May 5, 2007
CAPN10 UCSNP-43, UCSNP-19 and UCSNP-63 Polymorphisms and Metabolic Syndrome in Polycystic Ovary Syndrome
By Wiltgen, Denusa; Furtado, Lcia; Kohek, Maria B F; Spritzer, Poli Mara
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder with individual susceptibility determined by genetic and environmental risk factors. Recently, studies have evaluated the CAPN10 gene in PCOS patients, suggesting that different alleles may play a role in PCOS susceptibility. We performed a cross-sectional study with 88 southern Brazilian hirsute patients with PCOS or idiopathic hirsutism (IH) to assess the influence of CAPN10 genetic variants on clinical and biochemical features of metabolic syndrome. PCOS patients were defined by oligo/amenorrheic cycles ( 3.8 ng/ml), normal androgen levels, and without any known underlying disease (n = 29). Metabolic syndrome was defined according to the 2001 criteria of the National Cholesterol Education Program, Adult Treatment Panel III. UCSNP-43 polymorphism of CAPN10 was related to metabolic syndrome (p = 0.047) in PCOS; UCSNP-19 and UCSNP-63 were not associated with phenotypic traits in PCOS. These results provide evidence that CAPN10 gene UCSNP-43 polymorphisms may influence the PCOS metabolic phenotype. This should be further confirmed in large population-based studies.
Keywords: Insulin resistance, idiopathic hirsutism, metabolic syndrome, calpain-10, polycystic ovary syndrome
Polycystic ovary syndrome (PCOS) is a complex and prevalent endocrine disease characterized by ovulatory dysfunction and hyperandrogenism. In addition, PCOS patients present metabolic disturbances, such as dyslipidemia, insulin resistance and impaired glucose tolerance, which increase their risk of developing type 2 diabetes mellitus (T2DM) and possibly enhance their risk of cardiovascular disease [1-6]. However, the mechanisms involved in the etiology of PCOS and the association of PCOS with insulin resistance are still unclear. The current evidence indicates that PCOS is a multifactorial disease and that individual susceptibility is probably determined by multiple genetic and environmental risk factors.
Calpains are a family of non-lysosomal cysteine proteases found in all tissues. Experimental studies suggest that these enzymes could be implicated in adipocyte differentiation, modulation of insulin secretion and action, and free fatty acid metabolism [7,8], all of which are relevant for PCOS. The gene encoding calpain-10 (CAPN10), which consists of 15 exons spanning 31 kb, located on chromosome 2q37.3, was the first candidate gene for T2DM isolated by positional cloning. Horikawa and colleagues  found that three polymorphisms in CAPN10 (UCSNP-43, UCSNP-19 and UCSNP-63) defined a high-risk haplotype in Mexican-Americans and northern Europeans, implying a threefold increase in the risk for T2DM. Since Horikawa's study, several investigators have studied the association between the population frequency of polymorphic variants of CAPN10 and T2DM prevalence [10-17]. Recently, some studies have also evaluated the CAPN10 gene in PCOS patients [18-22]. The main focus of these studies was the haplotype/genotype distribution of the CAPN10 UCSNP- 43, UCSNP-19 and UCSNP-63 polymorphisms, without inquiring about the possible influence of each single polymorphic variant on the development of PCOS or its clinical characteristics.
Therefore, the aim of the present study was to assess the influence of these genetic variants of CAPN10 on the cluster of risk factors for cardiovascular disease referred to as metabolic syndrome in a southern Brazilian population of hirsute patients with PCOS or idiopathic hirsutism (IH).
The population consisted of 88 hirsute women recruited from the Gynecological Endocrinology Unit at the Hospital de Clnicas de Porto Alegre, Brazil. None had received any drugs known to interfere with hormone levels for at least 3 months before the study. PCOS patients (n = 59) were defined by oligo/amenorrheic cycles ( 3.8 ng/ml), normal androgen levels, and without any known underlying disease (n = 29). The study protocol was approved by the local ethics committee (Institutional Review Board-equivalent), and written informed consent was obtained from all subjects.
Anthropometric measurements included body weight, height, waist circumference, waist-to-hip ratio (WHR) (waist circumference recorded at the narrowest point or at the umbilicus; hip circumference at the level of the greater trochanter) and body mass index (BMI; current weight in kg divided by the square of height in m^sup 2^). Hirsutism was scored by the original method of Ferriman and Gallwey . Blood pressure was measured in the supine position after a 10-min rest. Metabolic syndrome was defined when at least three of the following five criteria were present: waist circumference > 88 cm, triglycerides (TG) ≥ 150 mg/dl, high- density lipoprotein cholesterol (HDL-C)
Hormonal and metabolic variables were assessed between days 2 and 10 of the menstrual cycle or on any day when the patients were amenorrheic. After an overnight fast, blood samples were drawn from an antecubital vein for determination of plasma lipids at baseline and glucose and insulin before and 2 h after an oral glucose tolerance test with 75-g glucose load. Blood samples were also drawn for hormone measurements: luteinizing hormone (LH), sex hormone- binding globulin (SHBG) and total testosterone (TT). All samples were obtained between 08.00 and 10.00 hours.
FAI was estimated by dividing TT (nmol/l) by SHBG (nmol/l) and multiplying by 100. Homeostasis model assessment quantitative insulin sensitivity check index (HOMA index) was calculated by multiplying insulin (IU/ml) by glucose (mmol/l) and dividing this product by 22.5 .
Total cholesterol (TC), HDL-C, TG and glucose were determined by colorimetric/enzymatic methods using the Modular P800 (Roche Diagnostics; Indianapolis, IN, USA). Low-density lipoprotein cholesterol (LDL-C) was determined indirectly using the formula: LDL- C = (TC-HDL-C + TG)/5. Serum LH was measured by a specific immunometric assay (Diagnostic Products Corporation, Los Angeles, CA, USA) with sensitivity of 0.05 mIU/ml and intraand inter-assay coefficients of variation (CVs) of 3.6 and 6.7%, respectively. Serum TT levels were measured with the radioimmunoassay method (ICN, Costa Mesa, CA, USA); intra- and inter-assay CVs were 10.0 and 11.6%, respectively. SHBG was measured by chemiluminescent enzyme immunoassay (Diagnostic Products Corporation) with sensitivity of 0.2 nmol/l, and intra- and inter-assay CVs of 6.1 and 8.0%, respectively. Serum insulin levels were measured with an electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany); sensitivity and intra- and inter-assay CVs were 0.20 IU/ml, 1.8 and 2.5%, respectively.
Single nucleotide polymorphism study
In addition to serum samples, whole blood samples were collected from all women. Genomic DNA was extracted from peripheral leukocytes following the technique described by Miller and associates .
Three single nucleotide polymorphisms (SNPs) of CAPN10 were analyzed: SNP-43, SNP-19 and SNP-63, as described by Haddad and co- workers . SNP-43 (CAPN10-g.4852G/A) was genotyped employing one common primer and two size-differentiated allele-specific primers. Nucleotide mismatches were introduced to improve allele specificity where necessary, and allele-specific primer concentrations were adjusted to compensate for the different efficiencies of the two amplifications in polymerase chain reaction (PCR) . PCR products for SNP-43 show a 134-bp allele and a 152-bp allele. SNP-19 (CAPN10- g.7920indel32bp) is an insertion/deletion polymorphism and after PCR amplification two alleles are obtained, with a difference of 32 bp (allele 1: 155 bp; allele 2: 187 bp). SNP-63 (CAPN10-g.1637C/T) was typed using the restriction fragment length polymorphism PCR technique (HhaI restriction enzyme; New England Biolabs, Inc, Beverly, MA, USA), producing a 162-bp fragment (allele 1) and a 192- bp fragment (allele 2).
The PCR reactions were performed using Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). Each reaction sample (50 l) contained 50 ng genomic DNA, 0.2 U Tag DNA polymerase, 5% dimethylsulfoxide, 1 PCR buffer [(NH^sub 4^)^sub 2^SO^sub 4^, 16 mmol/l; Tris-HCl, pH 8.4, 67 mmol/1; Tween20, 0.01%], 200 l \each dNTP and 1.5 mM MgCl^sub 2^. The primers used for amplification are those described by Haddad's group . Amplifications were performed using an automated thermocycler (MJ Research Inc., Watertown, MA, USA) under the following conditions: hot start, 12 min at 96C; 35 cycles of 30 s at 96C, 30 s with annealing temperatures of 56C for SNP-43, 60C for SNP-19 and 62C for SNP-63, and extension cycle of 30 s at 72C; and final extension cycle of 10 min at 72C. Digestion of SNP-63 PCR products was performed with 3 U Hhal for 2 h at 37C. The quality of the PCR products was assessed using 3% or 4% agarose gel electrophoresis.
Table I. Clinical, hormonal and metabolic characteristics of hirsute patients with polycystic ovary syndrome (PCOS) and idiopathic hirsutism (IH).
Results are presented as means standard deviation or median (interquartile range). Comparisons between group means were analyzed by Student's t test or one-way analysis of variance (ANOVA) followed by the Tukey test; comparisons between median values were analyzed using the Mann-Whitney U test or the Kruskal-Wallis test. The χ^sup 2^ test was used for comparison of allelic frequencies in PCOS and IH patients for all CAPN10 polymorphisms. All analyses were performed using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA). Data were considered to be significant at p
Table I shows the clinical, hormonal and metabolic profile of the hirsute patients. Hirsutism scores were similar in both groups, but PCOS patients presented greater BMI and waist circumference than IH patients. As expected, androgens and LH levels were also higher in patients with PCOS. While the prevalence of overweight and obese patients was similar in the two groups (67% in PCOS vs. 53% in IH, p = 0.251), 2-h glucose, fasting insulin, HOMA index and FAI were significantly higher in PCOS patients. These differences were maintained even when data were adjusted for BMI (p
The prevalence of hypertension was 23.74% in PCOS vs. 17.24% in IH, impaired glucose tolerance was 5% vs. 0.6%, and T2DM was 1.22% vs. 0.31%, respectively, in PCOS and IH.
All genotype frequencies were in Hardy-Weinberg equilibrium. The frequency of polymorphic alleles and genotype distribution at three loci within the CAPNlO gene (UCSNP-43, UCSNP-19 and UCSNP-63) were not significantly different in the PCOS and IH groups (Table II).
Table III shows data concerning the association between clustering of risk factors for cardiovascular disease (metabolic syndrome) and UCSNP-43 and UCSNP-19 polymorphisms in CAPN10. Although no association was observed when the entire sample of hirsute patients was analyzed, UCSNP-43 polymorphic allele A was associated with metabolic syndrome in the PCOS group. The polymorphic allele 2R of UCSNP-19 polymorphism showed a trend toward higher frequency in PCOS patients presenting metabolic syndrome, without however reaching statistical significance. No association was observed between phenotypic PCOS traits and the polymorphic T allele in UCSNP-63.
The present paper provides evidence that the polymorphic variant in CAPN10 UCSNP-43 is related to a higher prevalence of metabolic syndrome in PCOS patients. To our knowledge, this is the first report to describe this association.
Determination of the genetic background of PCOS has been a challenge to all investigators with an interest in the pathophysiology of this syndrome. The CAPN10 gene is a strong candidate to explain the phenotypic characteristics that are more prevalent in PCOS patients than in the general population, such as obesity-independent insulin resistance [36,37]. In the present cross- sectional study, hirsute patients who fulfilled the inclusion criteria were sequentially allocated to the study. Thus, because no selection was done, PCOS patients ended up being younger and more frequently obese than IH women. Moreover, the PCOS women presented higher insulin levels and HOMA index regardless of BMI. However, these clinical features strictly reflect the high-risk nature of PCOS metabolically speaking and underscore the relevance of searching for candidate genes for metabolic co-morbidities in these women.
Table II. Comparison of CAPN10 genotype frequencies in studies on patients with polycystic ovary syndrome (PCOS) and idiopathic hirsutism (IH).
While both PCOS and IH may share genetic factors, it is interesting to note that the observed absence of association between CAPN10 gene polymorphisms and clinical phenotype in our patients with IH is similar to that reported for non-IH control groups, as shown in Table II [20,22]. IH patients are an intriguing group of hyperandrogenic women who exhibit no increase in androgen secretion, but rather abnormalities in androgen metabolism and action [38,39]. In addition, their hormonal and metabolic profile is similar to that of non-hirsute healthy patients, while at the same time they have the same complaints and their hirsutism is similar to that of PCOS [25,29,31].
Although the association between CAPN10 gene polymorphisms and T2DM has been the preferred focus in population studies, secondary endpoints such as associations with dyslipidemia, hypertension and obesity have been also reported. In a Japanese population, polymorphic variants of UCSNP-44 and UCSNP-43 were related to dyslipidemia but not to T2DM . Moreover, in PCOS patients, the 1121 risk haplotype (UCSNP-44, -43, -19 and -63) was frequently found in dyslipidemic (TC >200 mg/dl) subjects . In addition, other studies have shown positive results for CAPN10 polymorphisms only in isolated components of the metabolic syndrome, such as higher TG levels and reduced adipose tissue mRNA expression of CAPN10, without association with obesity . Shima and colleagues  reported an association of the polymorphic deleted allele (2R) in the UCSNP-19 locus with elevated BMI and glycated hemoglobin in a Japanese population. In the present paper, we also found a higher but non-significant association of the polymorphic deleted allele (2R) in the UCSNP-19 locus with metabolic syndrome in PCOS patients. Further studies with additional patients are needed to confirm these results.
Table III. CAPN10 UCSNP-43 and UCSNP-19 polymorphisms and the presence of metabolic syndrome (MS).
Recently, a joint statement from the American Diabetes Association and the European Association for the Study of Diabetes questioned the usefulness of the 'metabolic syndrome' to detect risk of cardiovascular disease since the risk of all risk factors was additive and there was no special risk conferred by qualifying for an arbitrarily defined cluster . On the other hand, our data showing an association between higher prevalence of polymorphic variants of CAPN10 gene and metabolic syndrome supports the notion that a clustered syndrome diagnosis related to metabolic features, together with genetic alterations, would be more effective to identify young PCOS patients at increased risk for cardiovascular disease than the use of a single variable. Further studies on a control geographic population are also needed in order to estimate the frequencies of the CAPN10 gene marker in the Brazilian population.
In conclusion, the present data show an association between higher prevalence of the UCSNP-43 polymorphic allele of the CAPN10 gene and metabolic syndrome in PCOS women. These findings in a young population suggest that the presence of the UCSNP-43 polymorphism could be regarded as a marker for a subpopulation of PCOS patients at higher risk for metabolic co-morbidities.
The study was supported by grants from Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq), PRONEX 26/98 (Programa de Apoio aos Ncleos de Excelncia em Pesquisa), CAPES (Coordenao de Aperfeioamento de Pessoal de Nvel Superior) and FIPE- HCPA.
The paper was presented in part, as a free communication, at the 12th World Congress of Gynecological Endocrinology held in Florence, Italy in March 2006.
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DENUSA WILTGEN1,2, LCIA FURTADO2, MARIA B. F. KOHEK2,3, & POLI MARA SPRITZER1,2
1 Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clinicas de Pono Alegre, Porto Alegre, Brazil, 2 Laboratory of Molecular Endocrinology, Department of Physiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, and 3 Department of Physiological Sciences, Fundao Faculdade Federal de Cincias Mdicos de Porto Alegre, Porto Alegre, Brazil
(Received 17 July 2006; revised 19 January 2007; accepted 22 January 2007)
Correspondence: P. M. Spritzer, Department of Physiology, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite 500, 90050-170 Porto Alegre, RS, Brazil. Tel: 55 51 3316 3671. Fax: 55 51 3316 3656. E-mail: spritzerfaiufrgs.br
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