Necropsies and extensive histological evaluation for clinical and sub-clinical disease of approximately three hundred Portuguese Water dogs are available as part of an ongoing study to assess their state of health at the end of life. Throughout life these dogs enjoyed a variety of lifestyles and environments. Here we carry out retrospective quantitative assessments of life-time dietary input and physical activity for each dog. To do this, collagens from skull vault bone and from dentine have been analyzed for ratios of stable isotopes to determine differences in diet that individual dogs experienced during late or early life respectively. Robustness of skull bone (weight/unit of skull size) was used as a relative indicator of the amount of physical activity experienced during a dog’s lifetime. These environmental parameters were correlated with the frequency and severity of specific disease processes determined at necropsy. Both measures were shown to exert significant low-level (r < 25%) differential effects on specific diseases. The value of retrospective analysis of environmental influences is discussed.
Complex disease phenotypes are increasingly becoming the subject of whole genome analysis in well-structured populations. Genetic analysis of complex diseases is often constrained by the influence of environmental factors that may obscure genetic signals or play an important role in conditioning the disease process (e.g. carcinogens).
Retrospective, quantitative, assessment of environmental factors is, therefore, an important adjunct to quantitative genetic analysis.
The dog (C. familiaris) presents an excellent model system with which to analyze this concept in detail. This has been made possible by the availability of ca. 300 Portuguese Water dogs (PW dogs) that have been necropsied for state of health at the end of life [
The impact of exercise on health: Lieberman et al. [
The impact of nutrition on health: We have extracted nutritional data retrospectively by analyzing the stable isotope (non-radioactive) ratios of carbon, nitrogen and sulfur found in dentine collagen (laid down early in life) and from skull vault collagen (laid down and recycled throughout life [
In the material presented below, we correlate variation in skull robustness and stable isotopic signatures with specific histopathologies found at necropsy. This retrospective evaluation of variation in surrogates for exercise and diet suggests that specific effects of these environmental factors on age related diseases occur but are of relatively low magnitude.
Cadavers sent to the University of Utah by owners were necropsied as described previously [
Weights of cleaned skulls were measured without the mandible. 48 standardized points were measured using a 3D digitizer. All pairwise distances between these points were calculated and used for principal component analysis. The first principal component of these distances, explaining 64% of the total variation, was used as a measure of skull size. Skull weight was regressed onto skull size using the “lm” function of R [
Samples of collagen were purified from bone (skull vault) and canine teeth (dentine) following standard procedures [
Sample analyses. Collagen samples were analyzed using an isotope ratio mass spectrometer operated in continuous flow (CF-IRMS) mode in the Stable Isotope Ratio Facility for Environmental Research (SIRFER) at the University of Utah. For δ13C and δ15N analyses, 1 mg (± 10%) of material was used; for δ34S, 9 to 12 mg (±10%) were used. The collagen material was placed into small tin capsules that were then loaded into a zero-blank autosampler interfaced with an elemental analyzer (Carlo Erba) where they were combusted to produce N2 and CO2 or SO2. These gases were chromatographically separated and carried to the CF-IRMS (Finnigan MAT Delta S). All samples were analyzed alongside a set of internal laboratory reference materials that had been previously calibrated against international standards. Results for δ13C values are presented on the Vienna Pee Dee Belemnite (VPDB) scale, those for δ15N values on the AIR scale, and for δ34S values on the Vienna Canyon Diablo Troilite (VCDT) scale. The analytical precision (1), based on long-term measurements of internal laboratory reference materials for δ13C, δ15N and δ34S values, was 0.1‰, 0.2‰ and 0.4‰, respectively. Stable isotope values are reported using the standard δ-notation relative to an international standard in units “per mil” (‰) as follows: δ = 1000 (Rsample/Rstandard − 1), where Rsample and Rstandard are the molar ratios of the heavy to light isotopes of the sample and standard, respectively.
Correlations and permutations. Pearson product correlations “r” between two trait vectors (t1 and t2) were calculated with the “cor (t1, t2, use = “pair”)” function of R [
Heritability. Heritability was estimated using a generalized linear mixed model from the MCMCglmm package [
Skull weights from PW dogs were regressed on skull size (figure 1(a)), and values of residuals (robustness) determined (figure 1(b)). Based on pedigree and molecular (DNA marker) relatedness of the dogs (see Methods) we have measured the heritability of this trait. Although the range of residual values is large, we were unable to establish heritability suggesting that heritability, if it exists, is less than 23%. Correlating residuals with specific pathologies suggested that specific correlations might exist, but that correlations were low (~20%) and therefore the amount of variation involved was small, ~5%. Although analysis of histopathological change was extensive (see Methods and [