A hierarchical Bayesian model for predicting the functional consequences of amino-acid polymorphisms

Genetic polymorphisms in deoxyribonucleic acid coding regions may have a phenotypic effect on the carrier, e.g. by influencing susceptibility to disease. Detection of deleterious mutations via association studies is hampered by the large number of candidate sites; therefore methods are needed to narrow down the search to the most promising sites. For this, a possible approach is to use structural and sequence-based information of the encoded protein to predict whether a mutation at a particular site is likely to disrupt the functionality of the protein itself. We propose a hierarchical Bayesian multivariate adaptive regression spline (BMARS) model for supervised learning in this context and assess its predictive performance by using data from mutagenesis experiments on lac repressor and lysozyme proteins. In these experiments, about 12 amino-acid substitutions were performed at each native amino-acid position and the effect on protein functionality was assessed. The training data thus consist of repeated observations at each position, which the hierarchical framework is needed to account for. The model is trained on the lac repressor data and tested on the lysozyme mutations and vice versa. In particular, we show that the hierarchical BMARS model, by allowing for the clustered nature of the data, yields lower out-of-sample misclassification rates compared with both a BMARS and a frequen-tist MARS model, a support vector machine classifier and an optimally pruned classification tree.

The tagged microarray marker (TAM) method for high-throughput detection of single nucleotide and indel polymorphisms

The tagged microarray marker (TAM) method allows high-throughput differentiation between predicted alternative PCR products. Typically, the method is used as a molecular marker approach to determining the allelic states of single nucleotide polymorphisms (SNPs) or insertion-deletion (indel) alleles at genomic loci in multiple individuals. Biotin-labeled PCR products are spotted, unpurified, onto a streptavidin-coated glass slide and the alternative products are differentiated by hybridization to fluorescent detector oligonucleotides that recognize corresponding allele-specific tags on the PCR primers. The main attractions of this method are its high throughput (thousands of PCRs are analyzed per slide), flexibility of scoring (any combination, from a single marker in thousands of samples to thousands of markers in a single sample, can be analyzed) and flexibility of scale (any experimental scale, from a small lab setting up to a large project). This protocol describes an experiment involving 3,072 PCRs scored on a slide. The whole process from the start of PCR setup to receiving the data spreadsheet takes 2 d.

Polymorphisms in the apolipoprotein L1 gene and their effects on blood lipid and glucose levels in middle age males

Apolipoprotein L1 in plasma is associated with high- density lipoprotein. Novel APOL1 polymorphisms are investigated along with the association of two common haplotypes (Lys166Glu, Ile244Met, Lys271Arg) with circulating lipid and glucose levels. Although the amino acid substitutions occur in the amphipathic alpha helices region involved in lipid binding, these substitutions were found not to independently account for variability in circulating lipid and glucose levels in 149 middle age males.

Evidence for associations between common polymorphisms of estrogen receptor beta gene with homocysteine and nitric oxide

Background Homocysteine and asymmetric dimethylarginine (ADMA) affect nitric oxide (NO) concentration, thereby contributing to cardiovascular disease (CVD). Both amino acids can be reduced in vivo by estrogen. Variation in the estrogen receptor (ER) may influence homocysteine and ADMA, yet no information is available on associations with single nucleotide polymorphisms in the estrogen receptor genes ER alpha (PvuII and XbaI) and ER beta (1730G -> A and cx+56 G -> A). Objective To find relationships between common polymorphisms associated with cardiovascular disease and cardiovascular risk factors homocysteine and ADMA. Methods In a cross-sectional study with healthy postmenopausal women (n = 89), homocysteine, ADMA, nitric oxide metabolites (NOx), plasma folate and ER alpha and beta polymorphisms ER alpha PvuII, ER alpha XbaI; ER beta 1730G -> A (AluI), ER beta cx+56 G -> A (Tsp5091) were analyzed. Results Women who are homozygotic for ER beta cx+56 G -> A A/A exhibited higher homocysteine (p = 0.012) and NOx (p = 0.056) levels than wildtype or heterozygotes. NOx concentration was also significantly affected by ER beta 1730 G -> A polymorphism (p = 0.025). The ER beta (p < 0.001) and ER alpha (p < 0.001) polymorphisms were in linkage disequilibrium. Conclusions Women who are homozygotic for ER beta cx+S6 G -> A A/A may be at increased risk for cardiovascular disease due to higher homocysteine levels.

Polymorphisms in the apolipoprotein L1 gene and their effects on blood lipid and glucose levels in middle age males

Apolipoprotein L1 in plasma is associated with high- density lipoprotein. Novel APOL1 polymorphisms are investigated along with the association of two common haplotypes (Lys166Glu, Ile244Met, Lys271Arg) with circulating lipid and glucose levels. Although the amino acid substitutions occur in the amphipathic alpha helices region involved in lipid binding, these substitutions were found not to independently account for variability in circulating lipid and glucose levels in 149 middle age males.

Gene-nutrient interactions in the metabolic syndrome: single nucleotide polymorphisms in ADIPOQ and ADIPOR1 interact with plasma saturated fatty acids to modulate insulin resistance

Background: Progression of the metabolic syndrome (MetS) is determined by genetic and environmental factors. Gene-environment interactions may be important in modulating the susceptibility to the development of MetS traits. Objective: Gene-nutrient interactions were examined in MetS subjects to determine interactions between single nucleotide polymorphisms (SNPs) in the adiponectin gene (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) and plasma fatty acid composition and their effects on MetS characteristics. Design: Plasma fatty acid composition, insulin sensitivity, plasma adiponectin and lipid concentrations, and ADIPOQ, ADIPOR1, and ADIPOR2 SNP genotypes were determined in a cross-sectional analysis of 451 subjects with the MetS who participated in the LIPGENE (Diet, Genomics, and the Metabolic Syndrome: an Integrated Nutrition, Agro-food, Social, and Economic Analysis) dietary intervention study and were repeated in 1754 subjects from the LIPGENE-SU.VI.MAX (SUpplementation en VItamines et Mineraux AntioXydants) case-control study (http://www.ucd.ie/lipgene). Results: Single SNP effects were detected in the cohort. Triacylglycerols, nonesterified fatty acids, and waist circumference were significantly different between genotypes for 2 SNPs (rs266729 in ADIPOQ and rs10920533 in ADIPOR1). Minor allele homozygotes for both of these SNPs were identified as having degrees of insulin resistance, as measured by the homeostasis model assessment of insulin resistance, that were highly responsive to differences in plasma saturated fatty acids (SFAs). The SFA-dependent association between ADIPOR1 rs10920533 and insulin resistance was replicated in cases with MetS from a separate independent study, which was an association not present in controls. Conclusions: A reduction in plasma SFAs could be expected to lower insulin resistance in MetS subjects who are minor allele carriers of rs266729 in ADIPOQ and rs10920533 in ADIPOR1. Personalized dietary advice to decrease SFA consumption in these individuals may be recommended as a possible therapeutic measure to improve insulin sensitivity. This trial was registered at clinicaltrials.

ACC2 gene polymorphisms, metabolic syndrome, and gene-nutrient interactions with dietary fat.

Acetyl-CoA carboxylase β (ACC2) plays a key role in fatty acid synthesis and oxidation pathways. Disturbance of these pathways is associated with impaired insulin responsiveness and metabolic syndrome (MetS). Gene-nutrient interactions may affect MetS risk. This study determined the relationship between ACC2 polymorphisms (rs2075263, rs2268387, rs2284685, rs2284689, rs2300453, rs3742023, rs3742026, rs4766587, and rs6606697) and MetS risk, and whether dietary fatty acids modulate this in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). Minor A allele carriers of rs4766587 had increased MetS risk (OR 1.29 [CI 1.08, 1.58], P = 0.0064) compared with the GG homozygotes, which may in part be explained by their increased body mass index (BMI), abdominal obesity, and impaired insulin sensitivity (P < 0.05). MetS risk was modulated by dietary fat intake (P = 0.04 for gene-nutrient interaction), where risk conferred by the A allele was exacerbated among individuals with a high-fat intake (>35% energy) (OR 1.62 [CI 1.05, 2.50], P = 0.027), particularly a high intake (>5.5% energy) of n-6 polyunsaturated fat (PUFA) (OR 1.82 [CI 1.14, 2.94], P = 0.01; P = 0.05 for gene-nutrient interaction). Saturated and monounsaturated fat intake did not modulate MetS risk. Importantly, we replicated some of these findings in an independent cohort. In conclusion, the ACC2 rs4766587 polymorphism influences MetS risk, which was modulated by dietary fat, suggesting novel gene-nutrient interactions.

Leptin receptor polymorphisms interact with polyunsaturated fatty acids to augment risk of insulin resistance and metabolic syndrome in adults.

The leptin receptor (LEPR) is associated with insulin resistance, a key feature of metabolic syndrome (MetS). Gene-fatty acid interactions may affect MetS risk. The objective was to investigate the relationship among LEPR polymorphisms, insulin resistance, and MetS risk and whether plasma fatty acids, a biomarker of dietary fatty acids, modulate this. LEPR polymorphisms (rs10493380, rs1137100, rs1137101, rs12067936, rs1805096, rs2025805, rs3790419, rs3790433, rs6673324, and rs8179183), biochemical measurements, and plasma fatty acid profiles were determined in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). LEPR rs3790433 GG homozygotes had increased MetS risk compared with the minor A allele carriers [odds ratio (OR) = 1.65; 95% CI: 1.05–2.57; P = 0.028], which may be accounted for by their increased risk of elevated insulin concentrations (OR 2.40; 95% CI: 1.28–4.50; P = 0.006) and insulin resistance (OR = 2.15; 95% CI: 1.18–3.90; P = 0.012). Low (less than median) plasma (n-3) and high (n-6) PUFA status exacerbated the genetic risk conferred by GG homozygosity to hyperinsulinemia (OR 2.92–2.94) and insulin resistance (OR 3.40–3.47). Interestingly, these associations were abolished against a high (n-3) or low (n-6) PUFA background. Importantly, we replicated some of these findings in an independent cohort. Homozygosity for the LEPR rs3790433 G allele was associated with insulin resistance, which may predispose to increased MetS risk. Novel gene-nutrient interactions between LEPR rs3790433 and PUFA suggest that these genetic influences were more evident in individuals with low plasma (n-3) or high plasma (n-6) PUFA.

NOS3 gene polymorphisms are associated with risk markers of cardiovascular disease, and interact with omega-3 polyunsaturated fatty acids

Objective Omega-3 polyunsaturated fatty acids (n-3 PUFA) may protect against the development of cardiovascular disease (CVD). Genotype at key genes such as nitric oxide synthase (NOS3) may determine responsiveness to fatty acids. Gene–nutrient interactions may be important in modulating the development of CVD, particularly in high-risk individuals with the metabolic syndrome (MetS). Methods Biomarkers of CVD risk, plasma fatty acid composition, and NOS3 single nucleotide polymorphism (SNP) genotype (rs11771443, rs1800783, rs1800779, rs1799983, rs3918227, and rs743507) were determined in 450 individuals with the MetS from the LIPGENE dietary intervention cohort. The effect of dietary fat modification for 12 weeks on metabolic indices of the MetS was determined to understand potential NOS3 gene–nutrient interactions. Results Several markers of inflammation and dyslipidaemia were significantly different between the genotype groups. A significant gene–nutrient interaction was observed between the NOS3 rs1799983 SNP and plasma n-3 PUFA status on plasma triacylglycerol (TAG) concentrations. Minor allele carriers (AC + AA) showed an inverse association with significantly higher plasma TAG concentrations in those with low plasma n-3 PUFA status and vice versa but the major allele homozygotes (CC) did not. Following n-3 PUFA supplementation, plasma TAG concentrations of minor allele carriers of rs1799983 were considerably more responsive to changes in plasma n-3 PUFA, than major allele homozygotes. Conclusions Carriers of the minor allele at rs1799983 in NOS3 have plasma TAG concentrations which are more responsive to n-3 PUFA. This suggests that these individuals might show greater beneficial effects of n-3 PUFA consumption to reduce plasma TAG concentrations.

Polymorphisms in dopamine system genes are associated with individual differences in attention in infancy

Knowledge about the functional status of the frontal cortex in infancy is limited. This study investigated the effects of polymorphisms in four dopamine system genes on performance in a task developed to assess such functioning, the Freeze-Frame task, at 9 months of age. Polymorphisms in the catechol-O-methyltransferase (COMT) and the dopamine D4 receptor (DRD4) genes are likely to impact directly on the functioning of the frontal cortex, whereas polymorphisms in the dopamine D2 receptor (DRD2) and dopamine transporter (DAT1) genes might influence frontal cortex functioning indirectly via strong frontostriatal connections. A significant effect of the COMT valine158methionine (Val158Met) polymorphism was found. Infants with the Met/Met genotype were significantly less distractible than infants with the Val/Val genotype in Freeze-Frame trials presenting an engaging central stimulus. In addition, there was an interaction with the DAT1 3′ variable number of tandem repeats polymorphism; the COMT effect was present only in infants who did not have two copies of the DAT1 10-repeat allele. These findings indicate that dopaminergic polymorphisms affect selective aspects of attention as early as infancy and further validate the Freeze-Frame task as a frontal cortex task.

Single nucleotide polymorphisms at the ADIPOQ gene locus interact with age and dietary intake of fat to determine serum adiponectin in subjects at risk of the metabolic syndrome

Background: Adiponectin gene expression is modulated by peroxisome proliferator–activated receptor γ, which is a transcription factor activated by unsaturated fatty acids. Objective: We investigated the effect of the interaction between variants at the ADIPOQ gene locus, age, sex, body mass index (BMI), ethnicity, and the replacement of dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) or carbohydrates on serum adiponectin concentrations. Design: The RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) study is a parallel-design, randomized controlled trial. Serum adiponectin concentrations were measured after a 4-wk high-SFA (HS) diet and a 24-wk intervention with reference (HS), high-MUFA (HM), and low-fat (LF) diets. Single nucleotide polymorphisms at the ADIPOQ locus −11391 G/A (rs17300539), −10066 G/A (rs182052), −7734 A/C (rs16861209), and +276 G/T (rs1501299) were genotyped in 448 participants. Results: In white Europeans, +276 T was associated with higher serum adiponectin concentrations (n = 340; P = 0.006) and −10066 A was associated with lower serum adiponectin concentrations (n = 360; P = 0.03), after adjustment for age, BMI, and sex. After the HM diet, −10066 G/G subjects showed a 3.8% increase (95% CI: −0.1%, 7.7%) and G/A+A/A subjects a 2.6% decrease (95% CI: −5.6%, 0.4%) in serum adiponectin (P = 0.006 for difference after adjustment for the change in BMI, age, and sex). In −10066 G/G homozygotes, serum adiponectin increased with age after the HM diet and decreased after the LF diet. Conclusion: In white −10066 G/G homozygotes, an HM diet may help to increase adiponectin concentrations with advancing age. This trial was registered at clinicaltrials.gov as ISRCTN29111298.

PPARγ2 gene Pro12Ala and PPARα gene Leu162Val single nucleotide polymorphisms interact with dietary intake of fat in determination of plasma lipid concentrations

Background/Aims: The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of lipid metabolism, activated by unsaturated fatty acids. We investigated independent and interactive effects of PPARγ2 gene PPARG Pro12Ala (rs1801282) andPPARαgene PPARA Leu162Val (rs1800206) genotypes with dietary intake of fatty acids on concentrations of plasma lipids in subjects of whom 47.5% had metabolic syndrome. Methods: The RISCK study is a parallel design, randomised controlled trial. Plasma lipids were quantified at baseline after a 4-week high saturated fatty acids diet and after three parallel 24-week interventions with reference (high saturated fatty acids), high monounsaturated fatty acids and low-fat diets. Single nucleotide polymorphisms were genotyped in 466 subjects. Results: At baseline, the PPARG Ala12allele was associated with increased plasma total cholesterol (n = 378; p = 0.04), LDL cholesterol (p = 0.05) and apoB (p =0.05) after adjustment for age, gender and ethnicity. At baseline, PPARA Leu162Val × PPARG Pro12Ala genotype interaction did not significantly influence plasma lipid concentrations. After dietary intervention, gene-gene interaction significantly influenced LDL cholesterol (p =0.0002) and small dense LDL as a proportion of LDL (p = 0.005) after adjustments. Conclusions: Interaction between PPARG Pro12Ala and PPARA Leu162Valgenotypes may influence plasma LDL cholesterol concentration and the proportion as small dense LDL after a high monounsaturated fatty acids diet.

Neurotrophic gene polymorphisms and response to psychological therapy

Therapygenetics, the study of genetic determinants of response to psychological therapies, is in its infancy. Here, we investigate whether single-nucleotide polymorphisms in nerve growth factor (NGF) (rs6330) and brain-derived neutrotrophic factor (BDNF) (rs6265) genes predict the response to cognitive behaviour therapy (CBT). Neurotrophic genes represent plausible candidate genes: they are implicated in synaptic plasticity, response to stress, and are widely expressed in brain areas involved in mood and cognition. Allelic variation at both loci has shown associations with anxiety-related phenotypes. A sample of 374 anxiety-disordered children with white European ancestry was recruited from clinics in Reading, UK, and in Sydney, Australia. Participants received manualised CBT treatment and DNA was collected from buccal cells using cheek swabs. Treatment response was assessed at post-treatment and follow-up time points. We report first evidence that children with one or more copies of the T allele of NGF rs6330 were significantly more likely to be free of their primary anxiety diagnosis at follow-up (OR=0.60 (0.42–0.85), P=0.005). These effects remained even when other clinically relevant covariates were accounted for (OR=0.62 (0.41–0.92), P=0.019). No significant associations were observed between BDNF rs6265 and response to psychological therapy. These findings demonstrate that knowledge of genetic markers has the potential to inform clinical treatment decisions for psychotherapeutic interventions.