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BRIEF REPORT |
Departments of Internal Medicine (P.S., M.J.H.J.D., A.W.v.d.B., J.W.K., H.A.P.P., F.H.d.J., S.W.J.L.), Reproduction and Development (A.O.B.), and Epidemiology and Biostatistics (F.J.d.J., H.A.P.P., M.M.B.B.), Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Jan W. Koper, Department of Internal Medicine, Room Ee530C, Erasmus Medical Center, Dr. Molewaterplein 40, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: f.koper{at}erasmusmc.nl.
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Abstract |
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Objective: The aim was to study the separate and combined effects of these polymorphisms on body composition, adrenal androgen production, blood pressure, glucose metabolism, and the incidence of dementia in the healthy elderly population.
Design/Setting/Participants: The Rotterdam study (n = 6105) and the Frail Old Men study (n = 347) are population-based cohort studies in the elderly.
Main Outcome Measures: Genotype distributions and influences of (combined) genotypes on body mass index, adrenal androgen production, waist to hip ratio, systolic and diastolic blood pressure, fasting glucose levels, glucose tolerance test, and incidence of dementia were measured.
Results: No influence of the HSD11B1 83,557insA (allele frequencies 22.0 and 21.5%) and H6PD R453Q (allele frequencies 22.9 and 20.2%) variants was found for the different outcome measures that were investigated, either separately or when at least three alleles were affected.
Conclusions: Two population-based studies among Caucasian elderly showed no evidence for (combined) effects of two polymorphisms in the HSD11B1 and H6PD genes on body composition, adrenal androgen production, blood pressure, glucose metabolism, and incidence of dementia. Moreover, the high frequencies observed for these two polymorphisms do not correspond to the low incidence of CRD observed in the general population. Altogether, it is unlikely that these polymorphisms cause CRD.
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Introduction |
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Recently, Draper et al. (5) concluded from a study in kindreds with cortisone reductase deficiency (CRD) that a combination of mutations in HSD11B1 and H6PD interacts to cause CRD. They proposed a digenic triallelic mode of inheritance, in which three alleles from two (or more) loci are necessary for trait manifestation. We studied the role of HSD11B1 83,557insA and H6PD R453Q, either separate or combined, on body composition, adrenal androgen levels, blood pressure, glucose levels, and incidence of dementia in the elderly.
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Subjects and Methods |
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We genotyped a total of 6105 subjects for HSD11B1 83,557insA and H6PD R453Q from the Rotterdam study, a population-based, prospective cohort study of subjects aged 55 and older (7). In the Frail Old Men (FOM) study, a population-based cohort study in 403 independently living men age 70 yr and older (8), we genotyped 347 Caucasians. Both studies were approved by the Medical Ethics Committee of the Erasmus MC, and written informed consent was obtained from all participants.
DNA analysis
The appropriate Assay-by-Design mixes were designed, synthesized, and supplied by Applied Biosystems (Foster City, CA) (Table 1
). Five-microliter PCRs containing approximately 10 ng of DNA, 0.0625 µl 80x Assay-by-Design mix, 2.4375 µl water, and 2.5 µl Universal Master Mix (Applied Biosystems) were performed in 384-well plates. The reaction conditions were: 2 min 50 C, 10 min 95 C, followed by 40 cycles of 15 sec 92 C, and 60 sec 60 C. Plates were analyzed using the Applied Biosystems 7900HT Sequence Detection System and SDS version 2.0 software (Applied Biosystems).
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Body weight and height were measured, and body mass index (BMI) was defined as weight divided by the square of height (kilograms/meters2). The waist (umbilicus) and hip circumferences were measured, and the waist-hip ratio (WHR) was calculated. In the FOM study, total lean body mass and fat mass were measured by dual energy x-ray absorptiometry as previously described (9, 10).
Hormonal measurements
In a limited number of subjects of the Rotterdam study, plasma levels of androstenedione (n = 1608) and dehydroepiandrosterone sulfate (DHEAS) (n = 1654) were determined. Plasma levels of androstenedione and DHEAS were estimated in 12 separate batches of samples using coated-tube RIAs purchased from Diagnostic Systems Laboratories, Inc. (Webster, TX). Due to the relatively small volumes of plasma available, all values reported are single-sample estimations. Intraassay coefficients of variation, determined on the basis of duplicate results of internal quality control plasma pools with three different levels of each analyte, were less than 12% and 7% for androstenedione and DHEAS, respectively. Because interassay variations were relatively large (23% for androstenedione and 24% for DHEAS), results of all batches were normalized by multiplying all concentrations within a batch with a factor, which made results for the internal quality control pools comparable. This reduced interassay variations to 9% for androstenedione and 10% for DHEAS and was considered justified because the patterns of the results of these pools and the mean results for male and female sera in one assay batch were very similar.
In the FOM study, serum DHEA and DHEAS levels were determined by RIA (Diagnostics Products Corporation, Los Angeles, CA) in nearly all subjects (n = 346). The intraassay and interassay coefficients of variation for these assays were 3.8 and 2.1% and 8.6 and 5.1%, respectively.
Blood pressure measurement
Blood pressure was measured at the right upper arm in a sitting position with a random-zero sphygmomanometer. Persons using blood pressure-lowering drugs and persons without data on blood pressure-lowering drugs were excluded from the statistical analysis with regard to blood pressure.
Assessment of glucose metabolism
In the Rotterdam study, nonfasting serum blood samples were collected at baseline. Participants without diabetes mellitus (DM) also underwent a nonfasting glucose tolerance test (85% of the total population) (11). In the FOM study, only fasting glucose levels were measured.
Subjects with DM and subjects without data on DM were excluded from the statistical analysis with regard to glucose metabolism. In the Rotterdam study, DM was defined as the use of blood glucose-lowering medication or random serum glucose concentration of at least 11.1 mmol/liter, or both. In the FOM study, DM was defined as the use of blood glucose-lowering medication only.
Diagnosis of dementia
Dementia screening and diagnosis in the Rotterdam study followed a three-step protocol as previously described (12).
Combined genotype analysis
Because Draper et al. (5) proposed a digenic triallelic mode of inheritance for the manifestation of CRD, we compared carriers of at least three affected alleles with the rest of the study group. The combination of three or four affected alleles is referred to as "CRD genotype."
Statistical analysis
Data were analyzed using SPSS for Windows, release 10.1 (SPSS, Chicago, IL). All data are presented as means ± SEM. Statistical analyses on body composition, DHEA(S), androstenedione, blood pressure, and glucose metabolism were carried out by using the General Linear Model procedure and linear regression (Ptrend) and were corrected for age and sex. Analysis on DHEA(S) was also corrected for smoking. Analyses on body composition and DHEA(S) were stratified for sex. Statistical analysis on the incidence of dementia was performed using Cox proportional hazards models adjusted for age and sex.
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Results |
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In the Rotterdam study, 3704 (60.7%) persons were defined as wild type, 2110 (34.6%) as heterozygous carriers, and 291 (4.8%) as homozygous carriers (allele frequencies, 78.0% reference allele and 22.0% variant allele). In the FOM study, 217 (62.5%) subjects were defined as wild type, 111 (32.0%) as heterozygous carriers, and 19 (5.5%) as homozygous carriers (allele frequencies, 78.5% reference allele and 21.5% variant allele). Both populations were in Hardy-Weinberg equilibrium.
In the Rotterdam study, no associations with weight, BMI, waist circumference, hip circumference, or WHR were found. In female carriers, a trend toward higher length was found (wild types, 161.0 ± 0.14 cm; heterozygous carriers, 161.5 ± 0.19 cm; homozygous carriers, 161.8 ± 0.51 cm; PANOVA = 0.06; Ptrend = 0.02). There were no differences in adrenal androgen levels, blood pressure, glucose metabolism, or incidence of dementia. In the FOM study, no differences in weight, BMI, WHR, trunk fat mass, trunk lean mass, total lean mass, and total fat mass were found. However, there was an association with lower height in carriers (wild types, 173.4 ± 0.43 cm; heterozygous carriers, 172.2 ± 0.59 cm; homozygous carriers, 170.4 ± 1.44 cm; PANOVA = 0.049; Ptrend = 0.02). There were no differences in adrenal androgen levels, blood pressure, or glucose metabolism.
H6PD R453Q
In the Rotterdam study, 3655 (59.9%) subjects were defined as wild type, 2105 (34.5%) as heterozygous carriers, and 345 (5.7%) as homozygous carriers (allele frequencies, 77.1% reference allele and 22.9% variant allele). In the FOM study, we found 224 (64.6%) homozygous wild-type carriers, 106 (30.5%) heterozygous carriers, and 17 (4.9%) homozygous carriers (allele frequencies, 79.8% reference allele and 20.2% variant allele). Both populations were in Hardy-Weinberg equilibrium.
For the Rotterdam study, we found no associations with weight, height, BMI, or waist circumference. Male carriers had a smaller hip circumference compared with wild types (wild types, 98.7 ± 0.17 cm; heterozygous carriers, 98.2 ± 0.23 cm; homozygous carriers, 97.2 ± 0.60 cm; PANOVA = 0.02; Ptrend = 0.005). In female carriers, a significant trend toward higher WHR was found (wild types, 0.87 ± 0.002; heterozygous carriers, 0.87 ± 0.003; homozygous carriers, 0.88 ± 0.006; PANOVA = 0.10; Ptrend = 0.04). No differences in adrenal androgen levels, blood pressure, glucose metabolism, or incidence of dementia were found. In the FOM study, no statistically significant differences were found for the investigated outcome measures.
Combined genotype groups
In the Rotterdam study, 233 persons (3.8%) presented with the CRD genotype vs. 14 persons (4.0%) in the FOM study. In both the Rotterdam study and the FOM study, no statistically significant differences were found for the body composition parameters or other investigated outcome measures, including androgen levels.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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White (14) showed that the HSD11B1 83,557insA and H6PD R453Q SNPs occur more frequently than previously reported but found no differences for BMI, WHR, visceral adiposity, insulin sensitivity, testosterone, FSH or LH (females), the risk of PCOS, or an effect on urinary free cortisol/cortisone ratio or the corticosteroid metabolite ratios. However, Gambineri et al. (15) found that HSD11B1 83,557insA was significantly related to PCOS status, lower 0800–0830 h plasma cortisol, and higher cortisol response to ACTH1–24 in all women with PCOS, and with higher DHEAS levels, greater suppression of DHEAS by dexamethasone, and lower fasting plasma LDL-cholesterol levels in lean PCOS women.
In our study, we showed that HSD11B1 83,557insA and H6PD R453Q are relatively common in the elderly population. Because a considerable part in both of our elderly study groups (Rotterdam study, 3.8%; FOM study, 4.0%) is the carrier of at least three affected alleles, it is very unlikely that these SNPs interact to cause CRD. Moreover, we showed that carriers of at least three affected alleles do not have higher androgen production, which is the key factor causing the symptoms observed in patients with CRD.
In a recent study, Lavery et al. (6) showed that H6PDH knockout mice have a profound switch in 11ßHSD activity from oxoreductase to dehydrogenase, increasing the corticosterone clearance resulting in a reduction in circulating corticosterone levels. This demonstrated a critical requirement of H6PDH for 11ßHSD1 oxoreductase activity. However, in our study, no reliable effect of H6PD R453Q was found.
We conclude from our study in two independent elderly populations that we have not been able to detect any influence of the HSD11B1 83,557insA and H6PD R453Q SNPs, either separately or when using three or more affected alleles on body composition, adrenal androgen production, blood pressure, glucose levels, or incidence of dementia in the elderly. We also demonstrated that the presence of at least three affected alleles is relatively common in those two populations. Taking this together, it is unlikely that these SNPs cause CRD. However, because Lavery et al. (6) demonstrated the critical role of H6PDH for 11ßHSD1 oxoreductase activity, it is important to search for other possible functional SNPs in the HSD11B1 and H6PD genes.
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online October 24, 2006
1 P.S. and M.J.H.J.D. contributed equally to this work. 
Abbreviations: BMI, Body mass index; CRD, cortisone reductase deficiency; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; DM, diabetes mellitus; FOM, Frail Old Men; H6PDH, hexose-6-phosphate dehydrogenase; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; SNP, single nucleotide polymorphism; WHR, waist-hip ratio.
Received June 26, 2006.
Accepted October 18, 2006.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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cortisol conversion in subjects with central adiposity. J Clin Endocrinol Metab 84:1022–1027This article has been cited by other articles:
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  G. G. Lavery, E. A. Walker, A. Tiganescu, J. P. Ride, C. H. L. Shackleton, J. W. Tomlinson, J. M. C. Connell, D. W. Ray, A. Biason-Lauber, E. M. Malunowicz, et al. Steroid Biomarkers and Genetic Studies Reveal Inactivating Mutations in Hexose-6-Phosphate Dehydrogenase in Patients with Cortisone Reductase Deficiency J. Clin. Endocrinol. Metab., October 1, 2008; 93(10): 3827 - 3832. [Abstract] [Full Text] [PDF]
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