Epigenetics,heritability and longitudinal analysis

Background

Longitudinal data and repeated measurements in epigenome-wide association studies (EWAS) provide a rich resource for understanding epigenetics. We summarize 7 analytical approaches to the GAW20 data sets that addressed challenges and potential applications of phenotypic and epigenetic data. All contributions used the GAW20 real data set and employed either linear mixed effect (LME) models or marginal models through generalized estimating equations (GEE). These contributions were subdivided into 3 categories: (a) quality control (QC) methods for DNA methylation data; (b) heritability estimates pretreatment and posttreatment with fenofibrate; and (c) impact of drug response pretreatment and posttreatment with fenofibrate on DNA methylation and blood lipids.

Results

Two contributions addressed QC and identified large statistical differences with pretreatment and posttreatment DNA methylation, possibly a result of batch effects. Two contributions compared epigenome-wide heritability estimates pretreatment and posttreatment, with one employing a Bayesian LME and the other using a variance-component LME. Density curves comparing these studies indicated these heritability estimates were similar. Another contribution used a variance-component LME to depict the proportion of heritability resulting from a genetic and shared environment. By including environmental exposures as random effects, the authors found heritability estimates became more stable but not significantly different. Two contributions investigated treatment response. One estimated drug-associated methylation effects on triglyceride levels as the response, and identified 11 significant cytosine-phosphate-guanine (CpG) sites with or without adjusting for high-density lipoprotein. The second contribution performed weighted gene coexpression network analysis and identified 6 significant modules of at least 30 CpG sites, including 3 modules with topological differences pretreatment and posttreatment.

Conclusions

Four conclusions from this GAW20 working group are: (a) QC measures are an important consideration for EWAS studies that are investigating multiple time points or repeated measurements; (b) application of heritability estimates between time points for individual CpG sites is a useful QC measure for DNA methylation studies; (c) drug intervention demonstrated strong epigenome-wide DNA methylation patterns across the 2 time points; and (d) new statistical methods are required to account for the environmental contributions of DNA methylation across time. These contributions demonstrate numerous opportunities exist for the analysis of longitudinal data in future epigenetic studies.

     

Sea ice meiofauna distribution on local to pan‐Arctic scales

Arctic sea ice provides microhabitats for biota that inhabit the liquid‐filled network of brine channels and the ice–water interface. We used meta‐analysis of 23 published and unpublished datasets comprising 721 ice cores to synthesize the variability in composition and abundance of sea ice meiofauna at spatial scales ranging from within a single ice core to pan‐Arctic and seasonal scales. Two‐thirds of meiofauna individuals occurred in the bottom 10 cm of the ice. Locally, replicate cores taken within meters of each other were broadly similar in meiofauna composition and abundance, while those a few km apart varied more; 75% of variation was explained by station. At the regional scale (Bering Sea first‐year ice), meiofauna abundance varied over two orders of magnitude. At the pan‐Arctic scale, the same phyla were found across the region, with taxa that have resting stages or tolerance to extreme conditions (e.g., nematodes and rotifers) dominating abundances. Meroplankton, however, was restricted to nearshore locations and landfast sea ice. Light availability, ice thickness, and distance from land were significant predictor variables for community composition on different scales. On a seasonal scale, abundances varied broadly for all taxa and in relation to the annual ice algal bloom cycle in both landfast and pack ice. Documentation of ice biota composition, abundance, and natural variability is critical for evaluating responses to decline in Arctic sea ice. Consistent methodology and protocols must be established for comparability of meiofauna monitoring across the Arctic. We recommend to (1) increase taxonomic resolution of sea ice meiofauna, (2) focus sampling on times of peak abundance when seasonal sampling is impossible, (3) include the bottom 30 cm of ice cores rather than only bottom 10 cm, (4) preserve specimens for molecular analysis to improve taxonomic resolution, and (5) formulate a trait‐based framework that relates to ecosystem functioning.     

Inferring Patterns of Migration from Gene Frequencies under Equilibrium Conditions

A new maximum likelihood method to simultaneously estimate the parameters of any migration pattern from gene frequencies in stochastic equilibrium is developed, based on a model of multivariate genetic drift in a subdivided population. Motivated by simulations of this process in the simplified case of two subpopulations, problems related to the nuisance parameter q, the equilibrium gene frequency, are eliminated by conditioning on the observed mean gene frequency. The covariance matrix of this conditional distribution is calculated by constructing an abstract process that mimics the behavior of the original process in the subspace of interest. The approximation holds as long as there is limited differentiation between subpopulations. The bias and variance of estimates of long-range and short-range migration in a finite stepping stone model are evaluated by fitting the model to simulated data with known values of the parameters. Possible ecological extensions of the model are discussed.     

Harvest‐induced phenotypic selection in an island population of moose,Alces alces

Empirical evidence strongly indicates that human exploitation has frequently led to rapid evolutionary changes in wild populations, yet the mechanisms involved are often poorly understood. Here, we applied a recently developed demographic framework for analyzing selection to data from a 20‐year study of a wild population of moose, Alces alces. In this population, a genetic pedigree has been established all the way back to founders. We demonstrate harvest‐induced directional selection for delayed birth dates in males and reduced body mass as calf in females. During the study period, birth date was delayed by 0.81 days per year for both sexes, whereas no significant changes occurred in calf body mass. Quantitative genetic analyses indicated that both traits harbored significant additive genetic variance. These results show that selective harvesting can induce strong selection that oppose natural selection. This may cause evolution of less favorable phenotypes that become maladaptive once harvesting ceases.     

Effective size of density‐dependent populations in fluctuating environments

Reliable estimates of effective population size are of central importance in population genetics and evolutionary biology. For populations that fluctuate in size, harmonic mean population size is commonly used as a proxy for (multi‐) generational effective size. This assumes no effects of density dependence on the ratio between effective and actual population size, which limits its potential application. Here, we introduce density dependence on vital rates in a demographic model of variance effective size. We derive an expression for the ratio in a density‐regulated population in a fluctuating environment. We show by simulations that yearly genetic drift is accurately predicted by our model, and not proportional to as assumed by the harmonic mean model, where N is the total population size of mature individuals. We find a negative relationship between and N. For a given N, the ratio depends on variance in reproductive success and the degree of resource limitation acting on the population growth rate. Finally, our model indicate that environmental stochasticity may affect not only through fluctuations in N, but also for a given N at a given time. Our results show that estimates of effective population size must include effects of density dependence and environmental stochasticity.     

Dynamics of the Tec‐family tyrosine kinase SH3 domains

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains.     

Small-scale diel vertical migration of zooplankton in the High Arctic

Diel vertical migration (DVM) of zooplankton is considered less prominent at high latitudes where diel changes in irradiance are minimal during periods of midnight sun and polar night, leaving zooplankton without a temporal refuge and thus eliminating a key advantage of DVM. One of the shortcomings of previous DVM studies of zooplankton based on net sampling is that the depth resolution often has been too coarse to detect vertical migrations over short distances. We investigated DVM of zooplankton during August 2010 in drifting sea ice northeast of Svalbard (~81.5°N, ~30.5°E). Classical DVM behaviour (midnight rising, midday sinking) was observed between 20 and 80 m in young copepodite stages (CI–III) of Calanus finmarchicus and Calanus glacialis. The copepods Microcalanus spp., Pseudocalanus spp., Oithona atlantica, Oithona similis and Triconia borealis, alongside Eukrohnia hamata, Limacina helicina, and Fritillaria spp., all displayed signs of DVM. We conclude that zooplankton exhibit DVM in ice-covered waters over rather short distances to optimise food intake in the presence of predators.     

Age-specific optimal diets and optimal foraging tactics: A life-historic approach

By linking optimal foraging theory and optimal life history theory, we demonstrate that optimal diets, in general, may depend on the individual's age even when everything else remains the same. Older individuals (i.e., individuals with lower reproductive values) are predicted to have diets composed of highly nutritious food types that are possibly dangerous to pursue.     

Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes.

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.