Genetic susceptibility to ischemic stroke in Russians

The frequencies of alleles and genotypes for ten functionally significant single-nucleotide polymorphisms were determined in the FGA, FGB, APOE, LPL, ACE, and CMA1 genes for Russian ischemic stroke (IS) patients and for a control group of Russians similar in gender and age distribution. The groups showed no significant differences in the frequencies of individual alleles or genotypes for any polymorphism studied. However, complex analysis of genetic susceptibility by the APSampler algorithm demonstrated that carriership of the APOE (−491A) allele predisposed to IS (p = 0.044, OR 3.8, 95% CI 1.0–15.1). Correspondingly, the APOE (−491T/T) genotype was associated with resistance to IS (p = 0.044, OR 0.26, 95% CI 0.07–1.0). The carriership of FGB (−249C) allele together with this genotype enhanced its protective potential, reducing the p value of the combination twofold (OR 0.17, 95% CI 0.04–0.8). Two more protective combinations were identified: biallelic APOE (−427C) + LPL (1595G) and triallelic APOE (−491C) + LPL (1595G) + CMA1 (−1903G). In both cases, p = 0.0052, OR 0.18, and 95% CI 0.05–0.66. Altogether, involvement in the formation of IS risk in Russians was evidenced for alleles of four genes: APOE, FGB, LPL, and CMA1; the APOE involvement was demonstrated for alleles of two polymorphic loci: −491T and −427C. Linkage analysis suggested that these loci were involved in IS resistance independently of each other.     

Association study of renin-angiotensin system genes and hemostasis system genes with ischemic stroke among Russians of Central Russia

The analysis of alleles and genotypes frequencies of 14 SNP in genes of rennin-angiotensin system (REN, AGT, AGTR1, AGTR2, BKR2, ADRB2) and hemostasis system (FGB, F2, F5, F7, ITGB3, SERPINE1, MTHFR), as well as ACE insertion-deletion polymorphism in patients with stroke comparing to healthy controls matched by age, sex and ethnicity has been carried out. The genotyping procedure included the amplification of selected gene sequences following by hybridization of fluorescently labeled fragments with SNP-specific DNA probes. The analysis of allele frequencies of each gene separately revealed no statistically significant differences between groups of patients with stroke and healthy donors. Also the complex study has been performed to estimate the contribution of rennin-angiotensin system and hemostasis system genes to the genetic susceptibility to ischemic stroke among Russians from Central Russia using method MDR (Multifactor Dimensionality Reduction). The combination with increased risk for development of ischemic stroke was presented by complex genotype FGB G/- x ACE I/- x MTHFR C/- x SERPINE1 5G/5G (p = 0.03, OR = 2.4, 95% CI 1.1-5.3), which frequency was statistically significant higher in patients with stroke compared to healthy control.     

Association of polymophisms of renin-angiotensin and hemostasis system genes with ischemic stroke in Russians from central Russia

The allele and genotype frequencies of 14 SNPs of renin-angiotensin (REN, AGT, AGTR1, AGTR2, BKR2, and ADRB2) and hemostasis (FGB, F2, F5, F7, ITGB3, SERPINE1, MTHFR) system genes, as well as of the ACE insertion-deletion polymorphism, were analyzed in patients with ischemic stroke and in healthy controls matched by age, sex, and ethnicity. Genotyping was performed through the amplification of the selected gene sequences and subsequent hybridization of the labeled fragments with SNP-specific DNA probes on the biochip. There were no significant differences in the allele frequencies of individual genes between the groups of stroke patients and healthy donors. The contribution of the renin-angiotensin and hemostasis system genes to the genetic susceptibility to ischemic stroke in Russians from central Russia was also assessed using the MDR (Multifactor Dimensionality Reduction) approach. The genotype combination FGB G/- x ACE I/- x MTHFR C/- x SERPINE1 5G/5G, the frequency of which was significantly higher in patients with stroke than in healthy controls, was associated with an increased risk of ischemic stroke (P = 0.03, OR = 2.4, 95%CI 1.1?C5.3).     

A comprehensive association analysis of homocysteine metabolic pathway genes in Singaporean Chinese with ischemic stroke

Background

The effect of genetic factors, apart from 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphisms, on elevated plasma homocysteine levels and increasing ischemic stroke risk have not been fully elucidated. We conducted a comprehensive analysis of 25 genes involved in homocysteine metabolism to investigate association of common variants within these genes with ischemic stroke risk.

Methodology/Principal Findings

The study was done in two stages. In the initial study, SNP and haplotype-based association analyses were performed using 147 tagging Single Nucleotide Polymorphisms (SNPs) in 360 stroke patients and 354 non-stroke controls of Singaporean Chinese ethnicity. Joint association analysis of significant SNPs was then performed to assess the cumulative effect of these variants on ischemic stroke risk. In the replication study, 8 SNPs were selected for validation in an independent set of 420 matched case-control pairs of Singaporean Chinese ethnicity. SNP analysis from the initial study suggested 3 risk variants in the MTRR, SHMT1 and TCN2 genes which were moderately associated with ischemic stroke risk, independent of known stroke risk factors. Although the replication study failed to support single-SNP associations observed in the initial study, joint association analysis of the 3 variants in combined initial and replication samples revealed a trend of elevated risk with an increased number of risk alleles (Joint P _trend = 1.2×10⁻⁶).

Conclusions

Our study did not find direct evidence of associations between any single polymorphisms of homocysteine metabolic pathway genes and ischemic stroke, but suggests that the cumulative effect of several small to moderate risk variants from genes involved in homocysteine metabolism may jointly confer a significant impact on ischemic stroke risk.     

Lack of association of polymorphic variants of genes encoding zinc transporters with the risk of orofacial cleft-affected pregnancies

Maternal zinc deficiency seems to be a risk factor for orofacial clefts in offspring. This study was undertaken to investigate the involvement of polymorphic variants of genes for zinc transporters in the susceptibility of clefting. PCRRFLP analysis was used to analyze single nucleotide polymorphisms of SLC30A1 (rs7526700, rs2278651, rs611386), SLC30A4 (rs2453531, rs8029246), SLC30A5 (rs351444, rs164393, rs6886492), SLC39A1 (rs10127484, rs11264736), and SLC39A3 (rs759071, rs4806874, rs10415622) in mothers of children with non-syndromic cleft lip with or without cleft palate (CL/P) and control mothers. The allele, genotype, and haplotype distribution was found to be similar among case and control mothers. Also, the gene-by-gene interaction analysis conducted using the Multifactor Dimensionality Reduction approach revealed no significant interactive genetic effect on having a child with a cleft. In conclusion, our results demonstrated that the analyzed polymorphic variants of genes for zinc transporters are not implicated in abnormal palatogenesis in the investigated group of women from the Polish population.     

The role of polymorphic variants of gene of inducible NO-synthase NOS2 in brain infarction in patients with acute ischemic stroke

Cerebrovascular diseases including stroke are an important problem of public health. Stroke development depends on external factors and individual genetic specificity of patient. Excessive NO production by inducible NO-synthase (iNOS) damages brain tissue at various stages of the disease. The goal of this work was to study the role of 4 polymorphic variants of gene of inducible NO-synthase iNOS (-2447C/G, -1659C/T, -0,7(OTTA)n I/D, S608L (150C/T)) in brain infarction in patients with acute ischemic stroke. A statistically significant correlation between S608L (150C/T) polymorphism and infarction dynamics was observed during days 1-3 and 7-21 after infarction. These parameters correlated with neurological status estimated using the Orgogozo scale during days 1-7 of the disease development. It was demonstrated that genotype N150N was associated with ischemic focus propagation regardless of its volume and neurological status by Orgogozo scale in patients with acute stroke. It was also observed that genotype N150N had effect on ischemic damage during days 1-3 in case of low initial volume.     

Polymorphic variants of genes encoding interleukin-6 and fibrinogen, the risk of ischemic stroke and fibrinogen levels

Carriage frequencies of alleles and genotypes of polymorphous locus of -174G>C IL6 (rs1800795) were analyzed in the patients with ischemic stroke (IS) of Russian ethnic descent (200 cases) and in the control group of the same ethnic descent with similar sex and age (140 controls). Significant differences were identified in frequencies of carriage (in homo- or heterozygous form) of allele IL6*-174G (p = 0.0029, OR = 2.9, 95% CI: 1.4-5.8), which can be considered as risk factor for IS and in frequencies of IL6*-174C/C genotype carriage, correspondingly (p = 0.0029, OR = 0.35, 95% CI: 0.17-0.69). After sex stratification of patients and controls similar significant differences were observed only between female patients and controls, after age stratification the difference was observed only for the age group older 60 years. Complex analysis of association of SNP -174G>C IL6 alleles and genotypes carriage in combination with SNP 4266A>G (Thr312Ala) FGA (rs6050) (see symbol) -249C>T FGB (rs1800788) with IS revealed protective combinations IL6*-174C/C + FGA* 4266A (see symbol) IL6*-174C/C + FGB*-249C, which were slightly more significant than single protective genotype IL6*-174C/C associated with IS and their ORs didn't differ substantially from the single genotypes's OR value. At the same time the combinations of alternative allele IL6*-174G with the same FGB*-249C or FGA* 4266A alleles were revealed and their association significance levels as well as OR values were lower than the values for the single risk allele IL6*-174G. In case of the mutual carriage of IL6*-174G allele with FGA*4266A/A, FGB*-249C/C genotypes or with combinations of these alleles/genotypes the "neutralized" effect became stronger. In other words, we observed association of IS with allele/genotype combinations of genes IL6, FGA and FGB, in which IL6 plays key role and FGA and FGB have modulating function. In analysis of association of fibrinogen plasma levels with three analyzed polymorphous loci significant differences were not revealed.     

Allelic variants of polymorphic genes associated with a higher frequency of chromosome aberrations

Genotypic associations were studied for the frequency of chromosome aberrations in human peripheral blood lymphocytes. Cytogenetic analysis (1000 metaphase plate per individual) and genotyping at 19 sites of genes involved in detoxification and DNA repair were performed in a sample of 83 Chernobyl liquidators and a matched control sample of 96 volunteers. In either sample, the frequency of chromosome aberrations was higher in carriers of the minor alleles of the XPD gene (sites 2251T > G and 862G > A) and the positive genotypes of the GSTM1-GSTT1 genes. The highest frequency of chromosome aberrations was observed in carriers of a combined genotype including at least one minor allele of the XPD sites + at least one insertion in the GSTM1-GSTT1 genes. The high-risk genotype, which had a prevalence of 64%, was strongly associated with a higher frequency of chromosome aberrations in both volunteers (OR = 6.9, P = 0.008) and Chernobyl liquidators (OR = 5.6, P = 0.002).     

Polymorphic variants of the genes encoding intrleukin-6 and fibrinogen: Risk for ischemic stroke and fibrinogen levels

The occurrence of alleles and genotypes of polymorphic region −174G>C of the gene IL6 (rs1800795) in patients of Russian ethnicity with ischemic stroke (200 cases) and in the control of the same ethnicity similar in sex and age (140 control subjects) has been analyzed. Reliable differences were revealed in the carriage frequency of allele IL6*-174G in homozygous and heterozygous form (p = 0.0029, OR = 2.9, 95% CI: 1.4–5.8), which can be considered a risk factor for the development of ischemic stroke, and in the carriage frequencies of protective genotype IL6*-174C/C (p = 0.0029, OR = 0.35, 95% CI: 0.17–0.69), respectively. After the patients and the control subjects were divided by gender character, similar differences were observed only between women with ischemic stroke and those from the control group, and after division by age, they were only revealed in groups with members older than 60 years. Complex analysis of the association of ischemic stroke with the carriage of alleles and genotypes of IL6 SNP-174G>C in combination with SNP 4266A>G (Thr312Ala) of the gene FGA (rs6050) and -249C>T of the gene FGB (rs1800788) revealed protective combinations of IL6*-174C/C + FGA*4266A and IL6*-174C/C + FGB*-249C, which are slightly more reliably associated with ischemic stroke than the one protective genotype IL6*-174C/C and have nearly the same OR value. At the same time, combinations of alternative allele IL6*-174G and the same alleles FGA*4266A or FGB*-249C were revealed that are characterized by a decrease in both the significance level and the OR value as compared with the carriage of one risk allele IL6*-174G. When there is a combined carriage of the allele IL6*-174G with the genotypes FGA*4266A/A, FGB*-249C/C or with combinations of these alleles/genotypes, the neutralizing effect is enhanced. In other words, we observed the association of IL6, FGA and FGB allele combinations with ischemic stroke in which IL6 plays the key role and FGA and FGB have a modulating function. In analyzing the association of the alleles/genotypes of three polymorphic regions with the fibrinogen level in plasma, no reliable difference was revealed.     

Genetic association and functional studies of major polymorphic variants of MGMT

The DNA repair protein, O(6)-methylguanine DNA-methyltransferase (MGMT) prevents mutations and cell death that result from aberrant alkylation of DNA. The polymorphic variants Leu84Phe, Ile143Val, and Lys178Arg are frequent in the human population. We review here studies of these and other MGMT polymorphisms and their association with risk for lung, breast, colorectal and endometrial cancer with a consideration of gene-environment interactions. In addition, we review studies of the effects of polymorphic variation on alkyltransferase activity and expression. It is formally possible that polymorphic variation could modify functions of MGMT other than its alkyltransferase activity. While it was previously reported that an alkylated form of MGMT modifies Estrogen Receptor alpha activity, from our studies we conclude that this regulation is not a major function of MGMT. Overall, the effects of polymorphic variation on protein function are subtle, and further investigation is required to provide a comprehensive mechanism that explains the observed associations of these variants with risk for cancer.     

Rare variants in ischemic stroke: an exome pilot study

The genetic architecture of ischemic stroke is complex and is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes we undertook a pilot study to identify variation across the human exome (i.e., the coding exons across the entire human genome) in 10 ischemic stroke cases. Our efforts focused on evaluating the feasibility and identifying the difficulties in this type of research as it applies to ischemic stroke. The cases included 8 African-Americans and 2 Caucasians selected on the basis of similar stroke subtypes and by implementing a case selection algorithm that emphasized the genetic contribution of stroke risk. Following construction of paired-end sequencing libraries, all predicted human exons in each sample were captured and sequenced. Sequencing generated an average of 25.5 million read pairs (75 bp×2) and 3.8 Gbp per sample. After passing quality filters, screening the exomes against dbSNP demonstrated an average of 2839 novel SNPs among African-Americans and 1105 among Caucasians. In an aggregate analysis, 48 genes were identified to have at least one rare variant across all stroke cases. One gene, CSN3, identified by screening our prior GWAS results in conjunction with our exome results, was found to contain an interesting coding polymorphism as well as containing excess rare variation as compared with the other genes evaluated. In conclusion, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined.     

Analysis of the polymorphic variants of the PDE4D gene in patients with acute stroke in the Moldavian population

The risk of the ischemic stroke is mediated by both environmental and genetic factors. Recent studies of DeCode group identified the risk of polymorphisms for ischemic stroke in the phosphodiesterase 4D gene (PDE4D). The goal of this study was to explore the role of two variants of the gene encoding PDE4D [SNP41 (rs152312) and SNP87 (rs2910829)] in the Moldavian patients with ischemic stroke and in control. No significant association with ischemic stroke was observed with SNP41 and 87.     

No association between genetic variants in angiogenesis and inflammation pathway genes and breast cancer survival among Chinese women

Background: Angiogenesis and inflammation are implicated in breast cancer prognosis; however, the role of individual germline variation in related genes is unknown. Methods: A two-stage candidate pathway association study was conducted among 6983 Chinese women. Stage 1 included 2884 women followed for a median of 5.7 years; Stage 2 included 4099 women followed for a median of 4.0 years. Cox proportional hazards regression was used to estimate the effects of genetic variants on disease-free survival (DFS) and overall survival (OS). Results: Stage 1 included genotyping of 506 variants in 22 genes; analysis was conducted for 370 common variants. Nominally significant associations with DFS and/or OS were found for 20 loci in ten genes in Stage 1; variants in 19 loci were successfully genotyped and evaluated in Stage 2. In analyses of both study stages combined, nominally significant associations were found for nine variants in seven genes; none of these associations surpassed a significance threshold level corrected for the total number of variants evaluated in this study. Conclusions: No association with survival was found for 370 common variants in 22 angiogenesis and inflammation pathway genes among Chinese women with breast cancer. Impact: Our data do not support a large role for common genetic variation in 22 genes in breast cancer prognosis; research on angiogenesis and inflammation genes should focus on common variation in other genes, rare host variants, or tumor alterations.     

Association polymorphic variants of GRIN2B gene with paranoid schizophrenia and response to typical neuroleptics in Russians and Tatars from Bashkortostan Republic

An analysis of the association of paranoid schizophrenia seeking with polymorphic variants of GRIN2B was performed in order to identify genetic risk factors of disease development and genetic markers of the response to therapy by neuroleptics in Russian and Tatar patients from Bashkortostan Republic (BR). In the course of the analysis, we revealed the following: (1) genetic markers of increased risk of developing paranoid schizophrenia in various ethnic groups, including, in Tatars, the GRIN2B*T/*T genotype (p = 0.003; OR = 2.33) and GRIN2B*T allele (p = 0.001; OR = 2.36), rs1805247; in Russians, the GRIN2B*T/*T genotype (p = 0.038; OR = 2.12) and GRIN2B*T allele (p = 0.028; OR = 2.03), rs1805247, genotype GRIN2B*A/*A (p = 0.042; OR = 2.12), rs1805476; (2) genetic markers of the reduced risk of developing paranoid schizophrenia; (3) genetic markers of therapy response and the risk of side effects development during neuroleptics (haloperidol) treatment in Bashkortostan. The significant interethnic diversity of genetic factors related to the risk of this disease development was noted.

     

Analysis of the MTHFR gene linkage disequilibrium structure and association of polymorphic gene variants with coronary atherosclerosis

Analysis of the genome-specific linkage disequilibrium patterns in certain populations is a highly promising approach to the identification of functional variants that underlie susceptibility to complex diseases. In the present study, the linkage disequilibrium patterns of the methylenetetrahydrofolate reductase gene (MTHFR) were examined in a group of patients with coronary atherosclerosis (coronary artery disease, CAD) and in a control sample from the Russian population. It was demonstrated that in the samples from one population, which were differentiated by the presence or absence of CAD, the MTHFR linkage disequilibrium patterns had similar features. Association of the MTHFR rs7533315 and rs2066462 polymorphisms with CAD was demonstrated. In addition, the evolution of the haplotypes and their role in the formation of CAD in the Russian population was reconstructed. The data on the association between genetic variability in the MTHFR locus and pathogenetically important indices of lipid metabolism were obtained. The high informativeness of the haplotype approach in case-control tests for associations with CAD was demonstrated.     

Characterization of 48 polymorphic loci as potential markers for the risk of ischemic stroke

With the aim to determine specific genetic characteristics of the population of Moscow region, we determined allele frequencies of 48 polymorphic loci (SNP) associated with the increased risk of ischemic stroke development. The genotype frequency distribution for all the SNPs corresponds to Hardy–Weinberg equilibrium. Comparison of the allele frequencies with those obtained for Caucasian populations from the databases dbSNP and 1000 Genomes Project revealed significant differences for two SNPs (rs556621 and rs556512) and seven SNPs (rs556621, rs556512, rs1801133, rs1799983, rs5918, rs328, and rs2398162), respectively. The revealed genetic features of the population make it possible to increase the accuracy and reliability of the individual genetic risk assessment of development for multifactorial diseases in the examined population.     

Novel association of a PROC variant with ischemic stroke in a Chinese Han population

Protein C (PC) is a well-characterized anticoagulant enzyme. However, the association between PC and ischemic stroke (IS) remains controversial. The aim of the present study was to investigate whether any genetic variant in the human protein C gene (PROC) was associated with susceptibility to IS in the Chinese Han population. All exons and the 5′- and 3′-untranslated regions of PROC were initially sequenced to identify informative variants. Potential abnormal variants were analyzed in a population of 788 IS patients and 1,200 healthy controls. The analysis was stratified by stroke etiology, and the results were replicated in 262 IS patients and 288 healthy controls. Finally, functional studies were performed to evaluate the effects of the variant. A three-nucleotide duplication/deletion variant (c.574_576del) was identified and found to be significantly associated with IS (OR 2.56, 95 % CI 1.45-4.52, P = 0.001). Stratification by stroke etiology after adjustment for IS risk factors showed that this association persisted in the lacunar and cardioembolic subtypes (P < 0.001 and P = 0.008, respectively) but not in the atherothrombotic and undetermined subtypes (P = 0.070 and P = 0.998, respectively). The functional studies showed a significant difference in the anticoagulant activity of PC in c.574_576del carriers and non-carriers (P < 0.001). Our results suggested that the novel PROC c.574_576del variant is a possible genetic determinant of an increased risk of IS and diminished anticoagulant activity of PC.     

Multilocus association testing of quantitative traits based on partial least-squares analysis

Because of combining the genetic information of multiple loci, multilocus association studies (MLAS) are expected to be more powerful than single locus association studies (SLAS) in disease genes mapping. However, some researchers found that MLAS had similar or reduced power relative to SLAS, which was partly attributed to the increased degrees of freedom (dfs) in MLAS. Based on partial least-squares (PLS) analysis, we develop a MLAS approach, while avoiding large dfs in MLAS. In this approach, genotypes are first decomposed into the PLS components that not only capture majority of the genetic information of multiple loci, but also are relevant for target traits. The extracted PLS components are then regressed on target traits to detect association under multilinear regression. Simulation study based on real data from the HapMap project were used to assess the performance of our PLS-based MLAS as well as other popular multilinear regression-based MLAS approaches under various scenarios, considering genetic effects and linkage disequilibrium structure of candidate genetic regions. Using PLS-based MLAS approach, we conducted a genome-wide MLAS of lean body mass, and compared it with our previous genome-wide SLAS of lean body mass. Simulations and real data analyses results support the improved power of our PLS-based MLAS in disease genes mapping relative to other three MLAS approaches investigated in this study. We aim to provide an effective and powerful MLAS approach, which may help to overcome the limitations of SLAS in disease genes mapping.