Clinical factors such as B-type natriuretic peptide link to factor VII, endothelial NO synthase and estrogen receptor α polymorphism in elderly women

Main methods This is an observational study of 93 Japanese women with a mean age of 80.9 years recruited from outpatient clinics of Nagoya University and its related hospitals. Ten single nucleotide polymorphisms (SNPs) were studied. Each gene studied acts in or is related to either blood coagulation (factor V Leiden, prothrombin G20210A, factor XIII Val34Leu, factor VII Arg353Gln, MTHFR C677T, β-fibrinogen G-455A, PAI-1 4G/5G), metabolic syndrome-related pathways (PPARα Leu162Val), or endothelium/estrogen system (eNOS Glu298Asp, ERα IVS1-401). SNPs were analyzed for their relation to clinical values including lipids, B-type natriuretic peptide (BNP), fasting plasma glucose, tumor necrosis factor-α, interleukin-6, cyclic GMP, and nitric oxide metabolites. Key findings Comparisons between the distributions of different genotypes and clinical values showed three relationships. First, factor VII Arg353Gln and HDL-cholesterol (HDL-C) were linked to Arg/Arg carriers at higher levels ( P = .049). The HDL-C to LDL-cholesterol ratio supported this link ( P = .027). Second, eNOS Glu298Asp and triglycerides were linked to Glu/Glu carriers at higher levels ( P = .031). Third, ERα IVS1-401 and BNP were related to CC genotype at lower levels ( P = .031). Additionally, the last two relations showed that genotype does not influence the demarcation line of biomarkers, but the plasma/serum levels of biomarkers instead. Significance Correlations of factor VII Arg353Gln with HDL-C and eNOS Glu298Asp with triglycerides are new findings. Polymorphisms in the endothelium/estrogen system and the heart failure marker BNP are also correlated, with ERα IVS1-401 being the first identified marker. SNPs may be helpful for understanding the pathophysiology of atherosclerotic diseases in elderly women. Keywords Natriuretic peptide Estrogen receptor Factor V Leiden Endothelial nitric oxide synthase Single nucleotide polymorphism Thrombosis Introduction A thrombogenic state is an important risk factor for atherosclerotic diseases such as myocardial infarction and stroke. Thrombosis has been suggested to be an adverse effect associated with postmenopausal hormone-replacement therapy (HRT) ( Fisher et al. 1998; Ettinger et al. 1999; Rosendaal et al. 2002 ). Genetic mutation factors have been associated with an elevated risk associated with thrombosis. For example, factor V Leiden (also known as Arg506Gln, R506Q, or G1691A) has been associated with a 6.69-fold enhancement of the risk posed by thrombosis ( Cushman et al. 2004 ). This single nucleotide polymorphism (SNP) is a guanine (G) to adenine (A) transition at the second base of the codon for amino acid position 506 in exon 10 of the factor V (symbol; F5) gene. In total, 5% of Caucasians are carriers of this SNP, which is known to make the protein resistant to inactivation by activated protein C ( Bertina et al. 1994 ). This SNP is found in 20% of venous thrombosis patients ( Rosendaal et al. 1995 ). Regional differences exist; East Asians do not carry factor V Leiden ( Rees et al. 1995 ), which could be one of the reasons for the lower incidence of thrombosis in East Asians, although this remains unclear. Another noted SNP seen in Caucasians is prothrombin G20210A, which is a G to A transition at position 20210 of the prothrombin (symbol; F2) gene. It is observed among 6% of venous thrombosis patients ( Poort et al. 1996 ). For these two blood clotting factors, a meta-analysis has found a moderate association with coronary disease ( Ye et al. 2006 ), implying that, for these and other clotting factors, a genetic study should be performed. Several SNPs related to thrombosis and atherosclerotic risk have been proposed. Factor XIII Val34Leu shows a protective effect against venous thromboembolism ( Mikkola et al. 1994; Wells et al. 2006 ). Factor VII Arg353Gln (R353Q or G10976A) leads to a reduction of its protein levels with a lower risk of cardiovascular disease ( Green et al. 1991; Lane et al. 1992; Hunault et al. 1997; Girelli et al. 2000 ). MTHFR C677T leads to higher homocysteine levels and is thus a risk factor for coronary artery diseases ( Frosst et al. 1995; Ma et al. 1996 ). β-fibrinogen G-455A (HaeIII) is associated with higher plasma fibrinogen levels ( Thomas et al. 1991; Iso et al. 1995; van' t Hooft et al. 1999 ). PAI-1 4G/5G, which is the 4G allele related to elevation of circulating PAI-1, is associated with cardiovascular disease ( Dawson et al. 1991; Eriksson et al. 1995; Hoekstra et al. 2004 ). PPARα Leu162Val (L162V) is associated with increased transcriptional activation of itself and the elevation of serum lipid levels ( Vohl et al. 2000; Sapone et al. 2000 ). eNOS Glu298Asp (E298D) is associated with the possibility of impaired endothelial function ( Philip et al. 1999; Guzik et al. 2001 ). Lastly, ERα IVS1-401 (IVS1-397 or PvuII), which is a transition in the intervening sequence 1 at position-401, is associated with cardiovascular disease ( Herrington et al. 2002b; Nordström et al. 2003 ). In atherosclerotic lesions, lipid accumulation in the plaque intima ( Kramsch et al. 1971 ), increased cytokine levels ( Rus et al. 1991, 1996 ), and an increased BNP level are all detected ( Casco et al. 2002 ). It is believed that nitrite and nitrate, which are metabolites of nitric oxide (NOx) in vascular disease, are important factors that should be assessed ( Palmer et al. 1987 ). BNP and NO elevates intracellular cGMP level ( Palmer et al. 1987; Chinkers et al. 1989 ), indicating the importance of this second messenger. From a clinical point of view, it is important to identify biomarkers that can be used to predict both disease and longevity ( Nomura et al. 2002; Hayashi et al. 2007 ). However, the use of SNPs as biomarkers is not well understood to date. Our aim is to evaluate genetic factors in elderly women in order to identify biomarkers that correlate with SNPs. The experimental design of this report was restricted to an East Asian population that does not possess factor V Leiden, because it is an easier model for screening candidate factors associated with thrombosis or atherosclerotic risk. In addition, analyses focusing on postmenopausal elderly women are rarely performed, and thus may result in an improved fundamental understanding of older people. Material and methods Subjects We enrolled 104 Japanese female subjects over 60 years of age who were admitted to the Department of Geriatrics in an outpatient clinic of Nagoya University Hospital, Nagoya City (Japan), between May 2004 and January 2005. All subjects gave written informed consent. Proper authorization for the study was obtained from the Ethics Committee of the Nagoya University Graduate School of Medicine. As the data of serum or plasma collection or amount of assay for DNA analyses were insufficient in 11 patients, we finally analyzed the data of 93 patients. Serum or plasma collection and measurement Serum or plasma was collected from fasting blood samples after centrifugation at 3000 rpm at 4 °C, and stocked at − 30 °C until measurement. BNP and cGMP concentrations were derived from the blood sample analysis (SRL Laboratories, Japan) as measured by a specific immunoassay. Tumor necrosis factor-α and interleukin-6 were measured using the Quantikine HS Kit (R & D Systems, USA). NOx was determined by high-performance liquid chromatography ( Hayashi et al. 2007 ). Other clinical biochemical factors, such as LDL-C, HDL-C, and triglyceride levels, were also assessed. DNA isolation and genotyping DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN, Düsseldorf, Germany) and genotyped with the Mutector Dual Well Test Kit (TrimGen, Maryland, USA) according to the manufacturer's instructions. The Mutector kit was designed for mutation detection among known nucleotide substitutions using a 96-well strip plate, and can confirm all three genotypes (wild, mutant homozygous, and heterozygous types). Both positive and negative controls were provided by the manufacturer. In brief, a complimentary detection primer is designed and immobilized on the wells whose 3' end terminates just before the target base (i.e. order made by offering the specified sequence information). The polymerase chain reaction product from the preceding step is added to wells together with labeled nucleotides and extension primer for either mutant or wild strand. Primer for mutant strand makes extension when the target base is mutant type but not wild, and vice versa. As a result, labeled nucleotides are incorporated and a colorimetric reaction is observed and measured by 405 nm on a microplate reader. Selection of SNPs All ten analyzed SNPs have referential SNP cluster identification numbers (RefSNP ID) provided by the NCBI (National Center for Biotechnology Information, Maryland). They are: factor V Leiden (rs6025), prothrombin G20210A (rs1799963), factor XIII Val34Leu (rs3024472), factor VII Arg353Gln (rs6046), MTHFR C677T (rs1801133), β-fibrinogen G-455A (rs1800790), PAI-1 (-675) 4G/5G (rs1799889), PPARα Leu162Val (rs1800206), eNOS Asp298Glu (rs1799983), and ERα IVS1-401 (rs2234693). Statistical analyses The association of genotype distribution with clinical factors, represented as mean ± SEM, was analyzed using Microsoft Excel enhanced software with either an unpaired Student's t -test or a Mann–Whitney U -test, depending on histogram distribution. A chi-square test was used to describe the effect of genotype on biomarker levels dichotomized by the demarcation line. A probability value under 0.05 as determined by two-tail analyses was considered statistically significant. Results Study population and genotype distribution The patient profiles are shown in Table 1 . The observed population contains clinical backgrounds such as the following with some holding multiple diseases; angina ( n = 3), arteriosclerosis ( n = 2), cardiac insufficiency ( n = 5), cerebrovascular accident which contains a sequela ( n = 19), diabetes ( n = 11), hyperlipemia ( n = 14), hypertension ( n = 42), and none disease carriers ( n = 28) ( supplemental Table 1b ). The distribution of the different genotypes for each of the ten analyzed SNPs is shown in Table 2 . As suggested in the introduction, we confirmed that neither prothrombin G20210A nor factor V Leiden was present at detectable levels in our samples. Genotype frequencies were in Hardy–Weinberg equilibrium for all the SNPs, analyzed by chi-square test on observed versus expected genotype frequencies (all P > .05) ( supplemental Table 2b ). We proceeded with dominant model analyses to investigate the relation of genotypes to clinical values, and those which appeared to have significant differences are further addressed. Factor VII gene polymorphism and HDL-C As shown in Table 3 , the factor VII Arg353Gln polymorphism was linked to HDL-C. The GG genotype encoding for Arg/Arg was associated with higher HDL-C levels than the AG+AA genotypes ( P = .049). This finding is supported by comparison of the HDL-C to LDL-C ratio for the GG genotype (0.65 ± 0.10) and the AG+AA genotypes (0.39 ± 0.05), which were significantly different ( P = .027) ( Fig. 1 ). We did not observe a significant difference among LDL-C levels between genotypes, although there was a similar trend. Endothelial nitric oxide synthase gene polymorphism and triglycerides As shown in Table 4 , the eNOS Glu298Asp polymorphism was related to triglycerides. The GG genotype encoding for Glu/Glu was associated with higher triglyceride levels than the TG+TT genotypes ( P = .031). Since triglycerides is a clinical marker with an accepted demarcation line, that is 100 mg/dL, we used the chi-square test to assess whether the GG genotype and TG+TT genotypes has or has not had an effect on triglyceride levels dichotomized by this line. As shown in Fig. 2 , genotype does not influence the demarcation line but has an effect on the triglyceride levels ( P = .021). Estrogen receptor alpha gene polymorphism and BNP As shown in Table 5 , the ERα IVS1-401 polymorphism was related to the plasma BNP concentration, with the CC genotype being associated with a relatively lower plasma BNP concentration when compared to the TC+TT genotypes ( P = .031). Since BNP is a standard clinical marker for heart failure with a well-known demarcation line, that is 80 pg/mL, we used the chi-square test to assess whether the CC genotype and TC+TT genotypes has or has not had an effect on BNP levels dichotomized by this line. As shown in Fig. 2 , it was revealed that genotype does not influence the demarcation line but has an effect on the BNP levels ( P = .031). Discussion We restricted our subjects to postmenopausal elderly women (with a mean age of 80.9 years) who were free of factor V Leiden and prothrombin G20210A. In doing so, we were able to identify new correlations between the factor VII Arg353Gln polymorphism and HDL-C levels, and between the eNOS Glu298Asp polymorphism and triglyceride levels. We have demonstrated, for the first time, a statistically significant association between the ERα IVS1-401 polymorphism and plasma BNP concentration, suggesting that CC genotype carriers have relatively lower plasma BNP levels than TC+TT genotypes ( P = .031). To our knowledge, there are no other reports linking the heart failure marker BNP with an SNP in the estrogen system. Recently, an important meta-analysis of HRT ruled out the possibility that HRT increases HDL-C and reduces either LDL-C or the LDL-C to HDL-C ratio ( Salpeter et al. 2006 ). Concerning SNPs, a recent meta-analysis implied that the ERα IVS1-397 (which is synonymous with -401) polymorphism does not influence the HDL-C response to HRT ( Kjaergaard et al. 2007 ). Our focus is on elderly women, and we did not identify a relationship between ERα IVS1-401 and HDL-C levels, as shown in Table 5 . We did, however, observe a relationship between HDL-C and the factor VII Arg353Gln polymorphism, with the GG genotype being associated with higher HDL-C levels than the AG+AA genotypes ( P = .049). This finding was supported by comparison of the HDL-C to LDL-C ratio in each genotype in the same manner ( P = .027). This ratio was not significant for eNOS Glu298Asp (GG; 0.63 ± 0.03, TG+TT 0.55 ± 0.07), and ERα IVS1-401 (CC; 0.51 ± 0.06, TC+TT; 0.63 ± 0.10). It is interesting to consider that such an SNP could partially impact the circulating levels of HDL-C in elderly individuals, but not in younger individuals; however, there is currently no scientific evidence to support the influence of SNPs on clinical biomarker levels that are only present in the elderly. We have provided new evidence that the eNOS Glu298Asp polymorphism is related to triglyceride levels, with the GG genotype encoding for Glu/Glu being associated with higher triglyceride levels than the TG+TT genotypes ( P = .031). In addition, eNOS Glu298Asp genotype has an effect on triglyceride levels dichotomized by its demarcation line, according to the chi-square test ( P = .021). It is possible that the high frequency of the GG genotype (86%) has masked the results of statistical analyses presented in past reports ( Metzger et al. 2007 ). Because of the small sample study, ours has no power to analyze the differences in cardiovascular events between GG and TG+TT genotypes. Whether eNOS Glu298Asp is a functional SNP or not continues to be widely discussed. There has been report on eNOS Glu298Asp and cardiac disease, including hypertension, showing that T allele may be involved in a higher risk ( Srivastava et al. 2008 ). Importantly, this SNP resulting non-functional has also been reported in the yeast Pichia pastoris and in human endothelial cells ( Golser et al. 2003; McDonald et al. 2004 ). Based upon the inconsistently reported functionality of this polymorphism, we suggest that further research is required to clarify the cell types in which the eNOS Glu298Asp polymorphism is functional. In this report, the most novel finding obtained by focusing the analysis on elderly women is the relationship between the ERα IVS1-401 polymorphism and plasma BNP concentration. BNP is composed of 32 amino acids. It is synthesized in the heart ventricles, and is a well-known biomarker for heart failure ( Tsutamoto et al. 1999 ). Neither its transcriptional regulation nor its biochemical importance is well understood ( Daniels and Maisel 2007 ). For the ERα IVS1-401 polymorphism, the C allele, but not the T allele, is thought to result in elevated ERα expression ( Herrington et al. 2002a; Schuit et al. 2004 ). This C allele, results either positive or negative against protective effect on cardiovascular disease ( Hirschberg et al. 2009 ). Although estrogen up-regulates BNP mRNA and protein levels in rat neonatal cardiomyocytes ( Pedram et al. 2005 ) and ERα and BNP proteins are both produced in adult rat cardiomyocytes ( Nuedling et al. 1999; Pedram et al. 2005 ), there are no reports on the relation of polymorphism of ERα and BNP. Further, a previous study of ER knockout mice showed that ERβ encoded by the ESR2 gene, and not ERα, might be important for protection against heart failure ( Pelzer et al. 2005; Babiker et al. 2006; Pedram et al. 2008 ). ERβ is known to distribute in atherosclerotic area not normal artery nor ubiquitously and the study of SNP on ERβ in human is rare. The relation of the contribution to heart failure between ERα and ERβ, especially the relation to gene polymorphism needs to be clarified. Overall, the present results suggest that ERα IVS1-401 influences the estrogen/BNP cascade. It is remarkable that no such finding has been reported in the past, since both ERα and BNP are major factors, and produced in the same cells as discussed above. Therefore, it is possible that this study's population focus and sample collection is a key that may lead to other undiscovered SNPs that are specific to older people. There are several guidelines for relating BNP concentration to the severity of heart failure. Interestingly, according to our results, 12 of 13 samples from ERα IVS1-401 CC genotype carriers contained under 80 pg/mL BNP (as shown in Fig. 2 ), which is below the limit of 100 pg/mL and outside of the gray zone of 100–500 pg/mL ( Maisel et al. 2002; Brenden et al. 2006 ). The implication that the ERα IVS1-401 polymorphism has clinical importance on BNP levels, it needs to be further studied in larger case-control studies and in other countries, since our study size is limited and ethnic difference is untouched. We suggest that, when profiling elderly persons' clinical marker levels in order to judge their predisposition to specific diseases, SNP genotypes may play a role as prognostic factors in elderly. Limitation of the study This is a relatively small sample size, and a spurious association cannot be fully ruled out. Acknowledgements This study is supported by a Grant in Aid for Scientific Research from the Japan Society for the Promotion of Science. The sponsor had no role in the study design, methods, subject recruitment, data collection, analyses, or manuscript preparation. Appendix A Supplementary data Appendix A Supplementary data Supplementary data associated with this data can be found in the online version at 10.1016/j.lfs.2009.06.009 . References Babiker et al., 2006 F.A. Babiker D. Lips R. Meyer E. Delvaux P. Zandberg B. Janssen G. van Eys C. Grohé P.A. 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