Adducin 1 Research Articles

Single nucleotide polymorphism rs4961 in the adducin 1 gene is not associated with gastric cancer or preneoplastic cancer lesions.

Gastric cancer (GC) is the fourth most deadly cancer globally. The adducin 1 (ADD1) protein is involved in oncogenic signal transduction pathways in several types of cancer, and the rs4961 variant (c.1378 G>T, p.Gly460Trp) of the ADD1 gene is associated with salt-sensitive hypertension, renal cell cancer and breast cancer susceptibility; however, it has not been investigated in GC. The aim of the present study was to evaluate the association between the rs4961 variant and the development of GC and preneoplastic gastric lesions (PGLs) in a population from western Mexico. A total of 225 individuals who underwent an endoscopy were evaluated, of which 71 patients had histopathologically diagnosed GC and 53 patients had PGLs, with 101 patients used as controls. The rs4961 variant was genotyped by using PCR and DNA sequencing. The frequency of the mutated homozygous genotype (TT) of the rs4961 variant was <10% in the three evaluated groups, and the frequency of the minor allele (T) was <21% in the GC, PGL and control groups. Genotypic and allelic frequencies were similarly distributed in all of the studied groups (P>0.05). In summary, in the study population, the rs4961 variant was not associated with GC risk; however, its role in other populations and in other types of cancer is worthy of future research.

Mechanisms of Dangua Fang in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.

To explore the mechanism of Dangua Fang (, DGR) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics. Sprague-Dawley rats with normal glucose levels were randomly divided into three groups, including a conventional diet control group (Group A), high-fat-high-sugar diet model group (Group B), and DGR group (Group C, high-fat-high-sugar diet containing 20.5 g DGR). After 10 weeks of intervention, the fasting blood glucose (FBG), 2 h blood glucose [PBG; using the oral glucose tolerance test (OGTT)], hemoglobin A1c (HbA1c), plasma total cholesterol (TC), and triglycerides (TG) were tested, and the livers of rats were removed to calculate the liver index. Then, hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis followed by database search and bioinformatics analysis. Finally, cell experiments were used to verify the results of phosphoproteomics. Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4 (MAP4k4) and phosphorylated adducin 1 (ADD1) were detected using western blotting. DGR effectively reduced PBG, TG, and the liver index (P < 0.05), and significantly decreased HbA1c, TC, and hepatic portal TG (P < 0.01), showed significant hematoxylin and eosin (HE) staining, red oil O staining, and Masson staining of liver tissue. The total spectrum was 805 334, matched spectrum was 260 471, accounting for accounting 32.3%, peptides were 19 995, modified peptides were 14 671, identified proteins were 4601, quantifiable proteins were 4417, identified sites were 15 749, and quantified sites were 14659. Based on the threshold of expression fold change ( > 1.2), DGR up-regulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins, and down-regulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins, which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism. Therefore, DGR improved biological tissue processes, including information storage and processing, cellular processes and signaling, and metabolism. The metabolic functions regulated by DGR mainly include energy production and conversion, carbohydrate transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport, and catabolism. In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies. DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders, thereby regulating glycolipid metabolism through a multi-target and multi-method process.

Muscleblind-like 2 knockout shifts adducin 1 isoform expression and alters dendritic spine dynamics of cortical neurons during brain development.

Muscleblind-like 2 (MBNL2) plays a crucial role in regulating alternative splicing during development and mouse loss of MBNL2 recapitulates brain phenotypes in myotonic dystrophy (DM). However, the mechanisms underlying DM neuropathogenesis during brain development remain unclear. In this study, we aim to investigate the impact of MBNL2 elimination on neuronal development by Mbnl2 conditional knockout (CKO) mouse models. To create Mbnl2 knockout neurons, cDNA encoding Cre-recombinase was delivered into neural progenitors of Mbnl2flox/flox mouse brains by in utero electroporation. The morphologies and dynamics of dendritic spines were monitored by confocal and two-photon microscopy in brain slices and live animals from the neonatal period into adulthood. To investigate the underlying molecular mechanism, we further detected the changes in the splicing and molecular interactions of proteins associated with spinogenesis. We found that Mbnl2 knockout in cortical neurons decreased dendritic spine density and dynamics in adolescent mice. Mbnl2 ablation caused the adducin 1 (ADD1) isoform to switch from adult to fetal with a frameshift, and the truncated ADD1 failed to interact with alpha-II spectrin (SPTAN1), a critical protein for spinogenesis. In addition, expression of ADD1 adult isoform compensated for the reduced dendritic spine density in cortical neurons deprived of MBNL2. MBNL2 plays a critical role in maintaining the dynamics and homeostasis of dendritic spines in the developing brain. Mis-splicing of downstream ADD1 may account for the alterations and contribute to the DM brain pathogenesis.

Phospho‐proteomic Analysis of Protease‐activated Receptor‐1 Biased Signaling Reveals Novel Modulators of Endothelial Barrier Function

Dysregulation of the vascular endothelium is a hallmark of inflammation and promotes barrier disruption, vascular leakage and tissue edema. Vascular endothelial dysfunction is mediated by vasoactive agents and inflammatory mediators, many of which signal through G protein‐coupled receptors (GPCRs). A key GPCR inflammatory mediator is thrombin, a procoagulant protease generated in response to vascular injury and inflammation. Thrombin activates protease‐activated receptor‐1 (PAR1) in endothelial cells to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC) is an anticoagulant protease that activates PAR1 through a distinct mechanism and promotes endothelial barrier stabilization. The ability of distinct tethered ligands formed through cleavage of PAR1 by different proteases results in stabilization of unique receptor active conformational states that promote opposite signaling effects is termed biased signaling. Despite progress in understanding PAR1 bias signaling in endothelial cells, the proteins and pathways by which thrombin and APC regulate the endothelial barrier integrity are largely unknown. Here, we report the global phosphoproteome of thrombin and APC signaling using tandem‐mass‐tag (TMT) mass spectrometry and computational analyses. Our results reveal unique temporal dynamics profiles of thrombin and APC signaling and the identification of multiple enriched phosphoproteome associated biological functions including microtubules, adherens junctions, actin and signaling, all key modulators of endothelial barrier permeability. We selected candidates that showed statistically significant changes in phosphorylation following thrombin or APC stimulation, and validated the function of four proteins including: afadin (AFDN), CDC42 binding protein kinase alpha (BPA), adducin 1 (ADD1), and GPCR interacting protein 1 (GIT1) using an siRNA‐targeted approach. Using target‐specific siRNAs to deplete candidate proteins in endothelial cells, we found that depletion of afadin and adducin had statistically significant effects on endothelial barrier permeability. Afadin depletion led to an increase in thrombin‐induced permeability, while adducin depletion virtually ablated thrombin‐stimulated permeability. Immunofluorescence microscopy imaging of morphological changes in F‐actin stress fiber formation supported these results and revealed corresponding changes in thrombin‐induced gap formation consistent with the observed changes in permeability. In addition, depletion of candidate proteins also modulated thrombin‐stimulated p38 pro‐inflammatory signaling and APC‐induced AKT cytoprotective signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 bias signaling in endothelial cells.Support or Funding InformationThis work was supported by NIH/NIGMS R01 GM116597 and R35 GM127121 (J.T.), UC Tobacco‐related Disease Research Program Predoctoral Fellowship (C.C.R), and NIH/NIGMS K12 GM068524 (O.M.I., J.D.L.)

DNA Methylation of Candidate Genes (ACE II, IFN-γ, AGTR 1, CKG, ADD1, SCNN1B and TLR2) in Essential Hypertension: A Systematic Review and Quantitative Evidence Synthesis.

Physical, chemical, and social environments adversely affect the molecular process and results in cell signal transduction and the subsequent transcription factor dysregulation, leading to impaired gene expression and abnormal protein synthesis. Stressful environments such as social adversity, isolation, sustained social threats, physical inactivity, and highly methylated diets predispose individuals to molecular level alterations such as aberrant epigenomic modulations that affect homeostasis and hemodynamics. With cardiovascular disease as the leading cause of mortality in the US and blacks/African Americans being disproportionately affected by hypertension (HTN) which contributes substantially to these deaths, reflecting the excess mortality and survival disadvantage of this sub-population relative to whites, understanding the molecular events, including epigenomic and socio-epigenomic modulations, is relevant to narrowing the black-white mortality risk differences. We aimed to synthesize epigenomic findings in HTN namely (a) angiotensin-converting enzyme 2 (ACE II) gene, (b) Toll-like receptor 2 (TLR2) gene, (c) interferon γ (IFN-γ) gene, and (d) Capping Actin Protein, Gelosin-Like (CAPG) gene, adducin 1(ADD1) gene, (e) Tissue inhibitor of metalloproteinase 3 (TIMP3), (f) mesoderm specific transcript (MEST) loci, (g) sodium channel epithelial 1 alpha subunit 2 (SCNN1B), (h) glucokinase (CKG) gene (i) angiotensin II receptor, type1 (AGTR1), and DNA methylation (mDNA). A systematic review and quantitative evidence synthesis (QES) was conducted using Google Scholar and PubMed with relevant search terms. Data were extracted from studies on: (a) Epigenomic modulations in HTN based on ACE II (b) TLR2, (c) IFN-γ gene, (d) CAPG, ADD1, TIMP3, MEST loci, and mDNA. The random-effect meta-analysis method was used for a pooled estimate of the common effect size, while z statistic and I^2 were used for the homogeneity of the common effect size and between studies on heterogeneity respectively. Of the 642 studies identified, five examined hypermethylation while seven studies assessed hypomethylation in association with HTN. The hypermethylation of ACE II, SCNN1B, CKG, IFN-γ gene, and miR-510 promoter were associated with hypertension, the common effect size (CES) = 6.0%, 95% CI, −0.002–11.26. In addition, the hypomethylation of TLR2, IFN-γ gene, ADD1, AGTR1, and GCK correlated with hypertension, the CES = 2.3%, 95% CI, −2.51–7.07. The aberrant epigenomic modulation of ACE II, TLR2, IFN-γ, AGTR1, and GCK correlated with essential HTN. Transforming the environments resulting from these epigenomic lesions will facilitate early intervention mapping in reducing HTN in the US population, especially among socially disadvantaged individuals, particularly racial/ethnic minorities.

Phosphorylation of adducin-1 by cyclin-dependent kinase 5 is important for epidermal growth factor-induced cell migration

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neuron and plays an important role in neuronal physiology. Increasing evidence also indicates that Cdk5 may contribute to malignant progression of some types of cancers; however, the underlying mechanism remains elusive. In this study, we found that Cdk5 directly phosphorylated the actin-binding protein adducin-1 (ADD1) at T724 in vitro and in intact cells. The capability of the phosphomimetic T724D mutant to bind to actin filaments was lower than that of wild type ADD1 and the T724A mutant. Cdk5 co-localized with ADD1 at the lamellipodia upon epidermal growth factor (EGF) stimulation. The increased lamellipodia formation and cell migration of human breast cancer cells MDA-MB-231 by EGF were accompanied by Cdk5 activation and increased phosphorylation of ADD1 at T724. Depletion of Cdk5 in MDA-MB-231 cells abrogated the effects of EGF on ADD1 T724 phosphorylation, lamellipodia formation, and cell migration. Likewise, depletion of ADD1 suppressed the effects of EGF on lamellipodia formation, cell migration, and invasion, all of which were restored by FLAG-ADD1 WT and the T724D mutant, but not the T724A mutant. Together, our results suggest that phosphorylation of ADD1 at T724 by Cdk5 is important for EGF-induced cell migration and invasion.

Adducin‐1 is essential for spindle pole integrity through its interaction with TPX2

Bipolar spindle assembly is necessary to ensure the proper progression of cell division. Loss of spindle pole integrity leads to multipolar spindles and aberrant chromosomal segregation. However, the mechanism underlying the maintenance of spindle pole integrity remains unclear. In this study, we show that the actin‐binding protein adducin‐1 (ADD1) is phosphorylated at S726 during mitosis. S726‐phosphorylated ADD1 localizes to centrosomes, wherein it organizes into a rosette‐like structure at the pericentriolar material. ADD1 depletion causes centriole splitting and therefore results in multipolar spindles during mitosis, which can be restored by re‐expression of ADD1 and the phosphomimetic S726D mutant but not by the S726A mutant. Moreover, the phosphorylation of ADD1 at S726 is crucial for its interaction with TPX2, which is essential for spindle pole integrity. Together, our findings unveil a novel function of ADD1 in maintaining spindle pole integrity through its interaction with TPX2.

Gene and dietary calcium interaction effects on brachial–ankle pulse wave velocity

Understanding the lifestyle and genetic factors that affect pulse wave velocity (PWV) may provide clues to preventing atherosclerotic cardiovascular events. The aim of this study is to investigate genome-wide genetic and dietary calcium (Ca) intake interaction effects on brachial-ankle pulse wave velocity (baPWV). The baPWV was measured, and Ca intake was quantified by administering a food frequency questionnaire (FFQ) to 3198 participants, which included men and women (≥40 years) from the Korean Multi-Rural communities Cohort study (MRCohort). The interaction effects of dietary Ca intake and 19 single-nucleotide polymorphisms (SNPs) on baPWV were assessed using the general linear models. Dietary Ca intake was not significantly associated with baPWV or any type of SNP among the subjects herein. In men, however, the adducin1 (ADD1) rs4961_C SNP had a significant dietary Ca intake-dependent effect on mean baPWV (pinteraction=0.002). In women, the interaction of zinc finger proteins 618 (ZNF618) rs10817542_A with dietary Ca intake played a significant and key role in mean baPWV (pinteraction=0.001). In the results of ADD1 rs4961_C in men and ZNF618 rs10817542_A in women, the minor allele-lowest Ca intake tertile (T1) group had significantly higher mean baPWV value than other subgroups of Ca intake tertile-genotype cross-classification whereas genotype was not a significant effector on mean baPWV values among highest Ca intake subgroups (T3). The baPWV, a phenotype of arterial stiffness, can be modulated in subjects through regulation of dietary Ca intake, particularly in subjects with more vulnerable genotypes.

Kinome profiling of osteoblasts on hydroxyapatite opens new avenues on biomaterial cell signaling

In degenerative diseases or lesions, bone tissue replacement and regeneration are important clinical goals. The most used bone substitutes today are hydroxyapatite (HA) scaffolds. These scaffolds, developed over the last few decades, present high porosity and good osteointegration, but haven't completely solved issues related to bone defects. Moreover, the exact intracellular mechanisms involved in the response to HA have yet to be addressed. This prompted us to investigate the protein networks responsible for signal transduction during early osteoblast adhesion on synthetic HA scaffolds. By performing a global kinase activity assay, we showed that there is a specific molecular machinery responding to HA contact, immediately triggering pathways leading to cytoskeleton rearrangement due to activation of Adducin 1 (ADD1), protein kinase A (PKA), protein kinase C (PKC), and vascular endothelial growth factor (VEGF). Moreover, we found a significantly increased phosphorylation of the activating site Ser-421 in histone deacetylase 1 (HDAC1), a substrate of Cyclin-Dependent Kinase 5 (CDK5). These phosphorylation events are hallmarks of osteoblast differentiation, pointing to HA surfaces ability to promote differentiation. We also found that AKT was kept active, suggesting the maintenance of survival pathways. Interestingly, though, the substrate sequence of CDK5 also presented higher phosphorylation levels when compared to control conditions. To our knowledge, this kinase has never before been related to osteoblast biology, opening a new avenue of investigation for novel pathways involved in this matter. These results suggest that HA triggers a specific intracellular signal transduction cascade during early osteoblast adhesion, activating proteins involved with cytoskeleton rearrangement, and induction of osteoblast differentiation.

Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X

Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin-based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell-cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.

ACE and ADD1 gene in extra and intracranial atherosclerosis in ischaemic stroke

Objective: Hypertension is closely linked to ischaemic stroke (IS) and atherosclerosis, but there are no studies correlating the candidate hypertensive gene, namely angiotensin converting enzyme (ACE) and adducin 1 (ADD1) with magnetic resonance angiographic (MRA) abnormality, therefore this study was undertaken.Methods: Patients with magnetic resonance imaging (MRI) proven IS were included and their demography, stroke risk factors, and clinical findings were noted. Both intracranial (IC) and extracranial (EC) MRA were performed and more than 50% stenosis was considered significant. Angiotensin converting enzyme and ADD1 gene polymorphism was evaluated by polymerase chain reaction (PCR) both in patients and 188 controls.Results: Angiotensin converting enzyme and ADD1 polymorphism were performed in 151 patients whose median age was 60 years and 26·5% were females. Magnetic resonance angiography was abnormal in 77·5%; extracranial magnetic resonance angiography (ECMRA) in 58·3%, and intracranial magnetic resonance angiography (ICMRA) in 66·7%. The conventional risk factors were not different between the IS patients with and without MRA abnormalities. The presence of DD genotype (OR 3·86, 95% CI 0·78–2·28, P = 0·0001) and ADD1 GW genotype (OR 2·05, 95% CI 1·28–3·27, P = 0·003) significantly increased the risk of IS compared to controls. Both genotype and allele frequency of ACE and ADD1 were higher in MRA abnormal IS patients compared to controls; however, these were not significantly different between the patients with and without MRA abnormality.Conclusion: In IS patients, DD genotype and D allele of ACE gene and W allele of ADD1 gene were significantly related to MRA abnormality compared to controls but there was no association of ACE and ADD1 gene polymorphism in IS patients with MRA and without abnormality.

Computational Study of ADD1 Gene Polymorphism Associated with Hypertension

We have determined the non-synonymous single-nucleotide polymorphisms (nsSNPs) of α adducin 1 (ADD1) gene and its variations in different populations to understand its role in hypertension. Out of 1,113 SNPs, 9 are found to be non-synonymous, of which 7 showed significant damaging effect and one of them showed SNP variability with large differences among the minor allele frequency observed in various populations. The amino acid change found for rs4961 is from glycine to tryptophan, i.e., from an alkyl amino acid to an aromatic amino acid. This residual change is observed in the coiled region of the protein and is also predicted to be disordered by computational algorithm. Protein disorder plays an important role in structural and functional genomics. Hence, because of the complete change in side chains of the amino acid residues occurring in the coiled and disordered region of the protein, the structure of the protein might be altered and the function might be affected, leading to the risk for hypertension.

Accumulation of common polymorphisms is associated with development of hypertension: a 12-year follow-up from the Ohasama study

Hypertension is a complex multi-factorial and polygenic disorder. Nevertheless, most studies have focused on single-gene effects. Furthermore, a majority of these studies have been cross-sectional and diagnosed hypertension using conventional blood pressure (BP) measurements, which are known to be subject to biases, including the so-called white-coat effect. Thus, we performed a longitudinal association study to clarify the effects of polymorphism accumulation on the development of hypertension that is defined on the basis of self-measured BP at home (home BP). In 403 Japanese aged 40-79 years with home normotension (home BP <135/85 mm Hg, and not treated with antihypertensive medication at baseline), we examined the associations of 51 single-nucleotide polymorphisms (SNPs) classically nominated or reported to be associated with hypertension in the Japanese Millennium Genome Project for Hypertension with a 12-year risk of progression to home hypertension (home BP >or=135/85 mm Hg, or start of antihypertensive medication). Out of 51 SNPs, four significantly and independently predicted the risk of progression of home hypertension, even after adjustment for possible confounding factors, including baseline home BP value. These were rs3767489 near the regulator of G-protein signaling 2 (RGS2), rs4961 in adducin 1 (ADD1), rs2236957 in the calcium channel, voltage-dependent, alpha-2/delta-subunit 2 (CACNA2D2) and rs769214 in catalase (CAT). Accumulation of these SNPs significantly improved the predictive values for the development of home hypertension. In conclusion, this longitudinal study, which was based on home BP measurement, showed that accumulation of common polymorphisms reliably predicted the risk of future hypertension in the Japanese general population.

Angiotensin-Converting Enzyme and Adducin-1 Polymorphisms in Women With Preeclampsia and Gestational Hypertension

The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism and the Adducin-1 (ADD1) G460W nonsense single nucleotide polymorphism (SNP) have previously been associated to hypertension, whereas their association with preeclampsia (PE) and gestational hypertension (GH) is still controversial. We genotyped ACE I/D, ADD1 G460W, and ADD1 S586C polymorphisms in 672 unrelated pregnant women: 204 PE (81/204 mild PE), 56 GH, and 412 controls, evaluating both their single and combined effects on these pathologies. The genotype combination of the 3 polymorphisms was not statistically different in cases versus controls, nor were ACE and ADD1 polymorphisms in GH. Nevertheless, the distribution of ACE genotypes was different in PE. This was confirmed in mild PE, whereas no significance was found in severe PE. This could suggest that different factors may lead to mild and severe PE, with ACE polymorphism playing a more important role in the mild form.

Quantitative proteomic analysis of long‐term vasopressin actions in mpkCCD cells using SILAC and MRM

We employed 'stable isotope labeling with amino acids in cell culture' (SILAC) coupled LC‐MS/MS to identify proteins that change in abundance in response to long‐term vasopressin stimulation in mpkCCD cells. Cells were grown in media containing heavy isotope‐substituted or light non‐substituted amino acids, and either vasopressin analog dDAVP (100pM) or vehicle, respectively. After 5 days, heavy‐ and light‐labeled cell lysates were combined for quantitation by LC‐MS/MS. 65 proteins were increased and 56 were decreased in abundance in response to dDAVP. Increases in 5 proteins from the SILAC results were confirmed by immunoblotting: AQP2, ADD1 (adducin‐1), MAL2, AKAP12, and SLC9A3R1 (NHERF1). For antibody‐independent validation, proteins were selected for targeted quantitative proteomics using multiple‐reaction monitoring (MRM) on a triple quadrupole mass spectrometer. Increases in 4 proteins (AQP2, ASAP2 [ArfGAP with SH3 domain ankyrin repeat and PH domain 2], MON2 [yeast homolog involved in Golgi to endosome transport] and CLMN [calmin]) and a decrease in one protein (NALCN [sodium leak channel non‐selective]) were statistically validated. Overall 1,629 proteins were identified and deposited in a new online database (mpkCCD Proteome Database). These methodologies permit large‐scale targeted quantification, providing an antibody‐independent alternative to multiplexed immunoblotting.

Ten renin‐angiotensin system‐related gene polymorphisms in maximally treated Canadian Caucasian patients with heart failure

The progression and pharmacological response of heart failure-affected patients are subject to interindividual variability. It is also acknowledged that the genotype frequency of certain gene polymorphisms varies across different ethnic groups and that a difference in gene polymorphism frequencies between healthy and heart failure patients seems to exist. This study investigated associations between 10 gene polymorphisms of RAAS-related genes with an individual's susceptibility to heart failure. Our data suggest that the angiotensinogen (AGT) 235 single nucleotide polymorphism (SNP) may be associated with heart failure in our population and that the AGT(M174)-AGT(T235) haplotype, as well as the AGT/angiotensin-converting enzyme (ACE) gene combination, may play an important role in disease predisposition. Racial differences in survival outcomes point towards a genetic role in the pathophysiology of heart failure. Furthermore, contemporary evidence links genetics to heart failure (HF) predisposition. We tested for a difference in prevalence of 10 renin-angiotensin-aldosterone system (RAAS)-related gene polymorphisms between a homogenous population of HF patients and healthy controls. One hundred and eleven healthy volunteers and 58 HF patients were included in this study. The healthy control group consisted of males aged between 18 and 35 years old. The HF group consisted of patients (89.7% male) who were 63.8 +/- 7.9 years old, were in New York Heart Association (NYHA) class II-III and had a documented left ventricular ejection fraction (LVEF) <or= 40% within the previous 6 months. Despite being treated maximally for their condition with angiotensin-converting-enzyme (ACE)-inhibitors and beta-adrenoceptor blockers, they continued to be symptomatic and, as such, were a highly specialized and homogeneous patient population. Both groups were composed of Canadian Caucasians. The analyzed polymorphisms were: ACE (I/D), angiotensin-II-receptor-type-1 (AGTR1)(A1166C), angiotensinogen (AGT)(M235T and T174M), endothelial-nitric-oxide-synthase (eNOS)(T-786C and Glu298Asp), adrenergic-receptor-â2 (ADRB2)(Gln27Glu), bradykinin-receptor-beta2 (BDKRB2)(+9/-9), aldosterone-synthase (CYP11B2)(T-344C) and adducin-1 (ADD1)(Gly460Trp). RESULTS The AGT (T235) allele (P = 0.0025, OR 2.02, 95% CI 1.24, 3.30) was found to be more prevalent in our HF group. The AGT (174M)-AGT (235T) haplotype was also associated with the HF phenotype (P = 0.0069). Exploratory evaluation of gene-gene combinations revealed an indicative association of the AGT (T235)/ACE(D) combined polymorphisms in the HF group (P = 0.02, OR 2.12, 95% CI 1.11, 4.06). This study demonstrates that the SNPs of AGT may be associated with HF in our population and that the AGT/ACE gene combination may play an important role in disease predisposition.