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Background: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function., Methods: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents., Results: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance., Conclusions: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

Rationale: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r 2 >0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5' to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1)., Objective: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function., Methods and Results: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function., Conclusions: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

Objective: The grainyhead-like-2 (GRHL2) genetic variants were reported in age-related hearing impairment (ARHI) susceptibility in several case-control studies. However, their conclusions are conflicting; it is difficult to precisely assess the disease risk associated with the variants. Therefore we conduct the meta-analysis to discover the association of GRHL2 polymorphisms and the risk of ARHI., Methods: A related literature search was conducted in on-line databases, such as Wanfang database, China National Knowledge Infrastructure (CNKI), EMBASE, Web of Science, and PubMed (updated to August 30, 2018). We use Review Manager 5.0 and Stata SE 12.0 software to reckon the odds radio (OR), 95% confidence interval (CI) and P value in random- or fixed-effects model according to the I2 value in the heterogeneity test., Results: 2762 cases and 2321 controls in 5 articles were provided data to the meta-analysis. The pooled ORs (95% CI) of the rs10955255 polymorphism were 1.26 (1.05-1.50, P = .01), 1.33 (1.07-1.65, P = .01), and 1.32 (1.12-1.55, P = .0007) in the allele, homozygote and recessive model separately. Besides, a significant association was detected between rs1981361 in mixed population and the ARHI risk in the allele, heterozygote, and dominant genetic model respectively. Then subgroup analyses was performed by ethnicity, for rs10955255 meaningful associations were detected for the allele model, homozygote model, dominant model and recessive model in the Caucasian population but no relations in any of the 5 genetic models in Asian population., Conclusion: The meta-analysis indicated that the rs10955255 polymorphism could be an important risk factor for ARHI, especially in the Caucasians. The rs1981361 polymorphism may be a risk factor for ARHI in Asians. Larger scale researches are needed to further bring the consequences up to date.

Susceptibility to esophageal carcinoma (EC) is influenced by the interaction between genetic and environmental factors. To clarify the etiology of EC, several genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in PCLE1 and RFT2 genes as esophageal squamous cell carcinoma (ESCC) susceptibility loci in Asian populations. This study aimed to determine whether these SNPs also modify the risk of esophageal adenocarcinoma (EAC) and ESCC in western populations of Caucasian ethnicity. A European case-control study including 349 EC patients and 580 controls matched for age, sex, geographical location, and race was carried out. The SNPs rs2274223 in the PCLE1 gene at chromosome 10q23 and rs13042395 in the RFT2 gene at chromosome 20p13 were determined using PCR. Genotype distributions were compared between patients and controls, and odds ratios with 95% confidence intervals were calculated. The total EC group included 86 patients with ESCC and 258 patients with EAC. The distribution of PLCE1 and RFT2 genotypes did not differ between patients with EAC or ESSC, and the controls. In contrast to the modulation of the risk of ESCC in Asians, it is unlikely that the PLCE1 rs2274223 and RFT2 13042395 SNPs play a role in EAC or ESCC susceptibility in Dutch Caucasians.

Genome-wide association studies have revealed that many single nucleotide polymorphisms (SNPs) are associated with breast cancer, and yet the potential SNP-SNP interactions have not been well addressed to date. This study aims to develop a methodology for the selection of SNP-genotype combinations with a maximum difference between case and control groups. We propose a new chaotic particle swarm optimization (CPSO) algorithm that identifies the best SNP combinations for breast cancer association studies containing seven SNPs. Five scoring functions, that is, the percentage correct, sensitivity/specificity, positive predictive value/negative predictive value, risk ratio, and odds ratio, are provided for evaluating SNP interactions in different SNP combinations. The CPSO algorithm identified the best SNP combinations associated with breast cancer protection. Some SNP interactions in specific SNPs and their corresponding genotypes were revealed. These SNP combinations showed a significant association with breast cancer protection (P<0.05). The sensitivity and specificity of the respective best SNP combinations were all higher than 90%. In contrast to the corresponding non-SNP-SNP interaction combinations, the estimated odds ratio and risk ratio of the SNP-SNP interaction in SNP combinations for breast cancer were less than 100%. This suggests that CPSO can successfully identify the best SNP combinations for breast cancer protection. In conclusion, we focus on developing a methodology for the selection of SNP-genotype combinations with a maximum difference between case and control groups. The CPSO method can effectively identify SNP-SNP interactions in complex biological relationships underlying the progression of breast cancer.

Folate consumption is inversely associated with the risk of oral and pharyngeal cancer (OPC) and potentially interacts with alcohol drinking in the risk of OPC. Aldehyde dehydrogenase 2 (ALDH2) gene polymorphism is known to interact with alcohol consumption. The aim of this study was to investigate potential interaction between folate, alcohol drinking, and ALDH2 polymorphism in the risk of OPC in a Japanese population. The study group comprised 409 head and neck cancer cases and 1227 age-matched and sex-matched noncancer controls; of these, 251 cases and 759 controls were evaluated for ALDH rs671 polymorphism. Associations were assessed by odds ratios and 95% confidence intervals in multiple logistic regression models. We observed an inverse association between folate consumption and OPC risk. The odds ratio for high folate intake was 0.53 (95% confidence interval: 0.36-0.77) relative to low intake (P trend=0.003). This association was consistent across strata of sex, age, smoking, and ALDH2 genotypes. Interaction between folate consumption, drinking, and ALDH2 genotype was remarkable (three-way interaction, P<0.001). We observed significant interaction among folate, drinking, and ALDH2 genotype in the Japanese population.

A rare variant of chromosomal region 15q25.1, marked by rs16969968 (substitution 1354G>A in CHRNA5), was found to be associated with increased lung cancer and nicotine-dependence risk. We attempted to confirm the relationship of the polymorphism of the CHRNA5 gene and nicotine-dependence strength measured by the Fagerström test with the serum cotinine level in lung cancer and chronic obstructive pulmonary disease (COPD) patients and healthy individuals. Polymorphism of the CHRNA5 gene was analyzed using the PCR-based restriction fragment length polymorphism method in 97 lung cancer patients, 99 COPD patients, and 98 healthy individuals. The Fagerström test was used as an instrument for assessing the intensity of physical addiction. Cotinine serum level was measured using an enzyme-linked immunosorbent assay. The frequencies of AA, AG, and GG genotypes were 10.5, 47.3, and 42.2%, respectively. The polymorphism of CHRNA5 did not have a significant influence on the elevated risk of lung cancer and COPD. The percentage of smokers did not differ between groups of study participants with different genotypes. However, the presence of the GG genotype decreased the risk of nicotine addiction strength (hazard ratio=0.238; 95% confidence interval 0.066-0.857; P<0.05). Moreover, allele A was presented more frequently in participants with a high level of nicotine dependence and in participants with early addiction onset (P<0.05). Serum cotinine level was significantly correlated with the results of the Fagerström test (P<0.001). The carriers of allele A expressed significantly higher levels of cotinine when compared with the carriers of the GG genotype (P=0.05). We report for the first time the relationship between the polymorphism of the CHRNA5 gene and the strength of nicotine addiction measured by multiple factors including the Fagerström test score.

Chemopreventive dietary compounds, such as flavonols, may inhibit colorectal carcinogenesis partly by altering cytokine expression and attenuating inflammation. Single nucleotide polymorphisms (SNPs) in the promoter regions of genes encoding cytokines may influence flavonol-induced changes in cytokine expression and consequently cancer risk. Using logistic regression, we estimated odds ratios (OR) and 95% confidence intervals (CI) for the association between SNPs of interleukin (IL)-1β, 6, 8, and 10 alone or combined with flavonol intake or serum IL concentration changes, and adenoma recurrence in 808 participants from the intervention arm of the Polyp Prevention Trial, a 4-year intervention study evaluating the effectiveness of a low-fat, high-fiber, high-fruit and vegetable diet on adenoma recurrence. Overall, SNPs in genes encoding IL-1β, 6, 8, and 10 were not associated with their corresponding serum concentrations or adenoma recurrence. However, individuals homozygous for IL-10 -592 C (OR=2.23, 95% CI: 1.07-4.66, P(interaction)=0.03) orIL-10 -819 C (OR=2.18, 95% CI: 1.05-4.51, P(interaction)=0.05) had an elevated risk of high-risk adenoma recurrence when their serum IL-10 concentrations increased during the trial. In addition, IL-6 -174 GG in combination with above median flavonol intake (OR=0.14, 95% CI: 0.03-0.66) or with decreased IL-6 concentrations (OR=0.14, 95% CI: 0.03-0.65) reduced the risk of advanced adenoma recurrence, although the interaction term was not statistically significant. In conclusion, our results suggest that IL SNPs, in combination with a flavonol-rich diet or decreased serum IL, may lower the risk of adenoma recurrence.

Background: Genome-wide association studies (GWAS) on warfarin and acenocoumarol showed that interindividual dosage variation is mainly associated with single nucleotide polymorphisms (SNPs) in VKORC1 and to a lesser extent in CYP2C9 and CYP4F2. For phenprocoumon dosage, the genes encoding CYP3A4 and ApoE might play a role., Objective: To assess the association between common genetic variants within VKORC1, CYP2C9, CYP4F2, CYP3A4, and ApoE and phenprocoumon maintenance dosage, and to identify novel signals using GWAS., Methods: We selected all participants from the Rotterdam study who were treated with phenprocoumon. For each SNP, we tested the association between the above-mentioned genotypes and age, sex, body mass index, and target INR adjusted-phenprocoumon maintenance dosage., Results: Within our study population (N=244), VKORC1, CYP2C9, CYP4F2 genotypes together explained 46% of phenprocoumon maintenance dosage variation. Each additional VKORC1 variant allele reduced phenprocoumon maintenance dosage by 4.8 mg/week (P<0.0001) and each additional CYP2C9 variant allele by 2.2 mg/week (P=0.002). Each additional variant allele of CYP4F2 increased phenprocoumon dosage by 1.5 mg/week (P=0.022). Variant alleles of CYP3A41*B and ApoE showed no association with phenprocoumon dosage. Genome-wide significant SNPs were all related to VKORC1 activity. Best associated were two SNPs in complete linkage disequilibrium with each other and with SNPs within VKORC1: rs10871454 [Syntaxin 4A (STX4A)] and rs11150604 (ZNF646), each with a P value of 2.1×10⁻²². Each reduced phenprocoumon maintenance dosage weekly by 4.9 mg per variant allele., Conclusion: Similar to earlier findings with warfarin and acenocoumarol, phenprocoumon maintenance dosage depended on polymorphisms in the VKORC1 gene. CYP2C9 and CYP4F2 were of modest relevance.

Background and Objectives: Warfarin inhibits vitamin K epoxide reductase, of which microsomal epoxide hydrolase is a putative member. Several studies have found signals of association with warfarin maintenance dose in the EPHX1 gene. The aim of this study was to determine the effects of EPHX1 variants on warfarin maintenance dose in a multiethnic Asian population., Methods: We sequenced the exons of EPHX1 using PCR and direct sequencing in 279 patients consisting of three major ethnic groups receiving maintenance warfarin with a stable international normalized ratio. The effects of EPHX1 variants were assessed using multiple linear regression., Results: An association between an intronic SNP rs1877724 and warfarin maintenance dose was found, with homozygous variant carriers requiring approximately 0.5 mg/day lower than wild type and heterozygotes after adjustment for covariates. However, its contribution is small, explaining only an additional 0.8% of the dose variability. Rare variants were pooled but there was no association between their presence and warfarin maintenance dose. However, the presence of noncoding rare SNPs was significantly associated with warfarin maintenance dose., Conclusion: Despite a significant finding in rs1877724, which concurs with an earlier study, overall, genetic variants in EPHX1 do not have a clinically significant impact on warfarin dose requirements in our population.

Objectives: The G-protein-coupled receptor kinases (GRKs) GRK2 and GRK5 are important regulators of β-adrenergic signaling. This study characterized single-nucleotide polymorphisms (SNPs) in the GRK2 gene (ADRBK1) and determined if these and a GRK5 Gln41Leu polymorphism affect the blood pressure (BP) response to atenolol or hydrochlorothiazide or adverse cardiovascular outcomes in hypertensives., Methods: ADRBK1 regions were sequenced for 48 individuals. Putative functional SNPs were tested for mRNA expression differences in 96 lymphoblastoid cell line samples and 12 leukocyte samples from hypertensives. BP response to atenolol and hydrochlorothiazide by ADRBK1 SNPs and GRK5 Gln41Leu was tested in 418 patients from the Pharmacogenomic Evaluation of Antihypertensive Responses Study using linear regression. The influence of ADRBK1 SNPs and GRK5 Gln41Leu on death, myocardial infarction or stroke in treated hypertensives was evaluated in a case-control cohort (1 : 3) of the International Verapamil SR/Trandolapril Study GENEtic Substudy using logistic regression models., Results: A novel ADRBK1 promoter SNP was not associated with differential GRK2 expression. GRK5 Leu41 decreased the risk for adverse cardiovascular outcomes independent of treatment strategy (adjusted odds ratio 0.535, 95% confidence interval: 0.313-0.951, P=0.0222), but was not associated with BP response to antihypertensive medication. An ADRBK1 SNP (rs1894111 G>A) showed a signal for association with systolic and diastolic BP response to hydrochlorothiazide in Whites [diastolic BP: -11.29±3.74 (G/A) versus -4.26±4.79 mmHg (G/G), P=0.0034 and systolic BP: -18.37±14.90 (G/A), -8.11±7.55 mmHg (G/G), P=0.0191]., Conclusion: The GRK5 Leu41 allele protects from adverse cardiovascular outcomes in treated hypertensives.

Background: It is not known whether the genes involved with endurance performance during young adulthood are also involved with changes in performance. We examined the associations of gene variants with symptom-limited exercise test duration at baseline and decrease in duration over 20 years., Methods and Results: A total of 3783 (1835 black, 1948 white) and 2335 (1035 black, 1300 white) participants from the Coronary Artery Risk Development in Young Adults study were included in the baseline and 20-year models, respectively. Two hundred seventeen single-nucleotide polymorphisms (SNPs) in black participants and 171 in white participants from 17 genes were genotyped. In blacks, 5 SNPs in the ATP1A2, HIF1A, NOS3, and PPARGC1A loci tended to be associated (P<0.05) with baseline duration in a multivariate regression model. Blacks (n=99) with at least 4 of the most-favorable genotypes at these loci had an ≈2-minute longer baseline duration than those with only 2 such genotypes (P<0.0001). In whites, the HIF1A rs1957757 and PPARGC1A rs3774909 markers tended to be associated with baseline duration, but the association of a multimarker construct of the most-favorable genotypes at both SNPs with baseline duration was not statistically significant. In whites, 4 SNPs in the AGT, AMPD1, ANG, and PPARGC1A loci tended to be associated with decrease in exercise duration over 20 years, and those with all 4 favorable genotypes (n=40) had a 0.8-minute less decline in duration compared with those with none or 1 (n=232) (P<0.0001)., Conclusions: In multimarker constructs, alleles at genes related to skeletal muscle Na(+)/K(+) transport, hypoxia, and mitochondrial metabolism are associated with symptom-limited exercise test duration over time in adults.

Background: Population-based genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with cardiovascular disease or its risk factors. Genes in close proximity to these risk-SNPs are often thought to be pathogenetically important based on their location alone. However, the actual connections between SNPs and disease mechanisms remain largely unknown., Methods and Results: To identify novel susceptibility genes, we investigated how 166 SNPs previously found to be associated with increased cardiovascular risk and/or predisposing metabolic traits relate to the expression of nearby genes. Gene expression in 577 samples of aorta, liver, mammary artery, and carotid atherosclerotic plaque was measured using expression arrays. For 47 SNPs, the expression levels of proximal genes (located within 200 kb) were affected (P<0.005). More than 20 of these genes had not previously been identified as candidate genes for cardiovascular or related metabolic traits. SNP-associated gene effects were tissue-specific and the tissue specificity was phenotype-dependent., Conclusions: This study demonstrates several instances of association between risk-SNPs and genes immediately adjacent to them. It also demonstrates instances in which the associated gene is not the immediately proximal and obvious candidate gene for disease. This shows the necessity of careful studies of genetic marker data as a first step toward application of genome-wide association studies findings in a clinical setting.

Purpose: We examined associations among myostatin (MSTN) 2379 A > G and 163 G > A and follistatin (FST) -5003 A > T and -833 G > T single nucleotide polymorphisms (SNP) on the muscle size and the strength response to resistance training (RT)., Methods: Subjects (n = 645, age = 24.1 +/- 0.2 yr, body mass index [BMI] = 24.2 +/- 0.2 kg x m(-2)) self-disclosed themselves as Caucasian (78.9%), African American (3.6%), Asian (8.4%), Hispanic (5.0%), or Other (4.2%). They were genotyped for MSTN 2379 A > G (n = 645), MSTN 163 G > A (n = 639), FST -5003 A > T (n = 580), and FST -833 G > T (n = 603). We assessed dynamic (one repetition maximum [1RM]) and isometric (maximum voluntary contraction [MVC]) muscle strength and size (cross-sectional area [CSA]) of the elbow flexors before and after 12 wk of unilateral upper-arm RT. Repeated-measures ANCOVA tested associations among genetic variants and muscle phenotypes with age and BMI as covariates., Results: Baseline MVC was greater among African Americans who were carriers of the MSTN G(2379) allele (AG/GG, n = 15) than the A2379A homozygotes (n = 8; 64.2 +/- 6.8 vs 49.8 +/- 8.7 kg). African Americans who were carriers of the FST T(-5003) allele (n = 12) had greater baseline 1RM (11.9 +/- 0.7 vs 8.8 +/- 0.5 kg) and CSA (24.4 +/- 1.3 vs 19.1 +/- 1.2 cm(2)) than African Americans with the A-5003A genotype (n = 14; P < 0.05). No MSTN or FST genotype and muscle phenotype associations were found among the other ethnic groups (P >or= 0.05)., Conclusion: MSTN 2379 A > G and FST -5003 A > T were associated with baseline muscle strength and size among African Americans only. These ethnic-specific associations are hypothesis generating and should be confirmed in a larger sample of African Americans.

Identifying relevant cytotoxicity genes using an ex-vivo lymphoblastoid cell line (LCLs) model has distinct advantages for pharmacogenomic discovery studies of cancer chemotherapy, including standardized treatment conditions, availability of large numbers of samples, and publicly available genotypic data. However, there is little proof of principal data to confirm the promise of this approach. One of the known targets of 5-fluorouracil (5-FU) treatment is thymidylate synthase (TYMS). We hypothesized that genetic variants in TYMS would alter cytotoxicity because of 5-FU treatment using a LCL model system. LCLs from the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees (N=427) were treated with eight concentrations of 5-FU for 72 h, and cytotoxicity was determined using an Alamar Blue assay. For a subset of the 30 International Haplotype Mapping project (HapMap) trios, genotype data for 46 single-nucleotide polymorphism (SNP) variants encompassing the TYMS gene were downloaded from the HapMap website. Using a mixed models approach, each SNP was tested for association to 5-FU cytotoxicity in the subset of HapMap trios. Putatively associated SNPs (P<0.01), were then genotyped in the remaining LCLs in the CEPH pedigrees and tested for association. Two intronic SNPs in TYMS (rs2847153 and rs2853533) were significantly associated (P<0.01) with 5-FU cytotoxicity in the HapMap subset using the mixed models approach. After genotyping these SNPs in the full CEPH pedigrees, the associations with cytotoxicity showed a more reliable significance (P<0.0005), as a result of the increase in sample size. These results highlight the importance of the TYMS gene variants in response to 5-FU treatment. Furthermore, they provide additional biological validation of the relevance of LCLs as a model for pharmacogenomic gene discovery in cancer chemotherapy.

Objectives: In a previous investigation, we observed altered expression of sepiapterin reductase (SPR) in cultured neural cells chronically exposed to paroxetine. SPR is an enzyme, which catalyzes the final step in the synthesis of tetrahydrobiopterin (BH4). BH4 is an essential cofactor for synthesis of many neurotransmitters including serotonin. Given the pivotal role of SPR in neurotransmitter production, we sought to test the hypothesis that SPR would influence susceptibility to mood disorders and patient response to antidepressants., Methods: We tested for association of SPR promoter polymorphisms with antidepressant response in a well-characterized triad cohort of mood disorders. We evaluated the functional effect of these variants using the Dual-Luciferase Reporter Gene Assay System in two independent cell lines., Results: Two promoter single nucleotide polymorphisms (rs1876487 and rs2421095) in SPR were identified that occurred in three distinct haplotypes. We found a statistically significant association of haplotype pair 2,3 with bipolar I disorder [odds ratio: 5.47; 95% confidence interval: (1.68-17.88); P<0.005] and the personality measure self-transcendence (P = 0.020). Moreover, we found preliminary evidence that individuals with haplotype pair 2,3 responded better to the treatment with selective serotonin reuptake inhibitors. Reporter gene assays revealed a 1.4-fold to 1.6-fold decrease in the transcription rate of the two less common haplotypes (2 and 3) compared with haplotype 1, in the two cell lines investigated., Conclusion: This reduced transcription rate for SPR promoter haplotypes 2 and 3 may impact on BH4-mediated neurotransmitter production, thus suggesting a biological process through which SPR gene variants might influence antidepressant response and susceptibility to bipolar disorder.

Objective: We aimed to evaluate the association of non-response to antiepileptic pharmacotherapy with the frequency of variant alleles in the drug transporter genes ABCB1 and ABCC2 or in the CYP2C locus in young patients with epilepsy and an independent cohort of adults with drug-refractory epilepsy., Methods: A total of 221 pediatric or adolescent Caucasian patients with epilepsy (105 females; age: 14.5+/-6.54 years) were genotyped for nine putatively functionally relevant ABCB1, ABCC2, CYP2C8, CYP2C9, and CYP2C19 polymorphisms. In addition, 70 adult patients (35 females, age: 41.9+/-11.5 years) with drug-refractory epilepsy who had earlier undergone neurosurgical therapy were genotyped and partly (n = 22) investigated for hippocampal ABCB1 and ABCC2 mRNA expression. Finally, 242 healthy volunteers (167 females, age: 27.0+/-6.77 years) from the same region were included as controls., Results: The young cohort consisted of 103 (46.6%) responders and 118 (53.4%) non-responders to the first-line anticonvulsant. Carriers of the putatively low-expression ABCC2 -24T variant were significantly overrepresented among non-responders [odds ratio (OR) 2.15 (1.16-3.99); P = 0.016)]. This overrepresentation was confirmed by comparing young responders with adult drug-refractory patients [OR 3.36 (1.71-6.59); P<0.001]. Conversely, ABCB1 genotype distribution did not significantly differ between young responders and non-responders or adult drug-refractory patients. Excluding patients with febrile convulsions, heterozygous CYP2C8*4 [OR 0.35 (0.13-0.95); P = 0.038] and CYP2C9*3 [OR 0.34 (0.14-0.81); P = 0.015] variant allele carriers were underrepresented among non-responders. ABCC2 -24C>T genotype did not affect hippocampal ABCC2 expression, but was associated with increased ABCB1 expression (P = 0.034)., Conclusion: These data suggest a higher risk of antiepileptic drug failure in ABCC2 -24T allele carriers possibly because of compensatory upregulation of ABCB1.

Background: Pharmacogenetic research has shown that genetic variation may influence statin responsiveness. Statins exert a variety of beneficial effects beyond lipid lowering, including antithrombotic effects, which contribute to the risk reduction of cardiovascular disease. Statins have been shown to influence the expression of coagulation factors II, V, VII, XII and XIII., Aim: Data from a large randomized clinical trial of pravastatin, designed to show efficacy relative to usual care, were used to investigate whether a pharmacogenetic effect of polymorphisms in genes coding for coagulation factors II, V, VII, XII and XIII is associated with reduced fatal coronary heart disease (CHD) and nonfatal myocardial infarction, combined CHD and all-cause mortality., Methods: The Genetics of Hypertension Associated Treatment is an ancillary study of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial. The genotyped population in the lipid-lowering trial of Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial included 9624 participants randomly assigned to pravastatin or to usual care. The efficacy of pravastatin in reducing risk of all-cause mortality, CHD and nonfatal myocardial infarction and combined CHD, was compared among genotype strata by examining an interaction term in a proportional hazards model., Results: None of the polymorphisms were associated with the clinical outcomes. For the F7 (-323) ins/del polymorphism there was no interaction with pravastatin for either outcome. For both the F5 Arg506Gln G>A (rs6025) polymorphism and F7 Arg353Gln G>A (rs6046) polymorphism there were no interactions with pravastatin in relation to all-cause mortality, but there were significant interactions with combined CHD [interaction hazard ratio = 1.33, 95% confidence interval (1.01-1.76) and interaction hazard ratio = 1.92, 95% confidence interval (1.00-3.65), respectively]. There were no interactions between the polymorphisms in the other coagulation genes and pravastatin in relation to any outcome., Conclusion: Polymorphisms in anticoagulation genes (F5 and F7) seem to modify the efficacy of pravastatin in reducing risk of cardiovascular events.

Objectives: Oxidation of vitamin B12 by nitrous oxide leads to the inactivation of methionine synthase resulting in elevated plasma total homocysteine concentrations. Methionine synthase reductase is the only human enzyme that is able to reverse the oxidation of vitamin B12, which also occurs naturally by reactive oxygen species. A common polymorphism in methionine synthase reductase, MTRR 66A>G, is associated with reduced enzyme activity. Thus, we hypothesized that patients with this gene variant develop higher plasma total homocysteine concentrations after nitrous oxide anesthesia than wild-type patients., Methods: In this follow-up investigation of a previous gene association study, we prospectively included 140 healthy individuals undergoing elective surgery under general anesthesia that included 66% nitrous oxide. Peak postoperative plasma total homocysteine was the main outcome variable and was measured within 2 h after the end of anesthesia. The MTRR 66A>G genotype was determined after completion of the study. The association between genotype and peak postoperative total homocysteine was modeled with a general linear model., Results: No association between MTRR 66A>G and immediate postoperative plasma total homocysteine after nitrous oxide anesthesia was detected. All three groups, stratified by genotype (MTRR 66AA, AG, GG), shared similar baseline characteristics and increases in plasma total homocysteine. The average increase in plasma homocysteine was 2.4 mumol/l (+28%) in all three groups indicating the expected inactivation of methionine synthase by nitrous oxide through oxidation of vitamin B12, but no genetic effect., Conclusion: In conclusion, this study showed that the MTRR 66A>G gene variant is not associated with peak elevated postoperative plasma total homocysteine after nitrous oxide anesthesia. Whether the gene influences the rate of recovery of methionine synthase remains to be determined.

Host genetic factors play an important role in modifying the risk of human disease, including cancers of the upper gastrointestinal tract, with increasing interest in Toll-like receptor (TLR) signaling and the impact of genetic polymorphisms in these systems. The CD14-159C/T and the TLR9-1237T/C promoter polymorphisms have previously been shown to be associated with various inflammatory conditions including Helicobacter pylori-induced gastritis in Caucasian populations. In this study, we assessed the association of these two functional single nucleotide polymorphisms with gastric cancer in two independent Caucasian population-based case-control studies of upper gastrointestinal tract cancer, initially in 312 noncardia gastric carcinoma cases and 419 controls and then in 184 noncardia gastric carcinomas, 123 cardia carcinomas, 159 esophageal cancers, and 211 frequency-matched controls. Odds ratios were computed from logistic models and adjusted for potential confounding factors. No significant association was found between the CD14-159C/T and the TLR9-1237T/C promoter polymorphisms and increased risk of gastric cancer. Neither single nucleotide polymorphism has been assessed in a Caucasian gastric cancer case-control study before; although the CD14-159C/T polymorphism has been reported to show no apparent association with H. pylori-related gastric malignancy in a Taiwanese Chinese population. In conclusion, although our earlier preliminary studies suggested that the CD14-159C/T and the TLR9-1237T/C promoter polymorphisms increase the risk of precancerous outcomes, they do not seem to increase the risk of gastric cancer itself. This discrepancy merits further examination.

The objective of this study was to find out whether C-160A single nucleotide polymorphism of the promoter region of the E-cadherin gene might be a potential genetic marker for identifying individuals at risk for gastric cancer (GC). To test this hypothesis, 412 GC patients and 408 controls were analyzed statistically. A PCR-restriction fragment length polymorphism assay was adopted for C-160A single nucleotide polymorphism detection. No statistical differences were found among CC, CA, and AA genotypes and the risk of GC, even stratifying according to age, sex, and area of residence. Similarly, genotype was not associated with intestinal or diffuse histotypes, or with cardia or noncardia carcinomas. In conclusion, the C-160A polymorphism is not associated with GC risk in the Italian population.

Objectives: All microsomal cytochrome P450s enzymes, including those that metabolize the majority of clinically used drugs, require electron transfer through P450 oxidoreductase (POR). Mutations in human POR cause altered steroidogenesis and congenital malformations, but the clinical effects on drug metabolism are unclear. We examined the effects of POR sequence variants on two drug-metabolizing P450 enzymes, CYP1A2 and CYP2C19., Methods: Our previous sequencing of the human POR gene in POR-deficient patients and in 842 normal individuals identified 35 sequence variants. We expressed these 35 POR sequence variants in bacteria, reconstituted them with the CYP enzymes in vitro, and assayed their activities with human CYP1A2 and CYP2C19., Results: POR variants affected the activities of these enzymes to different extents. Disease-causing POR mutations A287P and R457H diminished catalysis by CYP1A2 and CYP2C19 to barely detectable levels. POR A503V, a polymorphism found in 28% of alleles in the normal population, had 85% of wild-type activity with CYP1A2 and 113% of wild-type activity with CYP2C19. Q153R, a disease-causing mutation that severely impaired steroidogenic activity and cytochrome c reduction, increased the activity of CYP1A2 to 144% and CYP2C19 activity to 284% of control., Conclusion: The activity of individual POR mutants may vary greatly depending on the electron recipient used to assay activity. Thus, the activity of a POR mutant to support catalysis by a particular P450 enzyme cannot be predicted by the activity of that POR mutant in an assay with a different P450 or with cytochrome c.

Polymorphisms in a number of genes encoding for DNA repair enzymes have been associated with altering the function of these enzymes and increasing risk of a number of cancers, including colon cancer. We have investigated the association between a common variant in polynucleotide kinase 3' phosphatase (PNKP), a putative DNA repair enzyme, and risk of adenoma recurrence in the Polyp Prevention Trial participants. We also investigated possible interaction or effect modification between carriage of the variant allele, dietary components and risk of adenoma recurrence. Unconditional logistic regression models were used to calculate the odds ratios and 95% confidence intervals for an association between the G/T polymorphism, PNKP T5644G and risk of adenoma recurrence. We observed no association between carriage of the variant allele and risk of adenoma recurrence. Furthermore, we found no effect modification between genotype, dietary components and risk of adenoma recurrence. The PNKP T5644G variant does not seem to be involved in adenoma recurrence in the Polyp Prevention Trial.

Objective: The ABCB1 haplotype combinations have been demonstrated to be associated with epilepsy treatment outcomes. The aim of this study is to investigate whether ABCB1 haplotype combinations would affect P-glycoprotein (Pgp) function and impact the clinical responses of antiepileptic drugs (AEDs)., Methods and Results: Transport of substrate rhodamine 123 and calcein-AM by human Pgp carrying 12 haplotype combinations of 1236C>T, 2677G>T/A and 3435C>T were assayed in the absence and presence of known inhibitors and AEDs. The inhibitory potency of the tested drugs from the dose-response relationships was cyclosporin A>verapamil> phenytoin> carbamazepine> lamotrigine>phenobarbital>valproic acid, levetiracetam, gabapentin. The silent polymorphisms combination (1236T-3435T) and triple haplotypes (1236T-2677A/T-3435T) resulted in profoundly less effective inhibition against substrates with significantly lower intracellular substrate concentration. These results confirmed that ABCB1 polymorphisms were associated with clinical responses of AEDs., Conclusion: Our findings demonstrated that human ABCB1 polymorphisms may alter the interactions between Pgp and substrates, and provided functional evidence for ABCB1 haplotypes-associated epilepsy treatment responses.

Purpose: Arterial stiffness, an independent risk factor for cardiovascular disease, increases with advancing age. Arterial stiffness is improved by regular exercise, but individual responses to exercise training are variable. Given that estrogen and estrogen receptor-alpha (ER-alpha) can induce vasodilation and can exert an antiatherosclerotic effect in vessels, we hypothesized that gene polymorphisms of ER-alpha might influence the ability of regular exercise to improve arterial stiffness in postmenopausal women., Methods: One hundred ninety-five healthy postmenopausal women (62 +/- 6 yr, mean +/- SD) participated in our cross-sectional study. We determined the genotype of single-nucleotide polymorphisms (SNP) at -401T/C of intron 1 of the ER-alpha gene. Arterial stiffness was measured by brachial-ankle pulse wave velocity (baPWV), and daily physical activity was estimated by a uniaxial accelerometer. Subjects were divided into active and inactive groups according to the median value (200 kcal.d(-1)) of energy expenditure., Results: baPWV in individuals with the TT variant of -401T/C genotype were significantly higher than for individuals with the TC+CC genotype. No significant differences in mean baPWV values were found between the active group and the inactive group (P = 0.09). A significant reduction of baPWV secondary to increased daily physical activity was observed in individuals with the TC+CC genotype but not in individuals with the TT genotype (TT/active: 1470 +/- 36 cm.s(-1); TT/inactive: 1457 +/- 34 cm.s(-1); TC+CC/active: 1359 +/- 21 cm.s(-1); TC+CC/inactive: 1433 +/- 24 cm.s(-1))., Conclusion: These results suggest that ER-alpha polymorphism affects the regular exercise-related reduction in arterial stiffness in healthy postmenopausal women.

Background: Experimental models suggest that immune cells recognize hepatitis C virus (HCV) through toll-like receptor (TLR)-2 and TLR4. We assessed the association between the single nucleotide polymorphism in genes that encode for these receptors and the outcome of liver transplantation for chronic HCV., Methods: A historical cohort of 92 liver transplant patients with chronic HCV were screened for TLR2 Arg753Gln and TLR4 Asp299Gly and Thr399Ile polymorphisms. The results were correlated with the predefined composite primary outcome of cirrhosis, retransplantation, and death. Statistical analysis was performed using Kaplan-Meier estimation and Cox proportional hazard model., Results: The mean patient age was 49+/-9 years. Sixty percent were male and 84% were white. Twelve (13%) patients had TLR2 Arg753Gln and 32 (35%) had TLR4 Asp299Gly and/or Thr399Ile polymorphism. During the mean follow-up period of 32 months after liver transplantation, the composite primary outcome occurred in 19 (24%) of 80 patients without TLR2 polymorphism, one (14%) of seven patients with heterozygous TLR2 polymorphism, and in all five (100%) patients with homozygous TLR2 polymorphism (P=0.0007). Time-to-event analysis showed a significant association between homozygous TLR2 polymorphism and the primary outcome (P<0.0001). After adjusting for donor age and azathioprine use, homozygous TLR2 mutation (RR 5.20 [1.65-13.9]; P=0.007) remained associated with the primary outcome. TLR4 polymorphisms were not associated with primary outcome., Conclusion: Homozygous TLR2 Arg753Gln polymorphism is associated with allograft failure and mortality after liver transplantation for chronic HCV. The potential clinical relevance of this observation should encourage studies to assess its biologic mechanism.

Background: The blood pressure (BP) and heart rate responses to exercise treadmill testing predict incidence of cardiovascular disease, but the genetic determinants of hemodynamic and chronotropic responses to exercise are largely unknown., Methods and Results: We assessed systolic BP, diastolic BP, and heart rate during the second stage of the Bruce protocol and at the third minute of recovery in 2982 Framingham Offspring participants (mean age 43 years; 53% women). With use of residuals from multivariable models adjusted for clinical correlates of exercise treadmill testing responses, we estimated the heritability (variance-components methods), genetic linkage (multipoint quantitative trait analyses), and association with 235 single-nucleotide polymorphisms in 14 candidate genes selected a priori from neurohormonal pathways for their potential role in exercise treadmill testing responses. Heritability estimates for heart rate during exercise and during recovery were 0.32 and 0.34, respectively. Heritability estimates for BP variables during exercise were 0.25 and 0.26 (systolic and diastolic BP) and during recovery, 0.16 and 0.13 (systolic and diastolic BP), respectively. Suggestive linkage was found for systolic BP during recovery from exercise (locus 1q43-44, log-of-the-odds score 2.59) and diastolic BP during recovery from exercise (locus 4p15.3, log-of-the-odds score 2.37). Among 235 single-nucleotide polymorphisms tested for association with exercise treadmill testing responses, the minimum nominal probability value was 0.003, which was nonsignificant after adjustment for multiple testing., Conclusions: Hemodynamic and chronotropic responses to exercise are heritable and demonstrate suggestive linkage to select loci. Genetic mapping with newer approaches such as genome-wide association may yield novel insights into the physiological responses to exercise.

Background: The study of genetic variation will promote our understanding of the differential predisposition to common diseases and variation in drug responses of individuals and ethnic populations. Such genetic variation is intrinsically structured into blocks of haplotypes in populations. Therefore, a comprehensive haplotype map based on the most abundant form of genetic variation, single nucleotide polymorphisms, will be useful. At the present time, however, our knowledge of the similarities and differences of haplotype structure among different ancestral populations is still inadequate., Methods: To determine whether common underlying haplotype patterns existed across ethnic populations, we analyzed data derived from African and European Americans for twenty-two genes spanning a total of 516 kb and the HapMap ENCODE data across 500 kb on chromosome 2p16.3 from three major world populations., Results and Conclusions: We observed that strong pairwise linkage disequilibrium (LD) between SNPs selected from populations having African ancestry was highly conserved across other non-African populations. Common haplotypes described by these LD-selected SNPs demonstrated a simple evolutionary structure with up to three major frameworks, which were likely ancestral backgrounds upon which more recent mutations have been superimposed. Also, haplotype block boundaries defined in populations having African ancestry revealed completely concordant recombinant haplotypes across all populations, providing a consistent definition of block structure. Finally, a large fraction of regulatory polymorphisms described in the literature appeared to tag these conserved haplotype frameworks, strongly suggesting their significance for disease association and pharmacogenetic studies.

Background: Brugada syndrome is associated with a high risk of sudden cardiac death and is caused by mutations in the cardiac voltage-gated sodium channel gene. Previously, the R282H-SCN5A mutation in the sodium channel gene was identified in patients with Brugada syndrome. In a family carrying the R282H-SCN5A mutation, an asymptomatic individual had a common H558R-SCN5A polymorphism and the mutation on separate chromosomes. Therefore, we hypothesized that the polymorphism could rescue the mutation., Methods and Results: In heterologous cells, expression of the mutation alone did not produce sodium current. However, coexpressing the mutation with the polymorphism produced significantly greater current than coexpressing the mutant with the wild-type gene, demonstrating that the polymorphism rescues the mutation. Using immunocytochemistry, we demonstrated that the R282H-SCN5A construct can traffic to the cell membrane only in the presence of the H558R-SCN5A polymorphism. Using fluorescence resonance energy transfer and protein fragments centered on H558R-SCN5A, we demonstrated that cardiac sodium channels preferentially interact when the polymorphism is expressed on one protein but not the other., Conclusions: This study suggests a mechanism whereby the Brugada syndrome has incomplete penetrance. More importantly, this study suggests that genetic polymorphisms may be a potential target for future therapies aimed at rescuing specific dysfunctional protein channels.

Drug-metabolizing enzymes are responsible for the activation or detoxification of cytotoxic drugs. Allelic variants are present with a variable frequency in different populations around the world and have an important role in the therapeutic index of such drugs. It is known that polymorphisms in thiopurine methyltransferase and dihydropyrimidine dehydrogenase have been associated with altered drug metabolism and increased risk of severe toxicity from 6-mercaptopurine and 5-fluorouracil, respectively. Additionally, a variant number of dinucleotide-repeat sequences in the promotor for uridine 5'-diphosphate glucuronosyltransferase 1A1 influences the glucuronidation of SN-38, the active metabolite of irinotecan, which is associated with severe toxicity, including diarrhea and neutropenia. In the same way, polymorphisms in thymidylate synthase have been associated with pyrimidine-associated toxicity and also with response to chemotherapy. The examples shown in this review demonstrate the usefulness of pre-screening patients for well-characterized polymorphism to identify the best-tolerated and most-effective treatment.

Background: There is marked heterogeneity in blood concentrations of tacrolimus following standard body-weight-based dosing. This is most apparent in black patients, who have a higher dose requirement when compared with other ethnic groups. Differences in intestinal P-glycoprotein and hepatic and intestinal cytochrome P4503A activity have been postulated as contributing to this problem., Methods: The dose-normalized blood concentrations of tacrolimus at 3 months after renal transplantation were related to CYP3AP1 and multiple drug resistance (MDR)-1 genotypes determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis., Results: We found that a single nucleotide polymorphism in the CYP3AP1 pseudogene (A/G(44)) that previously has been noted to be more common in African Americans and strongly associated with hepatic CYP3A5 activity correlated well with the tacrolimus dose requirement. A weaker association was found for a polymorphism in the MDR-1 gene, which influences intestinal P-glycoprotein expression., Conclusions: The CYP3AP1 genotype is a major factor in determining the dose requirement for tacrolimus, and genotyping may be of value in planning patient-specific drug dosing.