The challenges for molecular nutrition research 1: linking genotype to healthy nutrition

Nutrition science finds itself at a major crossroad. On the one hand we can continue the current path, which has resulted in some substantial advances, but also many conflicting messages which impair the trust of the general population, especially those who are motivated to improve their health through diet. The other road is uncharted and is being built over the many exciting new developments in life sciences. This new era of nutrition recognizes the complex relation between the health of the individual, its genome, and the life-long dietary exposure, and has lead to the realisation that nutrition is essentially a gene - environment interaction science. This review on the relation between genotype, diet and health is the first of a series dealing with the major challenges in molecular nutrition, analyzing the foundations of nutrition research. With the unravelling of the human genome and the linking of its variability to a multitude of phenotypes from " healthy'' to an enormously complex range of predispositions, the dietary modulation of these propensities has become an area of active research. Classical genetic approaches applied so far in medical genetics have steered away from incorporating dietary effects in their models and paradoxically, most genetic studies analyzing diet-associated phenotypes and diseases simply ignore diet. Yet, a modest but increasing number of studies are accounting for diet as a modulator of genetic associations. These range from observational cohorts to intervention studies with prospectively selected genotypes. New statistical and bioinformatics approaches are becoming available to aid in design and evaluation of these studies. This review discusses the various approaches used and provides concrete recommendations for future research.

Does genotype and equol-production status affect response to isoflavones? Data from a pan-European study on the effects of isoflavones on cardiovascular risk markers in post-menopausal women

The increase in CVD incidence following the menopause is associated with oestrogen loss. Dietary isoflavones are thought to be cardioprotective via their oestrogenic and oestrogen receptor-independent effects, but evidence to support this role is scarce. Individual variation in response to diet may be considerable and can obscure potential beneficial effects in a sample population; in particular, the response to isoflavone treatment may vary according to genotype and equol-production status. The effects of isoflavone supplementation (50hairspmg/d) on a range of established and novel biomarkers of CVD, including markers of lipid and glucose metabolism and inflammatory biomarkers, have been investigated in a placebo-controlled 2x8-week randomised cross-over study in 117 healthy post-menopausal women. Responsiveness to isoflavone supplementation according to (1) single nucleotide polymorphisms in a range of key CVD genes, including oestrogen receptor (ER) alpha and beta and (2) equol-production status has been examined. Isoflavones supplementation was found to have no effect on markers of lipids and glucose metabolism. Isoflavones improve C-reactive protein concentrations but do not affect other plasma inflammatory markers. There are no differences in response to isoflavones according to equol-production status. However, differences in HDL-cholesterol and vascular cell adhesion molecule 1 response to isoflavones v. placebo are evident with specific ER beta genotypes. In conclusion, isoflavones have beneficial effects on C-reactive protein, but not other cardiovascular risk markers. However, specific ER beta gene polymorphic subgroups may benefit from isoflavone supplementation.

Nutrition Society Silver Medal Lecture Nutrigenetics and personalised nutrition: how far have we progressed and are we likely to get there?

Nutrigenetics and personalised nutrition are components of the concept that in the future genotyping will be used as a means of defining dietary recommendations to suit the individual. Over the last two decades there has been an explosion of research in this area, with often conflicting findings reported in the literature. Reviews of the literature in the area of apoE genotype and cardiovascular health, apoA5 genotype and postprandial lipaemia and perilipin and adiposity are used to demonstrate the complexities of genotype-phenotype associations and the aetiology of apparent between-study inconsistencies in the significance and size of effects. Furthermore, genetic research currently often takes a very reductionist approach, examining the interactions between individual genotypes and individual disease biomarkers and how they are modified by isolated dietary components or foods. Each individual possesses potentially hundreds of 'at-risk' gene variants and consumes a highly-complex diet. In order for nutrigenetics to become a useful public health tool, there is a great need to use mathematical and bioinformatic tools to develop strategies to examine the combined impact of multiple gene variants on a range of health outcomes and establish how these associations can be modified using combined dietary strategies.

Nutrient gene interactions in lipid metabolism

Purpose of review To summarize recent findings relating to the impact of dietary fat composition on whole body lipid metabolism, and common gene variants on the blood lipid response to dietary fat change. Recent findings In recent years a more comprehensive understanding of the impact of polyunsaturated fat (PUFA) intake on the regulation of transcription factors involved in lipogenesis and fatty acid and lipoprotein metabolism has emerged. The evidence is suggestive of a greater potency of the long chain n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and in particular their oxidative products, relative to n-6 Pi In the area of nutrigenetics a number of common gene variants have been identified which may be important determinants of the blood lipid response to altered dietary fat composition. However, confirmation of associations in independent cohorts, and an understanding of the size effect of individual or combinations of genotypes, is often lacking. Summary Although in the future, genotyping holds the potential as a public health tool to target and personalize dietary advice, nutrigenetics is a relatively new science, and further research is needed to address the existing inconsistencies and knowledge gaps.

Differential effects of apolipoprotein E3 and E4 on markers of oxidative status in macrophages

ApoE is secreted by macrophages at the lesion site of the atherosclerotic plaque, where it is thought to play a protective role against atherosclerosis independently of its effects on lipid metabolism. Of the three common isoforms for apoE, apoE4 is associated with higher risk of cardiovascular disease (CVD). In vitro studies have shown that recombinant apoE may act as an antioxidant in an isoform-dependent manner (E2 > E3 > E4). The oxidative status of the macrophages plays a key role in the process of atherosclerosis. In the present study the possible differential actions of apoE3 and apoE4 on several parameters of oxidative status were determined in stably transfected murine macrophages (RAW 2647-apoE3 and apoE4). No differences between genotypes were observed after peroxide challenge in either protection against cytotoxicity or in cell membrane oxidation, and modest differences were observed in the non-enzymatic antioxidants (glutathione and a-tocopherol) in apoE3 v. apoE4 macrophages. Importantly, cells secreting apoE4 showed increased membrane oxidation under basal conditions, and produced more NO and superoxide anion radicals than the apoE3 macrophages after stimulation. The present data suggest that apoE genotype influences the oxidative status of macrophages, and this could partly contribute to the higher CVD risk observed in apoE4 carriers.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Soy-isoflavone-enriched foods and inflammatory biomarkers of cardiovascular disease risk in postmenopausal women: interactions with genotype and equol production

Background: Dietary isoflavones are thought to be cardioprotective because of their structural similarity to estrogen. The reduction of concentrations of circulating inflammatory markers by estrogen may be one of the mechanisms by which premenopausal women are protected against cardiovascular disease. Objective: Our aim was to investigate the effects of isolated soy isoflavones on inflammatory biomarkers [von Willebrand factor, intracellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), E-selectin, monocyte chemoattractant protein 1, C-reactive protein (CRP), and endothelin 1 concentrations]. Differences with respect to single-nucleotide polymorphisms in selected genes [estrogen receptor alpha (XbaI and PvuII), estrogen receptor beta [ER beta (AluI) and ER beta[cx] (Tsp5091), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), and cholesteryl ester transfer protein (TaqIB)] and equol production were investigated. Design: One hundred seventeen healthy European postmenopausal women participated in this randomized, double-blind, placebo-controlled, crossover dietary intervention trial. Isoflavone-enriched (genistein-to-daidzein ratio of 2:1;50 mg/d) or placebo cereal bars were consumed for 8 wk, with a washout period of 8 wk between the crossover. Plasma inflammatory factors were measured at 0 and 8 wk of each study arm. Results: Isoflavones improved CRP concentrations [odds ratio (95% Cl) for CRP values >1 mg/L for isoflavone compared with placebo: 0.43 (0.27, 0.69)]; no significant effects of isoflavone treatment on other plasma inflammatory markers were observed. No significant differences in the response to isoflavones were observed according to subgroups of equol production. Differences in the VCAM-1 response to isoflavones and to placebo were found with ER beta AluI genotypes. Conclusion: Isoflavones have beneficial effects on CRP concentrations, but not on other inflammatory biomarkers of cardiovascular disease risk in postmenopausal women, and may improve VCAM-1 in an ER beta gene polymorphic subgroup.

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.

Associations between apolipoprotein e genotype and circulating F-2-isoprostane levels in humans

Apolipoprotein E (apoE), an important determinant of plasma lipoprotein metabolism, has three common alleles (ε 2, ε 3, and ε 4). Population studies have shown that the risk of diseases characterized by oxidative damage, such as coronary heart disease and Alzheimer's disease, is significantly higher in ε 4 carriers. We evaluated the association between apoE genotypes and plasma F-2-isoprostane levels, an index of lipid peroxidation, in humans. Two hundred seventy-four healthy subjects (104 males, 170 females; 46.9 ± 13.0 yr; 200 whites, 74 blacks; 81 nonsmokers, 64 passive smokers, and 129 active smokers) recruited for a randomized clinical antioxidant intervention trial were included in this analysis. ApoE genotype was determined by PCR and restriction enzyme digestion. Free plasma F2-isoprostane was measured by GC-MS. Genotype groups were compared using multiple regression analysis with adjustment for sex, age, race, smoking status, body mass index, plasma ascorbic acid, and β-carotene. Subjects with ε 3/ε 4 and ε 4/ε 4 genotype (ε 4-carriers) and with ε 2/ε 3 and ε 3/ε 3 (non-ε 4-carriers) were pooled for analysis. In subjects with high cholesterol levels (total cholesterol above 200 mg/dl), plasma F-2-isoprostane levels were 29% higher in ε 4 carriers than in non-ε 4-carriers (P= 0.0056). High-cholesterol subjects that are ε 4 carriers have significantly higher levels of lipid peroxidation as assessed by circulating F-2-isoprostane levels.

Effects of genotype and environment on free amino acid levels in wheat grain: implications for acrylamide formation during processing

Acrylamide forms from free asparagine and reducing sugars during cooking, with asparagine concentration being the key parameter determining the formation in foods produced from wheat flour. In this study free amino acid concentrations were measured in the grain of varieties Spark and Rialto and four doubled haploid lines from a Spark x Rialto mapping population. The parental and doubled haploid lines had differing levels of total free amino acids and free asparagine in the grain, with one line consistently being lower than either parent for both of these factors. Sulfur deprivation led to huge increases in the concentrations of free asparagine and glutamine, and canonical variate analysis showed clear separation of the grain samples as a result of treatment (environment, E) and genotype (G) and provided evidence of G x E interactions. Low grain sulfur and high free asparagine concentration were closely associated with increased risk of acrylamide formation. G, E, and G x E effects were also evident in grain from six varieties of wheat grown at field locations around the United Kingdom in 2006 and 2007. The data indicate that progress in reducing the risk of acrylamide formation in processed wheat products could be made immediately through the selection and cultivation of low grain asparagme varieties and that further genetically driven improvements should be achievable. However, genotypes that are selected should also be tested under a range of environmental conditions.

Influence of temperature on pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) resistance to natural enemy attack

The ability to resist or avoid natural enemy attack is a critically important insect life history trait, yet little is understood of how these traits may be affected by temperature. This study investigated how different genotypes of the pea aphid Acyrthosiphon pisum Harris, a pest of leguminous crops, varied in resistance to three different natural enemies (a fungal pathogen, two species of parasitoid wasp and a coccinellid beetle), and whether expression of resistance was influenced by temperature. Substantial clonal variation in resistance to the three natural enemies was found. Temperature influenced the number of aphids succumbing to the fungal pathogen Erynia neoaphidis Remaudiere & Hermebert, with resistance increasing at higher temperatures (18 vs. 28degreesC). A temperature difference of 5degreesC (18 vs. 23degreesC) did not affect the ability of A. pisum to resist attack by the parasitoids Aphidius ervi Haliday and A. eadyi Stary Gonzalez & Hall. Escape behaviour from foraging coccinellid beetles (Hippodamia convergens Guerin-Meneville) was not directly influenced by aphid clone or temperature (16 vs. 21degreesC). However, there were significant interactions between clone and temperature (while most clones did not respond to temperature, one was less likely to escape at 16degreesC), and between aphid clone and ladybird presence (some clones showed greater changes in escape behaviour in response to the presence of foraging coccinellids than others). Therefore, while larger temperature differences may alter interactions between Acyrthosiphon pisum and an entomopathogen, there is little evidence to suggest that smaller changes in temperature will alter pea aphid-natural enemy interactions.

Genetic differentiation between hosts and locations in populations of latent Botrytis cinerea in southern England

This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria ´ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum of®- cinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154-bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9¤166), and by the rarity (3¤77) of a 112-bp allele at locus 7 that was common (58¤166) in isolates from other hosts. There was signi®cant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.

Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.)

Episodes of high temperature at anthesis, which in rice is the most sensitive stage to temperature, are expected to occur more frequently in future climates. The morphology of the reproductive organs and pollen number, and changes in anther protein expression, were studied in response to high temperature at anthesis in three rice (Oryza sativa L.) genotypes. Plants were exposed to 6 h of high (38 °C) and control (29 °C) temperature at anthesis and spikelets collected for morphological and proteomic analysis. Moroberekan was the most heat-sensitive genotype (18% spikelet fertility at 38 °C), while IR64 (48%) and N22 (71%) were moderately and highly heat tolerant, respectively. There were significant differences among the genotypes in anther length and width, apical and basal pore lengths, apical pore area, and stigma and pistil length. Temperature also affected some of these traits, increasing anther pore size and reducing stigma length. Nonetheless, variation in the number of pollen on the stigma could not be related to measured morphological traits. Variation in spikelet fertility was highly correlated (r=0.97, n=6) with the proportion of spikelets with ≥20 germinated pollen grains on the stigma. A 2D-gel electrophoresis showed 46 protein spots changing in abundance, of which 13 differentially expressed protein spots were analysed by MS/MALDI-TOF. A cold and a heat shock protein were found significantly up-regulated in N22, and this may have contributed to the greater heat tolerance of N22. The role of differentially expressed proteins and morphology during anther dehiscence and pollination in shaping heat tolerance and susceptibility is discussed.

Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.

A two-locus forensic match probability for subdivided populations

A two-locus match probability is presented that incorporates the effects of within-subpopulation inbreeding (consanguinity) in addition to population subdivision. The usual practice of calculating multi-locus match probabilities as the product of single-locus probabilities assumes independence between loci. There are a number of population genetics phenomena that can violate this assumption: in addition to consanguinity, which increases homozygosity at all loci simultaneously, gametic disequilibrium will introduce dependence into DNA profiles. However, in forensics the latter problem is usually addressed in part by the careful choice of unlinked loci. Hence, as is conventional, we assume gametic equilibrium here, and focus instead on between-locus dependence due to consanguinity. The resulting match probability formulae are an extension of existing methods in the literature, and are shown to be more conservative than these methods in the case of double homozygote matches. For two-locus profiles involving one or more heterozygous genotypes, results are similar to, or smaller than, the existing approaches.