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Nonreplication of the Type 5 17β-Hydroxysteroid Dehydrogenase Gene Association with Polycystic Ovary Syndrome

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine (M.O.G., M.R.J., H.J.A.), Department of Obstetrics and Gynecology (M.O.G., M.P., R.A.), and Medical Genetics Institute (M.O.G., Y-D.I.C.), Cedars-Sinai Medical Center, Los Angeles, California 90048; and Departments of Medicine (M.O.G., Y.-D.I.C., R.A.) and Obstetrics and Gynecology (R.A.), the David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Ricardo Azziz, M.D., M.P.H., M.B.A., Department of Ob/Gyn and Center for Androgen Related Disorders, Cedars-Sinai Medical Center, 8635 West Third Street, Suite 160W, Los Angeles, California 90048. E-mail: azzizr{at}cshs.org.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Increased androgen production is a primary feature of polycystic ovary syndrome (PCOS) and appears to be an inherited trait. The gene for the steroidogenic enzyme type 5 17β hydroxysteroid dehydrogenase (HSD17B5) was implicated as a candidate for the hyperandrogenemia of PCOS by a previous study that demonstrated an association of a single nucleotide polymorphism (SNP) in the promoter of this gene with PCOS.

Objective: The objective of the study was to replicate the previous report of association between the HSD17B5 gene and PCOS risk by genotyping the promoter SNP (as well as other SNPs in the region to provide improved coverage of the gene) in a large, well-characterized cohort suitable for replication study.

Design: Women with and without PCOS were genotyped for five SNPs in HSD17B5. SNPs and haplotypes were determined and tested for association with PCOS risk and phenotypic markers of PCOS.

Setting: Subjects were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham; controls were recruited from the surrounding community. Genotyping took place at Cedars-Sinai Medical Center in Los Angeles.

Participants: Participants included 287 white women with PCOS and 187 white controls.

Main Measurements: HSD17B5 genotype, PCOS risk, and testosterone levels were measured.

Results: No SNP or haplotype was significantly associated with PCOS risk, testosterone, or any of the traits tested.

Conclusions: These data suggest that polymorphisms in the HSD17B5 gene are not associated with PCOS risk or elevated testosterone as previously reported.

	Introduction

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Polycystic ovary syndrome (PCOS), characterized by hyperandrogenism, oligoovulation, and polycystic ovarian morphology, is a leading cause of infertility and affects 7% of women (1). Evidence for a genetic basis for the hyperandrogenemia of PCOS is found in the increased rates of hyperandrogenemia in the family members of PCOS probands, both with and without PCOS (2). In an effort to identify the genetic basis of hyperandrogenemia in PCOS, a number of genes encoding major enzymes of the androgen biosynthetic pathway have been examined and associations reported (3, 4, 5), although support for these associations in replication studies has not been unanimous.

Upon observation of an index case presenting with male range testosterone levels, severe type B insulin resistance due to the presence of antibodies for the insulin receptor, bilaterally enlarged ovaries, amenorrhea, and a male type response to a GnRH agonist test, Qin et al. considered the gene HSD17B5, encoding the steroidogenic enzyme type 5 17β-hydroxysteroid dehydrogenase, as a candidate for the hyperandrogenemia in this patient (6). Upon examination of the HSD17B5 gene in this index case, a variant (A to G) at position –71 of the promoter was identified. The investigators then examined this single nucleotide polymorphism (SNP) for association with PCOS in an association study in a mixed-ethnicity case-control cohort. Carriers of the –71G allele displayed increased PCOS risk, with an odds ratio (OR) of 1.66. A trend toward higher total testosterone in subjects homozygous for the –71G allele was noted (6).

HSD17B5 (also known as aldo-keto reductase family 1, member C3) is a member of the aldo-keto reductase family, a mixed function enzyme family. HSD17B5 catalyzes the conversion of androstenedione to testosterone. Regulation of the production of potent steroid hormones in steroidogenic tissues, including the ovary, is a proposed role of HSD17B5 (7, 8). In this study we investigated whether SNPs in HSD17B5 (–71 plus others) were associated with susceptibility to PCOS or the biochemical features of subjects with PCOS in a large, well-characterized cohort of Caucasian PCOS and control subjects.

	Subjects and Methods

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Subjects and phenotyping

A total of 287 consecutive unrelated non-Hispanic white patients with PCOS and 187 unrelated white control women were recruited from the Birmingham, Alabama, area as previously described (9). PCOS was diagnosed using the 1990 National Institutes of Health criteria (10). The comprehensive physical examination and hormonal evaluation of these subjects has been previously described in detail (11). All subjects gave written informed consent, and the study was performed according to the guidelines of the Institutional Review Boards of the University of Alabama and Cedars-Sinai Medical Center.

SNP genotyping and haplotype determination

SNPs were selected using frequency and validation data from the International HapMap database (12) with the aim of exploiting linkage disequilibrium (LD) for the study of the gene. HSD17B5 maps to chromosome 10p14-p15 and spans 13,613 bases. Genotyping of SNPs was by the 5'-exonuclease assay (Taqman MGB; Applied Biosystems, Foster City, CA) as previously described (13, 14). rs3763676 (SNP –71), rs12529 (glutamine to histidine at position 5), rs17396032, rs2518049, rs1937841, and rs11252946 were selected to represent the common haplotypes of the region. The PCR primers and TaqMan MGB probes are presented in supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org. The assay designed to genotype SNP rs11252946 was unable to produce usable data, so this SNP was dropped from the assay group. Duplicate genotyping of 96 samples for one SNP yielded 100% concordance. The genotyping success rate was 94.6%.

Haploview 3 (15) was used to determine haplotypes as well as haplotype blocks, using an accelerated expectation maximization algorithm. Haploview was used to calculate LD (the D' statistic) between each pairwise combination of all the SNPs. Haplotypes were assigned to individual subjects only when the assignment could be made with a greater than 95% certainty.

Statistical analysis

Unpaired t tests and ² tests were used to compare clinical characteristics between women with and without PCOS; quantitative trait values were log or square root transformed as appropriate to reduce nonnormality. Association between haplotype and quantitative phenotypic variables used analysis of covariance, adjusting for age and body mass index. Association with presence/absence of PCOS was evaluated using logistic regression, again adjusting for body mass index and age. Significance was taken at P < 0.017 to account for the effects of multiple testing, considering that we analyzed one linkage disequilibrium group of SNPs against three families of traits (PCOS diagnosis, androgens, metabolic traits), yielding a correction factor of three (i.e. three independent comparisons). Analyses were carried out using Statview 5.0 (SAS Institute, Cary, NC). Data are reported as median (interquartile range).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical features of the study cohort are presented in supplemental Table 2.

We genotyped five SNPs from the HSD17B5 gene region. Strong LD (D' > 0.95) was observed between each of the SNPs included in the analysis (Fig. 1). There were no significant associations between any SNP and phenotypes included in the analysis that met our nominal significance level of P = 0.017.

View larger version (64K): [in this window] [in a new window]   FIG. 1. LD among the SNPs genotyped in the HSD17B5 gene region. The LD plot displays D' values (percent) for each pair of SNPs in the box at the intersection of the diagonals from each SNP. The solid blocks indicate D' = 1 (100%) for the corresponding pair of variants. The SNPs comprise one haplotype block as indicated.

Six haplotypes composed of the SNPs –71G, rs12529, rs17396032, rs2518049, and rs1937841 were identified in the HSD17B5 gene that had a frequency more than 3%. We observed no significant association between haplotype and PCOS risk or any of the traits examined.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In this replication study, we were unable to demonstrate association between polymorphisms in the HSD17B5 gene region and PCOS risk or phenotypic markers of PCOS as had been previously reported. HSD17B5 was identified as a potential candidate gene in the hyperandrogenemia of PCOS due to its role in steroid production in the ovary (6).

The product of HSD17B5 is a member of the aldo-keto reductase gene superfamily 1, which includes at least four isoforms in humans (8). Aldo-keto reductases are monomeric 37-kDa proteins that utilize as cofactors the reduced forms of nicotinamide adenine dinucleotide (phosphate). HSD17B5 was first cloned in hepatic and prostate tissues (8); it is expressed in the ovary (7, 16), and tissue-specific functions suggest a number of roles for the enzyme (8). In steroidogenic tissues, the 17β-hydroxysteroid dehydrogenase function of HSD17B5 catalyzes the conversion of androstenedione to testosterone; however, this conversion is performed at an inefficient rate (k_cat/K_m of 4.54 min/mM substrate) in Escherichia coli (8), and it is unclear whether this is the primary enzyme responsible for this reaction in the ovary.

Whereas our primary goal was to replicate the –71G association with PCOS, to improve our ability to detect an effect, we expanded coverage of the gene by including five SNPs in our analysis of HSD17B5. Coverage of the gene was extensive but not complete due to inability to genotype rs11252946. By determining the LD structure of the region and whether there were additional SNPs in the region contributing to the reported association through LD, we were able to show that the reported SNP and in fact the HSD17B5 gene is not associated with PCOS risk.

The lack of replication of association seen in this study is representative of a larger issue in the field of PCOS genetics and may be the result of several interacting factors (1). The first and most significant is sample size and composition. Qin et al. (6) used a modest sample size of 121 PCOS subjects and 128 control subjects in their analysis. Compounding the issue of moderate sample size in that report was the heterogeneity of the sample. Of the subjects studied, only 59 of the cases (48.7%) and 67 of the controls (52.3%) were of Caucasian descent, with the remaining subjects being of African-American, Hispanic, and Asian descent. The report indicated that –71G carrier state conferred an OR of 1.66 in the entire cohort of mixed ethnicity; however, this OR was primarily driven by the subgroup of Caucasian subjects, within whom the OR was 2.25 (6). Using this OR because our cohort consists only of Caucasian subjects, our study had a power of 0.93 ( = 0.05) to detect this effect size (17).

The inclusion of functional in vitro data in the initial report (6) provided insight to the potential mechanism by which a functional variant might contribute to the pathophysiology of PCOS. The relatively modest increase in promoter activity (70%) by reporter constructs carrying the G allele in rat theca culture, although interesting, should be viewed with caution. Demonstration of functionality in vitro is often interpreted as evidence of important function in vivo. This functional variant with modest effect on the activity of an enzyme that may not significantly contribute to ovarian androgen output is unlikely to exert an effect on circulating testosterone levels in the in vivo environment, wherein multiple other influences on testosterone levels are operating. Furthermore, in the original report, the index case (–71G homozygote) had severely elevated androgen levels, whereas the 13 women with PCOS and eight controls homozygous for this allele in the association study did not have severe hyperandrogenemia (6). This suggests that other factors were present in the index case that contributed to the extreme hyperandrogenemia. In any case, if –71G were an important factor in hyperandrogenemia, association with testosterone levels should have been seen in our study. In light of the results presented here it would appear that HSD17B5 does not contribute to PCOS.

As acknowledged therein, the association study in the original report (6) was for discovery purposes, and a call for replication in a larger and well-characterized cohort was made, so it is this replication that we offer and suggest that the initial report of significance has not been replicated but was likely the result of small sample size and/or population stratification as a result of heterogeneity.

In cultured ovarian theca cells, HSD17B5 expression and function did not differ between PCOS and controls (16). In Spanish subjects, no association was identified between HSD17B5 SNPs and premature pubarche or markers of hyperandrogenemia; this study included several SNPs from the region as well as –71 (18).

The effect of small sample size and mixed ethnic composition and poor gene coverage on progress in the field of PCOS genetics is becoming more apparent as well-conducted large replication studies are attempted and not successful in confirming reports of susceptibility loci for PCOS risk (19, 20). The development of large, homogenous, and well-characterized cohorts is needed for rapid progress via adequately powered replication efforts. By addressing these issues, we have demonstrated that polymorphisms in the HSD17B5 gene are not associated with PCOS or component traits of PCOS in our large, homogenous, and well-characterized cohort, making it unlikely to be a susceptibility region for PCOS.

	Footnotes

Disclosure Statement: M.O.G., M.R.J., H.J.A., M.P., and Y.-D.I.C. have nothing to declare. R.A. has received consulting fees from Procter & Gamble, Merck & Co., and Organon.

First Published Online October 16, 2007

Abbreviations: LD, Linkage disequilibrium; OR, odds ratio; PCOS, polycystic ovary syndrome; SNP, single nucleotide polymorphism.

Received August 1, 2007.

Accepted October 4, 2007.

	References

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