Interactions between chromosomal and nonchromosomal elements reveal missing heritability

The measurement of any nonchromosomal genetic contribution to the heritability of a trait is often confounded by the inability to control both the chromosomal and nonchromosomal information in a population. We have designed a unique system in yeast where we can control both sources of information so that the phenotype of a single chromosomal polymorphism can be measured in the presence of different cytoplasmic elements. With this system, we have shown that both the source of the mitochondrial genome and the presence or absence of a dsRNA virus influence the phenotype of chromosomal variants that affect the growth of yeast. Moreover, by considering this nonchromosomal information that is passed from parent to offspring and by allowing chromosomal and nonchromosomal information to exhibit nonadditive interactions, we are able to account for much of the heritability of growth traits. Taken together, our results highlight the importance of including all sources of heritable information in genetic studies and suggest a possible avenue of attack for finding additional missing heritability.A fundamental problem in genetics is unraveling the link between genotype and phenotype. Ascertaining the heritability of a trait is a key step toward harnessing the predictive capacity of genetic information for human disease risk assessment and therapy (1). Knowledge of all of the elements contributing to heritability would facilitate the establishment of a causal relationship between the information that is passed down from generation to generation and the resulting phenotype. Genome-wide association studies (GWASs) have successfully identified many human polymorphisms that are associated with traits such as height, eye color, or susceptibility to common diseases, but these variants typically explain only a small proportion of the observed heritability of a trait (2, 3).A number of explanations for missing heritability have been suggested (2), including the existence of many weak variants with effects too small to achieve statistical significance (4), interactions between variants that cannot be identified with current studies (5), rare variants that were not identified by GWAS, and epigenetic effects (6–8). The contribution of nonchromosomal information to the missing heritability is rarely considered, despite the fact that there is a long history documenting the effect in many organisms of diverse cytoplasmic elements on phenotype. Recent work on a mouse model of Crohn disease supports a combinatorial model of complex disease traits in which the pathology requires the interaction between a specific mutation in the mouse and a specific strain of virus (9). Another recent study showed strong effects on the plant metabolome stemming from variation in mitochondrial and chloroplast genomes (10). In humans, the importance of nonchromosomal information has been supported by targeted analyses, but these studies have not analyzed its impact on heritability in a well-controlled context (11–13). Such nonchromosomal interactions might help explain why shared mutations in humans do not always produce the same phenotype, thus reducing the apparent heritability of a trait (14, 15).We sought to characterize explicitly how nonchromosomal modifiers collectively influence the heritability of a trait, colony size, in a system unique to yeast where we use a defined chromosomal genotype and vary the cytoplasmic genetic information. Yeast has at least four well-studied sources of inherited, nonchromosomal information: mitochondrial DNA, an endogenous dsRNA virus (16, 17), prions (18, 19), and a 2µ plasmid (20, 21).Our results show that the nonchromosomal contribution to heritability can be large, adding another dimension to the estimation of heritability in wild populations. Nonchromosomal information is not under the usual constraints of the nuclear genome. These nonchromosomal elements are extremely unstable: they mutate at higher frequencies than the DNA of the chromosomal genome, may be lost at high frequencies without loss of viability, and can vary in copy number from cell to cell. Thus, careful controls and measurements are necessary to characterize the effects of nonchromosomal modifiers.     

Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia

Platelet counts (PC) estimate bleeding risk in Immune Thrombocytopenia (ITP). We investigated whether measures of thromboelastometry and absolute immature platelet fraction (A‐IPF) would correlate better with acute bleeding score (ABS) than PC or mean platelet volume (MPV). Simultaneous determination of ABS, complete blood count and thromboelastometry was performed in 141 ITP patients; 112 underwent A‐IPF testing. Subgroup analyses were performed for paediatric subjects, PC <60 × 10⁹/l and <30 × 10⁹/l. PC significantly inversely correlated with ABS in all subjects, PC <30 × 10⁹/l and total paediatric cohort. MPV did not correlate with ABS in any subgroup. Thromboelastometry measures of clot firmness, but not PC, significantly correlated with ABS in all subjects with PC <60 × 10⁹/l, and children with PC <60 × 10⁹/l and <30 × 10⁹/l. A‐IPF demonstrated stronger correlation with ABS than did PC among all subjects, those with PC <60 × 10⁹/l, all children and children with PC <30 × 10⁹/l (r = ?0·37; r = ?0·34; r = ?0·44; r = ?0·60) versus ABS with PC (r = ?0·36; ns; r = ?0·32; ns). Stronger correlations of both thromboelastometry measures of clot firmness and A‐IPF than PC with ABS suggest factors beyond PC, i.e. related to platelet function, contribute to ITP bleeding pathophysiology. Thromboelastometry, A‐IPF and ABS can be incorporated into routine or acute visits.     

A seven day actigraphy-based study of rumination and sleep disturbance among young adults with depressive symptoms

Objectives

Trait ruminators exhibit significantly higher levels of sleep disturbance than those without this cognitive vulnerability. However, support for the sleep disruptive effects of state rumination, especially in the pre-sleep period, is rare, and hindered by methodological drawbacks such as self-report and single night assays of sleep. Finally, despite the pervasiveness of the ruminative response style among individuals with depression, the association between rumination and sleep disturbance has not been explored in this population. The present study employed a week-long daily sampling approach to examine the effects of naturally occurring pre-sleep rumination on self-reported and actigraphy-based sleep among individuals with high depressive symptomatology.

Methods

Forty-two university students (19.6 ± 3.2 yo;73.8% female), all of whom reported at least moderate levels of depressive symptoms, completed a short questionnaire after waking each morning for seven days. On this questionnaire, they self-reported sleep indices from the previous night and levels of engagement in pre-sleep rumination. Sleep was also monitored throughout this period via wrist actigraphy. Hierarchical-linear-modeling was used to examine the association between nightly rumination and sleep.

Results

Nightly variations in pre-sleep rumination were predictive of significantly longer actigraphy- and diary-based sleep onset latency (SOL). Notably, a 1 SD increase on the pre-sleep rumination scale was associated with an approximately 7 minute increase in actigraphy-based SOL, even after controlling for baseline sleep disturbance and depressive symptoms.

Conclusions

These data offer compelling evidence for the impact of pre-sleep rumination on sleep onset, providing insight into one potential mechanism that triggers sleep disturbance among individuals with depressive symptoms.     

Effect of food restriction and leptin supplementation on fetal programming in mice

Metabolic disease is a significant global health and economic problem. In a phenomenon referred to as fetal programming, offspring of underweight or overweight mothers have an increased incidence of adulthood obesity and metabolic disease. Undernourished individuals have decreased levels of leptin, a regulator of energy balance, whereas obese people develop hyperleptinemia and leptin resistance. We hypothesize that alterations in circulating leptin during pregnancy contribute to programming events caused by maternal nutritional status. To test this hypothesis, pregnant mice were randomly placed in one of three treatment groups: ad libitum feed plus saline injection (control, n = 5), 50% food restriction plus saline injection (restricted, n = 4), or 50% food restriction plus 1 mg/kg·d leptin injection (restricted, leptin treated, n = 4). Mice were treated from 1.5 to 11.5 d after conception and then returned to ad libitum feeding until weaning. At 19 wk after weaning, offspring were placed on a 45% fat diet and then followed up until 26 wk after weaning, at which time they were killed, and samples were collected for further analysis. Our results demonstrate that males are more negatively impacted by high-fat diet than females, regardless of maternal treatment. We provide evidence that differential response to leptin may mediate the sexual dimorphism observed in fetal programming in which male offspring are more affected by maternal undernutrition and female offspring by maternal overnutrition. We show that female offspring born to food-restricted, leptin-supplemented mothers are obese and insulin resistant. This may mimic fetal programming events seen in offspring of overweight women.     

From the Cover: Conditional control of gene function by an invertible gene trap in zebrafish

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.High throughput functional genomic and informatic methods have been developed to interrogate the genome and extract functional predictions about many genes at a time. However, careful phenotypic analysis of genetic mutants remains the sine qua non of reductionist biological science. In most experimental organisms, random mutagenesis is the preferred or only mutagenic technique available. DNA alkylating agents, transposable elements, or retroviruses are traditionally used in these organisms. A major limitation of these traditional genetic methods is that they reveal only the earliest and/or most prominent function of a gene as later functions are masked by the earlier phenotype, which is often lethality. To assess later functions, for example in metabolism, aging, or behavior, conditional alleles are required.The development of conditional alleles has proven a boon to studying gene function in temporally or spatially restricted contexts. Traditional conditional alleles disrupt gene function by changing the environment, for example by increasing the temperature. Engineered conditional alleles disrupt gene function by activating a recombination-mediated molecular switch that ablates gene function in one state, but has no functional consequences in the other state (1, 2). In the mouse, engineered conditional alleles can be generated by homologous recombination to insert the molecular switch at defined loci or by retroviral-mediated random insertion of the molecular switch (3, 4). The second approach leverages the orientation-dependent gene disruption of a gene trap and the ability of Flp/Cre recombinases to stably invert the gene trap. By strategically arranging dimers of heterotypical flp- and cre-recombinase binding sites flanking the gene trap, stable inversion is achieved in cis by recombinase-mediated Flip and Excision (FlEx) (5). However, this conditional gene-trap mutagen has not been validated at the organismal level.A distinct advantage of FlEx-based conditional gene-trap mutations is the possibility of stage- and tissue-specific rescue or knockout of the mutated genes. In zebrafish, several gene-trap mutagenesis methods have been developed (6, 7), including the “gene-break” (6, 8) and “FlipTrap” (9) technologies. We set out to test whether the FlEx-based conditional gene-trap mutagenesis approach functions at the organismal level in zebrafish. We show here that a highly mutagenic transposable element can be used for conditional analysis of essential genes.