Whole-genome characterization in pedigreed non-human primates using genotyping-by-sequencing (GBS) and imputation.

BACKGROUND: Rhesus macaques are widely used in biomedical research, but the application of genomic information in this species to better understand human disease is still in its infancy. Whole-genome sequence (WGS) data in large pedigreed macaque colonies could provide substantial experimental power for genetic discovery, but the collection of WGS data in large cohorts remains a formidable expense. Here, we describe a cost-effective approach that selects the most informative macaques in a pedigree for 30X WGS, followed by low-cost genotyping-by-sequencing (GBS) at 30X on the remaining macaques in order to generate sparse genotype data at high accuracy. Dense variants from the selected macaques with WGS data are then imputed into macaques having only sparse GBS data, resulting in dense genome-wide genotypes throughout the pedigree. RESULTS: We developed GBS for the macaque genome using a digestion with PstI, followed by sequencing of size-selected fragments at 30X coverage. From GBS sequence data collected on all individuals in a 16-member pedigree, we characterized high-confidence genotypes at 22,455 single nucleotide variant (SNV) sites that were suitable for guiding imputation of dense sequence data from WGS. To characterize dense markers for imputation, we performed WGS at 30X coverage on nine of the 16 individuals, yielding 10,193,425 high-confidence SNVs. To validate the use of GBS data for facilitating imputation, we initially focused on chromosome 19 as a test case, using an optimized panel of 833 sparse, evenly-spaced markers from GBS and 5,010 dense markers from WGS. Using the method of "Genotype Imputation Given Inheritance" (GIGI), we evaluated the effects on imputation accuracy of 3 different strategies for selecting individuals for WGS, including 1) using "GIGI-Pick" to select the most informative individuals, 2) using the most recent generation, or 3) using founders only.  We also evaluated the effects on imputation accuracy of using a range of from 1 to 9 WGS individuals for imputation. We found that the GIGI-Pick algorithm for selection of WGS individuals outperformed common heuristic approaches, and that genotype numbers and accuracy improved very little when using >5 WGS individuals for imputation. Informed by our findings, we used 4 macaques with WGS data to impute variants at up to 7,655,491 sites spanning all 20 autosomes in the 12 remaining macaques, based on their GBS genotypes at only 17,158 loci. Using a strict confidence threshold, we imputed an average of 3,680,238 variants per individual at >99 % accuracy, or an average 4,458,883 variants per individual at a more relaxed threshold, yielding >97 % accuracy. CONCLUSIONS: We conclude that an optimal tradeoff between genotype accuracy, number of imputed genotypes, and overall cost exists at the ratio of one individual selected for WGS using the GIGI-Pick algorithm, per 3-5 relatives selected for GBS. This approach makes feasible the collection of accurate, dense genome-wide sequence data in large pedigreed macaque cohorts without the need for more expensive WGS data on all individuals.

Evolution of Caenorhabditis mitochondrial genome pseudogenes and Caenorhabditis briggsae natural isolates.

Although most metazoan mitochondrial genomes are highly streamlined and encode little noncoding DNA outside of the "AT" region, the accumulation of mitochondrial pseudogenes and other types of noncoding DNA has been observed in a growing number of animal groups. The nematode species Caenorhabditis briggsae harbors two mitochondrial DNA (mtDNA) pseudogenes, named Psinad5-1 and Psinad5-2, presumably derived from the nad5 protein-coding gene. Here, we provide an in-depth analysis of mtDNA pseudogene evolution in C. briggsae natural isolates and related Caenorhabditis species. Mapping the observed presence and absence of the pseudogenes onto phylogenies suggests that Psinad5-1 originated in the ancestor to C. briggsae and its recently discovered outcrossing relative species Caenorhabditis sp. 5 and Caenorhabditis sp. 9. However, Psinad5-1 was not detected in Caenorhabditis sp. 9 natural isolates, suggesting a lineage-specific loss of this pseudogene in this species. Our results corroborated the previous finding that Psinad5-2 originated within C. briggsae. The observed pattern of mitochondrial pseudogene gain and loss in Caenorhabditis was inconsistent with predictions of the tandem duplication-random loss model of mitochondrial genome evolution and suggests that intralineage recombination-like mechanisms might play a major role in Caenorhabditis mtDNA evolution. Natural variation was analyzed at the pseudogenes and flanking mtDNA sequences in 141 geographically diverse C. briggsae natural isolates. Although phylogenetic analysis placed the majority of isolates into the three previously established major intraspecific clades of C. briggsae, two new and unexpected haplotypes fell outside of these conventional groupings. Psinad5-2 copy number variation was observed among C. briggsae isolates collected from the same geographic site. Patterns of nucleotide diversity were analyzed in Psinad5-1 and Psinad5-2, and confidence intervals were found to overlap values from synonymous sites in protein-coding genes, consistent with neutral expectations. Our findings provide new insights into the mode and tempo of mitochondrial genome and pseudogene evolution both within and between Caenorhabditis nematode species.

Genetic Architecture of Human Obesity Traits in the Rhesus Macaque.

OBJECTIVE: Whereas the metabolic consequences of obesity have been studied extensively in the rhesus macaque, corollary genetic studies of obesity are nonexistent. This study assessed genetic contributions to spontaneous adiposity in this species. METHODS: Phenotypic variation by age class and sex for BMI, waist to height ratio, waist to thigh ratio, and waist circumference was assessed in 583 macaques. Total and sex-specific heritability for all traits was estimated, including waist to thigh ratio adjusted for BMI, as well as genotypic and phenotypic correlations. In addition, functional genetic variation at BDNF, FTO, LEP, LEPR, MC4R, PCSK1, POMC, and SIM1 was assessed in four animals with extreme spontaneous adiposity. RESULTS: Trait heritability in the combined sample was low to moderate (0.14-0.32), whereas sex-specific heritability was more substantial (0.20-0.67). Heritability was greater in females for all traits except BMI. All traits were robustly correlated, with genetic correlations of 0.63 to 0.93 indicating substantial pleiotropy. Likely functional variants were discovered in the four macaques at all eight human obesity genes, including six missense mutations in BDNF, FTO, LEP, LEPR, and PCSK1 and, notably, one nonsense mutation in LEPR. CONCLUSIONS: A moderate polygenic contribution to adiposity in rhesus macaques was found, as well as mutations with potentially larger effects in multiple genes that influence obesity in humans.

Modelling disease risk for amyloid A (AA) amyloidosis in non-human primates using machine learning.

Objective: Amyloid A (AA) amyloidosis is found in humans and non-human primates, but quantifying disease risk prior to clinical symptoms is challenging. We applied machine learning to identify the best predictors of amyloidosis in rhesus macaques from available clinical and pathology records. To explore potential biomarkers, we also assessed whether changes in circulating serum amyloid A (SAA) or lipoprotein profiles accompany the disease. Methods: We conducted a retrospective study using 86 cases and 163 controls matched for age and sex. We performed data reduction on 62 clinical, pathological and demographic variables, and applied multivariate modelling and model selection with cross-validation. To test the performance of our final model, we applied it to a replication cohort of 2,775 macaques. Results: The strongest predictors of disease were colitis, gastrointestinal adenocarcinoma, endometriosis, arthritis, trauma, diarrhoea and number of pregnancies. Sensitivity and specificity of the risk model were predicted to be 82%, and were assessed at 79 and 72%, respectively. Total, low density lipoprotein and high density lipoprotein cholesterol levels were significantly lower, and SAA levels and triglyceride-to-HDL ratios were significantly higher in cases versus controls. Conclusion: Machine learning is a powerful approach to identifying macaques at risk of AA amyloidosis, which is accompanied by increased circulating SAA and altered lipoprotein profiles.

A Practical Approach for Designing Breeding Groups to Maximize Genetic Diversity in a Large Colony of Captive Rhesus Macaques (Macaca mulatta).

Limited guidance is available on practical approaches for maintaining genetic diversity in large NHP colonies that support biomedical research, despite the fact that reduced diversity in these colonies is likely to compromise the application of findings in NHP to human disease. In particular, constraints related to simultaneously housing, breeding, and providing ongoing veterinary care for thousands of animals with a highly complex social structure creates unique challenges for genetic management in these colonies. Because the composition of new breeding groups is a critical component of genetic management, here we outline a 3-stage protocol for forming new breeding groups of NHP that is aimed at maximizing genetic diversity in the face of frequent restrictions on age, sex, and numbers of animals per breeding group. As an example application of this protocol, we describe optimal combinations of rhesus macaques from an analysis of candidate animals available for breeding in July 2013, selected from among the approximately 4000 macaques maintained at the Oregon National Primate Research Center. In addition, a simulation study to explore the genetic diversity in breeding groups formed by using this protocol, indicated an approximate 10-fold higher genome uniqueness, 50% lower mean kinship, and an 84-fold lower mean inbreeding coefficient among potential offspring within groups, when compared with a suboptimal group design. We conclude that this protocol provides a practical and effective approach to breeding group design for colony managers who want to prevent the loss of genetic diversity in large, semiisolated NHP colonies.

Sex-specific heritability of spontaneous lipid levels in an extended pedigree of Indian-origin rhesus macaques (Macaca mulatta).

The rhesus macaque is an important model for human atherosclerosis but genetic determinants of relevant phenotypes have not yet been investigated in this species. Because lipid levels are well-established and heritable risk factors for human atherosclerosis, our goal was to assess the heritability of lipoprotein cholesterol and triglyceride levels in a single, extended pedigree of 1,289 Indian-origin rhesus macaques. Additionally, because increasing evidence supports sex differences in the genetic architecture of lipid levels and lipid metabolism in humans and macaques, we also explored sex-specific heritability for all lipid measures investigated in this study. Using standard methods, we measured lipoprotein cholesterol and triglyceride levels from fasted plasma in a sample of 193 pedigreed rhesus macaques selected for membership in large, paternal half-sib cohorts, and maintained on a low-fat, low cholesterol chow diet. Employing a variance components approach, we found moderate heritability for total cholesterol (h²=0.257, P=0.032), LDL cholesterol (h²=0.252, P=0.030), and triglyceride levels (h²=0.197, P=0.034) in the full sample. However, stratification by sex (N=68 males, N=125 females) revealed substantial sex-specific heritability for total cholesterol (0.644, P=0.004, females only), HDL cholesterol (0.843, P=0.0008, females only), VLDL cholesterol (0.482, P=0.018, males only), and triglyceride levels (0.705, P=0.001, males only) that was obscured or absent when sexes were combined in the full sample. We conclude that genes contribute to spontaneous variation in circulating lipid levels in the Indian-origin rhesus macaque in a sex-specific manner, and that the rhesus macaque is likely to be a valuable model for sex-specific genetic effects on lipid risk factors for human atherosclerosis. These findings are a first-ever report of heritability for cholesterol levels in this species, and support the need for expanded analysis of these traits in this population.