Characterization of complex, co-adapted skeletal biomechanics phenotypes: a needed paradigm shift in the genetics of bone structure and function.

The genetic architecture of skeletal biomechanical performance has tremendous potential to advance our knowledge of the biological mechanisms that drive variation in skeletal fragility and osteoporosis risk. Research using traditional approaches that focus on specific gene pathways is increasing our understanding of how and to what degree those pathways may affect population-level variation in fracture susceptibility, and shows that known pathways may affect bone fragility through unsuspected mechanisms. Non-traditional approaches that incorporate a new appreciation for the degree to which bone traits co-adapt to functional loading environments, using a wide variety of redundant compensatory mechanisms to meet both physiological and mechanical demands, represent a radical departure from the dominant reductionist paradigm and have the potential to rapidly advance our understanding of bone fragility and identification of new targets for therapeutic intervention.

Intracortical bone remodeling variation shows strong genetic effects.

Intracortical microstructure influences crack propagation and arrest within bone cortex. Genetic variation in intracortical remodeling may contribute to mechanical integrity and, therefore, fracture risk. Our aim was to determine the degree to which normal population-level variation in intracortical microstructure is due to genetic variation. We examined right femurs from 101 baboons (74 females, 27 males; aged 7-33 years) from a single, extended pedigree to determine osteon number, osteon area (On.Ar), haversian canal area, osteon population density, percent osteonal bone (%On.B), wall thickness (W.Th), and cortical porosity (Ct.Po). Through evaluation of the covariance in intracortical properties between pairs of relatives, we quantified the contribution of additive genetic effects (heritability [h (2)]) to variation in these traits using a variance decomposition approach. Significant age and sex effects account for 9 % (Ct.Po) to 21 % (W.Th) of intracortical microstructural variation. After accounting for age and sex, significant genetic effects are evident for On.Ar (h (2) = 0.79, p = 0.002), %On.B (h (2) = 0.82, p = 0.003), and W.Th (h (2) = 0.61, p = 0.013), indicating that 61-82 % of the residual variation (after accounting for age and sex effects) is due to additive genetic effects. This corresponds to 48-75 % of the total phenotypic variance. Our results demonstrate that normal, population-level variation in cortical microstructure is significantly influenced by genes. As a critical mediator of crack behavior in bone cortex, intracortical microstructural variation provides another mechanism through which genetic variation may affect fracture risk.

Reproductive status and sex show strong effects on knee OA in a baboon model.

OBJECTIVE: We aimed to characterize severity and occurrence of knee osteoarthritis (OA), and effects of age, sex, body mass, and reproductive status on population-level normal variation in this condition in the baboon, a natural model of human knee OA. METHODS: We visually inspected articular cartilage of distal right femora of 464 baboons (309 females, 155 males) and assigned an OA severity score (comparable to a modified Outerbridge score) from 1 = unaffected to 4 = advanced OA (eburnation). Presence/absence of osteophytes was recorded. We tested for significant effects of age, sex, body mass, and, in females, reproductive status (pre-, peri-, or post-menopausal) on OA. When appropriate, analyses were repeated on an age-matched subset (153 of each sex). RESULTS: Knee OA was more frequent and severe in older animals (P < 0.0001), but significant age variation was apparent in each severity grade. Sex differences within the younger and older age groups suggest that males develop knee OA earlier, but females progress more quickly to advanced disease. There is a strong relationship between reproductive status and OA severity grade in females (P = 0.0005) with more severe OA in peri- and post-menopausal female baboons, as in humans. CONCLUSIONS: Idiopathic knee OA is common in adult baboons. Occurrence and severity are influenced strongly by reproductive status in females, and by sex with regard to patterns of disease progression - providing an animal model to investigate sex-specific variation in OA susceptibility in which the environmental heterogeneity inherent in human populations is vastly reduced.

Genetical genomics of Th1 and Th2 immune response in a baboon model of atherosclerosis risk factors.

OBJECTIVE: CD4(+) T-cells mediate inflammation in atherosclerosis, but additive genetic effects on associated pathways of Th1 and Th2 immune response have not been described. We sought to characterize heritability, pleiotropy, and QTL effects on the expression of genes implicated in Th1 and Th2 immune response in a baboon model of risk factors for atherosclerosis. METHODS: We employed a maximum likelihood-based variance decomposition approach to estimate additive genetic effects on transcript levels generated from a gene expression profile of lymphocytes in 499 pedigreed baboons maintained on a basal diet. Transcript levels for 57 genes implicated in Th1 and Th2 immune response were selected for analysis based on significant heritability in this profile. Multipoint whole genome scans were conducted on heritable transcript levels to localize QTLs influencing these measures. To evaluate pleiotropic effects on transcript levels, we estimated genetic and phenotypic correlations among transcript measures, and assessed their correspondence using a Mantel test. Network analysis using GeneGo's MetaCore™ software was conducted to characterize known interaction among coded proteins. RESULTS: Heritabilities for candidate gene transcript levels ranged from 0.092-0.786 (median h(2)=0.278, P=4.72×10(-4)). Linkage analyses yielded significant evidence (LOD≥2.73) for 14 eQTLs (LOD score range 2.76-14.87, genome-wide P=4.9×10(-2)-1.03×10(-14)). Estimates of genetic correlation supported shared additive genetic effects incorporating all 57 transcripts (null hypothesis of ρ(G)=0 rejected at FDR≤0.05 for 522 of 1596 estimates), and accounted for most of the observed phenotypic correlation among transcripts (Mantel test, r([ρP],)([ρG])=0.781, P<0.0001). Network analysis revealed direct interactions among 54 of the 57 coded proteins. CONCLUSIONS: We conclude that major genetic effects influence expression levels of multiple genes implicated in Th1 and Th2 immune response. Additionally, we find that expression levels of these candidate genes are characterized by extensive pleiotropy, consistent with known interaction among their coded proteins, many of which are independently associated with atherosclerosis.

Heritability of lumbar trabecular bone mechanical properties in baboons.

Genetic effects on mechanical properties have been demonstrated in rodents, but not confirmed in primates. Our aim was to quantify the proportion of variation in vertebral trabecular bone mechanical properties that is due to the effects of genes. L3 vertebrae were collected from 110 females and 46 male baboons (6-32 years old) from a single extended pedigree. Cranio-caudally oriented trabecular bone cores were scanned with microCT then tested in monotonic compression to determine apparent ultimate stress, modulus, and toughness. Age and sex effects and heritability (h(2)) were assessed using maximum likelihood-based variance components methods. Additive effects of genes on residual trait variance were significant for ultimate stress (h(2)=0.58), toughness (h(2)=0.64), and BV/TV (h(2)=0.55). When BV/TV was accounted for, the residual variance in ultimate stress accounted for by the additive effects of genes was no longer significant. Toughness, however, showed evidence of a non-BV/TV-related genetic effect. Overall, maximum stress and modulus show strong genetic effects that are nearly entirely due to bone volume. Toughness shows strong genetic effects related to bone volume and shows additional genetic effects (accounting for 10% of the total trait variance) that are independent of bone volume. These results support continued use of bone volume as a focal trait to identify genes related to skeletal fragility, but also show that other focal traits related to toughness and variation in the organic component of bone matrix will enhance our ability to find additional genes that are particularly relevant to fatigue-related fractures.

Osteopenia and osteoporosis in adult baboons (Papio hamadryas).

BACKGROUND: Little is known about the degree to which baboons, an important animal model in skeletal research, spontaneously experience age-related osteopenia and osteoporosis. METHODS: We measured bone mineral density (BMD) in 667 baboons, assigned T-scores to older animals based on sex-specific young adult reference groups, and compared reproductive history in older females with low BMD to those with normal BMD. RESULTS: Approximately 25% of older baboon females were osteopenic. No females or males were osteoporotic. Neither parity nor interbirth interval spine clearly distinguished low vs. normal BMD groups. Intersite correspondence in low BMD was highest between sites in the same region rather than sites of the same bone type. CONCLUSION: As with humans, osteopenia is common among older females. The absence of osteoporotic animals may be due to colony maintenance resulting in truncation of the aged population and selection for healthier animals in the oldest ranges.

Bivariate linkage study of proximal hip geometry and body size indices: the Framingham study.

Femoral geometry and body size are both characterized by substantial heritability. The purpose of this study was to discern whether hip geometry and body size (height and body mass index, BMI) share quantitative trait loci (QTL). Dual-energy X-ray absorptiometric scans of the proximal femur from 1,473 members in 323 pedigrees (ages 31-96 years) from the Framingham Osteoporosis Study were studied. We measured femoral neck length, neck-shaft angle, subperiosteal width (outer diameter), cross-sectional bone area, and section modulus, at the narrowest section of the femoral neck (NN), intertrochanteric (IT), and femoral shaft (S) regions. In variance component analyses, genetic correlations (rho ( G )) between hip geometry traits and height ranged 0.30-0.59 and between hip geometry and BMI ranged 0.11-0.47. In a genomewide linkage scan with 636 markers, we obtained nominally suggestive linkages (bivariate LOD scores > or =1.9) for geometric traits and either height or BMI at several chromosomes (4, 6, 9, 15, and 21). Two loci, on chr. 2 (80 cM, BMI/shaft section modulus) and chr. X (height/shaft outer diameter), yielded bivariate LOD scores > or =3.0; although these loci were linked in univariate analyses with a geometric trait, neither was linked with either height or BMI. In conclusion, substantial genetic correlations were found between the femoral geometric traits, height and BMI. Linkage signals from bivariate linkage analyses of bone geometric indices and body size were similar to those obtained in univariate linkage analyses of femoral geometric traits, suggesting that most of the detected QTL primarily influence geometry of the hip.

Bone ALP and OC reference standards in adult baboons (Papio hamadryas) by sex and age.

BACKGROUND: Serum bone-specific alkaline phosphatase (bone ALP) and intact osteocalcin (OC) are markers of bone formation of interest because of easy measurability and potential utility as identifiers of those at risk for fractures associated with bone metabolism disorders. The baboon (Papio hamadryas) exhibits extensive biological similarities to humans making it particularly well suited to studies of bone maintenance and turnover. METHODS: We measured serum bone ALP and OC in 591 baboons. RESULTS: We report significant sex and age effects and present reference ranges and percentile distributions for these markers. CONCLUSIONS: This study is the first to characterize normal variation in bone ALP and OC levels in baboons and to assess the age and sex effects on this variation. The results provide much-needed reference standards to allow researchers to evaluate the status of their animals in cross-sectional studies and assess the meaning of changes in bone ALP and OC levels in longitudinal studies.

Cross-species replication of a serum osteocalcin quantitative trait locus on human chromosome 16q in pedigreed baboons.

Osteocalcin (OC), a serum marker of bone formation, in its intact form reflects osteoblast activity. It is of interest to clinicians and bone biologists due to easy measurability and potential utility as an identifier of those at risk for fracture and other complications associated with bone metabolism disorders. The only published linkage study in humans shows significant evidence for a quantitative trait locus (QTL) affecting OC levels on 16q. We used the baboon, a primate model for skeletal maintenance and turnover, to detect and quantify the effects of genes on serum OC levels and to localize chromosomal regions harboring the responsible loci. We assayed OC levels in 591 pedigreed animals, assessed OC heritability, and conducted a genomewide linkage scan for evidence of QTLs affecting this phenotype. Heritability in these baboons is 0.24. Suggestive linkage is evident with markers in a region homologous to human chromosome 16q. This first genomewide linkage scan in a nonhuman primate for QTLs affecting bone formation as reflected by OC levels provides cross-species replication of the QTL on chromosome 16q previously localized in humans. Given the concordance of results of the only two genome scans for this trait in two primate species, further studies of this region are warranted.

A quantitative trait locus for normal variation in forearm bone mineral density in pedigreed baboons maps to the ortholog of human chromosome 11q.

Baboons share many anatomical, physiological, and developmental characteristics with humans that make them excellent models for human bone maintenance and turnover. We conducted statistical genetic analyses, including a whole-genome linkage screen, of dual-energy x-ray absorptiometry-acquired measures of areal bone mineral density (aBMD), currently the most reliable single predictor of susceptibility to osteoporotic fracture in humans, from three forearm sites on the radius and ulna of 667 pedigreed baboons. We used a maximum likelihood-based variance decomposition approach to detect and quantify the effects of genes on normal variation in aBMD in the forearm of these baboons and to localize these effects to chromosomal regions. We estimated significant heritability for aBMD at all three sites and found evidence for a quantitative trait locus (QTL) contributing significantly to the genetic effects on this trait in a region of the baboon genome homologous to human chromosome 11q12-13. This first reported genome-wide linkage screen in a nonhuman primate for QTLs affecting forearm aBMD provides important cross-species replication of a QTL found in humans. The concordance of our results in a nonhuman primate with those reported for humans provides strong evidence that a gene (or genes) in this region affects normal variation in BMD.

Genotype-by-sex and environment-by-sex interactions influence variation in serum levels of bone-specific alkaline phosphatase in adult baboons (Papio hamadryas).

While more than 77% of the people in the US with osteoporosis are women, the contributions of genotype-by-sex (G x S) and environment-by-sex interactions to sex differences in osteoporosis risk factors have not been studied. To address this issue, we conducted a statistical genetic analysis of serum concentrations of bone-specific alkaline phosphatase (Bone ALP), a highly specific marker of osteoblast function that is elevated in persons with conditions like osteoporosis characterized by excessive bone turnover or rapid bone loss. We assayed Bone ALP from 657 pedigreed baboons using a commercially available ELISA kit. Using a maximum likelihood variance decomposition approach, we treated sex as an environmental milieu in which genes influencing Bone ALP levels are expressed. We modeled the genetic covariance in Bone ALP between all relative pairs conditional on their sex so that the covariance is the product of the kinship, the genetic correlation between trait levels in the two sexes, and the genetic variances in the two sexes. Sex-specific maximum likelihood estimates (MLE) of residual heritability for Bone ALP were greater for females than for males (h2 = 0.44 vs. h2 = 0.26, respectively), but likelihood ratio tests revealed only a marginally significant difference in sex-specific genetic variances (P = 0.057). In contrast, the between-sex genetic correlation (rhoG = 0.43) was significantly less than 1.0 (P = 0.037), and the difference in sex-specific environmental variances was highly significant (P = 0.00006). We report the first evidence for G x S interactions influencing variation in an osteoporosis risk factor. The diminished between-sex genetic correlation implies that different genes influence Bone ALP levels in the two sexes. The significant differences between environmental variances suggest that unmeasured factors, including those from the internal, biological environments of the two sexes, account for a greater proportion of the Bone ALP variation in males.

Osteon remodeling dynamics in Macaca mulatta: normal variation with regard to age, sex, and skeletal maturity.

As research into bone maintenance and turnover is accelerated and expanded due to public health concerns about osteoporosis and other age-related changes and pathologies of bone, nonhuman animal models are becoming increasingly important as they allow for enhanced experimental manipulation and environmental control relative to humans. Old World Monkeys, such as the rhesus macaque, share physiological and developmental characteristics that make these primates particularly well suited to such studies. The purpose of this study was to characterize normal age and sex variation in osteon remodeling dynamics in skeletally immature and mature rhesus macaques. Femoral cross-sections from 75 Macaca mulatta were examined to evaluate the effect of age, sex, and skeletal maturity on osteon remodeling dynamics in this popular research primate. Results indicate that sex has a significant effect on osteon area (On.Ar), but generally is not an important contributor to normal variation in intracortical remodeling dynamics. Age and skeletal maturity, however, contribute significantly to variation in osteon population density (OPD), activation frequency (Ac.f), and bone formation rate (BFR), as is the case in humans. This study is the first to characterize normal age and sex variation in osteon remodeling in growing and adult rhesus macaques and its results support the use of this animal as a model for age-related changes and pathologies in the human skeleton.

Bone mineral density reference standards in adult baboons (Papio hamadryas) by sex and age.

Osteoporosis is a progressive condition involving structural deterioration of bone tissue, leading to skeletal fragility and an increased susceptibility to fractures due to low bone mass and high rates of bone turnover. Areal bone mineral density (aBMD) serves as the most reliable predictor of susceptibility to osteoporotic fracture. The development of animal models, including Old World Monkeys, has been essential to studies of bone mineral density. These animals, including the baboon, exhibit many biological similarities with our own species relevant to the variation in age-related changes and pathology in bone that may make them an excellent model for studies of skeletal structure and maintenance in humans. The baboon has been shown to exhibit extensive biological similarities to humans regarding skeletal biology, but little is known about the range of normal variation in skeletal traits, such as bone mineral density, in this species. Our data, collected on baboons (Papio hamadryas) that are part of a large breeding colony at the Southwest Foundation for Biomedical Research and the Southwest National Primate Research Center (San Antonio, TX), involve 466 females and 210 males, ranging in age from 5.5 to 30 years. Student's t tests, bivariate correlations, and likelihood ratio tests show sex and age effects at all spinal sites. Age effects are minimal or absent in the forearm sites. This study is the first to characterize normal variation in aBMD in baboons, to assess the effect of age and sex on this variation, and to compare this variation to those data currently available from experimental control animals. As such, it provides much-needed reference standards that will allow researchers to evaluate the status of their animals in cross-sectional studies and more fully assess the meaning of aBMD changes in longitudinal studies.

Hematology and blood biochemistry in infant baboons (Papio hamadryas).

Although published normative reference standards for hematologic and clinical chemistry measures are available for adult baboons, their applicability to infants has not been addressed. We analyzed these measures in 110 infant baboons (55 females and 55 males) from a large breeding colony at the Southwest Regional Primate Research Center in San Antonio, Texas. The sample consists of olive baboons and olive/yellow baboon hybrids, 1 week to 12 months of age. We produced cross-sectional reference values and examined the effects of age, sex, and subspecies on these variables. Hematology reference ranges for infant baboons are similar to, but wider than, those for adults. Reference ranges for blood biochemistry measures are generally more dissimilar to adults, indicating that for many variables, reference ranges for adult baboons are not adequate for infants. Although sex and subspecies differences are rare, age accounts for more than 10% of the variance in many of the variables.

Pleiotropic effects on cardiovascular risk factors within and between the fourth and sixth decades of life: implications for genotype x age interactions.

We used an approach for detecting genotype x environment interactions to detect and characterize genotype x age interaction in longitudinal measures of three well known cardiovascular risk factors: total plasma cholesterol (TC), systolic blood pressure (SBP), and body weight (Wgt). Our objectives were to determine if the same gene or suite of genes influences quantitative variation in each of these phenotypes in the 4th and 6th decades of life, to assess the impact of additive gene effects in these two decades, and to evaluate the stability of pleiotropic relationships among these phenotypes. Using the Framingham Heart Study data, we constructed two cross-sectional samples comprising individuals on whom these phenotypes were measured at ages 30-39 years (Original Cohort: exam 1, Offspring Cohort: exam 2) and at ages 50-59 years (Original Cohort: exam 11, Offspring Cohort: exam 5). We also constructed a longitudinal sample from the cross-sectional sample members for whom measures on these traits were available at both ages (i.e., 4th and 6th decades of life). Patterns of pleiotropy, inferred from genetic correlations between traits, differ between the two age classes. Further, additive genetic variance in SBP during the 4th decade of life is attributable to a different gene or suite of genes than during the 6th. The magnitude of the effect increases for SBP. Variation in TC and Wgt appear to be influenced by the same gene or genes in both decades. The magnitude of the effect is stable for TC, but increases dramatically with age for Wgt.