The Relationship of eNOS, p22 phox, CETP and ESR1 Gene Polymorphisms Related to Endocrine-Metabolic Parameters and Metabolic Syndrome in Postmenopausal Women - A Sample Population Based Study

“C.I. Parhon” National Institute of Endocrinology, Bd. Aviatorilor 34-38, S1, 011863, Bucharest, Romania. “Carol Davila” University of Medicine and Farmacy, Str. Dionisie Lupu 37, S2, 020021, Bucharest, Romania. “Ana Aslan” National Institute of Geriatry and Gerontology, Str. Caldarusani 9, S1, Bucharest, Romania. “Nicolae Simionescu” Institute of Cellular Biology and Pathology, Str. B.P. Hasdeu 8, S5, 050568, Bucharest, Romania Genetic Lab SRL, Str. Capital Aviator Nicolae Drossu 9, S1, Bucharest, Romania.


INTRODUCTION
Aging is a complex, multifactorial process that dramatically influences human health and society. In this context, there is a perceived need to improve the quality of life for elderly population. The age-dependent endocrine changes, regulated by the central nervous system, are associated to dynamics of neuronal behavior, neuro-degeneration, cognition, biological rhythms, sexual behavior, and metabolism processes and also may have a great impact on life span and normal aging. The age of onset and rate of functional decline vary widely among the aging population, consistent to the regulatory role of genetic factors.
Over the past decades epidemiological research of so-called "complex" diseases has identified a number of risk factors for the common agerelated disorders. Genetic mechanisms that possibly trigger and define the rate at which we age have been well documented in lower organisms, but less is known in humans. Nowadays, a novel class of risk factors, genetic polymorphisms gained considerable interest. Associated studies have suggested the involvement of candidate genes in age-related disorders. Many of polymorphisms of these genes are associated with alteration of endothelial function such as endothelial NOS (eNOS) gene [1][2][3][4][5], oxidative stress such as p22phox gene-a subunit of NADPH-oxidase [6-9], lipid metabolism such as Cholesteryl Ester Transfer Protein (CETP) gene [10][11][12][13]. Also, variants in the estrogen receptor-α (ESR1) gene have been associated with components of the metabolic syndrome (MetS), including obesity, HDL/LDL cholesterol (HDL/LDL-C) metabolism, blood pressure (BP), and type 2 diabetes [14-16].
MetS is a constellation of characteristics that increases the risk for the development of diabetes (tpye 2 diabetes) and cardiovascular disease in postmenopausal women (PM) [17][18][19][20][21][22][23][24]. Many cross-sectional studies have shown an increased risk of metabolic syndrome in postmenopausal women which prevalence varies from 13.3 to 51% [25 -33]. The pathophysiology of MetS is not completely understood, but is thought to involve a complex interaction between the environment, genetic susceptibility, insulin resistance, and abnormal adipose tissue function [34][35][36][37].
The purpose of this study is to focus the endocrine-metabolic changes in postmenopausal women with metabolic syndrome and examine relationship with the polymorphisms of eNOS-G894T, p22(phox) (-930 A/G) , CETP TaqIB , ESR1 (PvuII and XbaI) genes. The SNPs (rs1799983, rs9932581,rs708272, rs9340799, rs2234693) of these genes were selected according to published literature that show to be associated with the susceptibility to components of MetS.
p22 phox protein is a major component of NADPH oxidase, a critical subunit that plays an essential role in NADPH oxidase activation in vascular cells. The −930A/G (rs9932581) polymorphism of p22 phox gene is located in the promoter region of CYBA at position −930 from the ATG codon and was reported to be associated with hypertension [8,39].
CETP mediates the exchange of lipids between anti-atherogenic high-density lipoprotein (HDL) and atherogenic apolipoprotein (apo) B containing lipoproteins and therefore plays a key role in human lipid metabolism. The gene coding for CETP encompasses 16 exons and is localized on chromosome 16q21. It has been demonstrated that the CETP gene is polymorphic and one of the most widely studied polymorphisms being TaqIB (rs708272). Most studies indicate that the TaqIB B1B1 genotype is associated with higher plasma CETP and lower HDL-C levels than the B2B2 genotype [10-14, 40-41].

Study Design and Subjects
Two-hundred-eighty apparently healthy women aged between 60 and 80 years participated. This study was a part of the GENAGE National Project that was first study related to GENETICS AND ENDOCRINE-METABOLIC-COGNITIVE CHANGES IN HUMAN AGING from Romania (2006)(2007)(2008). Exclusion criteria for all participants were: -Significant medical illness or organ failure, such as uncontrolled hypertension, diabetes, cardiac disease, cerebrovascular disease, chronic obstructive pulmonary disease, kidney and liver disease; -Significant neurologic disease that might affect cognition, such as Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis; -Significant current psychiatric illness, such as depression, schizophrenia; -Autoimmune disease; -Established genetic diseases; -Severe hormonal dysfunctions. This study was approved by Ethic Committee of our Institution and written informed consent from all enrolled subjects was obtained. Participants completed a questionnaire and were physically evaluated by physicians. Cognition was evaluated by the Mini Mental Status Examination (MMSE). MMSE is a brief test of several cognitive abilities with a maximum score of 30 points.
The postmenopausal women were classified into two groups, non-metabolic syndrome (212) and metabolic syndrome (68).
Key eligibility criteria of selection MetS postmenopausal women were according to National Cholesterol Education Program Adult Treatment Panel III [43][44]. The diagnostic criteria of MetS (ATPIII-IDF International Diabetes Federation) included: central obesity, defined as waist circumference of >88 cm and at least two of the following factors: increased serum triglyceride (TG) level: fasting value >150 mg/dl; reduced HDL-C: fasting concentration <50 mg/dl; elevated blood pressure: SBP>130 mmHg or DBP>85 mmHg; increased fasting plasma glucose (GLU): >100 mg/dl.

Clinical, Biochemical and Hormonal Measurements
Clinical and anthropometric parameters including blood pressure, weight and height, waist circumference were measured following the standard procedures. Body mass index (BMI) was calculated as weight/height 2 (Kg/m 2 ). Fasting blood samples were drawn by venipuncture between 7:00 and 9:00 h in order to evaluate the hematological, biochemical, hormonal profile and for extraction of genomic DNA. Hematological and biochemical parameters were measured by standardized methods by chemistry auto-analyzers: Beckman Coulter (hematology), Cobas Mira and Hitachi (biochemistry). Additional, C-reactive protein (CRP), apo B and apoA-I were also measured by chemistry auto-analyzers.

Genotyping
Genomic DNA was prepared from the whole blood using the Maxwell 16 Blood Purification Kit (Promega Inc.). PCR protocols and primers used for genotyping of eNOS-G894T, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22(phox) (-930 A/G), CETP TaqIB, ESR1 (PvuII and XbaI) were carried out according to the manufacturer's recommendations, and the products of hydrolysis were separated on agarose gel and visualized with ethidium bromide.
rs1799983 polymorphism of the missense G894T variant of eNOS gene was amplified by PCR with specific primers. The procedure to identify the polymorphism was the same as previously described [1]. The PCR amplified 248-bp fragments were further digested with two restriction enzymes, Ban II and Mbo I, and the products were separated by electrophoresis in 8% nondenaturing polyacrilamide gel, and stained with SYBR Green I. rs9932581 (-930 A/G) gene polymorphism in the promoter of human p22 phox was amplified by PCR with specific primers [39]. The PCR 650 bp fragments were digested with Bbv I, and the fragments were analyzed by 2% agarose gel electrophoresis.
rs708272 (TaqIB) genotyping: a fragment of 1420-bp was amplified by PCR using specific primers, as previously described [40]. The PCR products were digested with 10U/µl reaction TaqIB restriction enzyme (Promega GmbH, Mannheim, Germany). The digestion product was run on a 2% agarose gel stained with ethydium bromide and visualized under UV light. The CETP B1B1 genotype was identified as two bands of 750bp and 670bp, the B1B2 genotype as 750bp, 670bp and 1420bp, and the B2B2 genotype as the starting undigested 1420bp on the agarose gels. rs9340799 (XbaI) and rs2234693 (PvuII) were evaluated after a genotyping protocol using the AMPLIKIT-ESTR Test-Kit. Both polymorphisms of ESR1gene are in intron 1 and are separated by only 46 base pairs. The PvuII polymorphism is characterized by a T→C transition 397 nucleotides upstream in the intron 1(also known as c.454-497T→C) that obliterates the PvuII restriction site. The T allele has previously been called the p allele, whereas the C allele has been called the P allele, denoting the absence of the PvuII restriction site. The XbaI polymorphism marks an A→G transition 351 nucleotides upstream in intron 1 (also known as c.454-351A → G). Those with the G allele have an absent XbaI site that was previously called X in the literature, with the A allele denoted by x.

Statistical Analysis
The measured variables are presented as mean ±SEM. The genotype and allele frequency distributions were compared with the χ2 test and computed Hardy-Weinberg equilibrium for each SNP and group of participants. Variables with a non-symmetrical skewedness distribution were log transformed. The differences in measured variables between groups and among genotypes for each group were tested by with independent Samples T test and by analyses of variance ANOVA post-hoc Bonferroni test. The association analysis between the genotype subgroups were tested using ANOVA or nonparametric Mann-Whitney U and Kruskal-Wallis tests. Binary logistic regression analysis was performed to assess the associations between polymorphism and (components of) MetS. Logistic regression was useful to predict the presence or absence of a characteristic based on values of a set of predictor variables (metabolic/hormonal). It was estimated by a model using stepwise method-forward Wald. Statistical analyses were carried out using SPSS version 15.0 (for Windows) and P values less than .05 were considered statistically significant. Table 1 summarizes the baseline characteristics with significant differences between our participants with and without MetS. The group with metabolic syndrome had three or more MetS traits and therefore fulfilled the criteria for the MetS. As shown in Table 1, weight, waist circumference, systolic and diastolic blood pressure, WBC, triglycerides, LDL-C, TG/HDL-C ratio, apolipoprotein B, apoB/apoA-I ratio, fasting glucose, insulin, HOMA-IR, uric acid, were higher and HDL-C was lower in MetS group than in control group and therefore fulfilled the criteria for the MetS.

Discussion
On the basis of prior evidence for association of eNOS-G894T polymorphism with the metabolic syndrome and published associations with component traits of the metabolic syndrome, we evaluated 5 single nucleotide polymorphisms SNPs for associations with metabolic syndrome and its components in 280 postmenopausal women. In the entire cohort, apparently healthy women, 68 (32.08%) women fulfilled The Adult Treatment Panel III -NCEP criteria for MetS (mean age: 70.03 vs 69.00 years of postmenopausal women without MetS).     All five SNPs were significantly associated with one or more MetS traits (P ≤ .001 to P=.038) (annex 1). Two of 5 SNPs, p22phox (A/G) and ESR1 (XbaI) showed evidence of significant association with metabolic syndrome except that p22phox (A/G) polymorphism showed a protective effect against metabolic syndrome as summarized in Tables 2&3. Significant associations were detected between SNPs and endocrine-metabolic profile (Tab.4-7; annex 1-2). In addition, our study reported the significant higher serum levels of uric acid, apolipoprotein B, insulin, HOMA-IR, ratios of TG/HDL-C and apoB/apoA-I in MetS group compared to control group. Hyperuricemia has been associated with several metabolic and cardiovascular conditions, including MetS [46-47]. We found that the higher level of uric acid in MetS group compared to controls were correlated positively with CRP (P=.016), E2 (P=.001), FAI (P=.05), GHBP (P=.005), PTH (P<.001) and negatively with SHBG (P=.034), cortisol (P=.001), FT4 (P=.02). The increased levels of HOMA-IR and ratio of TG/HDL-C and of the apoB/apoA in MeS group are metabolic conditions for developing atherosclerosis, CVD and type-2 diabetes mellitus.
An analysis of endocrine axes showed significant high serum levels of E2, T3, GHBP, PTH, E2/E1 ratio and low levels of cortisol, SHBG, FSH, LH, IGFBP1, cortisol/DHEA ratio in MetS group as compared with control group (Table.1). The increase of estradiol is amplified by the decrease in gonadotropins (LH and FSH) and SHBG, and the subsequent increase in E2:E1 ratio. The higher level of T3 values was previously reported [20]. The lower levels of SHBG associated with higher levels of E2 in postmenopausal women with metabolic syndrome were also reported by Wienberg ME et al [33]. Unexpectedly, cortisol and gonadotropins values were lower in MetS group than in control group. Despite the fact that the ratio of cortisol/DHEA was significantly decreased no alteration of cortisol/DHEAS ratio (0.375±0.083 vs 0.421±0.032; P=0.611) was found. In the Pearson's correlation, cortisol values positively correlated with DHEA (P<0.001) and FSH (P=0.02). In literature, the reported association of cortisol/DHEAS ratio with metabolic syndrome was observed in Vietnam Experience Study from 584 veterans (age=38.74±2.52yr) [48]. We may speculate that cortisol decline is a consequence of endocrinemetabolic alterations in postmenopausal women older than 60 years with MetS and could contribute at accelerated aging.
We found an association between polymorphism of eNOS Glu298Asp and SBP, TG, apoB, HDL-C and uric acid, some features of the metabolic syndrome. The TT genotype carriers had higher levels of BP and glucose. We also found that the TT genotype carriers had higher levels of DHEAS, A4 and FT. Hyperandrogenism has also been postulated to be a marker for insulin resistance-key feature of the metabolic syndrome and a risk factor for cardiovascular disease [49-51]. Thus, defects in eNOS function may contribute for the development of metabolic syndrome as a risk factor for cardiovascular disease [2][3][4][5]52]. The increased prevalence of the metabolic syndrome after menopause may be a result of the metabolic consequences of central fat redistribution which may influence the tendency to develop the metabolic syndrome [25, [30][31]49].
The tests for association with metabolic syndrome showed a risk allele A of p22-phox -930A/G polymorphism that significantly interacted with metabolic syndrome, a significant increase in the A allele frequency was found in MeS subjects. We detected a protective effect against metabolic syndrome of both p22 (phox) 930 A/G and CETP TaqIB polymorphisms in presence of insulin and GHBP using the binary logistic regression. However, the higher levels of BMI and apoB/apoA ratio were detected in GG genotype carriers than in AA genotype individuals in metabolic syndrome. Overall, these findings indicate the susceptibility of p22-phox -930G allele for metabolic syndrome in accordance with some studies that demonstrated a functional role of this polymorphism [39,53]. Although other reports assessing the functional role of this polymorphism in NADPH oxidase activity yielded conflicting results [54]. Although the reasons by which the 930G protectively influenced against the development of MeS were not apparent in the present study, some explanations can be proposed. In this regard, aging is widely acknowledged to be associated with oxidative stress, typically referred to as the free radical theory of aging. In addition, oxidative stress is implicated in age-related disorders such as obesity, hyperglycemia, hyperlipidemia, and hypertension [6,9].
Cholesteryl ester transfer protein plays a key role in human lipid metabolism, and therefore CETP gene polymorphisms may alter susceptibility to MetS. In our study the tests for association of CETP TaqIB polymorphism no indicated a significant association with MetS while the analyses by binary logistic regression (MetS vs Controls) showed an OR<1 in presence TAS, GLU, TG, osteocalcin and ESR1 (T/C) gene polymorphism with OR>1. Also, CETP TaqIB polymorphism associated with MetS (OR<1) in presence of the p22phox (A/G) gene polymorphism whose OR was less than 1 and in presence of insulin and GHBP, both with OR>1. These results sustain that MetS is less likely to occur in carries with CETP TaqIB polymorphism. However it associated with one or more MetS traits as TAS, GLU, TG. Most studies have been shown that the B2B2 genotype is associated with higher HDL-C levels than the B1B1 genotype [11,13]. Wu Z et al concluded that their results lend support to the concept that increased HDL-C cannot be translated into a reduction in CAD risk [41]. In our MetS group, the levels of HDL-C, TAS, glucose, cholesterol, CRP, free testosterone were higher and TSH, cross laps were lower in the (B1B2) heterozygote carriers. We did not conclude any association of phenotypic features with indices of the metabolic syndrome in B2B2 genotype carriers because the studied group was relatively small.

ESR1 intronic polymorphisms variants, PvuII and
XbaI regarding as potential genetic predispositions to metabolic syndrome are controversial [42,55]. In animal models, ESR1 knockout mice have insulin resistance, impaired glucose tolerance, and obesity, indicating that variation in estrogen receptor signaling may have relevant metabolic effects. Studies in postmenopausal women have found associations between estrogen receptor genes (ESR1 or ESR2) and the metabolic syndrome components, particularly obesity and dyslipidemia [14-16,52]. We found that the presence of heterozygote AG showed a significant risk for MetS. The phenotype of ESR1 PvuII polymorphism in MetS group was partly to that of XbaI polymorphism. ESR1 PvuII (T/C) polymorphism associated with MetS in presence of HDL-C, LDL-C, and TG/HDL-C ratio. In MetS group, C allele carriers (TC+CC genotypes) had significantly increased levels of glucose and HOMA-IR. In addition, the PvuII (T/C) variant interacted with CETP-TaqIB. All together these findings sustain the functionality of these polymorphisms in metabolic syndrome.
To the best of our knowledge, the current study is the first to evaluate the interaction of candidate genes between hormonal status and metabolic syndrome in postmenopausal women. An important point is that each of the five SNPs has an association with one or more components of MetS. The defects of eNOS-G894T, p22(phox) (-930 A/G), CETP-TaqIB, ESR1 (PvuII and XbaI) genes may alter susceptibility to MetS, influencing diabetes and CVD risk through effects on components of the metabolic syndrome, specifically increased levels of glucose and HOMA-IR, increased ratios of TG/HDL-C and apoB/apoA.

CONCLUSION
Our results sustain an interaction between the studied polymorphisms and their phenotypes in conferring a higher susceptibility to the endocrine-metabolic changes involved in pathogenesis of MetS. The elevated values of TG/HDL-C and apoB/apoA ratios could be risk indicators for calculation cardiovascular risk in of MetS.