Replication of an association of variation in the FOXO3A gene with human longevity using both case–control and longitudinal data

Genetic variation in FOXO3A has previously been associated with human longevity. Studies published so far have been case–control studies and hence vulnerable to bias introduced by cohort effects. In this study we extended the previous findings in the cohorts of oldest old Danes (the Danish 1905 cohort, N = 1089) and middle‐aged Danes (N = 736), applying a longitudinal study design as well as the case–control study design. Fifteen SNPs were chosen in order to cover the known common variation in FOXO3A. Comparing SNP frequencies in the oldest old with middle‐aged individuals, we found association (after correction for multiple testing) of eight SNPs; 4 (rs13217795, rs2764264, rs479744, and rs9400239) previously reported to be associated with longevity and four novel SNPs (rs12206094, rs13220810, rs7762395, and rs9486902 (corrected P‐values 0.001–0.044). Moreover, we found association of the haplotypes TAC and CAC of rs9486902, rs10499051, and rs12206094 (corrected P‐values: 0.01–0.03) with longevity. Finally, we here present data applying a longitudinal study design; when using follow‐up survival data on the oldest old in a longitudinal analysis, we found no SNPs to remain significant after the correction for multiple testing (Bonferroni correction). Hence, our results support and extent the proposed role of FOXO3A as a candidate longevity gene for survival from younger ages to old age, yet not during old age.     

The FoxO3 gene and cause‐specific mortality

The G allele of the FOXO3 single nucleotide polymorphism (SNP) rs2802292 exhibits a consistently replicated genetic association with longevity in multiple populations worldwide. The aims of this study were to quantify the mortality risk for the longevity‐associated genotype and to discover the particular cause(s) of death associated with this allele in older Americans of diverse ancestry. It involved a 17‐year prospective cohort study of 3584 older American men of Japanese ancestry from the Honolulu Heart Program cohort, followed by a 17‐year prospective replication study of 1595 white and 1056 black elderly individuals from the Health Aging and Body Composition cohort. The relation between FOXO3 genotype and cause‐specific mortality was ascertained for major causes of death including coronary heart disease (CHD), cancer, and stroke. Age‐adjusted and multivariable Cox proportional hazards models were used to compute hazard ratios (HRs) for all‐cause and cause‐specific mortality. We found G allele carriers had a combined (Japanese, white, and black populations) risk reduction of 10% for total (all‐cause) mortality (HR = 0.90; 95% CI, 0.84–0.95; P = 0.001). This effect size was consistent across populations and mostly contributed by 26% lower risk for CHD death (HR = 0.74; 95% CI, 0.64–0.86; P = 0.00004). No other causes of death made a significant contribution to the survival advantage for G allele carriers. In conclusion, at older age, there is a large risk reduction in mortality for G allele carriers, mostly due to lower CHD mortality. The findings support further research on FOXO3 and FoxO3 protein as potential targets for therapeutic intervention in aging‐related diseases, particularly cardiovascular disease.     

Linkage disequilibrium analyses of natriuretic peptide precursor B locus reveal risk haplotype conferring high plasma BNP levels

BACKGROUND: Brain natriuretic peptide (BNP) has been widely used for the diagnosis and prognostic evaluation of chronic heart failure (CHF). In the present study, we performed association study of single nucleotide polymorphisms (SNPs) surrounding the natriuretic peptide precursor B (NPPB) gene with plasma BNP levels in 2970 adult Japanese. METHODS AND RESULTS: Association analysis between SNPs of the NPPB gene and plasma BNP revealed significant associations of the 8 SNPs surrounding the entire NPPB gene with plasma BNP levels. For instance, as to SNP rs198389 (T-381C), plasma BNP levels among the three genotypic categories, i.e., 2189 homozygous T-allele carriers (BNP 26.4+/-0.6pg/ml), 697 heterozygous carriers (35.0+/-1.1pg/ml), and 52 homozygous C-allele carriers (46.0+/-4.1pg/ml) indicated a co-dominant effect of the minor C-allele on elevating plasma BNP levels (P<0.0001). Linkage disequilibrium (LD) analysis among the 8 SNPs revealed that the region consisted of two, 5' major and 3' minor, LD blocks. Haplotype-based association analysis demonstrated that plasma BNP levels were associated closely with the haplotypes-1 and -2 of the major LD block. CONCLUSION: These results suggest that genetic variation at the primary locus NPPB gene, represented by definition of risk haplotypes, may be an important determinant of plasma BNP levels.     

FOXO3 gene polymorphisms influence the risk of acute lymphoblastic leukemia in Chinese children

Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed cancer in children and single-nucleotide polymorphisms (SNPs) in certain genes influence risk of ALL. Although FOXO3 had been demonstrated to be involved leukemia, the role of FOXO3 polymorphisms was still not clear. In the present study, we explored the association of FOXO3 SNPs with ALL risk in Chinese children. We genotyped four polymorphisms (rs17069665 A>G, rs4945816 T>C, rs4946936 C>T, and rs9400241 A>C) of FOXO3 in 425 ALL cases and 1339 health controls. The associations were estimated by odds ratios (ORs) with their 95% confidence intervals (CIs). Further analyses were performed to explore associations of rs17069665 and rs9400241 with ALL susceptibility in terms of age, gender, immunophenotype, minimal residual disease (MRD), and other clinical characteristics. We found rs17069665 related to the increased ALL risk (OR = 1.76; 95% CI = 1.02-3.04), rs9400241 related to decreased ALL risk (OR = 0.80; 95% CI = 0.64-0.99). The effects of rs17069665 on ALL risk were more predominant in males and children < 10 years, and patients with lower rates of platelet or neutrophil. As for rs9400241, the effects were more predominant in children < 10 years, and in patients with pre B ALL, positive MRD, anemia, or hepatomegaly. In conclusion, FOXO3 gene polymorphisms influence the risk of ALL in children and might be a potential biomarker for ALL susceptibility.     

Association of common genetic variation in the insulin/IGF1 signaling pathway with human longevity

The insulin/IGF1 signaling pathways affect lifespan in several model organisms, including worms, flies and mice. To investigate whether common genetic variation in this pathway influences lifespan in humans, we genotyped 291 common variants in 30 genes encoding proteins in the insulin/IGF1 signaling pathway in a cohort of elderly Caucasian women selected from the Study of Osteoporotic Fractures (SOF). The cohort included 293 long-lived cases (lifespan ≥ 92 years (y), mean ± standard deviation (SD) = 95.3 ± 2.2y) and 603 average-lifespan controls (lifespan ≤ 79y, mean = 75.7 ± 2.6y). Variants were selected for genotyping using a haplotype-tagging approach. We found a modest excess of variants nominally associated with longevity. Nominally significant variants were then replicated in two additional Caucasian cohorts including both males and females: the Cardiovascular Health Study and Ashkenazi Jewish Centenarians. An intronic single nucleotide polymorphism in AKT1 , rs3803304, was significantly associated with lifespan in a meta-analysis across the three cohorts (OR = 0.78 95%CI = 0.68–0.89, adjusted P  = 0.043); two intronic single nucleotide polymorphisms in FOXO3A demonstrated a significant lifespan association among women only (rs1935949, OR = 1.35, 95%CI = 1.15–1.57, adjusted P  = 0.0093). These results demonstrate that common variants in several genes in the insulin/IGF1 pathway are associated with human lifespan.     

FOXO 1a and FOXO 3a gene polymorphisms in association with metabolic syndrome

The prevalence and magnitude of childhood and adult obesity and diabetes are increasing dramatically. FOXO 1a and FOXO 3a will be evaluated in this study, in an effort to identify genetic polymorphisms in potential candidate genes that may be associated with body mass index (BMI), and metabolic syndrome (MS). Also to assess whether there is a relation between insulin sensitivity, and genotype, we will test the relation between fasting insulin, glucose, insulin resistance, insulin secretion and genotype.A total number of 248 presenting normal, overweight and obese individuals were recruited; 100 children and 148 adults of both sexes. They were divided by body mass index as follows, normal, overweight and obese. Lipid profile, fasting glucose and insulin HOMA-IR and HOMA-β index and RT-PCR for FOXO 1a and FOXO 3a were performed.An association was found among the studied group (children and adults) as regards foxo3a gene polymorphism and HOMA IR, HOMA B index and T-cholesterol (P = 0.022, 0.011 and 0.028, respectively), while there was only an association between LDL-C and foxo1a gene polymorphism among the studied group of children and adults (P = 0.023).In this study we demonstrated that FOXO3a mutant is correlated with HOMA-IR (marker of insulin resistance), HOMA-B index (marker of insulin secretion) and total cholesterol while as regards FOXO1a there was only an association between LDL cholesterol and mutant type of FOXO1a.     

TM6SF2 rs58542926 variant affects postprandial lipoprotein metabolism and glucose homeostasis in NAFLD

Mechanisms underlying the opposite effects of transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 C>T polymorphism on liver injury and cardiometabolic risk in nonalcoholic fatty liver disease (NAFLD) are unclear. We assessed the impact of this polymorphism on postprandial lipoprotein metabolism, glucose homeostasis, and nutrient oxidation in NAFLD. Sixty nonobese nondiabetic normolipidemic biopsy-proven NAFLD patients and 60 matched controls genotyped for TM6SF2 C>T polymorphism underwent: indirect calorimetry; an oral fat tolerance test with measurement of plasma lipoprotein subfractions, adipokines, and incretin glucose-dependent insulinotropic polypeptide (GIP); and an oral glucose tolerance test with minimal model analysis of glucose homeostasis. The TM6SF2 T-allele was associated with higher hepatic and adipose insulin resistance, impaired pancreatic β-cell function and incretin effect, and higher muscle insulin sensitivity and whole-body fat oxidation rate. Compared with the TM6SF2 C-allele, the T-allele entailed lower postprandial lipemia and nefaemia, a less atherogenic lipoprotein profile, and a postprandial cholesterol (Chol) redistribution from smaller atherogenic lipoprotein subfractions to larger intestinal and hepatic VLDL1 subfractions. Postprandial plasma VLDL1-Chol response independently predicted the severity of liver histology. In conclusion, the TM6SF2 C>T polymorphism affects nutrient oxidation, glucose homeostasis, and postprandial lipoprotein, adipokine, and GIP responses to fat ingestion independently of fasting values. These differences may contribute to the dual and opposite effect of this polymorphism on liver injury and cardiometabolic risk in NAFLD.     

Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers

Objective

Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis.

Study Design

Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis.

Results

After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs’ in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs.

Conclusions

We could demonstrate that common genetic variation in the PIK3CG locus, possibly via altered PIK3CG gene expression, determines plasma HDL-cholesterol concentrations. Since HDL2-, but not HDL3-, cholesterol is influenced by PIK3CG variants, PI3Kγ may play a role in HDL clearance rather than in HDL biogenesis. Even though the molecular pathways connecting PI3Kγ and HDL metabolism remain to be further elucidated, this finding could add a novel aspect to the pathophysiological role of PI3Kγ in atherogenesis.     

FOXO-independent suppression of programmed cell death by the PI3K/Akt signaling pathway in Drosophila

Signaling through the PI3K/Akt/FOXO pathway plays an important role in vertebrates in protecting cells from programmed cell death. PI3K and Akt have been similarly shown to be involved in survival signaling in the invertebrate model organism Drosophila. However, it is not known whether PI3K and Akt execute this function by controlling a pro-apoptotic activity of Drosophila FOXO. In this study, we show that elevated signaling through PI3K and Akt can prevent developmentally controlled death in the salivary glands of the fruit fly. We further show that Drosophila FOXO is not required for normal salivary gland death and that the rescue of salivary gland death by PI3K occurs independent of FOXO. These results give support to the notion that FOXOs have acquired pro-apoptotic functions after separation of the vertebrate and invertebrate lineages.     

Association analysis of common variants in FOXO3 with type 2 diabetes in a South Indian Dravidian population

Recent studies have identified common variants in forkhead box O3 gene (FOXO3) to be strongly associated with longevity in different populations. But studies have not been carried out to analyse the role of common variants in FOXO3 with type 2 diabetes. Since type 2 diabetes is an age related disorder and FOXO proteins play an important role in the regulation of metabolism, we studied the role of common variants in FOXO3 for association with type 2 diabetes. The study was carried out in 994 type 2 diabetic samples and 984 normoglycemic control samples from a South Indian Dravidian population. In our analysis, we found that there was no association between any of the selected SNPs in FOXO3 with type 2 diabetes. Analysis of these SNPs with diabetes related biochemical and clinical parameters also did not reveal any significant association. Haplotype association of SNPs in FOXO3 with type 2 diabetes was observed, but the frequency of the haplotypes was considerably lower and they do not remain significant after correction for multiple testing. In conclusion, we did not observe any association of SNPs in FOXO3 with type 2 diabetes and related parameters suggesting an entirely different mechanism by which these SNPs influence longevity. However additional studies in other populations are required to completely rule out the association of common variants in FOXO3 with type 2 diabetes.