The discovery of GGT1 as a novel gene for ischemic stroke conferring protection against disease risk in non-smokers and non-abusers of alcohol.

OBJECTIVES: Increased plasma gamma-glutamyl transferase (GGT1) has been identified as a robust and independent risk factor for ischemic stroke (IS), but the molecular mechanisms of the enzyme-disease association are unclear. The present study investigated whether polymorphisms in the GGT1 gene contribute to IS susceptibility. MATERIALS AND METHODS: DNA samples obtained from 1288 unrelated individuals (600 IS patients and 688 controls) were genotyped for common single nucleotide polymorphisms of GGT1 using the MassArray-4 platform. RESULTS: The rs5751909 polymorphism was significantly associated with decreased risk of ischemic stroke regardless sex and age (Pperm ≤ 0.01, dominant genetic model). The haplotype rs4820599A-rs5760489A-rs5751909A showed strong protection against ischemic stroke (OR 0.53, 95 %CI 0.36 - 0.77, Pperm ≤ 0.0001). The protective effect of SNP rs5751909 in the stroke phenotype was successfully replicated in the UK Biobank, SiGN, and ISGC cohorts (P ≤ 0.01). GGT1 polymorphisms showed joint (epistatic) effects on the risk of ischemic stroke, with some known IS-associated GWAS loci (e.g., rs4322086 and rs12646447) investigated in our population. In addition, SNP rs5751909 was found to be strongly associated with a decreased risk of ischemic stroke in non-smokers (OR 0.54 95 %CI 0.39-0.75, Pperm = 0.0002) and non-alcohol abusers (OR 0.43 95 %CI 0.30-0.61, Pperm = 2.0 × 10-6), whereas no protective effects of this SNP against disease risk were observed in smokers and alcohol abusers (Pperm < 0.05). CONCLUSIONS: We propose mechanisms underlying the observed associations between GGT1 polymorphisms and ischemic stroke risk. This pilot study is the first to demonstrate that GGT1 is a novel susceptibility gene for ischemic stroke and provides additional evidence of the genetic contribution to impaired redox homeostasis underlying disease pathogenesis.

Association between HSPA8 Gene Variants and Ischemic Stroke: A Pilot Study Providing Additional Evidence for the Role of Heat Shock Proteins in Disease Pathogenesis.

HSPA8 is involved in many stroke-associated cellular processes, playing a pivotal role in the protein quality control system. Here we report the results of the pilot study aimed at determining whether HSPA8 SNPs are linked to the risk of ischemic stroke (IS). DNA samples from 2139 Russians (888 IS patients and 1251 healthy controls) were genotyped for tagSNPs (rs1461496, rs10892958, and rs1136141) in the HSPA8 gene using probe-based PCR. SNP rs10892958 of HSPA8 was associated with an increased risk (risk allele G) of IS in smokers (OR = 1.37; 95% CI = 1.07-1.77; p = 0.01) and patients with low fruit and vegetable consumption (OR = 1.36; 95% CI = 1.14-1.63; p = 0.002). SNP rs1136141 of HSPA8 was also associated with an increased risk of IS (risk allele A) exclusively in smokers (OR = 1.68; 95% CI = 1.23-2.28; p = 0.0007) and in patients with a low fruit and vegetable intake (OR = 1.29; 95% CI = 1.05-1.60; p = 0.04). Sex-stratified analysis revealed an association of rs10892958 HSPA8 with an increased risk of IS in males (risk allele G; OR = 1.30; 95% CI = 1.05-1.61; p = 0.01). Thus, SNPs rs10892958 and rs1136141 in the HSPA8 gene represent novel genetic markers of IS.

Lipid-Associated GWAS Loci Predict Antiatherogenic Effects of Rosuvastatin in Patients with Coronary Artery Disease.

We have shown that lipid-associated loci discovered by genome-wide association studies (GWAS) have pleiotropic effects on lipid metabolism, carotid intima-media thickness (CIMT), and CAD risk. Here, we investigated the impact of lipid-associated GWAS loci on the efficacy of rosuvastatin therapy in terms of changes in plasma lipid levels and CIMT. The study comprised 116 CAD patients with hypercholesterolemia. CIMT, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) were measured at baseline and after 6 and 12 months of follow-up, respectively. Genotyping of fifteen lipid-associated GWAS loci was performed by the MassArray-4 System. Linear regression analysis adjusted for sex, age, body mass index, and rosuvastatin dose was used to estimate the phenotypic effects of polymorphisms, and p-values were calculated through adaptive permutation tests by the PLINK software, v1.9. Over one-year rosuvastatin therapy, a decrease in CIMT was linked to rs1689800, rs4846914, rs12328675, rs55730499, rs9987289, rs11220463, rs16942887, and rs881844 polymorphisms (Pperm < 0.05). TC change was associated with rs55730499, rs11220463, and rs6065906; LDL-C change was linked to the rs55730499, rs1689800, and rs16942887 polymorphisms; and TG change was linked to polymorphisms rs838880 and rs1883025 (Pperm < 0.05). In conclusion, polymorphisms rs1689800, rs55730499, rs11220463, and rs16942887 were found to be predictive markers for multiple antiatherogenic effects of rosuvastatin in CAD patients.

The Joint Link of the rs1051730 and rs1902341 Polymorphisms and Cigarette Smoking to Peripheral Artery Disease and Atherosclerotic Lesions of Different Arterial Beds.

Genome-wide association studies (GWAS) have discovered numerous single nucleotide polymorphisms (SNP) contributing to peripheral artery disease (PAD), but their joint effects with risk factors like cigarette smoking (CS) on disease susceptibility have not been systematically investigated. The present study looked into whether CS mediates the effects of GWAS loci on the development of PAD and atherosclerotic lesions in different arterial beds. DNA samples from 1263 unrelated individuals of Slavic origin including 620 PAD patients and 643 healthy subjects were genotyped by the MassArray-4 system for rs1051730, rs10134584, rs1902341, rs10129758 which are known as PAD-associated GWAS loci. The rs1051730 polymorphism was strongly associated with an increased risk of PAD (p = 5.1 × 10-6), whereas rs1902341 did not show an association with disease risk. The rs1051730 polymorphism was associated with increased plasma levels of LDL cholesterol (p = 0.001), and conferred a greater risk of PAD in cigarette smokers than in nonsmokers (p < 0.01). Interestingly, the rs1902341T allele was associated with an increased risk of PAD in smokers and a decreased disease risk in nonsmokers. SNPs and CS were both linked to unilateral and/or bilateral atherosclerotic lesions of peripheral vessels, as well as the abdominal aorta, coronary, and cerebral arteries. The studied polymorphisms exert pleiotropic and cigarette smoking-mediated effects on atherosclerotic lesions of different arterial beds.

The Impact of Genetic Polymorphisms in Glutamate-Cysteine Ligase, a Key Enzyme of Glutathione Biosynthesis, on Ischemic Stroke Risk and Brain Infarct Size.

The purpose of this pilot study was to explore whether polymorphisms in genes encoding the catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase, a rate-limiting enzyme in glutathione synthesis, play a role in the development of ischemic stroke (IS) and the extent of brain damage. A total of 1288 unrelated Russians, including 600 IS patients and 688 age- and sex-matched healthy subjects, were enrolled for the study. Nine common single nucleotide polymorphisms (SNPs) of the GCLC and GCLM genes were genotyped using the MassArray-4 system. SNP rs2301022 of GCLM was strongly associated with a decreased risk of ischemic stroke regardless of sex and age (OR = 0.39, 95%CI 0.24−0.62, p < 0.0001). Two common haplotypes of GCLM possessed protective effects against ischemic stroke risk (p < 0.01), but exclusively in nonsmoker patients. Infarct size was increased by polymorphisms rs636933 and rs761142 of GCLC. The mbmdr method enabled identifying epistatic interactions of GCLC and GCLM gene polymorphisms with known IS susceptibility genes that, along with environmental risk factors, jointly contribute to the disease risk and brain infarct size. Understanding the impact of genes and environmental factors on glutathione metabolism will allow the development of effective strategies for the treatment of ischemic stroke and disease prevention.

Genetic variation at the catalytic subunit of glutamate cysteine ligase contributes to the susceptibility to sporadic colorectal cancer: a pilot study.

BACKGROUND: Glutathione is a tripeptide detoxifying a variety of exogenous and endogenous free radicals and carcinogens, and a deficiency of glutathione is associated with an increased host susceptibility to oxidative stress, a pathological condition implicated in the development and progression of cancer. The catalytic subunit of glutamate-cysteine ligase (GCLC) is an enzyme responsible for the initial and rate-limiting step of glutathione biosynthesis. METHODS AND RESULTS: The aim of this pilot study was to investigate whether genetic variation at the GCLC gene contributes to the risk of colorectal cancer (CRC). DNA samples from 681 unrelated Russian individuals (283 patients with CRC and 398 age- and sex-matched healthy controls) were genotyped for six common functional SNPs of the GCLC gene (SNPs) such as rs12524494, rs17883901, rs606548, rs636933, rs648595 and rs761142 of the GCLC gene using the MassARRAY-4 system. We found that genotype rs606548-C/T is significantly associated with increased risk of CRC regardless of sex and age (OR 2.24; 95% CI 1.24-4.03; P = 0.007, FDR = 0.04). Moreover, ten GCLC genotype combinations showed association with the risk of CRC (P < 0.05). Functional SNP annotation enabled establishing the CRC-associated polymorphisms are associated with a decreased GCLC expression that may be attributed to epigenetic effects of histone modifications operating in a colon-specific manner. CONCLUSIONS: The present study was the first to show that genetic variation at the catalytic subunit of glutamate-cysteine ligase may contribute to the risk of colorectal cancer risk. However, further genetic association studies with a larger sample size are required to substantiate the role of GCLC gene polymorphisms in the development of sporadic colorectal cancer.

Comprehensive Statistical and Bioinformatics Analysis in the Deciphering of Putative Mechanisms by Which Lipid-Associated GWAS Loci Contribute to Coronary Artery Disease.

The study was designed to evaluate putative mechanisms by which lipid-associated loci identified by genome-wide association studies (GWAS) are involved in the molecular pathogenesis of coronary artery disease (CAD) using a comprehensive statistical and bioinformatics analysis. A total of 1700 unrelated individuals of Slavic origin from the Central Russia, including 991 CAD patients and 709 healthy controls were examined. Sixteen lipid-associated GWAS loci were selected from European studies and genotyped using the MassArray-4 system. The polymorphisms were associated with plasma lipids such as total cholesterol (rs12328675, rs4846914, rs55730499, and rs838880), LDL-cholesterol (rs3764261, rs55730499, rs1689800, and rs838880), HDL-cholesterol (rs3764261) as well as carotid intima-media thickness/CIMT (rs12328675, rs11220463, and rs1689800). Polymorphisms such as rs4420638 of APOC1 (p = 0.009), rs55730499 of LPA (p = 0.0007), rs3136441 of F2 (p < 0.0001), and rs6065906 of PLTP (p = 0.002) showed significant associations with the risk of CAD, regardless of sex, age, and body mass index. A majority of the observed associations were successfully replicated in large independent cohorts. Bioinformatics analysis allowed establishing (1) phenotype-specific and shared epistatic gene-gene and gene-smoking interactions contributing to all studied cardiovascular phenotypes; (2) lipid-associated GWAS loci might be allele-specific binding sites for transcription factors from gene regulatory networks controlling multifaceted molecular mechanisms of atherosclerosis.

Pharmacogenetic loci for rosuvastatin are associated with intima-media thickness change and coronary artery disease risk.

Aim: Polymorphisms at LPA, LDLR, APOE, APOC1, MYLIP and ABCG2 are attractive targets for assessment of their impact on lipid-lowering therapy with rosuvastatin. The present study investigated whether polymorphisms at these genes are associated with the risk of coronary artery disease (CAD) development, and reduction of atherogenic lipids and carotid intima-media thickness (CIMT) in CAD patients, taking rosuvastatin. Materials & methods: 190 CAD patients and 1697 subjects were enrolled in pharmacogenetic and genetic association study, respectively. SNP genotyping was done using the MassARRAY-4 system. Results:MYLIP rs6924995, rs3757354, APOC1 rs445925, LDLR rs6511720, APOE rs7412, ABCG2 rs2199936, rs1481012 variants were significantly associated with CAD susceptibility (p = 0.016, 0.0003, <0.0001, <0.0001, 0.013, 0.016, 0.0035, respectively), as well as with CIMT regression (except ABCG2 variants; p = 0.05, 0.039, 0.039, 0.016, 0.0065), and changes in plasma lipids during rosuvastatin therapy. Conclusion: The studied polymorphisms possess pleiotropic effects on plasma lipids and CIMT, CAD susceptibility, and determine lipid-lowering response to rosuvastatin.

Matrix metalloproteinases as target genes for gene regulatory networks driving molecular and cellular pathways related to a multistep pathogenesis of cerebrovascular disease.

The present study investigated a joint contribution of matrix metalloproteinases (MMPs) genes to ischemic stroke (IS) development and analyzed interactions between MMP genes and genome-wide associated loci for IS. A total of 1288 unrelated Russians (600 IS patients and 688 healthy individuals) from Central Russia were recruited for the study. Genotyping of seven single nucleotide polymorphisms (SNPs) of MMP genes (rs1799750, rs243865, rs3025058, rs11225395, rs17576, rs486055, and rs2276109) and eight genome-wide associated loci for IS were done using Taq-Man-based assays and MALDI-TOF mass spectrometry iPLEX platform, respectively. Allele - 799T at rs11225395 of the MMP8 gene was significantly associated with a decreased risk of IS after adjustment for sex and age (OR = 0.82; 95%CI, 0.70-0.96; P = 0.016). The model-based multifactor dimensionality reduction method has revealed 21 two-order, 124 three-order, and 474 four-order gene-gene (G×G) interactions models meaningfully (Pperm < 0.05) associated with the IS risk. The bioinformatic analysis enabled establishing the studied MMP gene polymorphisms possess a clear regulatory potential and may be targeted by gene regulatory networks driving molecular and cellular pathways related to the pathogenesis of IS. In conclusion, the present study was the first to identify an association between polymorphism rs11225395 of the MMP8 gene and IS risk. The study findings also indicate that MMPs deserve special attention as a potential class of genes influencing the multistep mechanisms of cerebrovascular disease including atherosclerosis in cerebral arteries, acute cerebral artery occlusion as well as the ischemic injury of the brain and its recovery.

A comprehensive study revealed SNP-SNP interactions and a sex-dependent relationship between polymorphisms of the CYP2J2 gene and hypertension risk.

This study investigated whether common polymorphisms of cytochrome P450 2J2 (CYP2J2), a major enzyme that controls the biosynthesis of vasoactive epoxyeicosatrienoic acids, are collectively involved in the molecular basis of essential hypertension (EH). A total of 2314 unrelated Russian subjects from the Kursk (discovery sample: 913 EH patients and 645 controls) and Belgorod (replication sample: 345 EH patients and 411 controls) regions were recruited for this study. Eight single nucleotide polymorphisms (SNPs), including rs890293, rs11572182, rs10493270, rs1155002, rs2280275, rs7515289, rs11572325, and rs10889162, of CYP2J2 were genotyped using the MassARRAY 4 system and TaqMan-based assays. Significant associations were identified among the SNPs rs890293 (OR = 2.17, 95%CI 1.30-3.65), rs2280275 (OR = 1.59, 95%CI 1.10-2.37) and rs11572325 (OR = 1.89, 95%CI 1.22-2.95) and the risk of EH in females from the Kursk population. Sixteen CYP2J2 genotype combinations only showed significant associations with EH risk only in females. A common haplotype, T-T-G-C-C-C-T-A, increased the risk of EH in females. The bioinformatic analysis enabled identification of the SNPs that possess regulatory potential and/or are located within the binding sites for multiple transcription factors that play roles in the pathways involved in hypertension pathogenesis. Moreover, the polymorphisms rs890293, rs2280275, and rs11572325 were found to be significantly associated with hypertension risk in the Belgorod population. In conclusion, the rs2280275 and rs11572325 SNPs of CYP2J2 may be considered novel genetic markers of hypertension, at least in Russian women. However, sex-specific associations between CYP2J2 gene polymorphisms and hypertension require further investigation to clarify the specific genetic and/or environmental factors that are responsible for the increased disease susceptibility of women compared to that of men.

A Novel Polymorphism in the Promoter of the CYP4A11 Gene Is Associated with Susceptibility to Coronary Artery Disease.

Enzymes CYP4A11 and CYP4F2 are involved in biosynthesis of vasoactive 20-hydroxyeicosatetraenoic acid and may contribute to pathogenesis of coronary artery disease (CAD). We investigated whether polymorphisms of the CYP4A11 and CYP4F2 genes are associated with the risk of CAD in Russian population. DNA samples from 1323 unrelated subjects (637 angiographically confirmed CAD patients and 686 age- and sex-matched healthy individuals) were genotyped for polymorphisms rs3890011, rs9332978, and rs9333029 of CYP4A11 and rs3093098 and rs1558139 of CYP4F2 by using the Mass-ARRAY 4 system. SNPs rs3890011 and rs9332978 of CYP4A11 were associated with increased risk of CAD in women: OR = 1.26, 95% CI: 1.02-1.57, P = 0.004, and Q = 0.01 and OR = 1.45, 95% CI: 1.13-1.87, P = 0.004, and Q = 0.01, respectively. Haplotype G-C-A of CYP4A11 was associated with increased risk of CAD (adjusted OR = 1.41, 95% CI: 1.12-1.78, and P = 0.0036). Epistatic interactions were found between rs9332978 of CYP4A11 and rs1558139 of CYP4F2 (Pinteraction = 0.025). In silico analysis allowed identifying that SNP rs9332978 is located at a binding site for multiple transcription factors; many of them are known to regulate the pathways involved in the pathogenesis of CAD. This is the first study in Europeans that reported association between polymorphism rs9332978 of CYP4A11 and susceptibility to coronary artery disease.

Polymorphisms of CYP2C8, CYP2C9 and CYP2C19 and risk of coronary heart disease in Russian population.

Epoxyeicosatrienoic acids (EETs) are important vasoactive products of arachidonic acid metabolism with a wide range of biological actions in the cardiovascular system. The present study investigated whether single nucleotide polymorphisms (SNP) of genes coding cytochrome P450 2C subfamily, enzymes involved in biosynthesis of EETs, are associated with the risk of coronary heart disease (CHD). A total of 1255 unrelated Russian subjects comprising 561 patients with angiographically diagnosed CHD and 694 age- and sex-matched healthy subjects were included in the study. DNA samples from all study participants were genotyped for six common SNPs rs7909236, rs1934953 of CYP2C8, rs9332242, rs4918758 and rs61886769 of CYP2C9 and rs4244285 of CYP2C19 using by the Mass-ARRAY 4 system. SNP rs4918758 of CYP2C9 was associated with decreased risk of CHD (codominant model) at a borderline significance with odds ratio adjusted for sex and age 0.61 (95% CI: 0.41-0.92, P=0.038, Q=0.20). SNP rs9332242 of CYP2C9 showed a trend towards association with increased CHD risk in cigarette smokers (P=0.049, Q=0.29). Log-likelihood ratio test (LRT) pointed out epistatic interactions between rs9332242 and rs61886769 of CYP2C9 (codominant model, Pinteraction=0.02), however, this P-value did not survive after correction for multiple tests. Bioinformatic analysis revealed a regulatory potential for a majority of the investigated SNPs. Our preliminary results demonstrate that polymorphisms of genes encoding CYP2C subfamily represent potential genetic markers of CHD susceptibility. Further studies are required to substantiate the contribution of these genes to the disease risk.

The contribution of CYP2C gene subfamily involved in epoxygenase pathway of arachidonic acids metabolism to hypertension susceptibility in Russian population.

Numerous studies demonstrated an importance of cytochrome P-450 epoxygenase pathway of arachidonic acids metabolism for the pathogenesis of essential hypertension (EH). The present study was designed to investigate whether common single-nucleotide polymorphisms (SNP) of CYP2C gene subfamily such as CYP2C8 (rs7909236 and rs1934953), CYP2C9 (rs9332242), and CYP2C19 (rs4244285) are associated with susceptibility to EH in Russian population. A total of 816 unrelated Russian individuals comprising 425 EH patients and 391 normotensive controls were included into the study. Genotyping of SNPs was performed using the MassARRAY 4 system. SNP rs7909236 of CYP2C8 was significantly associated with increased risk of EH (OR adjusted for sex and age was 2.99 95% CI 1.39-6.44, P = 0.005). SNPs rs1934953 CYP2C8 and rs4244285 of CYP2C19 showed association with EH risk but at a borderline statistical significance (P ≤ 0.04). Combination of genotypes CYP2C8 rs7909236 TT and CYP2C19 rs4244285 GG was associated with increased EH risk (OR 3.34 95%CI 1.48-7.51, P = 0.004). Genotype-phenotype correlation analysis showed that the levels of CYP2C8 mRNA were significantly correlated with SNP rs7909236 (P = 0.01). in silico functional prediction analysis revealed the functionality of majority of investigated SNPs. Thus, genes of CYP2C subfamily are important genetic determinants of susceptibility to essential hypertension in Russians.

Altered erythrocyte membrane protein composition mirrors pleiotropic effects of hypertension susceptibility genes and disease pathogenesis.

OBJECTIVE: The study was designed to assess the effects of polymorphisms in genes associated with essential hypertension on the variation of erythrocyte membrane proteins (EMPs) in hypertensive patients. METHODS: Major EMPs content was analyzed in blood from 1162 unrelated Russians (235 hypertensive patients, 176 healthy controls, and 751 random individuals from the Central Russia population). Essential hypertension patients were genotyped for 11 polymorphisms of essential hypertension susceptibility genes including ADD1 (rs4961), GNB3 (rs5443, rs16932941), NOS3 (rs1799983, rs2070744), ACE (rs5186), AGTR1 (rs5186), AGT (rs699, rs4762), MR (rs5534), and TGFB1 (rs1800471). EMP contents and their relationship with the genetic loci were analyzed using various statistical tests. RESULTS: Sex-specific differences in EMP contents between the cases and controls were observed. Regardless of sex, hypertensives exhibited mainly decreased levels of alpha (SPTA1) and beta-spectrin (SPTB) and increased levels of glucose transporter (GLUT1) as compared with healthy subjects (P ≤ 0.001). EMP correlated differently in essential hypertension patients and controls. Almost 70% of the joint variation in the EMP levels is explained by five gender-specific principal components. The essential hypertension susceptibility genes showed considerable effects on the levels of spectrins and glucose transporter. A joint variation of the genes explained about half the total polygenic variance in the GLUT1, SPTA1, and SPTB levels in hypertensives. CONCLUSIONS: The study showed that essential hypertension susceptibility genes are the important factors of the inherited EMP variation, and their pleitropic effects may be mirrored in the altered expression of genes encoding cytoskeletal proteins and those related to intracellular glucose metabolism.