41%) as overweight according to BMI (< or >= 25 kg/m2, respectively). PCOS status and lean/overweight status were found not to be statistically related in our population ({chi}2; P = 0.353).

Table 1Go gives the mean ± SD values of hormonal and metabolic parameters in these patients according to their lean/overweight and PCOS status.


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  		TABLE 1. Hormonal and metabolic parameters in PCOS, non-PCOS, lean, and overweight hirsute women

 
PCOS and non-PCOS patients had similar mean BMIs. PCOS patients had lower SHBG levels and higher non-SHBG-T and A levels than non-PCOS patients, whereas all other variables were not significantly different between the two groups. The Glc/Ins ratio, shown to be a useful tool to assess insulin sensitivity in PCOS women (29), did not differ between our PCOS and non-PCOS subjects. Overweight patients had lower SHBG levels and higher non-SHBG-T, A, Glc, Ins, and Glc/Ins values compared with lean patients.

Influence of hormonal and metabolic parameters and SHBG gene polymorphisms on plasma SHBG concentration

Table 2Go shows the influence of each parameter on plasma SHBG concentration (column 1) and the relationships between the other parameters.


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  		TABLE 2. Relationships between plasma SHBG levels, hormonal and metabolic parameters, and SHBG gene polymorphisms in 303 hirsute women

 
D327N allele frequencies and genotype distribution in hirsute women

BbsI-digested PCR products of human SHBG gene exon 8 (290 bp) resolved into two fragments of 223 and 67 bp in W/W subjects, into three fragments of 290, 223, and 67 bp in W/v subjects, and were not digested in v/v subjects (Fig. 1Go). In our population, 82.2% (n = 249) had a W/W genotype, 17.2% (n = 52) were W/v, and two unrelated patients were homozygous for the variant SHBG allele, in agreement with the Hardy-Weinberg law ({chi}2 = 0.179; P > 0.05). Frequencies were 0.908 and 0.092 for the wild-type and the variant allele, respectively (Table 3Go).



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  		FIG. 1. Restriction fragment length polymorphism analysis of SHBG exon 8, from three individual genomic DNA samples. PCR products were digested with BbsI to detect D327N mutation. Digestion resulted in two bands (223 and 67 bp) in W/W, three fragments (290, 223, and 67 bp) in W/v, and no digested band (290 bp) in v/v. Fragments were resolved by electrophoresis on 2% agarose gel and stained by ethidium bromide. DNA markers and their sizes (bp) are indicated on the left.

 

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  		TABLE 3. Allele frequencies and genotype distribution of the D327N polymorphism in hyperandrogenic women (n = 303)

 
Influence of D327N polymorphism on plasma SHBG concentration

Plasma SHBG level differed according to D327N genotype, whether in W/W (31.1 ± 16.1 nmol/liter; n = 249), W/v (36.9 ± 15.9 nmol/liter; n = 52), or v/v (43.5 ± 3.5 nmol/liter; n = 2) patients (one-way ANOVA: P = 0.039 and P = 0.010 for trend, according to the number of D327N v alleles). Among the metabolic parameters studied, D327N polymorphism was related to fasting glucose level (4.94 ± 0.70 mmol/liter in W patients vs. 4.66 ± 0.39 mmol/liter in v patients; Student’s t test: P = 0.042) and to Glc/Ins ratio (0.52 ± 0.46 in W/W patients vs. 0.74 ± 0.57 in W/v patients; Student’s t test: P = 0.036).

Distribution of pentanucleotide repeat alleles in hirsute women

The various alleles for the PNRP in the 5'-flanking region of the SHBG gene were assigned by GeneScan analysis after PCR amplification in 245 patients. Amplified products resolved into fragments of 135–160 bp with an increment of 5 bp, revealing the existence of at least six alleles with between six and 11 TAAAA repeats. Alleles with six, eight, and nine repeats were the most common, with respective frequencies of 0.278, 0.422, and 0.186, in contrast to the low frequency of alleles with seven and 11 repeats (0.033 and 0.020, respectively). The PNR allele frequencies were in Hardy-Weinberg equilibrium, the observed distribution being in good agreement with that expected ({chi}2 = 14.71; P > 0.05; Table 4Go).


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  		TABLE 4. Observed and expected distributions of patients and mean ± SD SHBG levels according to TAAAA repeat genotypes in the 5' flanking region of the SHBG gene in hirsute women (n = 245)

 
PNRP and SHBG level

Differences in plasma SHBG levels between the 18 different TAAAA genotypes were not significant on one-way ANOVA in our population (P = 0.298). However, because similar polymorphisms in the 5' region of other genes have been shown to influence transcription (30, 31, 32), and because no consensual model has been yet described to explain the underlying molecular mechanism, the effect of some genotypes and interactions between alleles could not be excluded. Thus we explored further the possible role of the various alleles that could be masked in the one-way ANOVA involving all alleles. Comparing plasma SHBG levels in the subpopulation of homozygous patients revealed a significant difference among the various homozygous genotypes (one-way ANOVA; P = 0.010). Post hoc comparisons (Fisher’s protected least-significant test) revealed that the overall difference was due to SHBG levels being higher in the two 10/10 patients (60.0 ± 14.1 nmol/liter) than in the 6/6 (34.9 ± 16.2 nmol/liter; n = 21; P = 0.033), 8/8 (28.5 ± 15.8 nmol/liter; n = 44; P = 0.007), and 9/9 patients (21.5 ± 13.0 nmol/liter; n = 8; P = 0.003) and higher in the 6/6 than in the 9/9 patients (P = 0.043). None of the hormonal and metabolic variables studied were found to be related to any TAAAA variables.

Relationship between D327N and PNR polymorphisms

A linkage disequilibrium between the two SHBG gene polymorphisms was demonstrated by the highly significant relationship between the presence of the D327N v allele and eight TAAAA repeats; 93% (instead of the 66% expected) of the patients bearing the D327N v allele had at least one allele with eight TAAAA repeats ({chi}2 test: P < 0.0001). The only two subjects homozygous for the v allele were also homozygous for the allele with eight TAAAA repeats.

Multivariate analysis of the effects of hormonal, metabolic, and SHBG gene polymorphism on SHBG level

Examination of the individual relationships between the various parameters studied (Table 2Go) led us to perform multivariate analysis to find the independent SHBG level predictors. Qualitative variables that were significantly related to SHBG levels in monofactorial analysis (PCOS status, lean/overweight status, or presence of allele v of the D327N polymorphism) were included in multifactor ANOVA. All three variables induced a significant effect on SHBG level (three-way ANOVA: PCOS, P = 0.016; allele v, P = 0.009; lean/overweight, P < 0.0001). These effects were independent, because none of the statistical interactions were significant (Fig. 2Go).



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  		FIG. 2. Influence of PCOS, weight status, and D327N polymorphism on serum SHBG concentration in hirsute women. SHBG concentrations are given as mean ± SD. The number of patients in each group is indicated at the bottom of the corresponding column.

 
To further study the potential predictors of SHBG level, multivariate analysis was performed by multiple linear regression with SHBG level as the dependant variable. Because of obvious colinearity between some of the variables, the independent variables to enter in the multiple regression were selected according to their individual relationships (Table 2Go) and their physiopathological signification. Three kinds of parameter could be identified: androgenic, metabolic, and genetic. Among the androgenic parameters, non-SHBG-T and PCOS status were related to SHBG in univariate analysis. The reason for the relationship between non-SHBG-T and SHBG is, however, debatable. Because the non-SHBG-T assay is based on the precipitation of SHBG and SHBG-bound T by ammonium sulfate (26), the relationship between SHBG and non-SHBG-T could be due to a methodological rather than a physiological link. For this reason we chose to include PCOS status rather than non-SHBG-T in the multivariate analysis. The metabolic parameters BMI, Glc, Ins, and Glc/Ins ratio were all found to be related on univariate analysis, BMI being the most significant predictor of SHBG level. Among the genetic variables, the number of D327N v alleles and eight-TAAAA repeat alleles were found to be closely related on univariate analysis, the number of D327N v alleles being related to SHBG level. It was therefore decided to include PCOS status (non-PCOS = 0, PCOS = 1), BMI, and the number of D327N v alleles in the multivariate linear regression model. All three parameters were included in the model by a stepwise procedure, giving rise to a significant prediction, each of the parameters contributing significantly to the model, as indicated in Table 5Go.


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  		TABLE 5. Model fitting results for stepwise multiple linear regression of SHBG levels according to BMI, PCOS status, and D327N polymorphism in hirsute women

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Plasma concentrations of SHBG are highly variable in the general healthy population (33). In addition to well known factors that regulate circulating SHBG (1, 2), some studies have emphasized the influence of genetics, showing that SHBG binding capacity is less variable within groups of brothers than among nonbrothers (19). In addition, a significant heritability (genetic and family-environmental) of SHBG blood levels has been demonstrated (20, 21). However, only a few polymorphisms of the SHBG gene have as yet been reported. A single-nucleotide polymorphism with worldwide distribution (34) introduces an additional consensus coding sequence for an N-glycosylation site at codon 327. In our population of hirsute patients, the allele frequency for this polymorphism was as expected (24, 34, 35, 36). We found that hirsute patients with D327N have significantly increased SHBG levels, this result being independent of the effect of both PCOS status and BMI, suggesting that the additional carbohydrate chain on the SHBG variant molecule may indeed delay metabolic clearance from plasma, thereby contributing to an increase in SHBG levels as we reported earlier in an animal model (24).

Recently, Becchis et al. (37) have reported that the v allele is more frequently observed in patients bearing positive estrogen and progesterone receptor breast tumors and in breast cancer patients diagnosed when over 50 yr of age, suggesting a link between the estrogen dependence of breast cancer and the presence of the variant SHBG. However, this remains controversial, inasmuch as a similar recent study in Polish and Nordic populations failed to reach the same conclusions (38).

In characterizing the upstream region of the human SHBG promoter, Hogeveen et al. (25) found a pentanucleotide repeat (TAAAA) within an alu sequence that binds a 46-kDa liver-enriched protein. These authors reported that the number of TAAAA repeats was highly variable within the normal population and, more importantly, affected the transcriptional activity of the SHBG promoter in vitro. The sequence had a silencing effect on transcription, which was further dependent on the presence of an SP-1 binding site downstream of the PNR.

In our population of hirsute women, the SHBG gene had at least six alleles with between six and 11 TAAAA repeats, with six, eight, and nine repeats being the most common. Investigating the role of each allele of the TAAAA repeat polymorphism on SHBG levels was more complex than for D327N because of the number of the different genotypes (as many as 18 in our population) and the low frequency of some of them. One-way ANOVA including all the PNR genotypes did not reveal any significant effect. Because the combinations of the various alleles might have masked the effects of some of them, we studied their effects in homozygous patients and found significant differences in SHBG levels between 6/6 and 9/9 homozygous subjects, with 8/8 subjects having levels between the two. These results should be interpreted with caution. Indeed, they diverge from those found in vitro by Hogeveen et al. (25), who reported lower transcription activity in a human SHBG promoter-luciferase reporter construct containing six TAAAA repeats than in similar reporters construct containing seven to 10 TAAAA repeats, when tested in a human hepatoma cell line (HepG2). This apparent discrepancy could be related to the fact that HepG2 cell lines, although secreting SHBG, have some embryonic characters and also that a D69A mutation of the hepatocyte nuclear factor-4{alpha} (HNF-4{alpha}) protein, which may impair the function of this crucial transcription factor for the SHBG gene, has been recently reported (39). We prefer the hypothesis that the strong disequilibrium linkage between the D327N v allele and the eight-TAAAA repeat in our population of hirsute women unmasked the respective effect of these polymorphisms on SHBG levels. The significance of these polymorphisms would be better investigated in a population of normal subjects.

Adenine- and thymine-rich pentanucleotide repeats are frequently found along the human genome. Variations in the number of PNRs in several human genes have already been reported, such as in promoter genes for apolipoprotein(a) [APO(a)] (40), glutathione S-transferase (GSTP1) (41), fatty acid ethyl ester synthase-III (FAEES-III) (42), and P450 side chain cleavage enzyme (CYP11A1) (32), as well as in introns such as tumor-suppressor gene p53 (43) or the ß-globin gene (44, 45). As for the SHBG gene, these PNRs have been mapped several times in the close vicinity of alu sequences (40, 41, 44, 46). The repeats are highly variable in number, depending on the gene considered, from four to 11 for CYP11A1, APO(a), and ß-globin genes to more than 20 in GSTP1 and FAEES-III. The exact functions of these sequences are as yet unknown, but there is some evidence for their being involved in the regulation of gene transcription. For example, in the GSTP1 promoter, an ATAAA repeat polymorphism has been mapped to a boundary that separates methylated and unmethylated regions, gene expression being directed by the unmethylated region (41).

The involvement of a TTTTA repeat within the APO(a) gene promoter in the control of lipoprotein(a) [Lp(a)] levels has been extensively studied. Most of these studies have reported a significant influence on Lp(a) plasma concentration (30, 31, 47). In contrast, in vitro studies of the transcription activity of promoter fragments with different numbers of repeats have produced discordant results on HepG2 cells (30, 40). Interestingly, the frequency of the PNRP allele and its relationship with Lp(a) concentration are heterogeneous among populations of different origins. Although this PNRP is partly associated with Lp(a) concentration variation in Caucasians, this influence is absent in Black Africans, suggesting that other promoter sequences could be involved (31). Our data show that six to nine repeats of the SHBG promoter TAAAA motif were associated with basal SHBG level. Similar associations have been reported for the TTTTA repeats of the APO(a) gene promoter and Lp(a) concentrations in Caucasians (31). In our population of hirsute patients, we failed to find any significant difference in non-SHBG-T levels between carriers for the different SHBG alleles. One explanation could be that their mean SHBG level, even with significant differences between genotypes, remained within reference limits for normal women. These results suggest that hirsutism is probably related to increases in androgen secretion and skin metabolism and that a decreased SHBG level is not enough to produce increased hair growth. This is further corroborated by the fact that many obese (48) or type-2 diabetic women [Cousin, P., and M. Pugeat, unpublished data from the MEDIA (Ménopause et Diabète) study] show no evidence of excess hair growth, although a low SHBG level is generally observed with a highly significant correlation to fat mass.

Our population mainly comprised white French women, who may have shared a similar genetic background; thus it cannot be excluded that some unknown common genetic factors may be influencing SHBG gene expression in these patients. Indeed, it has been suggested that several interacting loci are linked to SHBG levels (21); a large scan analysis identified an interesting linkage between region 1q44 and SHBG levels in both Black and white subjects, along with several other loci that may also have linkage, suggesting that many genes may regulate SHBG concentration (49). It would therefore be of prime interest to study the distribution of SHBG gene polymorphisms, and possible syntenic genetic factors associated with the SHBG locus, in larger subgroups of populations of different ethnic backgrounds.

In conclusion, the existence of significant associations of polymorphisms within coding sequences, and potentially within regulatory sequences, of the SHBG gene on the one hand and circulating SHBG levels on the other suggest that polymorphism may be a part of the genetic background of SHBG variation in humans. Because several diseases, including osteoporosis (50, 51), cognitive dysfunction (52), type-2 diabetes (53), cardiovascular disease (17, 54), and breast cancer (55) have been linked to plasma SHBG levels, probably through androgen as well as estrogen availability, additional studies need to be carried out to achieve a better understanding of the part played by SHBG among risk factors for general public health disorders.


   Acknowledgments
 
We are indebted to Dr. Geoffrey L. Hammond and Kevin N. Hogeveen for helpful discussions and to Dr. Henri Déchaud and Dr. Francine Claustrat for hormone assays. The secretarial assistance of Monique Benas and the technical help of Christine Baret and Claude Ducros were also greatly appreciated.


   Footnotes
 
This work was supported by a generous grant from the Fondation de France; P.C. was supported by a postdoctoral fellowship from the Ligue Nationale Contre le Cancer.

This work was presented in part at the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, 2002 (poster P607).

Abbreviations: A, Androstenedione; APO, apolipoprotein; BMI, body mass index; DHAS, dehydroepiandrosterone sulfate; Glc, glucose; GSTP, glutathione S-transferase; Ins, insulin; Lp, lipoprotein; PCOS, polycystic ovary syndrome; PNRP, pentanucleotide repeat polymorphism; T, testosterone; v, variant; W, wild-type.

Received October 4, 2002.

Accepted October 31, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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