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A Common Polymorphism in the CYP3A7 Gene Is Associated with a Nearly 50% Reduction in Serum Dehydroepiandrosterone Sulfate Levels

Departments of Internal Medicine (P.S., A.W.v.d.B., J.W.K., H.A.P.P., F.H.d.J., S.W.J.L.), Clinical Chemistry (R.H.N.v.S., M.v.d.W., J.L.), Reproduction and Development (A.O.B.), and Epidemiology and Biostatistics (H.A.P.P.), Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. Jan W. Koper, Department of Internal Medicine, Room Ee585, Erasmus Medical Center, Dr. Molewaterplein 40, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: f.koper{at}erasmusmc.nl.

	Abstract

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Context: CYP3A7, expressed in the human fetal liver and normally silenced after birth, plays a major role in the 16-hydroxylation of dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and estrone. Due to a replacement of part of the CYP3A7 promoter with a sequence identical with the same region in the CYP3A4 promoter (referred to as CYP3A7*1C), some individuals still express a variant of the CYP3A7 gene later in life.

Objective: The objective of this study was to examine the effect of the CYP3A7*1C polymorphism on serum steroid hormone levels.

Design, Setting, Participants: Two population-based cohort studies were performed. Study group 1 consisted of 208 subjects randomly selected from the Rotterdam Study, and study group 2 consisted of 345 elderly independently living men.

Main Outcome Measures: Serum DHEA(S), androstenedione, estradiol, estrone, and testosterone levels were the main outcome measures.

Results: In study groups 1 and 2, heterozygous CYP3A7*1C carriers had almost 50% lower DHEAS levels compared with homozygous carriers of the reference allele [study group 1, 1.74 ± 0.25 vs. 3.33 ± 0.15 µmol/liter (P = 0.02); study group 2, 2.09 ± 0.08 vs. 1.08 ± 0.12 µmol/liter (P < 0.001)]. No differences in circulating DHEA, androstenedione, estradiol, or testosterone levels were found. However, in study group 2, serum estrone levels were lower in heterozygous CYP3A7*1C carriers compared with homozygous carriers of the reference allele (0.11 ± 0.002 vs. 0.08 ± 0.006 nmol/liter; P < 0.001).

Conclusion: The CYP3A7*1C polymorphism causes the persistence of enzymatic activity of CYP3A7 during adult life, resulting in lower circulating DHEAS and estrone levels.

	Introduction

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CYTOCHROME P450 ENZYMES are important for the metabolism of many endogenous compounds, procarcinogens, and drugs. The CYP3A subfamily is one of the major subfamilies of the CYP superfamily expressed in the human liver and comprises CYP3A4, CYP3A5, and CYP3A7. The CYP3A genes are located adjacent to each other on chromosome band 7q21, but are differentially regulated (1). CYP3A4 is the most abundant form of CYP3A (30% of total CYP), whereas CYP3A5 accounts for approximately 20% of the CYP3A content in adult liver and is known to be polymorphically expressed (2, 3). Of the oxidative enzymes, it has been shown that CYP3A7 accounts for up to 50% of the total fetal hepatic CYP content and up to 87–100% of total fetal hepatic CYP3A content (4, 5). Among the endogenous substrates and exogenous chemicals, the fetal/neonatal CYP3A7 has a high catalytic activity for the 16-hydroxylation of estrone (E1) and dehydroepiandrosterone (DHEA) (6, 7). Presumably, these latter effects of CYP3A7 are necessary during development to protect the fetus against placental production of estradiol (E2) from DHEA (8, 9). DHEA has a short half-life, with a high metabolic clearance rate (2000 liters/d), whereas DHEAS circulates in relatively large quantities and undergoes delayed metabolism (10). During adult life, DHEA is known to be a precursor steroid in the (peripheral) production of androgens and estrogens (11). CYP3A7 expression sharply decreases or stops shortly after birth, although some individuals still express CYP3A7 into adulthood due to replacement of an approximately 60-bp stretch [nucleotides (nt) –129 to –188] of the CYP3A7 promoter with a sequence identical with the same region in the CYP3A4 promoter. This genotype is referred to as CYP3A7*1C (12).

The aim of our study was to clarify the role of the CYP3A7*1C polymorphism in the regulation of serum DHEAS levels and the effects of DHEAS levels on the serum levels of other steroid hormones in the elderly. For this, we genotyped a group of 208 elderly subjects and a group of 345 elderly men (all Caucasian) for this polymorphism and determined serum DHEA(S) concentrations as well as serum androstenedione, E1, E2, and testosterone levels.

	Subjects and Methods

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Study group 1

We genotyped a total of 212 subjects who were randomly selected from the Rotterdam Study, a longitudinal population-based cohort study (comprising 7983 subjects) in a suburb of Rotterdam, The Netherlands, in which the determinants of chronic disabling diseases in the elderly are studied (13). The medical ethics committee of the Erasmus Medical Center approved this study, and all participants gave written informed consent. Serum DHEAS, androstenedione, E2, and testosterone levels were measured.

Four subjects were excluded from the statistical analysis: two females taking estrogen-containing medication (because of the effects of oral contraceptives on serum DHEAS levels), one male with a very high estrogen level, and one male with a pathologically low baseline cortisol level. The age in the resulting study group (n = 208) varied between 53 and 82 yr (98 men and 110 women with mean ages of 67.7 ± 0.6 and 66.1 ± 0.6 yr, respectively).

Study group 2

A group of 345 independently living men, all Caucasian, aged 73 yr or older (mean age, 77.6 ± 0.2 yr), were genotyped, and serum DHEAS, DHEA, E1, E2, and testosterone levels were measured. Participants were recruited by letters of invitation, which were sent to the oldest male inhabitants of Zoetermeer, a medium-sized town in The Netherlands. All subjects provided informed consent to participate in the study, which was approved by the medical ethics committee of the Erasmus Medical Center. Subjects were judged sufficiently healthy to participate in the study if they were physically and mentally able to visit the study independently. No additional health-related criteria were used.

Genotyping of CYP3A7*1C

Within the replacement of a 60-bp stretch (nt –129 to –188) of the CYP3A7 promoter sequence, 7 bp changed compared with the reference CYP3A7 allele. Genotyping of the CYP3A7*1C was performed by PCR-restriction fragment length polymorphism based upon the T-167G variant. A PCR amplification was performed in a 50-µl reaction volume, using approximately 10 ng genomic DNA, 1x PCR Buffer II (PerkinElmer, Wellesley, MA), 1.25 mM MgCl₂, 0.2 mM each of the deoxynucleotide triphosphates (Roche, Indianapolis, IN), 1.25 U AmpliTaq Gold (PerkinElmer), and 40 pmol each of forward primer 5'-CCATAGAGACAAGAGGAGA-3' and reverse primer 5'-CTGAGTCTTTTTTTCAGCAGC-3'. Amplification consisted of an initial denaturation step (7 min at 94 C), followed by 35 cycles (each consisting of 1 min at 94 C, 1 min at 58 C, and 1 min at 72 C), and ending with an extension cycle (7 min at 72 C). For restriction analysis, 10 µl from the PCR amplification was digested for 2 h at 37 C in a final volume of 15 µl containing 1x restriction buffer and 5 U SspI (New England Biolabs, Beverly, MA). The digested fragments were separated by electrophoresis on a 3% agarose gel with ethidium bromide staining. The fragments produced were 244 and 126 bp for the wild-type sequence; 370, 244, and 126 bp for heterozygous sequences; and 370 bp for homozygous variant sequences.

Hormone measurements

Serum DHEAS, DHEA, E1, E2, and androstenedione levels were determined by RIA (Diagnostics Products Corp., Los Angeles, CA). The intraassay coefficients of variation (CV) for these assays were 5.3% or less, 3.8%, 5.6%, 7.0% or less, and 8.3%, respectively. The interassay CVs were 7.0% or less, 8.6%, 10.2%, 8.1%, and 9.2% respectively. Testosterone was measured with a noncommercial RIA (intra- and interassay CVs, 5.6% and 9.0%) (14) in study group 1 and with an RIA in study group 2 (Diagnostics Products Corp.; intra- and interassay CVs, 8.1% and 10.5%).

Anthropometric measurements

Body weight and height of the subjects were measured, and body mass index was defined as weight (kilograms) divided by the square of height (meters).

Statistical analysis

Data were analyzed using SPSS for Windows, release 10.1 (SPSS, Inc., Chicago, IL). Data are expressed as the mean ± SEM. Statistical analysis was carried out using the general linear model procedure, and results were adjusted for age and, and if necessary, sex, body mass index, alcohol use, and smoking habits. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We studied the CYP3A7*1C polymorphism in two independent populations consisting of 208 healthy elderly subjects (study group 1) and 345 independently living males (study group 2) and found 6.7% (n = 14) and 8.4% (n = 29) heterozygous carriers, respectively. Allele frequencies for study groups 1 and 2 were 96.6% reference allele and 3.4% variant allele, and 95.8% reference allele and 4.2% variant allele, respectively. Both populations were found to be in Hardy-Weinberg equilibrium.

In study group 1, the CYP3A7*1C polymorphism was associated with lower serum DHEAS levels (P = 0.02) when corrected for age, sex, alcohol use, and smoking habits. Heterozygous carriers of the CYP3A7*1C polymorphism had 47.7% lower mean serum DHEAS levels compared with the wild type (1.74 ± 0.25 vs. 3.33 ± 0.15 µmol/liter, respectively; Fig. 1). Because all female subjects in our study group were postmenopausal, males and females were analyzed together. Separate analysis for males and females followed the same trend, although no significance was reached, presumably due to the small numbers of subjects in both groups [males: wild type, 4.08 ± 0.22 µmol/liter; heterozygous mutant, 2.34 ± 0.71 µmol/liter (P = 0.18); females: wild type, 2.63 ± 0.16 µmol/liter; heterozygous mutant, 1.51 ± 0.19 µmol/liter (P = 0.07)]. Serum androstenedione, E2, as well as testosterone levels did not differ between homozygous carriers of the reference allele and heterozygous carriers of the CYP3A7*1C polymorphism (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Serum DHEAS concentrations in wild-type and heterozygous mutant carriers of the CYP3A7*1C polymorphism in a group of 208 healthy elderly individuals. The P value was adjusted for age, sex, alcohol use, and smoking habits. *, P = 0.02 vs. reference allele. Values are the mean ± SEM.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Serum DHEAS and E1 concentrations in wild-type and heterozygous mutant carriers of the CYP3A7*1C polymorphism in a group of 345 independently living elderly men. The P value for DHEAS was adjusted for age and smoking habits; the P value for E1 was adjusted for age. *, P < 0.001; **, P < 0.001 (vs. reference allele). Values are the mean ± SEM.

	Discussion

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Much controversy exists about the potential significance of (lowered) DHEA(S) levels in the aging process (15). In both groups, no changes in circulating androgen and E2 levels were observed in the individuals who had possibly lived a life with decreased DHEAS levels due to the presence of this polymorphism. However, according to de Ronde et al. (16), strong correlations exist between DHEAS levels and E2, E1, androstenedione, and testosterone levels in both men and women. The significantly lower serum E1 levels observed in heterozygous CYP3A7*1C carriers in study group 2 can be explained by the high catalytic activity of CYP3A7 for this estrogen (7).

Previous studies suggested that lower(ed) DHEAS levels might be associated with a higher mortality (17). Therefore, it is possible that selective survival bias already occurred in our study populations. We found allele frequencies of 3.4% (study group 1) and 4.2% (study group 2) for the variant CYP3A7 allele. In a separate study, a frequency of 3.2% was found in 500 healthy adult Caucasian blood donors (van Schaik, R. H. N., M. van der Werf, J. Lindemans, unpublished observations). This is in agreement with the frequencies observed in previous studies performed by Kuehl et al. (18) and Burk et al. (19) (3.0% and 3.5%, respectively). However, no description of these study populations in terms of age was given.

In conclusion, we found that a common heterozygous variant allele of the CYP3A7 gene results in a nearly 50% reduction in DHEAS levels in two populations of healthy elderly individuals. However, no indications were found that such lowered levels are associated with an acceleration of the aging process, suggesting that even these lower(ed) DHEAS levels are sufficiently high to enable the steroid to act as an adequate precursor for peripheral estrogen and androgen formation. However, these intriguing data are still preliminary and deserve additional studies.

	Footnotes

 
This work was supported by Netherlands Organization for Scientific Research (NWO) grant 903-43-093.

First Published Online June 28, 2005

¹ P.S. and R.H.N.v.S. contributed equally to this work.

Abbreviations: CV, Coefficient of variation; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; E1, estrone; E2, estradiol; nt, nucleotide.

Received February 11, 2005.

Accepted June 21, 2005.

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