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Departments of Medicine (D.A.E., M.H., X.T., J.I., N.J.C., G.I.B.), Human Genetics (N.J.C., G.I.B.), and Biochemistry and Molecular Biology (P.E.H.S., Y.H., G.I.B.), and the Howard Hughes Medical Institute (P.E.H.S., Y.H., 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, 5841 South Maryland Avenue, MC 1027, Chicago, Illinois 60637. E-mail: . dehrmann{at}medicine.bsd.uchicago.edu
Abstract
Polycystic ovary syndrome (PCOS) is associated with an increased risk of impaired glucose tolerance and type 2 diabetes. Recent evidence suggests that variation in the gene encoding the cysteine protease calpain-10 influences susceptibility to type 2 diabetes. The present study was undertaken to determine whether variation in this gene is associated with quantitative traits pertinent to the pathogenesis of PCOS and diabetes.
We studied 212 women with PCOS (124 white of European ancestry, 57 African-American, 13 Hispanic, 13 Asian-American, and 5 Middle-Eastern). Each subject was genotyped for 3 DNA polymorphisms in the calpain-10 gene associated with type 2 diabetes (SNP-43, -19, and -63). The white and African-American subjects were examined for association of these polymorphisms with phenotypic features of PCOS and type 2 diabetes. There were not enough individuals in the other groups for similar genotype/phenotype analyses.
Nineteen (9%) of the 212 women with PCOS were diabetic and were not included in the genotype/phenotype analyses. Twelve (63%) of these subjects were African-American. Phenotypic traits in nondiabetic white probands did not differ whether analyzed for each individual SNP (SNP-43, -19, -63) or haplotype combination. Nor was there association of SNP-43, -19, or -63 with any of the phenotypic features of type 2 diabetes or PCOS in nondiabetic African-Americans. However, nondiabetic African-Americans with the 112/121-haplotype combination had significantly higher insulin levels, in response to an oral glucose challenge, as reflected in the area under the insulin curve (257,021 ± 95,384 vs. 136,240 ± 11,468 pmol/min; P = 0.03), compared with those with other haplotypes. This finding was particularly notable because the 112/121 subjects were less obese. The difference between groups in area under the insulin response curve remained significant (P = 0.002 by analysis of covariance) after adjustment for body mass index. In addition to its association with insulin levels in African-Americans, the 112/121-haplotype combination was associated with an approximate 2-fold increase in risk of PCOS in both African-Americans and whites.
POLYCYSTIC OVARY SYNDROME (PCOS) affects nearly 10% of reproductive age women (1, 2) making it one of the most common endocrine disorders in this age group. In addition to its impact on reproductive function, PCOS confers a substantial risk for development of impaired glucose tolerance and type 2 diabetes mellitus (3, 4). The mechanisms underlying this predisposition to diabetes are not fully understood. It has been well established, however, that both insulin resistance and defects in insulin secretion are present in nondiabetic women with PCOS (5, 6).
Recent studies suggest that variation in the gene encoding the cysteine protease calpain-10 are associated with insulin resistance (7, 8) and influence genetic susceptibility to type 2 diabetes (9, 10). Because PCOS and type 2 diabetes share a number of etiologic factors (11, 12, 13), the present study was undertaken with the aim of determining whether variation in the calpain-10 gene is associated with quantitative traits related to the pathogenesis of PCOS and type 2 diabetes.
Subjects and Methods
Subjects
Subjects were recruited from the Endocrinology Clinics of the University of Chicago. All were at least 2 yr post menarche and not greater than 40 yr of age. A diagnosis of PCOS required (14): 1) the presence of oligo/amenorrhea; 2) hyperandrogenemia, with a plasma free T level 34.7 pM or greater; 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 PRL. In addition to meeting these diagnostic criteria for PCOS [often referred to as the NIH consensus criteria (15)], 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 T level after administration of dexamethasone (14). For at least 2 months before study, subjects had not taken steroid preparations (including oral contraceptives) or medications known to alter insulin secretion and/or action. After a diagnosis of PCOS was confirmed in the proband, all first-degree relatives were asked to participate, with a blood sample taken for preparation of DNA and measurement of glycohemoglobin; female relatives had an additional sample of blood obtained for hormonal measures (described below). The Institutional Review Board of the University of Chicago approved all studies, and written informed consent was obtained from each subject. A previous report (16) includes clinical data for a subset of the subjects reported on in the present study.
Oral glucose tolerance test (OGTT)
All individuals, with the exception of those known to be diabetic, had an OGTT. After an overnight fast, blood samples were obtained at times -15 and 0 min. A glycohemoglobin level was also obtained at time 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 (17). 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 mM, between 7.8 and 11.1 mM, or 11.1 mM or higher, respectively.
Hormonal measures
Serum was obtained from each PCOS subject and their female first-degree relatives for measurement of total T, free T, SHBG, and dehydroepiandrosterone sulfate (DHAS).
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 (18) with a lower limit of sensitivity of 20 pM and an average intraassay coefficient of variation of 6%. The cross-reactivity of proinsulin in the RIA for insulin is approximately 40%. Plasma C-peptide was measured as previously described (18). The lower limit of sensitivity of the assay is 0.02 pmol/ml, and the intraassay coefficient of variation averaged 6%.
Plasma T was measured using a kit from Diagnostic Products (Los Angeles, CA). The free fraction of plasma T and the concentration of SHBG were measured by a competitive protein binding assay (19). The intra- and interassay coefficients of variation were 3.8 and 8.7%, respectively. DHAS was measured by RIA using a kit from Diagnostic Systems Laboratories, Inc. (Webster, TX).
Molecular genetic studies
Genomic DNA was isolated from peripheral blood lymphocytes. DNA polymorphisms in the calpain-10 gene were typed using PCR-based methods (9). SNP-43, CAPN10-g.4852G/A, was genotyped by direct sequencing of the PCR product. The region of intron 3 containing SNP-43 was amplified using forward primer 5'-GCTGGCTGGTGACATCAGTG-3' and reverse primer 5'-TCAGGTTCCATCTTTCTGCCAG-3' (product size, 475 bp). PCR was carried out using Taq DNA polymerase, 5% dimethylsulfoxide (DMSO), and 1.0 mM MgCl2 with denaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec and extension at 72 C for 30 sec, and a final extension at 72 C for 10 min. The PCR product was then sequenced using the internal primer 5'-GTGAGCCTCTGGCATTGAGC-3' and an ABI Prism 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). The G- and A-alleles of SNP-43 were designated as alleles 1 and 2 in haplotypes including this DNA polymorphism. SNP-19, CAPN10-g.7920indel32bp, is a 2-allele insertion/deletion (indel) polymorphism resulting from the presence of two or three copies of a 32-bp sequence. The region of intron 6 containing SNP-19 was amplified using forward primer 5'-GTTTGGTTCTCTTCAGCGTGGAG-3' and reverse primer 5'-CATGAACCCTGGCAGGGTCTAAG-3' (product size, 155 or 187 bp). PCR was carried out using Taq DNA polymerase, 5% DMSO, and 1.0 mM MgCl2 with denaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec and extension at 72 C for 30 sec, and a final extension at 72 C for 10 min. The PCR products were separated by electrophoresis on a 3% agarose gel and visualized by ethidium bromide staining: allele 1 (two repeats) is 155 bp and allele 2 (three repeats) is 187 bp. SNP-63, CAPN10-g.16378C/T, was genotyped by direct sequencing of the PCR product. The region of intron 13 containing SNP-63 was amplified using forward primer 5'-GAACCAGTGCTTGGCAGCTCAC-3' and reverse primer 5'-GCAGTGCGTGGTGCCTGAAGG-3' (product size, 478 bp). PCR was carried out using eLONGase DNA polymerase (Invitrogen, Baltimore, MD), 5% DMSO, and 1.8 mM MgCl2 with denaturation at 94 C for 1 min followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 58 C for 30 sec and extension at 68 C for 1 min, and final extension at 68 C for 10 min. The PCR product was sequenced using the reverse primer. The C- and T-alleles of SNP-43 were designated as alleles 1 and 2, as described previously.
Statistical analysis
All statistical analyses were performed using StatView software (SAS Institute, Inc., 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. P < 0.05 was considered significant. All data are presented as mean ± SE.
The odds ratios for calpain-10 haplotype and PCOS were calculated using 422 unaffected first-degree relatives of PCOS probands, excluding mothers and sibs with PCOS. Each unaffected first-degree relative was genotyped for SNP-43, -19, and -63; and haplotypes were assigned as described above. There were 115 unaffected African-American first-degree relatives and 307 unaffected white first-degree relatives.
Results
Clinical characteristics of the study population
We enrolled 212 PCOS subjects [124 (58%) white women of European ancestry, 57 (27%) African-Americans, 13 (6%) Hispanics, 13 (6%) Asian-Americans, and 5 (3%) women of Middle Eastern origin]. Because of the small number of subjects of Hispanic, Asian-American, and Middle Eastern ancestry, we analyzed only white and African-American subjects. The clinical and hormonal characteristics of these 181 PCOS probands (124 white and 57 African-American) are shown in Table 1
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PCOS and the calpain-10 gene
We tested three DNA polymorphisms (SNP-43, -19, and -63) in the calpain-10 gene for association with phenotypic traits related to PCOS (levels of total and free T, SHBG, and DHAS) and to type 2 diabetes (age, BMI, glycohemoglobin level, glucose and insulin levels during the OGTT, and HOMA index). White and African-American groups were analyzed separately because the allele frequencies of these polymorphisms differed between the two groups: SNP-43 (allele 1) 0.65 and 0.80, (allele 2) 0.35 and 0.20, respectively; SNP-19 (allele 1) 0.36 and 0.66, (allele 2) 0.64 and 0.34, respectively; and SNP-63 (allele 1) 0.90 and 0.53, (allele 2) 0.10 and 0.47, respectively.
Genotype/phenotype relationships were examined only in nondiabetic subjects to avoid the confounding effects of diabetes on measures of insulin secretion and action. Nondiabetic subjects stratified by SNP-genotype did not differ significantly in any phenotypic measure, except white subjects with the SNP-43 1/1 genotype were older than those with either the 1/2 or 2/2 genotype (32.3 ± 1.1 vs. 28.8 ± 0.8 yr; P < 0.01).
Four major haplotypes, defined by SNP-43, -19, and -63, were observed in both whites and African-Americans (Table 2), although these frequencies differed between these two groups. The 112/121-haplotype combination, which is associated with a 3-fold risk of type 2 diabetes in Mexican-Americans and Northern Europeans (9), was not associated with increased risk of type 2 diabetes in either white or African-American women with PCOS, although there was little power to detect an effect on diabetes risk, because of the small number of women with PCOS and type 2 diabetes (7 whites and 12 African-Americans) in our study group. Among nondiabetic white PCOS probands, there were no significant differences in any clinical or hormonal measures between the group of individuals with the 112/121-haplotype combination and all other haplotype combinations observed (Table 3). However, among the nondiabetic African-American PCOS probands (Table 4), those with the 112/121-haplotype combination had a significantly higher area under the insulin curve (25,7021 ± 95,384 vs. 136,240 ± 11,468 pmol/min; P = 0.03) during an OGTT. This finding is particularly notable because the 112/121 subjects had a lower BMI. The insulin area under the curve, when adjusted for BMI, was still highly significant (P = 0.002, by analysis of covariance).
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None of the calpain-10 alleles or haplotypes were observed to be overtransmitted to PCOS subjects from heterozygous parents, by the transmission/disequilibrium test (TDT) (20).
Discussion
PCOS and type 2 diabetes are both common conditions that share a number of phenotypic features (3, 6, 11). Given the recent evidence that variation in the gene encoding calpain-10 is associated with insulin resistance (7, 8) and susceptibility to type 2 diabetes (9, 10), we sought to determine whether variation in this gene is associated with quantitative traits related to type 2 diabetes in women with PCOS.
Our results suggest that the calpain-10 gene does not affect PCOS-associated hormonal and metabolic measures, with the possible exception of insulin levels in nondiabetic African-Americans. This latter finding must be interpreted with caution and needs to be confirmed in a series of patients examined prospectively.
In a study of similar design, Haddad et al. (21) used single-locus TDT to examine the effect of the calpain-10 gene on PCOS, in a group of women in the United Kingdom, and found no overtransmission of alleles or haplotypes at calpain-10. We also observed no overtransmission of alleles or haplotypes at calpain-10. However, the TDT has poor power to detect potential effects of the variation, at the calpain-10 gene, on disease susceptibility because increased risk of disease is observed only in individuals with two different haplotypes. Thus, we would not expect to observe overtransmission of either of the at-risk haplotypes except in matings in which the other at-risk haplotype could also be transmitted, which reduces the power of the approach because of differences in the frequencies of the at-risk haplotypes. Because of the low power of the TDT, we further examined the possibility that the at-risk haplotype combination identified as increasing risk for type 2 diabetes affected risk of PCOS, by calculating odds ratios for PCOS patients relative to their unaffected family members and, in the case of our white subjects, relative to a set of data from unrelated controls from several Northern European populations. Both the white and African American PCOS subjects with the high-risk haplotype combination showed a 2-fold increase in risk for PCOS. Larger studies or meta-analyses will be needed to confirm this result and to establish the effect of the calpain-10 gene on the development of PCOS.
Acknowledgments
Footnotes
This work was supported by grants from the U.S. Public Health Service (DK-20595, DK-47486, DK-55889, and RR-00055), a Research Award from the American Diabetes Association (to D.A.E.), and a gift from the Blum-Kovler Foundation.
Abbreviations: BMI, Body mass index; CI, confidence interval; DHAS, dehydroepiandrosterone sulfate; DMSO, dimethylsulfoxide; HOMA, homeostasis model assessment; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; TDT, transmission/disequilibrium test.
Received August 8, 2001.
Accepted December 26, 2001.
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