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Obesity is generally protective against osteoporosis and bone fracture. However, recent studies indicate that the influence of obesity on the skeleton is complex and can be detrimental. We evaluated the effects of a high-fat, obesogenic diet on the femur and radius of 1100 mice (males and females) from the Large-by-Small advanced intercross line (F34 generation). At age 5 months, bone morphology was assessed by microCT and mechanical properties by three-point bending. Mice raised on a high-fat diet had modestly greater cortical area, bending stiffness, and strength. Size-independent material properties were unaffected by a high-fat diet, indicating that diet influenced bone quantity but not quality. Bone size and mechanical properties were strongly correlated with body mass. However, the increases in many bone traits per unit increase in body mass were less in high-fat diet mice than low-fat diet mice. Thus, although mice raised on a high-fat diet have, on average, bigger and stronger bones than low-fat-fed mice, a high-fat diet diminished the positive relationship between body mass and bone size and whole-bone strength. The findings support the concept that there are diminishing benefits to skeletal health with increasing obesity. © 2019 American Society for Bone and Mineral Research.

Obesity is generally protective against osteoporosis and bone fracture. However, recent studies indicate that the influence of obesity on the skeleton is complex and can be detrimental. We evaluated the effects of a high-fat, obesogenic diet on the femur and radius of 1100 mice (males and females) from the Large-by-Small advanced intercross line (F(34) generation). At 5 months age, bone morphology was assessed by microCT and mechanical properties by three-point bending. Mice raised on a high-fat diet had modestly greater cortical area, bending stiffness and strength. Size-independent material properties were unaffected by a high-fat diet, indicating that diet influenced bone quantity but not quality. Bone size and mechanical properties were strongly correlated with body mass. However, the increases in many bone traits per unit increase in body mass were less in high-fat diet mice than low-fat diet mice. Thus, although mice raised on a high-fat diet have, on average, bigger and stronger bones than low-fat fed mice, a high-fat diet diminished the positive relationship between body mass and bone size and whole-bone strength. The findings support the concept that there are diminishing benefits to skeletal health with increasing obesity.

Nonalcoholic fatty liver disease, a condition in which excess fat accumulates in the liver, is strongly associated with the metabolic syndrome, including obesity and other related conditions. This disease has the potential to progress from steatosis to steatohepatitis, fibrosis, and cirrhosis. The recent increase in the prevalence of the metabolic syndrome is largely driven by changes in diet and activity levels. Individual variation in the response to this obesogenic environment, however, is attributable in part to genetic variation between individuals, but very few mammalian genetic loci have been identified with effects on fat accumulation in the liver. To study the genetic basis for variation in liver fat content in response to dietary fat, liver fat proportion was determined using quantitative magnetic resonance imaging in 478 mice from 16 LG/J X SM/J recombinant inbred strains fed either a high-fat (42% kcal from fat) or low-fat (15% kcal from fat) diet. An analysis of variance confirmed that there is a genetic basis for variation in liver fat content within the population with significant effects of sex and diet. Three quantitative trail loci that contribute to liver fat content also were mapped.

Variations in diabetic phenotypes are caused by complex interactions of genetic effects, environmental factors, and the interplay between the two. We tease apart these complex interactions by examining genome-wide genetic and epigenetic effects on diabetes-related traits among different sex, diet, and sex-by-diet cohorts in a Mus musculus model. We conducted a genome-wide scan for quantitative trait loci that affect serum glucose and insulin levels and response to glucose stress in an F(16) Advanced Intercross Line of the LG/J and SM/J intercross (Wustl:LG,SM-G16). Half of each sibship was fed a high-fat diet and half was fed a relatively low-fat diet. Context-dependent genetic (additive and dominance) and epigenetic (parent-of-origin imprinting) effects were characterized by partitioning animals into sex, diet, and sex-by-diet cohorts. We found that different cohorts often have unique genetic effects at the same loci, and that genetic signals can be masked or erroneously assigned to specific cohorts if they are not considered individually. Our data demonstrate that the effects of genes on complex trait variation are highly context-dependent and that the same genomic sequence can affect traits differently depending on an individual's sex and/or dietary environment. Our results have important implications for studies of complex traits in humans.

Autoresuscitation (AR) is a highly conserved response among mammals, which allows survival from transient extreme hypoxia. During hypoxia, bradycardia, and hypoxic gasping develop after a brief period of hyperactivity. Normally, AR occurs if oxygen is restored during the gasping period where an initial heart rate increase is rapidly followed resumption or eupneic breathing. Humans and other mammals can survive multiple immediately repeated AR. A defective AR capacity has been implicated in Sudden Infant Death Syndrome. We had reported earlier that inbred strains of mice such as BALB/cJ could survive a characteristic number of immediately repeated AR trials, but that SWR/J mice failed to AR from a single hypoxic episode. We now report that strains closely related to SWR/J, FVB/N and SJL/J exhibit partial resuscitation defects relative to BALB/cJ or other mouse strains, establishing a genetic basis for variation in AR failure. The AR trial phenotype of BALB/cJ x SWR/J intercross F(1) and F(2) mice was consistent with BALB/cJ dominance and a discrete number of loci. Genome-wide mapping conducted with 60 intercross F(2) animals linked two loci to the number of AR trials survived, including one sex-specific locus with male expression, consistent with the observed 50% male bias for Sudden Infant Death Syndrome in humans. A locus carried on SWR/J chromosome 10 seems to be particularly important in AR failure and was confirmed in a partial consomic line. These results establish a genetic basis for AR failure phenotype in mice, with relevance to Sudden Infant Death Syndrome.

Maternal effects on offspring phenotypes occur because mothers in many species provide an environment for their developing young. Although these factors are correctly "environmental" with respect to the offspring genome, their variance may have both a genetic and an environmental basis in the maternal generation. Here, reciprocal crosses between C57BL/6J and 10 LGXSM recombinant inbred (RI) strains were performed, and litters were divided at weaning into high-fat and low-fat dietary treatments. Differences between reciprocal litters were used to measure genetic maternal effects on offspring phenotypes. Nearly all traits, including weekly body weights and adult blood serum traits, show effects indicative of genetic variation in maternal effects across RI strains, allowing the quantitative trait loci involved to be mapped. Although much of the literature on maternal effects relates to early life traits, we detect strong and significant maternal effects on traits measured at adulthood (as much as 10% of the trait variance at 17 or more weeks after weaning). We also found an interaction affecting adult phenotype between the effects of maternal care between RI strain mothers and C57BL/6J mothers and a later environmental factor (dietary fat intake) for some age-specific weights.

As perceived by Darwin, evolutionary adaptation by the processes of mutation and selection is difficult to understand for complex features that are the product of numerous traits acting in concert, for example the eye or the apparatus of flight. Typically, mutations simultaneously affect multiple phenotypic characters. This phenomenon is known as pleiotropy. The impact of pleiotropy on evolution has for decades been the subject of formal analysis. Some authors have suggested that pleiotropy can impede evolutionary progress (a so-called 'cost of complexity'). The plausibility of various phenomena attributed to pleiotropy depends on how many traits are affected by each mutation and on our understanding of the correlation between the number of traits affected by each gene substitution and the size of mutational effects on individual traits. Here we show, by studying pleiotropy in mice with the use of quantitative trait loci (QTLs) affecting skeletal characters, that most QTLs affect a relatively small subset of traits and that a substitution at a QTL has an effect on each trait that increases with the total number of traits affected. This suggests that evolution of higher organisms does not suffer a 'cost of complexity' because most mutations affect few traits and the size of the effects does not decrease with pleiotropy.

The rodent genus Peromyscus is the most numerous and species rich mammalian group in North America. The naturally occurring diversity within this genus allows opportunities to investigate the genetic basis of adaptation, monogamy, behavioral and physiological phenotypes, growth control, genomic imprinting, and disease processes. Increased genomic resources including a high quality genetic map are needed to capitalize on these opportunities. We produced interspecific hybrids between the prairie deer mouse (Peromyscus maniculatus bairdii) and the oldfield mouse (Peromyscus polionotus) and scored meiotic recombination events in backcross progeny. A genetic map was contructed by genotyping of backcross progeny at 185 gene-based and 155 microsatellite markers representing all autosomes and the X chromosome. Comparison of the constructed genetic map with the molecular maps of Mus and Rattus and consideration of previous results from interspecific reciprocal whole chromosome painting allowed most linkage groups to be unambiguously assigned to specific Peromyscus chromosomes. Based on genomic comparisons, this Peromyscus genetic map covers approximately 83% of the Rattus genome and 79% of the Mus genome. This map supports previous results that the Peromyscus genome is more similar to Rattus than Mus. For example, coverage of the 20 Rattus autosomes and the X chromosome is accomplished with only 28 segments of the Peromyscus map, but coverage of the 19 Mus autosomes and the X chromosome requires 40 chromosomal segments of the Peromyscus map. Furthermore, a single Peromyscus linkage group corresponds to about 91% of the rat and only 76% of the mouse X chromosomes.

Obesity, in addition to being associated with metabolic diseases, such as diabetes, has also been found to lower the risk of osteoporotic fractures. The relationship between obesity and bone trabecular structure is complex, involving responses to mechanical loading and the effects of adipocyte-derived hormones, both directly interacting with bone tissue and indirectly through central nervous system signaling. Here we examine the effects of sex, a high fat diet, and genetics on the trabecular density and structure of the lumbar and caudal vertebra and the proximal tibia along with body weight, fat pad weight, and serum leptin levels in a murine obesity model, the LGXSM Recombinant Inbred (RI) mouse strains. The sample included 481 mice from 16 RI strains. We found that vertebral trabecular density was higher in males while the females had higher tibial trabecular density. The high fat diet led to only slightly higher trabecular density in both sexes despite its extreme effects on obesity and serum leptin levels. Trait heritabilities are moderate to strong and genetic correlations among trabecular features are high. Most genetic variation contrasts strains with large numbers of thick, closely-spaced, highly interconnected, plate-like trabeculae with a high bone volume to total volume ratio against strains displaying small numbers of thin, widely-spaced, sparsely connected, rod-like trabeculae with a low bone volume to total volume ratio. Genetic correlations between trabecular and obesity-related traits were low and not statistically significant. We mapped trabecular properties to 20 genomic locations. Only one-quarter of these locations also had effects on obesity. In this population obesity has a relatively minor effect on trabecular bone morphology. Key Words: bone; trabecular morphology; obesity; quantitative trait loci; mice

Variation in serum cholesterol, free-fatty acids, and triglycerides is associated with cardiovascular disease (CVD) risk factors. There is great interest in characterizing the underlying genetic architecture of these risk factors, because they vary greatly within and among human populations and between the sexes. We present results of a genome-wide scan for quantitative trait loci (QTL) affecting serum cholesterol, free-fatty acids, and triglycerides in an F(16) advanced intercross line of LG/J and SM/J (Wustl:LG,SM-G16). Half of the population was fed a high-fat diet and half was fed a relatively low-fat diet. Context-dependent genetic (additive and dominance) and epigenetic (imprinting) effects were characterized by partitioning animals into sex, diet, and sex-by-diet cohorts. Here we examine genetic, environmental, and genetic-by-environmental interactions of QTL overlapping previously identified loci associated with CVD risk factors, and we add to the serum lipid QTL landscape by identifying new loci.

Although the current obesity epidemic is of environmental origin, there is substantial genetic variation in individual response to an obesogenic environment. In this study, we perform a genome-wide scan for quantitative trait loci (QTLs) affecting obesity per se, or an obese response to a high-fat diet in mice from the LG/J by SM/J Advanced Intercross (AI) Line (Wustl:LG,SM-G16). A total of 1,002 animals from 78 F16 full sibships were weaned at 3 weeks of age and half of each litter placed on high- and low-fat diets. Animals remained on the diet until 20 weeks of age when they were necropsied and the weights of the reproductive, kidney, mesenteric, and inguinal fat depots were recorded. Effects on these phenotypes, along with total fat depot weight and carcass weight at necropsy, were mapped across the genome using 1,402 autosomal single-nucleotide polymorphism (SNP) markers. Haplotypes were reconstructed and additive, dominance, and imprinting genotype scores were derived every 1 cM along the F16 map. Analysis was performed using a mixed model with additive, dominance, and imprinting genotype scores, their interactions with sex, diet, and with sex-by-diet as fixed effects and with family and its interaction with sex, diet, and sex-by-diet as random effects. We discovered 95 trait-specific QTLs mapping to 40 locations. Most QTLs had additive effects with dominance and imprinting effects occurring at two-thirds of the loci. Nearly every locus interacted with sex and/ or diet in important ways demonstrating that gene effects are primarily context dependent, changing depending on sex and/or diet.

The term “differential dominance” describes the situation in which the dominance effects at a pleiotropic locus vary between traits. Directional selection on the phenotype can lead to balancing selection on differentially dominant pleiotropic loci. Even without any individual overdominant traits, some linear combination of traits will display overdominance at a locus displaying differential dominance. Multivariate overdominance may be responsible, in part, for high levels of heterozygosity found in natural populations. We examine differential dominance of 70 mouse skeletal traits at 92 quantitative trait loci (QTL). Our results indicate moderate to strong additive and dominance effects at pleiotropic loci, low levels of individual-trait overdominance, and universal multivariate overdominance. Multivariate overdominance affects a range of 6% to 81% of morphospace, with a mean of 32%. Multivariate overdominance tends to affect a larger percentage of morphospace at pleiotropic loci with antagonistic effects on multiple traits (42%). We conclude that multivariate overdominance is common and should be considered in models and in empirical studies of the role of genetic variation in evolvability.

Quantitative trait locus (QTL) mapping techniques are frequently used to identify genomic regions associated with variation in phenotypes of interest. However, the F(2) intercross and congenic strain populations usually employed have limited genetic resolution resulting in relatively large confidence intervals that greatly inhibit functional confirmation of statistical results. Here we use the increased resolution of the combined F(9) and F(10) generations (n = 1455) of the LG,SM advanced intercross to fine-map previously identified QTL associated with the lengths of the humerus, ulna, femur, and tibia. We detected 81 QTL affecting long-bone lengths. Of these, 49 were previously identified in the combined F(2)-F(3) population of this intercross, while 32 represent novel contributors to trait variance. Pleiotropy analysis suggests that most QTL affect three to four long bones or serially homologous limb segments. We also identified 72 epistatic interactions involving 38 QTL and 88 novel regions. This analysis shows that using later generations of an advanced intercross greatly facilitates fine-mapping of confidence intervals, resolving three F(2)-F(3) QTL into multiple linked loci and narrowing confidence intervals of other loci, as well as allowing identification of additional QTL. Further characterization of the biological bases of these QTL will help provide a better understanding of the genetics of small variations in long-bone length.

Quantitative trait locus (QTL) studies of a skeletal trait or a few related skeletal components are becoming commonplace, but as yet there has been no investigation of pleiotropic patterns throughout the skeleton. We present a comprehensive survey of pleiotropic patterns affecting mouse skeletal morphology in an intercross of LG/J and SM/J inbred strains (N = 1040), using QTL analysis on 70 skeletal traits. We identify 798 single-trait QTL, coalescing to 105 loci that affect on average 7–8 traits each. The number of traits affected per locus ranges from only 1 trait to 30 traits. Individual traits average 11 QTL each, ranging from 4 to 20. Skeletal traits are affected by many, small-effect loci. Significant additive genotypic values average 0.23 standard deviation (SD) units. Fifty percent of loci show codominance with heterozygotes having intermediate phenotypic values. When dominance does occur, the LG/J allele tends to be dominant to the SM/J allele (30% vs. 8%). Over- and underdominance are relatively rare (12%). Approximately one-fifth of QTL are sex specific, including many for pelvic traits. Evaluating the pleiotropic relationships of skeletal traits is important in understanding the role of genetic variation in the growth and development of the skeleton.

Pleiotropy is an aspect of genetic architecture underlying the phenotypic covariance structure. The presence of genetic variation in pleiotropy is necessary for natural selection to shape patterns of covariation between traits. We examined the contribution of differential epistasis to variation in the intertrait relationship and the nature of this variation. Genetic variation in pleiotropy was revealed by mapping quantitative trait loci (QTLs) affecting the allometry of mouse limb and tail length relative to body weight in the mouse-inbred strain LG/J by SM/J intercross. These relationship QTLs (rQTLs) modify relationships between the traits affected by a common pleiotropic locus. We detected 11 rQTLs, mostly affecting allometry of multiple bones. We further identified epistatic interactions responsible for the observed allometric variation. Forty loci that interact epistatically with the detected rQTLs were identified. We demonstrate how these epistatic interactions differentially affect the body size variance and the covariance of traits with body size. We conclude that epistasis, by differentially affecting both the canalization and mean values of the traits of a pleiotropic domain, causes variation in the covariance structure. Variation in pleiotropy maintains evolvability of the genetic architecture, in particular the evolvability of its modular organization.

Do body size components, such as weights of internal organs and long bone lengths, with different functions and different developmental histories also have different genetic architectures and pleiotropic patterns? We examine murine quantitative trait loci (QTL) for necropsy weight, four long bone lengths, and four organ weights in the LG/J × SM/J intercross. Differences between trait categories were found in number of QTL, dominance, and pleiotropic patterns. Ninety-seven QTLs for individual traits were identified: 52 for long bone lengths, 30 for organ weights, and 15 for necropsy weight. Results for long bones are typically more highly significant than for organs. Organ weights were more frequently over- or underdominant than long bone lengths or necropsy weight. The single-trait QTLs map to 35 pleiotropic loci. Long bones are much more frequently affected in groups while organs tend to be affected singly or in pairs. Organs and long bones are found at the same locus in only 11 cases, 8 of which also include necropsy weight. Our results suggest mainly separate genetic modules for organ weights and long bone lengths, with a few loci that affect overall body size. Antagonistic pleiotropy, in which a locus has opposite effects on different characteristics, is uncommon.

Obesity is one of the most serious threats to human health today. Although there is general agreement that environmental factors such as diet have largely caused the current obesity pandemic, the environmental changes have not affected all individuals equally. To model gene-by-environment interactions in a mouse model system, our group has generated an F16 advanced intercross line (AIL) from the SM/J and LG/J inbred strains. Half of our sample was fed a low-fat (15% energy from fat) diet while the other half was fed a high-fat (43% energy from fat) diet. The sample was assayed for a variety of obesity- and diabetes-related phenotypes such as growth rate, response to glucose challenge, organ and fat pad weights, and serum lipids and insulin. An examination in the F16 sample of eight adiposity quantitative trait loci previously identified in an F2 intercross of SM/J and LG/J mouse strains reveals locus-by-diet interactions for all previously mapped loci. Adip7, located on proximal chromosome 13, demonstrated the most interactions and therefore was selected for fine mapping with microsatellite markers. Three phenotypic traits, liver weight in male animals, serum insulin in male animals, and reproductive fat pad weight, show locus-by-diet interactions in the 127-kb region between markers D13Mit1 and D13Mit302. The phosphofructokinase (PFK) C (Pfkp) and the pitrilysin metalloprotease 1 (Pitrm1) genes are compelling positional candidate genes in this region that show coding sequence differences between the parental strains in functional domains.

Levels of human obesity have increased over the past 20 years worldwide, primarily due to changes in diet and activity levels. Although environmental changes are clearly responsible for the increasing prevalence of obesity, individuals may show genetic variation in their response to an obesogenic environment. Here, we measure genetic variation in response to a high-fat diet in a mouse model, an F16 Advanced Intercross Line derived from the cross of SM/J and LG/J inbred mouse strains. The experimental population was separated by sex and fed either a high-fat (42% of energy from fat) or low-fat (15% of energy from fat) diet. A number of phenotypic traits related to obesity and diabetes such as growth rate, glucose tolerance traits, organ weights and fat pad weights were collected and analysed in addition to serum levels of insulin, free fatty acids, cholesterol and triglycerides. Most traits are different between the sexes and between dietary treatments and for a few traits, including adult growth, fat pad weights, insulin and glucose tolerance, the dietary effect is stronger in one sex than the other. We find that fat pad weights, liver weight, serum insulin levels and adult growth rates are all phenotypically and genetically correlated with one another in both dietary treatments. Critically, these traits have relatively low genetic correlations across environments (average r=0·38). Dietary responses are also genetically correlated across these traits. We found substantial genetic variation in dietary response and low cross environment genetic correlations for traits aligned with adiposity. Therefore, genetic effects for these traits are different depending on the environment an animal is exposed to.

Reply to: J. Hermisson & A. P. McGregor , 10.1038/nature07452 (2008) In our paper on pleiotropic scaling and the cost of complexity1, we presented evidence for three findings: first, most genes affect a small number of traits (the degree of pleiotropy is low); second, the total effect of a quantitative trait locus (QTL) increases with the degree of pleiotropy, refuting the constant total effect model2,3; and third, the increase in total effect (defined as , where Ai is the effect on character i, that is, half the difference between the genotypic values of the homozygous genotypes) seems to be stronger than predicted by the superposition model4,5 of pleiotropic effects. Hermisson and McGregor 6 point out that the last result could be due to multiple mutations being mapped to the same QTL, but only if these mutations affect overlapping sets of traits. We agree that this is a possibility that we could not address with the data at hand.