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Departments of Endocrinology (J.I.B.-C., F.A.-B., M.L.-R., J.S., H.F.E.-M.) and Molecular Genetics (J.L.S.M.), Hospital Ramón y Cajal, E-28034 Madrid, Spain; and Division of Endocrinology and Metabolic Diseases (P.M.), University of Verona, I-37126 Verona, Italy
Address all correspondence and requests for reprints to: Héctor F. Escobar-Morreale, M.D., Ph.D., Department of Endocrinology, Hospital Ramón y Cajal, Carretera de Colmenar Km 9'1, E-28034 Madrid, Spain. E-mail: hescobarm.hrc{at}salud.madrid.org.
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Abstract |
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Objective: The objective was to study these mutations in PCOS.
Design: The design was a case-control study.
Setting: The study was conducted in an academic hospital.
Participants: A total of 116 PCOS patients and 76 nonhyperandrogenic controls participated.
Main Outcome Measures: Genotype distributions and influence of genotypes on clinical and biochemical variables and, in 28 patients and 12 controls, estimates of 11ßHSD oxoreductase activity were the main outcome measures.
Results: Four controls and five patients presented three of four mutant alleles in H6PD R453Q and HSD11B1 83557insA, which is the genotype observed in some subjects with CRD. Estimates of 11ßHSD oxoreductase activity were measured in six of these nine women, ruling out CRD. Moreover, H6PD R453Q and HSD11B1 83557insA genotypes, either separately or in combination, did not influence 11ßHSD oxoreductase activity. The distribution of H6PD R453Q genotypes (R/R, R/Q, and Q/Q) was different in patients and controls (42% of controls and 63% of PCOS patients were R/R; 53% of controls and 31% of PCOS patients were R/Q; and 5% of controls and 6% of PCOS patients were Q/Q; 
2 = 9.1; P = 0.011). Patients homozygous for R453 alleles presented with increased cortisol and 17-hydroxyprogesterone levels, compared with carriers of Q453 alleles, but these differences were not observed in controls. On the contrary, HSD11B1 83557insA genotypes were not associated with PCOS and did not influence any phenotypic variable.
Conclusions: Digenic triallelic genotypes of the H6PD R453Q variant and HSD11B1 83557insA mutation do not always cause CRD. On the contrary, the H6PD R453Q variant is associated with PCOS and might influence its phenotype by influencing adrenal activity.
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Introduction |
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In CRD, the oxoreductase activity of 11ßHSD1 is impaired, reducing the generation of cortisol (F) from cortisone (1). Draper et al. (1) hypothesize that this occurs because the mutations in HSD11B1 result in reduced enzyme expression and because the mutations in H6PD impair the generation of reduced nicotinamide adenine dinucleotide phosphate within the lumen of the endoplasmic reticulum, because reduced nicotinamide adenine dinucleotide phosphate might be essential in driving 11ßHSD1 oxoreductase activity (1).
Given that the phenotype of women with CRD resembles that of polycystic ovary syndrome (PCOS) (2), and 11ßHSD1 activity may be reduced in PCOS patients (3), H6PD and HSD11B1 can be considered candidate genes to explain the inheritance of PCOS.
In the present study, we have genotyped our series of PCOS patients and nonhyperandrogenic controls for CRD mutations.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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One hundred sixteen PCOS patients and 76 nonhyperandrogenic controls were included. PCOS was defined by oligoovulation, clinical and/or biochemical hyperandrogenism, and exclusion of hyperprolactinemia (serum prolactin < 24 ng/ml), nonclassic congenital adrenal hyperplasia [ACTH-stimulated 17-hydroxyprogesterone (17OHP) levels <10 ng/ml (4)], and androgen-secreting tumors (5). In these patients, evidence for oligoovulation was provided by chronic oligomenorrhea or amenorrhea, by luteal phase progesterone less than 4 ng/ml, or by basal body temperature charts.
The control group was composed of healthy lean female volunteers and consecutive patients attending the clinical practice of one the authors (H.F.E.-M.) for treatment of obesity. None of the controls had signs or symptoms of hyperandrogenism, menstrual dysfunction, or history of infertility. The degree of obesity was established depending on the body mass index (BMI) of the women and classified as being lean (BMI < 25 kg/m2), overweight (BMI, 25–29.9 kg/m2), or obese (BMI 
30 kg/m2) (6).
Data from some patients and controls, regarding different aspects of the pathophysiology of hirsutism, have been previously published (7). The patients and controls had not taken hormonal medications, including contraceptive pills, for the last 6 months. All the subjects were Caucasian.
The ethics committee of the Hospital Ramón y Cajal approved the study, and informed consent was obtained from each patient and control.
Hormone profiles
Studies were performed between d 5 and 10 of the menstrual cycle, or during amenorrhea, after excluding pregnancy by appropriate testing. Hirsutism was quantified by a modified Ferriman-Gallwey score. Between 0800 and 0900 h, after a 12-h overnight fast, an indwelling iv line was placed in a forearm vein, and after 15–30 min, basal blood samples were obtained for the measurement of total testosterone, dehydroepiandrosterone sulfate (DHEAS), SHBG, glucose, and insulin. Immediately after taking basal samples, a 250-µg iv bolus of 1–24 ACTH (Synacthen, Ciba-Geigy, Basel, Switzerland) was injected, and blood samples were obtained at 0 and 60 min for the measurement of F, 11-deoxycortisol (S), 17OHP, and 
4-androstenedione (4-A). In 27 controls, the ACTH test was not performed. Samples were immediately centrifuged, and serum was separated and frozen at –20 C until assayed.
The technical characteristics of the assays employed for hormone measurements have been reported elsewhere (8, 9). The free testosterone concentration was calculated as described by Vermeulen et al. (10). Insulin resistance in the fasting state was estimated from glucose, and insulin levels using homeostasis model assessment (HOMAIR) (11). In 28 patients and 12 controls, 11ßHSD oxoreductase activity was estimated from timed urine samples obtained in a retrospective nonrandom fashion. The ratios of tetrahydrocortisol (THF) plus 
-THF (alloTHF) to tetrahydrocortisone (THE) were measured by the method of Schmidt et al. (12). This method is based on anion-exchange chromatographic separation, purification on diethylaminoethyl Sephadex A-25 microcolumns, and final analysis of the individual steroids by capillary column gas-liquid chromatography.
DNA analysis
Genomic DNA was obtained from whole blood using a Nucleon BACC3 DNA isolation kit (Amersham, Buckingham, UK). Regarding CRD mutations, subsequent analysis by Draper et al. (13) showed that the H6PD 620ins29bp621 is extremely rare in the general population and that the two mutations in intron 3 of HSD11B1 are in complete linkage disequilibrium, reducing the genotype analysis to the combination of H6PD R453Q and HSD11B1 83557insA.
PCR and restriction fragment length polymorphism assays were used. For the H6PD R453Q variant, genomic DNA was amplified using the following primers: forward, 5'-ACT ACG CCT ACA GCC CTC TGC-3', and reverse, 5'-CCA GGA GGC CAG CAA GTT CTC-3'. A mismatch in the forward primer (underlined) was used to create a PstI target when the rare Q453 allele was present. The HSD11B1 83557insA variant was amplified with primers 5'-GCT GTT TCT CTT ACC TCC TCC-3' and 5'-TGA GCA TGT CTA GTC CTC CTG-3' and digested with XcmI, which cuts the PCR product only when the insertion is present.
Power analysis
A priori power analysis of the differences in genotype frequencies between PCOS patients and controls was conducted using the G*Power software (14). Our sample size permitted the detection of effect sizes for the difference between frequencies (w) of 0.20 and 0.22 for the 2 test with 1 or 2 degrees of freedom, respectively. By convention, effects sizes for the differences between frequencies are considered very small or trivial when less than 0.10, small from 0.10–0.30, moderate from 0.30–0.50, and large when greater than 0.50 (15). Therefore, our sample size permitted the detection of small differences in the genotype frequencies between PCOS patients and controls. However, very small differences may have not been detected in our study because of the relatively small sample size.
Statistical analysis
Results are expressed as means ± SD. The Kolmogorov-Smirnov statistic was applied to continuous variables. Logarithmic or square root transformations were applied as needed to ensure normal distribution of the variables. The mean values were compared by general linear models in which age was introduced as a covariate to correct for the difference in age between patients and controls. To evaluate the association between discontinuous variables, we used the 2 test and Fisher’s exact test as appropriate. Logistic regression was used as described below. Analyses were performed using SPSS10 for the Macintosh (SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.
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Results |
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Patients were younger compared with controls (25 ± 6 vs. 31 ± 8 yr; P < 0.001), but there were no differences in the mean BMI (29.8 ± 8.3 vs. 29.1 ± 7.8 kg/m2; P > 0.2) or in the distribution of both groups depending on the degree of obesity [for PCOS, n = 42 (36%) for lean, n = 26 (22%) for overweight, and n = 48 (41%) for obese; for controls: n = 28 (37%) for lean, n = 15 (20%) for overweight, and n = 33 (43%) for obese; 2 = 0.205; P > 0.2).
The comparison of clinical and biochemical variables of PCOS patients and nonhyperandrogenic controls, and the influence of the degree of obesity in these variables, are shown in Table 1
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4-A, DHEAS levels, fasting insulin, and HOMAIR and decreased SHBG levels.
The degree of obesity influenced serum total and free testosterone levels, SHBG and basal 4-A concentrations, ACTH-stimulated F, S, and 4-A levels, fasting glucose and insulin concentrations, and HOMAIR (Table 1
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Prevalence of the genotype characteristic of CRD in PCOS patients and controls and influence of the H6PD R453Q and HSD11B1 83557insA variants on the (THF + alloTHF)/THE ratio
Only four controls (5%) and five patients (4%) presented three of four mutant alleles (no subject had four mutant alleles) of the H6PD R453Q and HSD11B1 83557insA variants, which is the genotype characteristic of patients with CRD, suggesting that this combination of mutations is relatively common in women but is not associated specifically with PCOS.
Moreover, the (THF + alloTHF)/THE ratios were available for six of these women (four patients and two controls). None of these women had a (THF + alloTHF)/THE ratio below or close to 0.05, which is the cutoff value for diagnosing CRD (16), suggesting that still unknown factors are needed in addition to the bigenic triallelic mode of inheritance to induce this rare disorder. Moreover, their (THF + alloTHF)/THE ratios were not statistically different compared with those of the women carrying other H6PD and HSD11B1 genotype combinations (Fig. 1).
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). Therefore, we were unable to demonstrate that the different H6PD and HSD11B1 genotypes studied here influenced 11ßHSD oxoreductase activity, yet it is important to highlight that our study was not designed for this purpose and that the small sample size analyzed for this activity precluded any conclusion in this regard, other than ruling out CRD in the women presenting three of four mutant alleles of the H6PD R453Q and HSD11B1 83557insA variants in our series.
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The distribution of H6PD R453Q genotypes (R/R, R/Q, and Q/Q) was different in patients and controls [42% of controls (n = 32) and 63% of PCOS patients (n = 73) were R/R; 53% of controls (n = 40) and 31% of PCOS patients (n = 36) were R/Q; and 5% of controls (n = 4) and 6% of PCOS patients (n = 7) were Q/Q; 2 = 9.1; P = 0.011].
On the contrary, HSD11B1 83557insA alleles were distributed similarly in PCOS patients and controls [71% of controls (n = 54) and 66% of PCOS patients (n = 76) were wild-type/wild-type; 26% of controls (n = 20) and 32% of PCOS patients (n = 37) were wild-type/83557insA; and 3% of controls (n = 2) and 3% of PCOS patients (n = 3) were 83557insA/83557insA; 2 = 0.69; P > 0.2].
When women were classified as having zero, one, two, or three variant alleles of H6PD R453Q and HSD11B1 83557insA, no differences were found among PCOS patients and controls [28% of controls (n = 21) and 41% of PCOS patients (n = 47) had four wild-type alleles; 55% of controls (n = 42) and 43% of PCOS patients (n = 50) had one variant allele; 12% of controls (n = 9) and 12% of PCOS patients (n = 14) had two variant alleles; and 5% of controls (n = 4) and 4% of PCOS patients (n = 5) had three variant alleles; 2 = 3.66; P > 0.2].
Influence of the H6PD and HSD11B1 genotypes on clinical and biochemical variables
Given that obesity influenced some of the variables studied here, BMI in addition to age was introduced as a covariate in these analyses. When considering patients and controls as a whole, women homozygous for H6PD R453 alleles presented, compared with women carrying one or two H6PD Q453 alleles, with a statistically significant increase in hirsutism scores (10 ± 8 vs. 6 ± 7; P = 0.016), basal F (16 ± 5 vs. 14 ± 5 µg/dl; P = 0.012), basal 17OHP (1.0 ± 0.6 vs. 0.7 ± 0.4 ng/ml; P = 0.005), and ACTH-stimulated 17OHP (2.9 ± 1.3 vs. 2.3 ± 0.9 ng/ml; P = 0.004) and a tendency (P < 0.10) toward an increase in basal 4-A concentrations (3.6 ± 1.5 vs. 3.2 ± 1.1 ng/ml; P = 0.096) and ACTH-stimulated F levels (30 ± 7 vs. 28 ± 8 µg/dl; P = 0.072).
Some of these findings were not actually unexpected given that R453 alleles were more frequent in PCOS patients, but when restricting the analysis to these women, the increase observed in basal F and in basal and ACTH-stimulated 17OHP levels in subjects homozygous for H6PD R453 alleles compared with heterozygous and homozygous H6PD Q453 carriers persisted (Table 2). On the contrary, such differences did not reach statistical significance when studying the control group separately (Table 2). These results point to an effect of the H6PD R453Q variant on adrenal steroidogenesis only in PCOS patients.
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) or when considering PCOS patients and controls as a whole (data not shown).
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To evaluate the possible interaction between both polymorphisms, logistic regression was used. The dependent variable was coded 1 for PCOS and 0 for nonhyperandrogenic controls and, for each polymorphism, having the wild-type genotype or carrying one or two mutated alleles were introduced as independent variables using the backward stepwise (likelihood ratio) method. The degree of obesity was introduced as a covariate.
The model retained only the H6PD R453Q polymorphism, although this variant contributed only in a modest 5.6% to having PCOS or being a control in our series (R2 = 0.056; 2 = 8.060; P = 0.005). Carrying one or two H6PD Q453 alleles protected against PCOS (odds ratio = 0.43; 95% confidence interval, 0.24- 0.77), whereas carrying one or two HSD11B1 83557insA alleles did not have any effect (odds ratio = 1.23; 95% confidence interval, 0.65–2.32), nor did the interaction between H6PD R453Q and HSD11B1 83557insA genotypes (odds ratio = 0.90; 95% confidence interval, 0.25–3.21).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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However, our present results also demonstrate that digenic triallelic genotypes of the H6PD R453Q variant and the HSD11B1 83557insA mutation do not result by themselves in CRD. Of the nine women with these genotypes in our series, four were controls and did not have symptoms of CRD. Moreover, of the six women (two controls and four PCOS patients) presenting with digenic triallelic genotypes in whom 11ßHSD oxoreductase activity was estimated by the urinary (THF + alloTHF)/THE ratios, none had ratios below or even near 0.05, and therefore none of them actually showed CRD.
This is not really unexpected, considering that to date only 11 cases of CRD have been diagnosed (16), and it was highly unlikely that nine of the 192 women in our series, and 17 of the 696 women in the series by Draper et al. (13), had this extremely rare disorder. Therefore, other still unknown factors, in addition to digenic triallelic genotypes of the H6PD R453Q variant and the HSD11B1 83557insA mutation, must play a major role in the pathogenesis of CRD (16).
Furthermore, we have not been able to detect any influence of the H6PD R453Q variant and the HSD11B1 83557insA mutation, either separately or in combination, on 11ßHSD oxoreductase activity as estimated by the urinary (THF + alloTHF)/THE ratio. However, this result has to be considered with caution because our present study was not designed for this purpose, and the small sample size together with the multiple genotype combinations present, might have obscured any small influence of the H6PD R453Q variant and the HSD11B1 on this activity.
The H6PD Q453 alleles are less frequent in PCOS patients compared with nonhyperandrogenic controls and might influence adrenal activity. Compared with PCOS patients homozygous for wild-type alleles, patients homo- or heterozygous for H6PD Q453 alleles had lower concentrations of F and adrenal steroid precursors, particularly, lower basal and ACTH-stimulated 17OHP levels. Considering that adrenal hyperactivity is a frequent finding in PCOS (17, 18), the H6PD R453Q variant may play a modifying role in the PCOS phenotype by ameliorating adrenal hyperactivity in carriers of Q453 alleles. However, according to the results of the logistic regression analysis, H6PD R453 alleles contributed only modestly to PCOS, in conceptual agreement with a polygenic etiology for this disorder, in which favoring and protective variants interfere with environmental factors resulting in the PCOS phenotype (19).
Our data also suggest that the effects of the H6PD R453Q variant on PCOS are independent of the HSD11B1 83557insA mutation, given that the genotypes of the latter were equally distributed in patients and controls, did not influence any phenotypic variable, and in the logistic regression model, had no direct effect or interaction with the H6PD R453Q variant on the occurrence of PCOS.
In conclusion, digenic triallelic genotypes of the H6PD R453Q variant and HSD11B1 83557insA mutation are not the sole cause of CRD, and other unknown factors must be involved given that PCOS patients and nonhyperandrogenic controls presenting with these genotype combinations do not show evidence of this rare disorder. Yet the H6PD R453Q variant is associated with PCOS and might influence its phenotype by influencing adrenal activity.
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Acknowledgments |
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Footnotes |
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Results from this work were presented at the 86th Annual Meeting of The Endocrine Society, New Orleans, LA, June 2004.
First Published Online April 12, 2005
Abbreviations: 4-A, 4-Androstenedione; BMI, body mass index; CRD, cortisone reductase deficiency; DHEAS, dehydroepiandrosterone sulfate; F, cortisol; HOMAIR, homeostasis model assessment for insulin resistance; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; 17OHP, 17-hydroxyprogesterone; PCOS, polycystic ovary syndrome; S, 11-deoxycortisol; THE, tetrahydrocortisone; THF, tetrahydrocortisol.
Received August 2, 2004.
Accepted April 4, 2005.
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