Fitness Of Genotypes Research Articles

Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus–infected patients†

Boceprevir is a hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease inhibitor that is currently being evaluated in combination with peginterferon alfa-2b and ribavirin in phase 3 studies. The clinical resistance profile of boceprevir is not characterized in detail so far. The NS3 protease domain of viral RNA was cloned from HCV genotype 1-infected patients (n = 22). A mean number of 47 clones were sequenced before, at the end, and after treatment with 400 mg boceprevir twice or three times daily for 14 days for genotypic, phenotypic, and viral fitness analysis. At the end of treatment, a wild-type an NS3 protease sequence was observed with a mean frequency of 85.9%. In the remaining isolates, five previously observed resistance mutations (V36M/A, T54A/S, R155K/T, A156S, V170A) and one mutation (V55A) with unknown resistance to boceprevir were detected either alone or in combination. Phenotypic analysis in the HCV replicon assay showed low (V36G, T54S, R155L; 3.8- to 5.5-fold 50% inhibitory concentration [IC(50)]), medium (V55A, R155K, V170A, T54A, A156S; 6.8- to 17.7-fold IC(50)) and high level (A156T; >120-fold IC(50)) resistance to boceprevir. The overall frequency of resistant mutations and the level of resistance increased with greater declines in mean maximum HCV RNA levels. Two weeks after the end of treatment, the frequency of resistant variants declined and the number of wild-type isolates increased to 95.5%. With the exception of V36 and V170 variants all resistant mutations declined by more than 50%. Mathematical modeling revealed impaired replicative fitness for all single mutations, whereas for combined mutations a relative increase of replication efficiency was suggested. During boceprevir monotherapy, resistance mutations at six positions within the NS3 protease were detected by way of clonal sequence analysis. All mutations are associated with reduced replicative fitness estimated by mathematical modeling and show cross-resistance to telaprevir.

A new test for genotype–fitness associations reveals a single microsatellite allele that strongly predicts the nature of tuberculosis infections in wild boar

There is increasing interest in the basis of commonly observed heterozygosity-fitness correlations (HFCs). Two models appear possible, a genome-wide effect due to inbreeding depression, and a single-locus effect due to chance linkage to a gene(s) experiencing balancing selection. Recent studies suggest that the latter tends to be more important in the majority of studies, but tests for the presence of single-locus effects tend to be rather weak. One of the problems is that the linkage disequilibrium between a microsatellite and a nearby gene experiencing balancing selection is never likely to be 100%. With this in mind, we conduct stochastic simulations aimed at determining the conditions under which single-locus HFCs may develop. We also suggest a new approach that could offer improved detection of HFCs but which also offers a more general method for detecting genotype-fitness correlations. Our method is based on looking for the maximum possible strength of association between genotype and fitness, and then asking whether randomized data sets are able to generate similarly strong associations. This method is tested on both simulated and real data. In both cases, our method generates greater levels of significance than current tests. Applied to previously published data from wild boar affected by tuberculosis, the method uncovers a strong single-allele association that is strongly predictive of whether the disease is localized or spreads throughout the body. We further suggest a simple method for dealing with the problem of population structure, and believe this approach will help to identify genomic regions associated with fitness.

Intense Habitat-Specific Fisheries-Induced Selection at the Molecular Pan I Locus Predicts Imminent Collapse of a Major Cod Fishery

Predation is a powerful agent in the ecology and evolution of predator and prey. Prey may select multiple habitats whereby different genotypes prefer different habitats. If the predator is also habitat-specific the prey may evolve different habitat occupancy. Drastic changes can occur in the relation of the predator to the evolved prey. Fisheries exert powerful predation and can be a potent evolutionary force. Fisheries-induced selection can lead to phenotypic changes that influence the collapse and recovery of the fishery. However, heritability of the phenotypic traits involved and selection intensities are low suggesting that fisheries-induced evolution occurs at moderate rates at decadal time scales. The Pantophysin I (Pan I) locus in Atlantic cod (Gadus morhua), representing an ancient balanced polymorphism predating the split of cod and its sister species, is under an unusual mix of balancing and directional selection including current selective sweeps. Here we show that Pan I alleles are highly correlated with depth with a gradient of 0.44% allele frequency change per meter. AA fish are shallow-water and BB deep-water adapted in accordance with behavioral studies using data storage tags showing habitat selection by Pan I genotype. AB fish are somewhat intermediate although closer to AA. Furthermore, using a sampling design covering space and time we detect intense habitat-specific fisheries-induced selection against the shallow-water adapted fish with an average 8% allele frequency change per year within year class. Genotypic fitness estimates (0.08, 0.27, 1.00 of AA, AB, and BB respectively) predict rapid disappearance of shallow-water adapted fish. Ecological and evolutionary time scales, therefore, are congruent. We hypothesize a potential collapse of the fishery. We find that probabilistic maturation reaction norms for Atlantic cod at Iceland show declining length and age at maturing comparable to changes that preceded the collapse of northern cod at Newfoundland, further supporting the hypothesis. We speculate that immediate establishment of large no-take reserves may help avert collapse.

Apoe Genotype, Aerobic Fitness, And Cerebral Cortical Activation During Working Memory Challenge In Middle-aged Adults

PURPOSE: APOE e4 genotype is linked to accelerated age-related cognitive decline and is an established risk for Alzheimer's disease. Cardiovascular fitness may counteract this liability based on animal studies that reveal remarkable neurobiological benefits in the brain. The present study determined if cardiovascular fitness is positively associated with neurocognitive integrity in e4 carriers during memory challenge. METHODS: After providing informed consent and health history, 22 men and women aged 50-70 years completed cognitive screening by interview and the Rockport Walk test to determine aerobic fitness. Genotyping preceded a battery of cognitive tests (APOE e4 carriers, n=11.) Working memory challenge included presentation of a modified Sternberg Letter task (stimuli of 5- or 7-letters) during non-invasive magnetoencephalographic (MEG) brain imaging. Averaged M170 amplitude and latency, derived from global MEG activity recorded from 157 sites, were separately and hierarchically regressed on age, education, genotype, aerobic fitness (estimated VO2max [ml/kg/min]), and the interaction between genotype and aerobic fitness. We predicted that aerobic fitness would be positively related to M170 amplitude and negatively related to M170 latency, and that the interaction of APOE genotype would explain additional unique variance, particularly as the task became more difficult (i.e., 7-letter condition). RESULTS: The Rockport Walk test produced an estimated VO2max of 32.2 ml/kg/min (± 8.6). M170 amplitude in response to 5- letter memory probe revealed a slope of.95 and R2 value of.56, p=.001 for aerobic fitness. M170 amplitude in response to the 7-letter memory probe revealed a slope of.72 and R2 value of.42, p=.02 for aerobic fitness. Latency was not related to any of the independent variables. CONCLUSIONS: Aerobic fitness is positively associated with cortical activation during memory challenge across varying levels of load, regardless of APOE genotype.

Looking for answers to questions about heat stress: researchers are getting warmer

While the public continues to debate the reality of global warming, scientists should prepare to deal with the consequences of this very real phenomenon. Our planet has warmed by 0·13 ° C per decade since 1956, and will likely warm more rapidly in the decades to come (Intergovernmental Panel on Climate Change 2007). This global measure of thermal change belies the more pronounced and less predictable changes that are occurring on smaller scales (Walther et al . 2002). For example, the greater frequency and intensity of heat waves in many areas ranks among the more threatening aspects of anthropogenic climate change. Will acclimation or adaptation enable the persistence of populations that face such thermal stresses? And, if populations do persist, will their densities and compositions change radically? To answer these questions, we must understand a suite of phenomena, ranging from the mechanistic basis of thermotolerance to the demographic and ecological consequences of thermal plasticity (Angilletta 2009). This issue of Functional Ecology contains four papers that improve our understanding of these phenomena. To enhance your appreciation of this collection, I briefly highlight the significance of each paper while laying out the broader issues concerning the biological impacts of heat stress. During rapid warming, the performance of an organism depends on its capacity to protect and modify its cellular structures. Brief exposure to extreme heat often causes greater thermotolerance within hours, a phenomenon referred to as

Compensatory mutations are repeatable and clustered within proteins.

Compensatory mutations improve fitness in genotypes that contain deleterious mutations but have no beneficial effects otherwise. As such, compensatory mutations represent a very specific form of epistasis. We show that intragenic compensatory mutations occur non-randomly over gene sequence. Compensatory mutations are more likely to appear at some sites than others. Moreover, the sites of compensatory mutations are more likely than expected by chance to be near the site of the original deleterious mutation. Furthermore, compensatory mutations tend to occur more commonly in certain regions of the protein even when controlling for clustering around the site of the deleterious mutation. These results suggest that compensatory evolution at the protein level is partially predictable and may be convergent.

Effect of variation in self-incompatibility on pollen limitation and inbreeding depression in Flourensia cernua (Asteraceae) scrubs of contrasting density.

Selection may favour a partial or complete loss of self-incompatibility (SI) if it increases the reproductive output of individuals in the presence of low mate availability. The reproductive output of individuals varying in their strength of SI may also be affected by population density via its affect on the spatial structuring and number of S-alleles in populations. Modifiers increasing levels of self-compatibility can be selected when self-compatible individuals receive reproductive compensation by, for example, increasing seed set and/or when they become associated with high fitness genotypes. The effect of variation in the strength of SI and scrub density (low versus high) on seed set, seed germination and inbreeding depression in seed germination (delta(germ)) was investigated in the partially self-incompatible species Flourensia cernua by analysing data from self-, cross- and open-pollinated florets. Examination of 100 plants in both high and low scrub densities revealed that 51% of plants were strongly self-incompatible and 49 % varied from being self-incompatible to self-compatible. Seed set after hand cross-pollination was higher than after open-pollination for self-incompatible, partially self-incompatible and self-compatible plants but was uniformly low for strongly self-incompatible plants. Strongly self-incompatible and self-incompatible plants exhibited lower seed set, seed germination and multiplicative female fitness (floral display x seed set x seed germination) in open-pollinated florets compared with partially self-incompatible and self-compatible plants. Scrub density also had an effect on seed set and inbreeding depression: in low-density scrubs seed set was higher after open-pollination and delta(germ) was lower. These data suggest that (a) plants suffered outcross pollen limitation, (b) female fitness in partially self-incompatible and self-compatible plants is enhanced by increased mate-compatibility and (c) plants in low-density scrubs received higher quality pollen via open-pollination than plants in high-density scrubs.

Key determinants of resistance durability to plant viruses: Insights from a model linking within- and between-host dynamics

The emergence of new genotypes of parasites involves several evolutionary, epidemiological and ecological processes whose individual effects and interactions are difficult to disentangle using experimental approaches. Here, a model is proposed to investigate how these processes lead to the emergence of plant viral genotypes breaking down qualitative resistance genes. At the individual plant scale, selection, drift and mutation processes shape the evolution of viral populations from a set of differential equations. The spatial segregation of virus genotypes in their hosts is also considered. At the host population scale, the epidemiological dynamics is given by an individual-based algorithm. Global sensitivity analyses allowed ranking the ten demo-genetic and epidemiological parameters of the model according to their impact on the mean and variance of the risk of breakdown of a plant resistance. Demo-genetic parameters (number and nature of mutations involved in breakdown, fitness of mutant genotypes) had the largest impact on the mean breakdown risk, whereas epidemiological parameters had more influence on its standard deviation. It is discussed how these results can be used to choose the potentially most durable resistance genes among a pool of candidates. Finally, our analyses point out the parameters which should be estimated more precisely to improve durability predictions.

Tracking costs of virulence in natural populations of the wheat pathogen, Puccinia striiformis f.sp.tritici

BackgroundCosts of adaptation play an important role in host-parasite coevolution. For parasites, evolving the ability to circumvent host resistance may trade off with subsequent growth or transmission. Such costs of virulence (sensu plant pathology) limit the spread of all-infectious genotypes and thus facilitate the maintenance of genetic polymorphism in both host and parasite. We investigated costs of three virulence factors in Puccinia striiformis f.sp.tritici, a fungal pathogen of wheat (Triticum aestivum).ResultsIn pairwise competition experiments, we compared the fitness of near-isogenic genotypes that differed by a single virulence factor. Two virulence factors (vir4, vir6) imposed substantial fitness costs in the absence of the corresponding resistance genes. In contrast, the vir9 virulence factor conferred a strong competitive advantage to several isolates, and this for different host cultivars and growing seasons. In part, the experimentally derived fitness costs and benefits are consistent with frequency changes of these virulence factors in the French pathogen population.ConclusionOur results illustrate the variation in the evolutionary trajectories of virulence mutations and the potential role of compensatory mutations. Anticipation of such variable evolutionary outcomes represents a major challenge for plant breeding strategies. More generally, we believe that agro-patho-systems can provide valuable insight in (co)evolutionary processes in host-parasite systems.

Quasispecies theory for horizontal gene transfer and recombination

We introduce a generalization of the parallel, or Crow-Kimura, and Eigen models of molecular evolution to represent the exchange of genetic information between individuals in a population. We study the effect of different schemes of genetic recombination on the steady-state mean fitness and distribution of individuals in the population, through an analytic field theoretic mapping. We investigate both horizontal gene transfer from a population and recombination between pairs of individuals. Somewhat surprisingly, these nonlinear generalizations of quasispecies theory to modern biology are analytically solvable. For two-parent recombination, we find two selected phases, one of which is spectrally rigid. We present exact analytical formulas for the equilibrium mean fitness of the population, in terms of a maximum principle, which are generally applicable to any permutation invariant replication rate function. For smooth fitness landscapes, we show that when positive epistatic interactions are present, recombination or horizontal gene transfer introduces a mild load against selection. Conversely, if the fitness landscape exhibits negative epistasis, horizontal gene transfer or recombination introduces an advantage by enhancing selection towards the fittest genotypes. These results prove that the mutational deterministic hypothesis holds for quasispecies models. For the discontinuous single sharp peak fitness landscape, we show that horizontal gene transfer has no effect on the fitness, while recombination decreases the fitness, for both the parallel and the Eigen models. We present numerical and analytical results as well as phase diagrams for the different cases.

The Generation and Maintenance of Genetic Variation by Frequency-Dependent Selection: Constructing Polymorphisms Under the Pairwise Interaction Model

Frequency-dependent selection remains the most commonly invoked heuristic explanation for the maintenance of genetic variation. For polymorphism to exist, new alleles must be both generated and maintained in the population. Here we use a construction approach to model frequency-dependent selection with mutation under the pairwise interaction model. The pairwise interaction model is a general model of frequency-dependent selection at the genotypic level. We find that frequency-dependent selection is able to generate a large number of alleles at a single locus. The construction process generates multiallelic polymorphisms with a wide range of allele-frequency distributions and genotypic fitness relationships. Levels of polymorphism and mean fitness are uncoupled, so constructed polymorphisms remain permanently invasible to new mutants; thus the model never settles down to an equilibrium state. Analysis of constructed fitness sets reveals signatures of heterozygote advantage and positive frequency dependence.

Temperature‐dependent costs of parasitism and maintenance of polymorphism under genotype‐by‐environment interactions

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.

Synthetic variability and genotype fitness

In two previous articles the analysis has chiefly dealt with phenotypic effects in cereal development, centering on the feature of spike fertility conditioned by different climatic treatments. In the present survey a series of generative, semigenerative and vegetative traits have been considered. The experiments have given information on the role of genotype, photoperiod and thermoperiod, as well as of the binary and three-factor interactions, together with features of replication, on trait development and performance, covering inherent and environmentally conditioned variability. The analytical procedures imply an establishment of synthetic multidimensional variables, with variance deductions of significant effects of diverse genotype and environment factors. Three kinds of materials are involved: (1) the barley variety Bonus, its X-ray mutant Mari, and their reciprocal monohybrids, (2) Bonus barley and Drabant spring wheat in a mutual comparison of growth and performance, and (3) a climatological study of four (five) barley varieties, two of six-row type, and two (three) of two-row type. The plant materials were grown under controlled conditions in four (five) different photoperiods and two (three) thermoperiods.

Phenotypic Plasticity, Costs of Phenotypes, and Costs of Plasticity

Why are some traits constitutive and others inducible? The term costs often appears in work addressing this issue but may be ambiguously defined. This review distinguishes two conceptually distinct types of costs: phenotypic costs and plasticity costs. Phenotypic costs are assessed from patterns of covariation, typically between a focal trait and a separate trait relevant to fitness. Plasticity costs, separable from phenotypic costs, are gauged by comparing the fitness of genotypes with equivalent phenotypes within two environments but differing in plasticity and fitness. Subtleties associated with both types of costs are illustrated by a body of work addressing predator-induced plasticity. Such subtleties, and potential interplay between the two types of costs, have also been addressed, often in studies involving genetic model organisms. In some instances, investigators have pinpointed the mechanistic basis of plasticity. In this vein, microbial work is especially illuminating and has three additional strengths. First, information about the machinery underlying plasticity--such as structural and regulatory genes, sensory proteins, and biochemical pathways--helps link population-level studies with underlying physiological and genetic mechanisms. Second, microbial studies involve many generations, large populations, and replication. Finally, empirical estimation of key parameters (e.g., mutation rates) is tractable. Together, these allow for rigorous investigation of gene interactions, drift, mutation, and selection--all potential factors influencing the maintenance or loss of inducible traits along with phenotypic and plasticity costs. Messages emerging from microbial work can guide future efforts to understand the evolution of plastic traits in diverse organisms.

The effect of fodder plant genotype on the variation of larval fitness traits in genotype classes of green oak leafroller moth

The effect of genetic variation of oak (Quercus pubescens L. and Q. petraea L.) on the genotype fitness components in green oak leafroller moth larvae (Tortrix viridana L.) at esterase (Est-4) and protease (Pts-4) loci was studied. The samples of larvae were collected from nine oak trees, whose genetic variation was assayed by RAPD-PCR using primer OPA14. The contributions of the factors of Yoak species/genotype and green oak leafroller moth genotype and their interaction to the variation of important size-related traits of the larvae were evaluated by two-way ANOVA. It was shown that the same larval genotype can display maximum fitness on the trees of one species or genotype and minimal, on the trees of other species or genotype. The interactions between the oak genotype and green oak leafroller moth genotype factors lead to the relationships that appear in statistically significant associations between genotype classes of green oak leafroller moth and oak. These results are discussed from the standpoint of a recently developed new field, community or ecosystem genetics.

Limits to local adaptation in six populations of the annual plantDiodia teres

* Local adaptation is common, but tests for adaptive differentiation frequently compare populations from strongly divergent environments, making it unlikely that any influence of stochastic processes such as drift or mutation on local adaptation will be detected. Here, the hypothesis that local adaptation is more likely to develop when the native environments of populations are more distinct than when they are similar was tested. * A reciprocal transplant experiment including two populations from each of three habitats was conducted to determine the pattern of local adaptation. In addition to testing for local adaptation at the population level, the hypothesis was tested that local adaptation is more common between populations from different habitats than between populations from the same habitat. * Local adaptation was not common, but more evidence was found of local adaptation between populations from different habitats than between populations from the same habitat. Two instances of foreign genotype fitness advantage confirm that stochastic processes such as drift can limit local adaptation. * These results are consistent with the hypothesis that stochastic processes can inhibit local adaptation but are more likely to be overwhelmed by natural selection when populations occur in divergent environments.

Proportions of different habitat types are critical to the fate of a resistance allele

We describe a simple deterministic theoretical framework for analysing the gene frequency evolution of two alternative alleles at a single genetic locus in a habitat comprising two environments in which the genotypes have different relative fitnesses. We illustrate this for adaptation of pest insects, where one allele (resistance to toxins expressed in transgenic crops) is favoured in one environment (transgenic plants) and the other allele (susceptibility to toxins) is favoured in the other environment (‘refuges’ of non-transgenic plants). The evolution of allele frequencies depends on selection pressure because of relative sizes of the environments and relative fitnesses of the genotypes in each environment. We demonstrate that there are critical threshold proportions for habitat division that determine equilibrium allele frequencies. The stability of the system depends on relationships between the relative genotype fitnesses. In some cases, the division of the habitat in exactly the threshold proportions removes selection pressure and maintains polymorphism at all allele frequencies.

Economic implications of using Japanese Black sires carrying recessive genes associated with genetic defects

The objective of this study was to calculate cumulative discounted expressions (CDE) for Japanese Black sires carrying a single defective allele in a herd by applying the gene-flow method to investigate the expression pattern of homozygous recessive genotype and to evaluate the monetary loss of using these sires. A single biallelic locus was considered with A representing the dominant allele and a representing the recessive allele. The gene-flow method was modified to consider the fitness of homozygous recessive genotype. Input parameters representing a typical situation in a Japanese Black cattle herd were used to calculate the CDE and the loss of using carrier sires. The effects of initial allele frequency and fitness on the CDE were determined for Aa and AA sires. The CDE of Aa sires were larger than those of AA sires under all initial allele frequencies and fitness. The difference in the CDE between using Aa and AA sires was largest when fitness was 0 (lethal recessive condition). The differences in the loss between Aa and AA sires were larger with increasing initial allele frequencies in lethal recessive condition. Applying the method used in this study to defects reported in Japanese Black cattle and with a population size of 628 000, the difference in the loss between using Aa and AA sires was US$48 575 800 and US$74 418 000 in the case of Band-3 and Claudin-16 deficiencies, respectively. The approach used in this study could be applied to other genetic defects in different breeds and species.

Fitness Variation of Borrelia burgdorferi Sensu Stricto Strains in Mice

Lyme borreliosis in North America is caused by the tick-borne spirochete Borrelia burgdorferi, a zoonotic bacterium that is able to persistently infect a wide range of vertebrate species. Given the pronounced strain structure of B. burgdorferi in the northeastern United States, we asked whether the fitness of the different genotypes varies among susceptible vertebrate hosts. The transmission dynamics of two genetically divergent human isolates of B. burgdorferi, BL206 and B348, were analyzed experimentally in white-footed mice and in C3H/HeNCrl mice over a time period of almost 3 months. We found that the initially high transmission efficiency from white-footed mice to ticks declined sharply for isolate B348 but remained considerably high for isolate BL206. In contrast, in C3H/HeNCrl mice, high transmission efficiency persisted for both isolates. Our findings provide proof-of-principle evidence for intrinsic fitness variation of B. burgdorferi strains in vertebrate host species, perhaps indicating the beginnings of adaptive radiation.

Effect of Endothelin 1 Genotype on Blood Pressure Is Dependent on Physical Activity or Fitness Levels

Contributions of the DNA sequence variation at the endothelin 1 locus to the risk of hypertension and to endurance training-induced changes in blood pressure were investigated in the Aerobics Center Longitudinal Study and the Health, Risk Factors, Exercise Training and Genetics Family Study cohorts. We identified 586 normotensive control subjects and 607 incident hypertensive case subjects from the Aerobics Center Longitudinal Study cohort (all whites) who were normotensive and healthy at their first clinic visit. The case subjects were diagnosed with hypertension during an average follow-up of 9.5 years, whereas the control subjects remained normotensive. The allele and genotype frequencies of 5 endothelin 1 haplotype tagging single nucleotide polymorphisms did not differ significantly between the case and control subjects. However, we observed a significant (P=0.0025) interaction between the endothelin 1 rs5370 (G/T; Lys198Asn) genotype and cardiorespiratory fitness level on the risk of hypertension: among low-fit subjects, the rs5370 minor allele (T; 198Asn) was associated with higher risk of hypertension (odds ratio: 1.95; 95% CI: 1.36 to 2.81; P=0.0003), whereas the risk did not differ among genotypes in high-fit subjects. In the white Health, Risk Factors, Exercise Training and Genetics subjects (N=480), the rs5370 T allele was associated with blunted systolic blood pressure (P=0.0046) and pulse pressure (P=0.0016) responses to a 20-week endurance training program. The Lys198Asn variant of the endothelin 1 locus is associated with blood pressure phenotypes in whites. However, the expression of the genotype effect is modulated by physical activity or cardiorespiratory fitness level. Our study provides an illustrative example of how physical activity and fitness level modifies the associations between a candidate gene and outcome phenotype.