“Alas poor Yorick”: What retrospective analysis of canine skulls can tell us about the impact of environmental factors on health ()
1. INTRODUCTION
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 [1]. These dogs, raised by different owners located throughout the continental United States and Canada have experienced a variety of life styles (exercise and diet) primarily determined by the breeders in whose kennels they originated and by the subsequent owners with whom they lived. In this paper, we use data from the skulls of necropsied dogs to retrospectively examine environmental influences on the frequency and severity of disease. For this purpose we have analyzed the correlation of two sets of metrics—skull robustness (weight/unit of skull size) and stable isotope ratios of skull and dentine collagens—with the frequency or severity of histopathological changes observed at necropsy.
The impact of exercise on health: Lieberman et al. [2] used pigs run on tread mills to demonstrate that skull robustness increased with increasing amounts of exercise. They interpreted their results to suggest that varying amounts of exercise effected bone growth in all parts of the mammalian skeleton. PW dogs exhibit a wide range of variation in skull weights between skulls of comparable size. We have therefore used the robusticity of the skull as a method to retrospectively evaluate exercise during a dog’s life.
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 [3]). Ratios of stable isotopes have already proved valuable for inferring diets of primitive peoples and extant animal species, as well as geographical locations of habitats and migrations of marine and terrestrial animals. Ratios of 13C/12C, 15N/14N and 34S/32S are determined by the primary source of these elements. Carbon isotope ratios (δ13C) reflect the isotope ratios of the primary dietary carbon source and indicate the proportion of C3 plants (lower δ13C values in e.g., temperate grasses, fruits, vegetables, wheat, soybean) or C4 plants (higher values in e.g., tropical grasses, corn, sugar cane) consumed directly or indirectly as a component in animal food. Nitrogen isotope ratios (δ15N) reflect the original nitrogen source (low values for N fixation for plants grown on synthetic fertilizers, and higher values for plants using other natural reactive species such as natural nitrate). Sulfur isotope ratios (δ34S) have primarily been used to differentiate diets of terrestrial origin (lower δ34S values) from marine-derived ones (higher values). Subsequently, discrimination between isotopes (primarily for nitrogen) continues to occur at each metabolic cycle during the rise in trophic level (by soil microbes, during plant metabolism and within the animals that feed on these plants). Thus the ultimate isotope ratio of a consumer will be determined in large part by the original nutrient source of the food ingested and the trophic level of the food ingested [4-6]. This will vary according to diet and to the location of the raw materials used in preparing that diet, for example the use of marine as contrasted with terrestrial sources of protein. Therefore, measuring stable carbon, nitrogen and sulfur isotopes in collagenous tissues provides an integrated and retrospective view of protein sources being consumed.
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.
2. MATERIALS AND METHODS
2.1. Necropsy Data
Cadavers sent to the University of Utah by owners were necropsied as described previously [1]. Anatomical and pathological evaluation included 53 whole body or organ weights or dimensions, and one-to-several histological studies of each of 27 tissues. Additional samples were collected where gross lesions (e.g. tumors) were visible in non-designated tissues. Common histological observations were scored for each tissue on a severity scale of 0 (none), 1 (mild), 2 (moderate), 3 (severe). Summary scores for pathologies observed in multiple tissues (e.g. Fibrosis) were calculated for each dog by averaging the severity score over all affected tissues.
2.2. Skull “Robustness” Data
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 [7]. The residuals from this regression were used as a measure of skull robustness.
2.3. Isotope Data
Samples of collagen were purified from bone (skull vault) and canine teeth (dentine) following standard procedures [8]. Briefly, the calcified bone matrix was dissolved with HCL and the residual collagen matrix subsequently purified and dried. Teeth were crushed into small pieces decalcified and purified in a similar manner to the bone sample, following standard protocols [2]. Dry material was separated for isotope analyses. Skull vault and dentine collagen samples from three dogs were analyzed for purity by mass spectrometry. All samples yielded peptide fingerprints identifying the protein samples as collagen.
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.
2.4. Statistical Methods
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 [7]. Significance was established using 100,000 - 1,000,000 permutations where one trait was randomized with respect to the other. P-values are reported as the fraction of permutation trials for which the absolute value of r was greater than or equal to the non-permuted absolute value of r.
Heritability. Heritability was estimated using a generalized linear mixed model from the MCMCglmm package [9] of R [7]. The MCMCglmm function was run using the marker validated pedigree for the Portuguese Water dog population and the following settings: nitt = 1,000,000, thin = 195, burnin = 25,000.
3. RESULTS
3.1. Skull Robustness
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 [1]), only a few correlations were potentially significant. Table 1 presents the most significant correlations observed. Not unexpectedly, less robust skulls were correlated significantly with several pathologies. An unexpected result was the small, but significant correlation of more robust skulls with the frequency of sarcomas.
3.2. Stable Isotope Ratios
Figure 2 presents isotope ratios for C, N and S from