Kunjie Hua

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

Genetic analysis of complex traits in the emerging Collaborative Cross

The Collaborative Cross (CC) is a mouse recombinant inbred strain panel that is being developed as a resource for mammalian systems genetics. Here we describe an experiment that uses partially inbred CC lines to evaluate the genetic properties and utility of this emerging resource. Genome-wide analysis of the incipient strains reveals high genetic diversity, balanced allele frequencies, and dense, evenly distributed recombination sites-all ideal qualities for a systems genetics resource. We map discrete, complex, and biomolecular traits and contrast two quantitative trait locus (QTL) mapping approaches. Analysis based on inferred haplotypes improves power, reduces false discovery, and provides information to identify and prioritize candidate genes that is unique to multifounder crosses like the CC. The number of expression QTLs discovered here exceeds all previous efforts at eQTL mapping in mice, and we map local eQTL at 1-Mb resolution. We demonstrate that the genetic diversity of the CC, which derives from random mixing of eight founder strains, results in high phenotypic diversity and enhances our ability to map causative loci underlying complex disease-related traits.

Dopaminergic Dysregulation in Mice Selectively Bred for Excessive Exercise or Obesity

Dysregulation of the dopamine system is linked to various aberrant behaviors, including addiction, compulsive exercise, and hyperphagia leading to obesity. The goal of the present experiments was to determine how dopamine contributes to the expression of opposing phenotypes, excessive exercise and obesity. We hypothesized that similar alterations in dopamine and dopamine-related gene expression may underly obesity and excessive exercise, as competing traits for central reward pathways. Moreover, we hypothesized that selective breeding for high levels of exercise or obesity may have influenced genetic variation controlling these pathways, manifesting as opposing complex traits. Dopamine, dopamine-related peptide concentrations, and gene expression were evaluated in dorsal striatum (DS) and nucleus accumbens (NA) of mice from lines selectively bred for high rates of wheel running (HR) or obesity (M16), and the non-selected ICR strain from which these lines were derived. HPLC analysis showed significantly greater neurotransmitter concentrations in DS and NA of HR mice compared to M16 and ICR. Microarray analysis showed significant gene expression differences between HR and M16 compared to ICR in both brain areas, with changes revealed throughout the dopamine pathway including D1 and D2 receptors, associated G-proteins (e.g., Golf), and adenylate cyclase (e.g., Adcy5). The results suggest that similar modifications within the dopamine system may contribute to the expression of opposite phenotypes in mice, demonstrating that alterations within central reward pathways can contribute to both obesity and excessive exercise.

Genetic architecture of voluntary exercise in an advanced intercross line of mice

Exercise is essential for health, yet the amount, duration, and intensity that individuals engage in are strikingly variable, even under prescription. Our focus was to identify the locations and effects of quantitative trait loci (QTL) controlling genetic predisposition for exercise-related traits, utilizing a large advanced intercross line (AIL) of mice. This AIL (G(4)) population originated from a reciprocal cross between mice with genetic propensity for increased voluntary exercise [high-runner (HR) line, selectively bred for increased wheel running] and the inbred strain C57BL/6J. After adjusting for family structure, we detected 32 significant and 13 suggestive QTL representing both daily running traits (distance, duration, average speed, and maximum speed) and the mean of these traits on days 5 and 6 (the selection criteria for HR) of a 6-day test conducted at 8 wk of age, with many co-localizing to similar genomic regions. Additionally, seven significant and five suggestive QTL were observed for the slope and intercept of a linear regression across all 6 days of running, some representing a combination of the daily traits. We also observed two significant and two suggestive QTL for body mass before exercise. These results, from a well-defined animal model, reinforce a genetic basis for the predisposition to engage in voluntary exercise, dissect this predisposition into daily segments across a continuous time period, and present unique QTL that may provide insight into the initiation, continuation, and temporal pattern of voluntary activity in mammals.

Host genetics and diet, but not immunoglobulin A expression, converge to shape compositional features of the gut microbiome in an advanced intercross population of mice

BackgroundIndividuality in the species composition of the vertebrate gut microbiota is driven by a combination of host and environmental factors that have largely been studied independently. We studied the convergence of these factors in a G10 mouse population generated from a cross between two strains to search for quantitative trait loci (QTLs) that affect gut microbiota composition or ileal Immunoglobulin A (IgA) expression in mice fed normal or high-fat diets.ResultsWe found 42 microbiota-specific QTLs in 27 different genomic regions that affect the relative abundances of 39 taxa, including four QTL that were shared between this G10 population and the population previously studied at G4. Several of the G10 QTLs show apparent pleiotropy. Eight of these QTLs, including four at the same site on chromosome 9, show significant interaction with diet, implying that diet can modify the effects of some host loci on gut microbiome composition. Utilization patterns of IghV variable regions among IgA-specific mRNAs from ileal tissue are affected by 54 significant QTLs, most of which map to a segment of chromosome 12 spanning the Igh locus. Despite the effect of genetic variation on IghV utilization, we are unable to detect overlapping microbiota and IgA QTLs and there is no significant correlation between IgA variable pattern utilization and the abundance of any of the taxa from the fecal microbiota.ConclusionsWe conclude that host genetics and diet can converge to shape the gut microbiota, but host genetic effects are not manifested through differences in IgA production.

Voluntary Exercise and Its Effects on Body Composition Depend on Genetic Selection History

Little is known about how genetic variation affects the capacity for exercise to change body composition. We examined the extent to which voluntary exercise alters body composition in several lines of selectively bred mice compared to controls. Lines studied included high runner (HR) (selected for high wheel running), M16 (selected for rapid weight gain), Institute of Cancer Research (ICR) (randomly bred as control for M16), M16i (an inbred line derived from M16), HE (selected for high percentage of body fat while holding body weight constant), LF (selected for low percentage of body fat), C57BL/6J (common inbred line), and the F1 between HR and C57BL/6J. Body weight and body fat were recorded before and after 6 days of free access to running wheels in males and females that were individually caged. Total food intake was measured during this 6‐day period. All pre‐ and postexercise measures showed significant strain effects. While HR mice predictably exercised at higher levels, all other selection lines had decreased levels of wheel running relative to ICR. The HR × B6 F1 ran at similar levels to HR demonstrating complete dominance for voluntary exercise. Also, all strains lost body fat after exercise, but the relationships between exercise and changes in percent body were not uniform across genotypes. These results indicate that there is significant genetic variation for voluntary exercise and its effects on body composition. It is important to carefully consider genetic background and/or selection history when using mice to model effects of exercise on body composition, and perhaps, other complex traits as well.

Exercise, weight loss, and changes in body composition in mice: phenotypic relationships and genetic architecture

The regulation of body weight and composition is complex, simultaneously affected by genetic architecture, the environment, and their interactions. We sought to analyze the complex phenotypic relationships between voluntary exercise, food consumption, and changes in body weight and composition and simultaneously localize quantitative trait loci (QTL) controlling these traits. A large (n = 815) murine advanced intercross line (G(4)) was created from a reciprocal cross between a high-running line and the inbred strain C57BL/6J. Body weight and composition (% fat, % lean) were measured at 4, 6, and 8 wk of age. After measurements at 8 wk of age, mice were given access to running wheels, during which food consumption was quantified and after which body weight and composition were assessed to evaluate exercise-induced changes. Phenotypic correlations indicated that the relationship between exercise and overall change in weight and adiposity depended on body composition before the initiation of exercise. Interval mapping revealed QTL for body weight, % fat, and % lean at 4, 6, and 8 wk of age. Furthermore, QTL were observed for food consumption and changes in weight, % fat, and % lean in response to short-term exercise. Here we provide some clarity for the relationship between weight loss, reduction in adiposity, food consumption, and exercise. Simultaneously, we reinforce the genetic basis for body weight and composition with some independent loci controlling growth at different ages. Finally, we present unique QTL providing insight regarding variation in weight loss and reduction in adiposity in response to exercise.

A Multi-Megabase Copy Number Gain Causes Maternal Transmission Ratio Distortion on Mouse Chromosome 2

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.

A Novel Intronic Single Nucleotide Polymorphism in the Myosin heavy polypeptide 4 Gene Is Responsible for the Mini-Muscle Phenotype Characterized by Major Reduction in Hind-Limb Muscle Mass in Mice

Replicated artificial selection for high levels of voluntary wheel running in an outbred strain of mice favored an autosomal recessive allele whose primary phenotypic effect is a 50% reduction in hind-limb muscle mass. Within the High Runner (HR) lines of mice, the numerous pleiotropic effects (e.g., larger hearts, reduced total body mass and fat mass, longer hind-limb bones) of this hypothesized adaptive allele include functional characteristics that facilitate high levels of voluntary wheel running (e.g., doubling of mass-specific muscle aerobic capacity, increased fatigue resistance of isolated muscles, longer hind-limb bones). Previously, we created a backcross population suitable for mapping the responsible locus. We phenotypically characterized the population and mapped the Minimsc locus to a 2.6-Mb interval on MMU11, a region containing ∼100 known or predicted genes. Here, we present a novel strategy to identify the genetic variant causing the mini-muscle phenotype. Using high-density genotyping and whole-genome sequencing of key backcross individuals and HR mice with and without the mini-muscle mutation, from both recent and historical generations of the HR lines, we show that a SNP representing a C-to-T transition located in a 709-bp intron between exons 11 and 12 of the Myosin heavy polypeptide 4 (Myh4) skeletal muscle gene (position 67,244,850 on MMU11; assembly, December 2011, GRCm38/mm10; ENSMUSG00000057003) is responsible for the mini-muscle phenotype, Myh4Minimsc. Using next-generation sequencing, our approach can be extended to identify causative mutations arising in mouse inbred lines and thus offers a great avenue to overcome one of the most challenging steps in quantitative genetics.

Parent-of-origin effects on voluntary exercise levels and body composition in mice

Despite the health-related benefits of exercise, many people do not engage in enough activity to realize the rewards, and little is known regarding the genetic or environmental components that account for this individual variation. We created and phenotyped a large G(4) advanced intercross line originating from reciprocal crosses between mice with genetic propensity for increased voluntary exercise (HR line) and the inbred strain C57BL/6J. G(4) females (compared to males) ran significantly more when provided access to a running wheel and were smaller with a greater percentage of body fat pre- and postwheel access. Change in body composition resulting from a 6-day exposure to wheels varied between the sexes with females generally regulating energy balance more precisely in the presence of exercise. We observed parent-of-origin effects on most voluntary wheel running and body composition traits, which accounted for 3-13% of the total phenotypic variance pooled across sexes. G(4) individuals descended from progenitor (F(0)) crosses of HRfemale symbol and C57BL/6Jmale symbol ran greater distances, spent more time running, ran at higher maximum speeds/day, and had lower percent body fat and higher percent lean mass than mice descended from reciprocal progenitor crosses (C57BL/6Jfemale symbol x HRmale symbol). For some traits, significant interactions between parent of origin and sex were observed. We discuss these results in the context of sex dependent activity and weight loss patterns, the contribution of parent-of-origin effects to predisposition for voluntary exercise, and the genetic (i.e., X-linked or mtDNA variations), epigenetic (i.e., genomic imprinting), and environmental (i.e., in utero environment or maternal care) phenomena potentially modulating these effects.