The Association Study Of Polymorphisms Of Gene Mfn2 And EH, Response To ACEI And Left Ventricular Structure And Function In The Northern Han Chinese

Background The gene Mfn2(mitofusion-2, also named Hyperplasiasuppressor gene, HSG) was initially isolated using differential display technology fromvascular smooth muscle cells(VSMCs) of the spontaneously hypertensive rats(SHRs) and Wistar Kyoto rats(WKY). Its expression was reduced in VSMCs of SHRs and increased in VSMCs of WKY, which suggested that the Mfn2 gene may be a hypertension-related gene. Further research has found that overexpression of Mfn2 gene can inhibit the proliferation of VSMCs through Ras-Raf-ERK/MAPK signaling, subsequently incurring the cell cycle at G0/G1, which implied that Mfn2 was an important factor of suppressing the VSMCs proliferation.Recent experimental data indicated that loss of Mfn2 can lead to insulin resistance and endoplasmic reticulum(ER) stress. Given that VSMCs proliferation, insulin resistance and ER stress are strongly associated with essentialhypertension(EH), so we inferred that Mfn2 gene may be one of important EH candidate genes and the common polymorphisms of Mfn2 gene were associated with risk for EH. Angiotensin-converting enzyme inhibitors(ACEIs) are one of the first-line agents for treating EH. Their effect in reducing blood pressure(BP) has been recognized worldwide. However, the BP response to ACEIs varies markedly among hypertensive patients. It has been suggested that the genetic factors may explain more than 50% of the variation in BP response to ACEIs. To date, there have been many studies investigating the relationship between genetic variants and BP response to ACEIs. As a target for ACEIs, Angiotensin II(Ang II) could play an important role in the modulating the contraction and hypertrophy of VSMCs through Ras-Raf-ERK1/2 signalling except regulating the sodium-water balance. A series of experiments in vitro and in vivo demonstrated that Mfn2 can combine with Ras to inhibit Ang II-induced activity of extracellular signal-regulated kinase(ERK1/2), consequently performing a function for suppressing cells proliferation. Based on the relative evidence, we postulated that Ang II interacted with Mfn2 to influence the BP through modulating the contraction hypertrophy of vascular smooth muscle cells. Inaddition, the expression level of Mfn2 gene was negatively associated with the extent of aortic remodeling of SHR. Therefore, we inferred that Mfn2 can be an indirect target for ACEIs and the polymorphisms of Mfn2 gene may be associated with the BP response to ACEIs. Prolonged high blood pressure can lead to changes of cardia structure and function including the occurrence of left ventricular hypertrophy(LVH). The epidemiologic evidence suggests that LVH are independent risk factors for cardiovascular morbidity and mortality. It has been suggested that, besides blood pressure levels, LVH and changes of left ventricular structure and function were determined by many other factors such as gender, age, history of hypertension, drugs treatment, obesity, diabetes and genetic factors. Among the non-blood pressure factors, the influence of genetic factors for left ventricular myocardium may explain about 60%. A series of experiments in vitro and in vivo indicated that the expression of Mfn2 gene was down-regulated in the cardiac hypertrophy models and overexpression of Mfn2 gene attenuated the development of LVH. Therefore, we inferred that Mfn2 gene may be associated with LVH and changes of left ventricular structure and function. Because of the important role of Mfn2 in the modulation of vascular smooth muscle proliferation, insulin resistance, endoplasmic reticulum, ERK1/2 signaling and cardiomyocyte hypertrophy, we inferred that Mfn2 gene polymorphisms were associated with essential hypertension, the BP response to ACEI and left ventricular structure and function. The candidate gene approach was used to investigate the relationship between Mfn2 gene polymorphisms and essential hypertension, the BP response to ACEI and left ventricular structure and function.Objective 1. To evaluate the relationship between the rs2336384, rs2295281, rs17037564, rs2236057, rs2236058 and rs3766741 polymorphisms of the Mfn2 gene and EH risk among the northern Han Chinese population, which may provide genetic theory for the earlyrisk evaluation of essential hypertension. 2. To assess the association between the six polymorphisms of gene Mfn2 and BP response to ACEIs in northern Han Chinese, which may provide theory referencefor individually using ACEIs in clinical practice. 3. To investigate whether there are associations between the polymorphisms of gene Mfn2 and left ventricular structure and function in the new-diagnosed hypertensive patients, which may provide the reference for screening of clinical biomarkers for cardiac damage of hypertensive patients.Methods 1. All individuals in this case-control study were of northern Han Chinese origin. A total of 1244 unrelated participants comprising 626 hypertensive patients and 618 normotensive control subjects were recruited from September 2013 to July 2014 in this study. We collected the clinical characteristics and blood samples of all participants. We genotyped the SNPs using the Taq Man assay. Genotypes were discriminated by analyzing the fluorescene levels of PCR products using ABI PRISM 7900 HT Sequence Detector. The association between the Mfn2 gene SNPs and EH risk was evaluated through univariate analyses, stratification analyses, linkage disequilibrium and haplotype analyses. The SPSS 17.0 software was used to perform the statistical analyses. Linkage disequilibrium and haplotype analysis was conducted using the Haploview and PLINK software. 2. 126 new-onset hypertensive(mild to moderate) patients were recruited in this study. They are all Han Chinese and permanent residents in the Huilongguan Community of Beijing. Oral Benazepril was given to reduce blood pressure. The antihypertensive efficacy was estimated based on the BP changes for eight weeks. The candidate method was used to investigate the relationship between single nucleotide polymorphisms of gene Mfn2 and the BP response to Benazepril. The genotype of six tagging SNPs was detected using the Taq Man-MGB method. The Hardy-Weinberg equilibrium was tested using the network software. We used the SPSS 17.0 statistical software to conduct the statistical analyses. 3. 355 new-diagnosed hypertensive patients who had mild to moderate hypertension were recruited in this study. We performed an association studybetween six tagging single nucleotide polymorphisms of gene Mfn2(rs2236384, rs2295281, rs17037564, rs2236057, rs2236058 and rs3766741) and the echocardiographic indexes of left ventricular structure and function in the northern Han Chinese population. We collected the baseline data by questionaires and obtained theechocardiographic index of left ventricular structure and function. Genotype was taken with the Taq Man-MGB method. The echocardiographic index of left ventricular structure and function were compared among the genotypes of six tagging SNPs of gene Mfn2. Multivariate regression analysis was used to adjust for the covariates including age, gender, BMI, SBP and DBP.Results 1. 1The genotype and allele frequency distribution of SNP rs2236384, rs2236058 and rs3766741 was statistically different between the hypertensive cases and control subjects(P<0.05). No significant difference in the genotype and allele frequency distribution of SNP rs2295281, rs17037564 and rs2236057 was observed between the case group and control group.2 Logistic regression analysis was performed after adjustment for the potential covariates and showed that rs2336384 polymorphism was significantly associated with an increased risk for essential hypertension in additive genetic model(OR=1.273, 95%CI[1.023-1.584], P=0.031) and in dominant genetic model(OR=1.617, 95%CI[1.155-2.264], P=0.005). For rs2236057 and rs3766741, significant association can be found in dominant genetic model(OR=1.418, 95%CI[1.033-1.948], P=0.031; OR=0.639,95%CI[0.433-0.943], P=0.024). There was also significantly association between rs2236058 and EH in additive genetic model(OR=0.802, 95%CI[0.649-0.991], P=0.041) and in recessive genetic model(OR=0.662, 95%CI[0.463-0.946], P=0.023). No significant association between rs2295281 or rs17037564 polymorphism and hypertension risk was observed.3 Subgroup analyses were performed by gender and showed that rs2336384 polymorphism was significantly associated with hypertension risk in additive genetic model(OR=1.407, 95%CI[1.005-1.97], P=0.047) and indominant genetic model(OR=1.89, 95%CI[1.63-3.071], P=0.01) in males, but not in females. For rs2236057, significant association can be found in dominnat genetic model(OR=1.665, 95%CI[1.052-2.635], P=0.03) in males, but not in the subgroup of females. In addition, there was significantly associated with a decreased risk for hypertension in recessive genetic model(OR=0.576, 95%CI[0.342-0.967], P=0.05) in males, but not in females. As for rs2295281, rs17037564 or rs3766741 polymorphism, no significant association was found in either subgroup(P>0.05).4 The six polymorphisms of the Mfn2 gene were in tight linkage disequilibrium with each other. Five haplotypes were detected in the haplotype analyses. The C-C-A-A-C-C(rs2336384-rs2295281-rs17037564-rs2236057-rs2236058-rs3766741)haplotype was a risk haplotype(OR=1.24, P=0.047), while the A-C-A-G-G-G was observed to be a protective haplotype(OR=0.642, P=0.024). The other haplotypes were not associated with hypertension risk in this case-control study. 2. 1The Ridit tests showed that there was no significant difference in the BP response to Benapril between the genotypes of six SNPs of gene Mfn2.2The non-parameters tests showed that there was no significant difference in the decline inâ–³SBP and â–³DBPbetween the genotypes of six SNPs of gene Mfn2(P>0.05).3After adjusting for the covariates(age, gender, BMI, baseline BP) that may influence BP response to ACEIs, the multiple linear regression analyses showed that there was no significant association between the six polymorphisms of gene Mfn2 and the decline in â–³SBP and â–³DBP(P>0.05). 3. 1The genotype distribution of 6 tagging SNPs of gene Mfn2 was in agreement with the Hardy-Weinberg equilibrium. In our study, there were only 22 cases with left ventricular hypertrophy(9 cases for males, 13 cases for females) according to the diagnostic criteria: LVMI>134g/m2 for males or LVMI>110g/m2 for females.2The ANOVA analysis showed that there are no statistical differences in these echocardiographic indexes(LVEDD, LVESD, IVST, PWT, RWT, LVM, LVMI, EF, LA, E/A) which could reflect the structure and function of left ventricularamong the genotypes of 6 SNPs of gene Mfn2.3The multivariate regression analysis showed that there were no associations between the Mfn2 gene polymorphisms and left ventricular structure and function after adjusting for the covariates(age, gender, BMI, SBP, DBP).Conclusion 1. The rs2336384, rs2236057, rs2236058 and rs3766741 polymorphisms of the Mfn2 gene were associated with essential hypertension risk in the northern Han Chinese population. The C allele of rs2336384 and the A allele of rs2236057 was observed to be a risk factor for EH, while the GG genotype of rs2236058 and G allele of rs3766741 was found to be protective.Subgroup analyses were performed by gender and found that rs2336384, rs2236057 or rs2236058 polymorphism was associated with EH risk in males, but not in females.The rs2336384, rs2295281, rs17037564, rs2236057, rs2236058 and rs3766741 polymorphisms of the Mfn2 gene were in close linkage disequilibrium.The C-C-A-A-C-C haplotype was a risk haplotype for hypertension, while the A-C-A-G-G-G haplotype was found to be protective. 2. The polymorphisms of gene Mfn2 were not significantly associated with the BP response to ACEI(Benazepril), which implied that Mfn2 gene polymorphisms could not be used as genetic markers to guide the individual treatment with ACEI(Benazepril) in clinical practice. These results should be verified in the studies with larger sample size and longer follow-up. 3. The results of the present study suggest that the 6 tagging SNPs of gene Mfn2(located in the introns, with the MAFs?5%) were not associated with the left ventricular structure and function in the northern Han Chinese population, but these results should be verified in the study with larger sample size in different populations.