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Association of the (AU)AT-Rich Element Polymorphism in PPP1R3 with Hormonal and Metabolic Features of Polycystic Ovary Syndrome

Departments of Biochemistry and Molecular Biology (S.Y.A., G.I.B.), Medicine (M.H., G.I.B., D.A.E.), and Human Genetics (G.I.B.), The University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: David A. Ehrmann, M.D., Department of Medicine, Section of Endocrinology, The University of Chicago Pritzker School of Medicine, 5841 South Maryland Avenue, MC 1027, Chicago, Illinois 60637. E-mail: dehrmann{at}medicine.bsd.uchicago.edu.

	Abstract

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Insulin resistance, a key factor in the pathogenesis of polycystic ovary syndrome (PCOS), is associated with a reduction in activation of muscle glycogen synthase. A 5-bp insertion-deletion polymorphism in the (AU)AT-rich element (ARE) within the 3'-untranslated region of the gene encoding the muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PPP1R3) has been associated with insulin resistance and type 2 diabetes. The present study was undertaken to examine the relationship of the ARE polymorphism with clinical and hormonal characteristics of women with PCOS. We studied 186 women with PCOS who had undergone a standard 75-g oral glucose tolerance test and measurement of serum androgen and SHBG levels. Among the largest cohort of nondiabetic subjects (Caucasian, n = 112), the presence of the deletion allele (ARE-2) was associated with insulin resistance and hyperandrogenemia. There was no association of the ARE polymorphism with body mass index or blood glucose concentration during the oral glucose tolerance test. Subjects who were homozygous for the insertion allele (ARE-1/1) had a mean insulin area under the curve (99,116 ± 6,625 pmol/liter·min) that was significantly lower than that in either the heterozygous (ARE-1/2) (132,195 ± 12,340 pmol/liter·min) or homozygous (ARE-2/2) (164,661 ± 24,219 pmol/liter·min) deletion groups. In addition, ARE-1/1 subjects had significantly lower serum concentrations of dehydroepiandrosterone sulfate compared with ARE-2/2 subjects (4.2 ± 0.3 vs. 6.6 ± 0.7 µmol/liter) and a trend toward lower levels of free testosterone (78.8 ± 6.5 vs. 114.1 ± 30.8 pmol/liter). Studies of diabetic and nondiabetic PCOS women of other racial and ethnic backgrounds will be necessary to assess the impact of this and other variants in PPP1R3 upon the phenotype and natural history of women with PCOS.

	Introduction

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POLYCYSTIC OVARY SYNDROME (PCOS) is a common disorder characterized by menstrual irregularity, hyperandrogenism, and an increased risk for development of type 2 diabetes mellitus (1, 2). The increased risk of diabetes results, at least in part, from the fact that women with PCOS are insulin resistant (3, 4), typically to a degree that exceeds that observed in weight-matched control women (5, 6).

Insufficient activation of muscle glycogen synthase is characteristic of insulin resistance (7). Glycogen-bound type-1 protein phosphatase (PP1G), the glycogen-associated form of protein phosphatase-1 (PP1), is derived from skeletal muscle and binds to muscle glycogen with high affinity, thereby enhancing dephosphorylation of glycogen-bound substrates for PP1 such as glycogen synthase and glycogen phosphorylase kinase (8). Phosphorylation at Ser48 of the PP1G subunit (gene symbol, PPP1R3) in response to insulin increases PP1 activity (8, 9).

A number of variants in PPP1R3 lead to reduced expression of PP1G and therefore to insulin resistance (10). In particular, a 5-bp insertion-deletion polymorphism in the AU(AT)-rich element (ARE-1 and its deletion variant ARE-2) within the 3'-untranslated region (UTR) of PPP1R3 has been associated with insulin resistance and type 2 diabetes in both Pima Indian (10) and Japanese (11) populations. The present study was undertaken to examine the relationship of the ARE-2 polymorphism in PPP1R3 to the hormonal and clinical characteristics of women with PCOS.

	Subjects and Methods

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Subjects

Subjects were recruited from the endocrinology clinics of the University of Chicago (Chicago, IL). All were at least 2 yr post menarche and less than 40 yr of age. A diagnosis of PCOS required 1) the presence of oligo/amenorrhea; 2) hyperandrogenemia, defined by a supranormal plasma free testosterone level (34.7 pmol/liter); 3) hyperandrogenism, as evidenced by infertility, hirsutism, acne, or androgenetic alopecia; and 4) exclusion of nonclassic 21-hydroxylase deficiency congenital adrenal hyperplasia, Cushing’s syndrome, hypothyroidism, or significant elevations in serum prolactin (12). In addition to meeting these diagnostic criteria for PCOS, often referred to as the National Institutes of Health consensus criteria (13), each subject had hormonal evidence of ovarian androgen overproduction documented by an abnormal 17-hydroxyprogesterone response to GnRH agonist administration or a supranormal plasma free testosterone level after administration of dexamethasone (12). All subjects had not taken steroid preparations (including oral contraceptives) or medications known to alter insulin secretion and/or action for at least 2 months before the study. A negative screening pregnancy test was required before the study.

All studies were approved by the Institutional Review Boards of the University of Chicago, and a written informed consent was obtained from each subject.

Oral glucose tolerance test (OGTT)

Except for those individuals known to be diabetic, all subjects had an OGTT. After an overnight 12-h fast, blood samples were obtained at times –15 and 0 min. Dextrose (75 g) was then administered orally, and blood samples were obtained at 30, 60, 90, and 120 min for measurement of glucose and insulin concentrations. Glucose tolerance status was based on the plasma glucose concentration at 2 h using criteria of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus of the American Diabetes Association (14). Specifically, a diagnosis of normal glucose tolerance, impaired glucose tolerance, or diabetes was assigned if the glucose level at 2 h was less than 7.8, between 7.8 and 11.1, or 11.1 mmol/liter or more, respectively.

Hormonal measures

Serum was obtained from each subject at time 0 min during the OGTT for measurement of glycohemoglobin, total testosterone, free testosterone, SHBG, and dehydroepiandrosterone sulfate (DHAS).

Molecular genetic studies

The genotypes of the ARE in the 3'-UTR of PPP1R3 were determined by sizing the fluorescent-labeled PCR products on an ABI Prism 377 DNA Sequencer (ABI, Foster City, CA). The ARE region was amplified using the forward primer, 5'-ATGGACAATGGCAGAGGA-CT-3', and the reverse primer, 5'-TGAAAAGGTTGAAATATTTGATCAA-3', in the presence of [F]deoxynucleotide triphosphate (PerkinElmer, Norwalk, CT), yielding a product of 108 bp. The insertion-deletion variant (ARE-2) is 5 bp shorter than the wild-type sequence; this allows the two alleles to be resolved visually on a polyacrylamide gel. The PCR conditions were as follows: hot start at 95 C for 5 min, then 35 cycles of 95 C for 30 sec, 58 C for 30 sec, and 72 C for 30 sec, with a final extension at 72 C for 10 min.

Assay methods

Plasma glucose was measured immediately using a glucose analyzer (model 2300 STAT, YSI, Inc., Yellow Springs, OH). The coefficient of variation of this method is less than 2%. Glycohemoglobin was measured by boronate affinity chromatography with an intraassay coefficient of variation of 4% (Bio-Rad Laboratories, Inc., Hercules, CA). Serum insulin was assayed by a double-antibody technique (15) with a lower limit of sensitivity of 20 pmol/liter and an average intraassay coefficient of variation of 6%. The cross-reactivity of proinsulin in the RIA for insulin is approximately 40%. The lower limit of sensitivity of the assay is 0.02 pmol/ml, and the intraassay coefficient of variation averaged 6%. Plasma testosterone was measured using a kit from Diagnostic Products (Los Angeles, CA). The free fraction of plasma testosterone and the concentration of SHBG were measured by a competitive protein-binding assay as previously described (16). The intra- and interassay coefficients of variation were 3.8 and 8.7%, respectively. DHAS was measured by RIA using a commercial kit (Diagnostics Systems Laboratories, Inc., Webster, TX).

Statistical analysis

Allele frequencies were compared using StatView software (SAS Institute, Cary, NC). Between-group comparisons were made using ANOVA or analysis of covariance with logarithmic transformation of data not normally distributed and post hoc correction for multiple comparisons. For all analyses, P < 0.05 was considered significant. All data are presented as the mean ± SE.

	Results

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One hundred and eighty-six PCOS patients were studied: 116 (62.4%) Caucasian of European ancestry, 47 (25.2%) African-American, 13 (7%) of Asian ancestry, and 10 (5.4%) Hispanic. Fourteen (12%) of the subjects were diabetic, consistent with the increased prevalence of diabetes in PCOS (1, 2). African-Americans accounted for seven (50%) of the 14 diabetes cases; the remaining half was comprised of four (29%) Caucasians, two (14%) Hispanics, and one (7%) Asian.

Ninety-two (49.5%) of the subjects were ARE-1/1, 72 (38.7%) were heterozygous for the deletion/rearrangement variant (ARE-1/2), and 22 (11.8%) subjects had two ARE-2 alleles (ARE-2/2) (Table 1). The frequency of ARE-2 in the study group was 0.31183, varying from 0.29310 in Caucasians to 0.4000 in the smaller Hispanic group. These allelic frequencies are lower than those reported in Pima Indians (10) but similar to those in aboriginal Canadians (17), Japanese (11), and Swedish (18) subjects.

Compared with Caucasians, African-American subjects had significantly higher levels of insulin during the OGTT, both at the 2-h time point (1494.4 ± 228.9 vs. 1131.8 ± 77.2 pmol/liter) and for area under the curve (167,839 ± 21,648 vs. 117,573 ± 6,493 pmol/liter·min).

To avoid the confounding effects of the diabetic state on hormonal and glycemic measures, only nondiabetic subjects were included in analyses examining these measures regarding the PPP1R3 genotype (Table 2). There were too few subjects with the ARE-2 allele in the other racial groups to allow similar comparisons to be made.

Finally, concordant with the lower insulin responses to an oral glucose challenge, ARE-1/1 subjects had significantly lower serum concentrations of DHAS compared with ARE-2/2 subjects (4.2 ± 0.3 vs. 6.6 ± 0.7 µmol/liter) and a trend toward lower levels of free testosterone (78.8 ± 6.5 vs. 114.1 ± 30.8 pmol/liter).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The etiology of PCOS is complex and incompletely understood (4, 20). However, it is generally recognized that insulin resistance is a key component in the pathogenesis of the disorder (4), and several reports have documented the association of PCOS with genes influencing insulin action (21, 22). The 5-bp insertion-deletion polymorphism in the ARE-2 within the 3'-UTR of PPP1R3 has been implicated in the pathogenesis of other insulin-resistant conditions (19), including type 2 diabetes (10, 11) and impaired glucose tolerance (18). To determine whether ARE-2 is associated with the pathogenesis of PCOS, we typed 186 PCOS probands for the 5-bp deletion polymorphism in the ARE within the 3'-UTR of PPP1R3.

We found that nondiabetic Caucasian women with PCOS who had two copies of the ARE-2 allele had higher insulin levels during a 2-h OGTT than those who had two copies of the wild-type ARE-1 allele. This difference could not be accounted for by differences in BMI. These results are consistent with other studies in non-PCOS subjects, indicating the ARE-2 allele is associated with a varying degree of insulin sensitivity (10, 11, 19). Our results suggest that the ARE-2 allele may attenuate insulin sensitivity in Caucasian women with PCOS. ARE-2 may destabilize the mRNA of PPP1R3 (10), slowing the initial steps in the insulin action cascade. Perhaps at a greater allelic frequency, the deletion polymorphism would enhance this delayed action and lead to the insulin resistance as reported in Pima Indians (10). Larger studies of PCOS women of different ethnic backgrounds will be necessary to assess the impact of variants in PPP1R3 upon the risk of type 2 diabetes in this extremely insulin-resistant group of subjects. Finally, because it was shown here that wild-type African-American PCOS women had substantially higher levels of insulin compared with Caucasians to maintain blood glucose homeostasis, a more extensive study with a large cohort of African-Americans is needed to explore whether variants in PPP1R3 play a role in this process.

	Acknowledgments

 
We thank the patients for kindly agreeing to participate in these studies. In addition, the expert nursing care provided by the University of Chicago Clinical Research Center is acknowledged.

	Footnotes

 
This work was supported by the U.S. Public Health Service (Grants DK-20595, DK-44840, DK-47486, and RR-00055), a Clinical Research Award (to D.A.E.) from the American Diabetes Association, and a gift from the Blum-Kovler Foundation.

G.I.B. is an Investigator of the Howard Hughes Medical Institute.

Abbreviations: BMI, Body mass index; DHAS, dehydroepiandrosterone sulfate; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; PP1, protein phosphatase-1; PP1G, glycogen-bound PP1; UTR, untranslated region.

Received July 9, 2003.

Accepted February 23, 2004.

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