A Common 34C>G variant at the peroxisome proliferator-activated receptor-[gamma]2 gene in Chilean women with polycystic ovary syndrome and controls/Polimorfismo 34C>G del gen del receptor activado por proliferadores peroxisomales-[gamma]2 en mujeres Chilenas con sindrome de ovario poliquistico y controles.
KEY WORDS: Polycystic ovary syndrome; Peroxime proliferator-activated receptor; Pro12Ala polymorphism.
RESUMEN: El polimorfismo 34C>G del gen del receptor activado por proliferadores peroxisomales-[gamma]2 (PPAR-[gamma]2) ha sido relacionado con el sindrome de ovario poliquistico (SOP). Sin embargo, los resultados obtenidos entre poblaciones son contradictorios. En el presente estudio se investigo la posible asociacion entre el polimorfismo 34C>G del gen PPAR-[gamma]2 y SOP, en mujeres chilenas. Fueron analizadas 50 mujeres no relacionadas con diagnostico de SOP (29.1 ?8.1 anos) y 75 mujeres controles (29.3 ?9.3 anos). Se evaluaron las concentraciones sericas de lipidos, glucosa y acido urico mediante metodos enzimaticos-colorimetricos. La genotipificacion de la variante 34C>G del gen PPAR-[gamma]2 fue realizada mediante la tecnica de PCRRFLP. Los datos muestran que las mujeres con SOP presentan elevados niveles de glucosa, colesterol total, trigliceridos, LDL-C y acido urico; y bajos niveles de HDL-C al ser comparadas con las mujeres controles (p<0.05). La frecuencia del alelo mutado 34G fue 9% en las mujeres con SOP y 12% en las mujeres controles (p=0.589). La odds ratio para PCOS asociada al alelo 34G fue 0.73 (I.C. 95% = 0.31 - 1.69) confirmando la ausencia de asociacion. En conclusion, nuestros datos sugieren que el polimorfismo 34C>G del gen PPAR-[gamma]2 no esta relacionado a SOP, en mujeres chilenas. PALABRAS CLAVE: Sindrome de ovario poliquistico; Receptor activado por proliferadores peroxisomales; Polimorfismo Pro12Ala.
Polycystic Ovary Syndrome (PCOS) constitutes an important health public problem in young women, with a prevalence of 5-10% depending on their ethnic background. This disorder involves the combination of chronic anovulation, clinical and endocrinological signs of hyperandrogenism, evident hyperinsulinemia and polycystic ovaries (Norman et al., 2007). In addition, several studies have been associated this disorder with metabolic abnormalities as dyslipidemia, diabetes, hypertension, obesity and cardiovascular diseases (Cussons et al., 2006, 2007).
Although the inheritance mode of PCOS is still uncertain, multiple genetic factors including mutations and polymorphisms to several genes have been associated with PCOS and its phenotypic traits (Diamanti-Kandarakis & Piperi, 2005; Haap et al., 2005; Luque-Ramirez et al., 2006; Unluturk et al., 2007); specially, the genes involved in the energy homeostasis as the peroxisome proliferator activated receptor-[gamma](PPAR[gamma]).
The PPAR[gamma]gene is located on 3p25, comprises nine exons and extends over more than 100 kb of genomic DNA (Fajas et al., 1997). Three different PPAR[gamma]mRNAs have been characterized in humans generated by alternative splicing. Among these, the PPAR[gamma]2 is a protein predominantly expressed in adipose tissue, and has been considerate a regulator of adipocyte differentiation and glucose homeostasis (Beaven & Tontonoz, 2006).
Several genetic variants have been described in the PPAR-[gamma]2 gene (Unluturk et al.). However, there are two PPAR-[gamma]2 gene polymorphisms that have been systematically investigated in various populations. The first is the silent 478C>T substitution which resides in the exon 6 and the second is the missense 34C>G mutation resulting in a change of prolina by alanina at codon 12 (Pro12Ala) of the exon 2 (Yen et al., 1997; Meirhaeghe & Amouyel, 2004).
Numerous studies have been conducted with the objective to evaluate the possible relationships between 34C>G mutation (Pro12Ala) of the PPAR-[gamma]2 gene and dyslipidemia (Zietz et al., 2002; Tai et al., 2004), obesity (Masud & Ye, 2003), type 2 diabetes (Hara et al., 2000) and insulin sensitivity (Ek et al., 2001; Meshkani et al., 2007). Since PCOS and type 2 diabetes share certain phenotypic features such as obesity and insulin resistance, several studies have been also investigated the association between PCOS and 34C>G variant at the PPAR-[gamma]2 gene (Hara et al., 2002; Korhoren et al., 2003; Orio et al., 2003; San Millan et al., 2004; Hahn et al., 2005; Tok et al., 2005; Wang et al., 2006; Antoine et al., 2007). However, these studies have yielded conflicting results.
Considering a significant interethnic allele frequency variation for 34C>G polymorphism across populations, the aim of the present study was to investigate the frequency and possible association between 34C>G variant at the PPAR-[gamma]2 gene and the presence of PCOS in Chilean women.
MATERIAL AND METHOD
Subjects. A total of 125 unrelated Chilean women were studied. Fifty were patients with PCOS (16 - 43 years old) and 75 were non-PCOS women (controls, 20 - 50 years old) with normal menstrual cycles (< 32 days) without hirsutism, acne, or male-type alopecia, and not taking hormonal medications. All women were recruited from the Obstetrics and Gynaecology Service of the Hernan Henriquez Hospital of Temuco city, Chile.
The diagnosis of PCOS was assigned using the 1990 National Institute of Health criteria, which define PCOS as ovulatory dysfunction plus hirsutism and/or hyperandrogenemia, with exclusion of other disorders (Zawadzki & Dunaif, 1992). Polycystic ovary syndrome was diagnosed after exclusion of androgen-producing tumors, nonclassic 21-hydroxylase-deficient adrenal hyperplasia, hyperprolactinemia, active thyroid disease, and Cushing's syndrome. Ovulatory dysfunction was defined as menstrual cycles >45 days in length, or a progesterone level <2 ng/mL on days 22-24 of the menstrual cycle, in conjunction with a monophasic basal body temperature chart.
Demographic data and history of hypertension, diabetes mellitus, and hypercholesterolemia were assessed in each subject. Subjects with a history of diabetes or basal glycemia [greater than or equal to] 126 mg/dl were defined as diabetic. We calculated the BMI [body weight (kg) divided by square of height (m)] to assess obesity.
The study protocol was approved by the Ethics Committee of the University of La Frontera, and all subjects gave written informed consents according basic principle of biomedical investigation enumerated in the Helsinki Declaration.
Laboratory measurements. Biochemical measurements were determined from blood sample collected after overnight (>12h) fast. Triglycerides (TG) and total cholesterol (TC) levels were assayed by enzymatic colorimetric methods (Fossati & Prencipe, 1982; Fossati & Medicci, 1987). High-density lipoprotein cholesterol (HDL-C) concentrations were measured by enzymatic assay after phosphotungstic acid and magnesium precipitation (Burstein et al., 1970). Low density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation when the triglyceride concentrations did not exceed 4.8 mmol/l (Friedewald et al., 1972). Serum glucose and uric acid levels were also determined by enzymatic methods (Barham & Trinder, 1972; Fossati et al., 1980).
DNA analysis. Genomic DNA was extracted from blood leukocytes by salting out procedure optimized by Salazar et al. (1998). The PPAR-[gamma]2 34C>G polymorphism was identified according to conditions described by Tavares et al. (2005). A 244-bp fragment was amplified by PCR in a final volume of 50 [micro]l containing 50 ng of genomic DNA, 100 nM of each primer, 200 mM of each dNTP, 1 unit of Taq DNA polymerase and PCR buffer (KCl 50 mM, 2 mM Mg[Cl.sub.2], 20 mM (N[H.sub.4])2S[O.sub.4], 75 mM Tris-HCl, pH 9.0). After initial denaturation at 98[degrees]C for 3 min, the amplification was performed in 30 cycles consisting of 1 min at 94[degrees]C, 1 min at 62[degrees]C and 1 min at 72[degrees]C. A final extension of 10 min at 72[degrees]C completed the reaction.
PCR products were submitted to FnuDII cleavage (5U) in a total reaction volume of 20[micro]l. Enzymatic digestions were carried out at 37[degrees]C over night. The fragments were separated on 3% agarose gel for about 60 min at 100V and stained with 0.5mg/dl of ethidium bromide, and visualized on a UV transilluminator. The correct assessment of genotype for 34C>G (Pro12Ala) polymorphism at the PPAR[gamma]2 gene was evaluated using a homozygous sample for restriction site as a positive control. In addition, all gels were reread blindly by two persons without any change, and 10% of the analyses were randomly repeated.
Statistical analysis. Statistical analysis was carried out using the Sigma Stat Software, v. 2.0 (Jandel Sci., San Rafael, CA). Data are presented as mean [+ or -] SD. Differences between the means of the 2 continuous variables were evaluated by Student's t-test. The allelic frequencies and genotype distribution were estimated by gene counting. Differences between noncontinuous variables, genotype distribution and allele frequency were tested by chi-square analysis ([chi square]). The Odds Ratio (OR) for PCOS and their 95% confidence interval (CI) associated with the 34G (Ala) variant was also calculated. Statistical significance was at P<0.05.
The clinical characteristics of women enrolled in the study are summarizes in Table I. The serum total cholesterol, triglycerides, LDLC, glucose and uric acid concentrations were higher in the PCOS women (P<0.05). In addition, PCOS patients presented a lower HDL-C levels (P <0.05) and higher systolic blood pressure (P=0.049) and BMI values (P< 0.001) when compared to control women.
The genotype distribution and the relative allele frequencies of the Pro12Ala polymorphism at the PPAR-[gamma]2 gene in PCOS patients and controls are shown in Table II. Genotype frequencies did not deviate from the Hardy-Weinberg equilibrium in controls ([chi square] =1.01; P = NS) and in PCOS women ([chi square] = 1.06; P = NS).
The frequency of G (Ala) allele for 34C>G polymorphism at the PPAR-[gamma]2 was 9% in PCOS patients and 12% in control women (P <0.05, Table II). The Odds ratio for polycystic ovary syndrome among carriers of G allele was 0.73 (95% CI = 0.31 - 1.69, P=NS) confirming the absence of association. No significant differences were observed between PCOS carriers of the PPAR-[gamma]2 C alleles (wild) and that of G variant alleles with regard to the anthropometric or metabolic parameters investigated (Table III). On the other hand, was observed that healthy women carrying the 34G allele presented higher serum concentrations of total cholesterol (p=0.047) and LDL-C (p=0.027).
Polycystic ovary syndrome is a metabolic disorder resulting from the interaction of genetic predisposition and environmental risk factors (Crosignani & Nicolosi, 2001; Carmina, 2003). In the current study, we examined the possible association between 34C>G (Pro12Ala) polymorphism at the PPAR-[gamma]2 gene and the presence of polycystic ovary syndrome in Chilean women.
When we evaluated the clinical and laboratory characteristics in PCOS patients and controls, we observed that the BMI values, total cholesterol, triglycerides, LDL-C, fasting glucose and uric acid concentrations were significantly greater in PCOS patients. On the other hand, the serum levels of HDL cholesterol were lower in this group when compared to controls. These data confirm the well-know associations between PCOS and traditional cardiovascular risk factors (Quinonez et al., 2000; Cussons et al., 2006; Shroff et al., 2007).
Our data also shown that the 34C>G polymorphism at the PPAR-[gamma]2 gene was not associated with PCOS in Chilean women. The frequency of G (Ala) allele for 34C>G polymorphism at the PPAR-[gamma]2 was 9% in PCOS patients and 12% in control women. This result is in accordance with other previous studies (Urbanek et al., 1999; Orio et al.; San Millan et al.; Tok et al.; Wang et al.; Antoine et al.). On the other hand, Korhoren et al. (2003) in Finnish population found a significant association between 34C>G polymorphism and PCOS. In another study, Hara et al. (2002) showed that Caucasian PCOS patients with 34G allele are more insulin sensitive than those with 34C allele. Recently, Hahn et al. also reported that the 34C>G polymorphism is associated with increased insulin sensitivity as well as lower hirsutism scores in PCOS women. Similarly, Yilmaz et al. (2005) suggest that 34C>G polymorphism may be protective against insulin resistance and might prevent the development of diabetes mellitus, in the first-degree relatives of subjects with PCOS.
These contradictory results may be explained, at least in part, by undetected ethnic admixture in cases or controls that falsely distort allele frequencies in some situations (Colhoun et al., 2003). Several studies have been described a significant interethnic allelic variation for 34C>G polymorphism of the PPAR-[gamma]2 gene across populations, with a higher allele frequency in Caucasians and minor frequency in Asian subjects (Ruiz-Narvaez, 2005).
In addition, was reported that there are a widely spectrum of allele frequencies within and between populations, irrespective of disease status (Cardon & Palmer, 2003). PCOS is a common and complex disorder. Similar to other multifactorial traits such as type 2diabetes and obesity, the complexity of the underlying genetic model as well as potential gene-gene and gene-environment interactions pose a difficulty for genetic analysis (Unluturk et al.). Other major limitations of genetic studies in PCOS are lack of universally accepted diagnostic criteria and definition, relatively small sample size of the study populations, and variable penetrance and expressivity.
In summary, our data suggest that the 34C>G polymorphism of the PPAR-[gamma]2 gene is not associated with increased susceptibility to PCOS in Southern Chilean women.
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Prof. Dr. Luis A. Salazar
Departamento de Ciencias Basicas
Facultad de Medicina
Universidad de La Frontera
Av. Francisco Salazar 01145
Temuco - CHILE
* Neftali Guzman; * Leonardo Erices; ** Patricio Valdes & * Luis A. Salazar
* Laboratorio de Biologia Molecular & Farmacogenetica, Departamento de Ciencias Basicas, Facultad de Medicina, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile.
** Departamento de Obstetricia & Ginecologia, Facultad de Medicina, Universidad de La Frontera, Av. Manuel Montt 112, Casilla 54-D, Temuco, Chile. This study was supported by Grants from Direccion de Investigacion y Desarrollo, Universidad de La Frontera (DIDUFRO EP 120338), Chile.
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GUZMAN, N.; ERICES, L.; VALDES, P. & SALAZAR, L. Polimorfismo 34C>G del gen del receptor activado por proliferadores peroxisomales-[gamma]2 en mujeres chilenas con sindrome de ovario poliquistico y controles. Int. J. Morphol., 25(4):867-873, 2007.
Table I. Clinical and metabolic characteristics of the study population. PCOS (50) Controls (75) Age, years 29.1 [+ or -] 8.1 29.3 [+ or -] 9.3 BMI, kg/[m.sup.2] 33.0 [+ or -] 8.2 23.3 [+ or -] 2.6 SBP, mmHg 117.8 [+ or -] 9.9 110.4 [+ or -] 11.0 DBP, mmHg 76.0 [+ or -] 9.7 69.8 [+ or -] 9.8 Fasting glucose, mg/dl 103 [+ or -] 18 90 [+ or -] 16 Total cholesterol, mg/dl 201 [+ or -] 36 180 [+ or -] 32 Triglycerides, mg/dl 153 [+ or -] 73 82 [+ or -] 43 HDL-C, mg/dl 43 [+ or -] 7.5 54 [+ or -] 11.8 LDL-C, mg/dl 127 [+ or -] 41 108 [+ or -] 32 Uric acid, mg/dl 4.7 [+ or -] 1.0 3.7 [+ or -] 0.6 P * Age, years 0.919 BMI, kg/[m.sup.2] < 0.001 SBP, mmHg 0.049 DBP, mmHg 0.066 Fasting glucose, mg/dl < 0.001 Total cholesterol, mg/dl < 0.040 Triglycerides, mg/dl < 0.001 HDL-C, mg/dl < 0.001 LDL-C, mg/dl 0.002 Uric acid, mg/dl < 0.001 Number of individuals in parenthesis; BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure; PCOS, polycystic ovary syndrome; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. * Student's t-test. Table II. Genotype distribution and relative allele frequencies of 34C>G (Pro12Ala) polymorphism at the PPAR-[gamma] 2 gene in Chilean women with PCOS and controls. Genotypes Alleles CC CG GG C G PCOS 84% 14% 2% 0.91 0.09 (50) (42) (7) (1) Controls 79% 19% 2% 0.88 0.12 (75) (59) (14) (2) [chi square] = 0.26; 1 df;[chi square] = 0.29; 1 df; p = 0.610 * p = 0.589 Number of individuals in parenthesis; PCOS: polycystic ovary syndrome; df, degree of freedom; * CC vs. CG + GG Table III. Clinical and laboratory characteristics (mean [+ or -] SD) of Chilean women with PCOS and controls according to different genotypes of 34C>G (Pro12Ala) polymorphism of the PPAR-[gamma] 2 gene. PCOS CC CG/GG P * (42) (8) Age, years 26 [+ or -] 7 30 [+ or -] 8 0.238 BMI, kg/[m.sup.2] 32 [+ or -] 7 34 [+ or -] 6 0.606 SBP, mmHg 124 [+ or -] 8 116 [+ or -] 8 0.192 DBP, mmHg 78 [+ or -] 4 77 [+ or -] 6 0.725 Glucose, mg/dl 104 [+ or -] 19 96 [+ or -] 11 0.283 TC, mg/dl 206 [+ or -] 43 173 [+ or -] 19 0.076 HDL-C, mg/dl 43 [+ or -] 8 44 [+ or -] 7 0.695 LDL-C, mg/dl 131 [+ or -] 43 104 [+ or -] 13 0.125 TG, mg/dl 158 [+ or -] 73 128 [+ or -] 74 0.362 Uric acid, mg/dl 4.7 [+ or -] 1.0 4.9 [+ or -] 1.4 0.703 Controls CC CG/GG P * (59) (16) Age, years 32 [+ or -] 6 27 [+ or -] 3 0.208 BMI, kg/[m.sup.2] 23 [+ or -] 3 24 [+ or -] 2 0.322 SBP, mmHg 110 [+ or -] 11 107 [+ or -] 10 0.510 DBP, mmHg 70 [+ or -] 9 68 [+ or -] 12 0.566 Glucose, mg/dl 88 [+ or -] 19 91 [+ or -] 19 0.723 TC, mg/dl 170 [+ or -] 27 196 [+ or -] 33 0.047 HDL-C, mg/dl 54 [+ or -] 11 59 [+ or -] 12 0.292 LDL-C, mg/dl 94 [+ or -] 26 120 [+ or -] 27 0.027 TG, mg/dl 88 [+ or -] 42 81 [+ or -] 34 0.658 Uric acid, mg/dl 3.7 [+ or -] 0.7 3.8 [+ or -] 0.5 0.792 Number of individuals in parenthesis, BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure; PCOS, polycystic ovary syndrome; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides. * P values from Student's t test.