The CARD8 inflammasome dictates HIV/SIV pathogenesis and disease progression.

While CD4+ T cell depletion is key to disease progression in people living with HIV and SIV-infected macaques, the mechanisms underlying this depletion remain incompletely understood, with most cell death involving uninfected cells. In contrast, SIV infection of "natural" hosts such as sooty mangabeys does not cause CD4+ depletion and AIDS despite high-level viremia. Here, we report that the CARD8 inflammasome is activated immediately after HIV entry by the viral protease encapsulated in incoming virions. Sensing of HIV protease activity by CARD8 leads to rapid pyroptosis of quiescent cells without productive infection, while T cell activation abolishes CARD8 function and increases permissiveness to infection. In humanized mice reconstituted with CARD8-deficient cells, CD4+ depletion is delayed despite high viremia. Finally, we discovered loss-of-function mutations in CARD8 from "natural hosts," which may explain the peculiarly non-pathogenic nature of these infections. Our study suggests that CARD8 drives CD4+ T cell depletion during pathogenic HIV/SIV infections.

Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Complete Genomic Assembly of Mauritian Cynomolgus Macaque Killer Ig-like Receptor and Natural Killer Group 2 Haplotypes.

Mauritian-origin cynomolgus macaques (MCMs) serve as a powerful nonhuman primate model in biomedical research due to their unique genetic homogeneity, which simplifies experimental designs. Despite their extensive use, a comprehensive understanding of crucial immune-regulating gene families, particularly killer Ig-like receptors (KIR) and NK group 2 (NKG2), has been hindered by the lack of detailed genomic reference assemblies. In this study, we employ advanced long-read sequencing techniques to completely assemble eight KIR and seven NKG2 genomic haplotypes, providing an extensive insight into the structural and allelic diversity of these immunoregulatory gene clusters. Leveraging these genomic resources, we prototype a strategy for genotyping KIR and NKG2 using short-read, whole-exome capture data, illustrating the potential for cost-effective multilocus genotyping at colony scale. These results mark a significant enhancement for biomedical research in MCMs and underscore the feasibility of broad-scale genetic investigations.

Comparative molecular genomic analyses of a spontaneous rhesus macaque model of mismatch repair-deficient colorectal cancer.

Colorectal cancer (CRC) remains the third most common cancer in the US with 15% of cases displaying Microsatellite Instability (MSI) secondary to Lynch Syndrome (LS) or somatic hypermethylation of the MLH1 promoter. A cohort of rhesus macaques from our institution developed spontaneous mismatch repair deficient (MMRd) CRC with a notable fraction harboring a pathogenic germline mutation in MLH1 (c.1029C

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

SVhound: detection of regions that harbor yet undetected structural variation.

BACKGROUND: Recent population studies are ever growing in number of samples to investigate the diversity of a population or species. These studies reveal new polymorphism that lead to important insights into the mechanisms of evolution, but are also important for the interpretation of these variations. Nevertheless, while the full catalog of variations across entire species remains unknown, we can predict which regions harbor additional not yet detected variations and investigate their properties, thereby enhancing the analysis for potentially missed variants. RESULTS: To achieve this we developed SVhound ( https://github.com/lfpaulin/SVhound ), which based on a population level SVs dataset can predict regions that harbor unseen SV alleles. We tested SVhound using subsets of the 1000 genomes project data and showed that its correlation (average correlation of 2800 tests r = 0.7136) is high to the full data set. Next, we utilized SVhound to investigate potentially missed or understudied regions across 1KGP and CCDG. Lastly we also apply SVhound on a small and novel SV call set for rhesus macaque (Macaca mulatta) and discuss the impact and choice of parameters for SVhound. CONCLUSIONS: SVhound is a unique method to identify potential regions that harbor hidden diversity in model and non model organisms and can also be potentially used to ensure high quality of SV call sets.

De novo Mutations in Domestic Cat are Consistent with an Effect of Reproductive Longevity on Both the Rate and Spectrum of Mutations.

The mutation rate is a fundamental evolutionary parameter with direct and appreciable effects on the health and function of individuals. Here, we examine this important parameter in the domestic cat, a beloved companion animal as well as a valuable biomedical model. We estimate a mutation rate of 0.86 × 10-8 per bp per generation for the domestic cat (at an average parental age of 3.8 years). We find evidence for a significant paternal age effect, with more mutations transmitted by older sires. Our analyses suggest that the cat and the human have accrued similar numbers of mutations in the germline before reaching sexual maturity. The per-generation mutation rate in the cat is 28% lower than what has been observed in humans, but is consistent with the shorter generation time in the cat. Using a model of reproductive longevity, which takes into account differences in the reproductive age and time to sexual maturity, we are able to explain much of the difference in per-generation rates between species. We further apply our reproductive longevity model in a novel analysis of mutation spectra and find that the spectrum for the cat resembles the human mutation spectrum at a younger age of reproduction. Together, these results implicate changes in life-history as a driver of mutation rate evolution between species. As the first direct observation of the paternal age effect outside of rodents and primates, our results also suggest a phenomenon that may be universal among mammals.

Paternal age in rhesus macaques is positively associated with germline mutation accumulation but not with measures of offspring sociability.

Mutation is the ultimate source of all genetic novelty and the cause of heritable genetic disorders. Mutational burden has been linked to complex disease, including neurodevelopmental disorders such as schizophrenia and autism. The rate of mutation is a fundamental genomic parameter and direct estimates of this parameter have been enabled by accurate comparisons of whole-genome sequences between parents and offspring. Studies in humans have revealed that the paternal age at conception explains most of the variation in mutation rate: Each additional year of paternal age in humans leads to approximately 1.5 additional inherited mutations. Here, we present an estimate of the de novo mutation rate in the rhesus macaque (Macaca mulatta) using whole-genome sequence data from 32 individuals in four large pedigrees. We estimated an average mutation rate of 0.58 × 10-8 per base pair per generation (at an average parental age of 7.5 yr), much lower than found in direct estimates from great apes. As in humans, older macaque fathers transmit more mutations to their offspring, increasing the per generation mutation rate by 4.27 × 10-10 per base pair per year. We found that the rate of mutation accumulation after puberty is similar between macaques and humans, but that a smaller number of mutations accumulate before puberty in macaques. We additionally investigated the role of paternal age on offspring sociability, a proxy for normal neurodevelopment, by studying 203 male macaques in large social groups.

Primate phylogenomics uncovers multiple rapid radiations and ancient interspecific introgression.

Our understanding of the evolutionary history of primates is undergoing continual revision due to ongoing genome sequencing efforts. Bolstered by growing fossil evidence, these data have led to increased acceptance of once controversial hypotheses regarding phylogenetic relationships, hybridization and introgression, and the biogeographical history of primate groups. Among these findings is a pattern of recent introgression between species within all major primate groups examined to date, though little is known about introgression deeper in time. To address this and other phylogenetic questions, here, we present new reference genome assemblies for 3 Old World monkey (OWM) species: Colobus angolensis ssp. palliatus (the black and white colobus), Macaca nemestrina (southern pig-tailed macaque), and Mandrillus leucophaeus (the drill). We combine these data with 23 additional primate genomes to estimate both the species tree and individual gene trees using thousands of loci. While our species tree is largely consistent with previous phylogenetic hypotheses, the gene trees reveal high levels of genealogical discordance associated with multiple primate radiations. We use strongly asymmetric patterns of gene tree discordance around specific branches to identify multiple instances of introgression between ancestral primate lineages. In addition, we exploit recent fossil evidence to perform fossil-calibrated molecular dating analyses across the tree. Taken together, our genome-wide data help to resolve multiple contentious sets of relationships among primates, while also providing insight into the biological processes and technical artifacts that led to the disagreements in the first place.

Copy number variants and fixed duplications among 198 rhesus macaques (Macaca mulatta).

The rhesus macaque is an abundant species of Old World monkeys and a valuable model organism for biomedical research due to its close phylogenetic relationship to humans. Copy number variation is one of the main sources of genomic diversity within and between species and a widely recognized cause of inter-individual differences in disease risk. However, copy number differences among rhesus macaques and between the human and macaque genomes, as well as the relevance of this diversity to research involving this nonhuman primate, remain understudied. Here we present a high-resolution map of sequence copy number for the rhesus macaque genome constructed from a dataset of 198 individuals. Our results show that about one-eighth of the rhesus macaque reference genome is composed of recently duplicated regions, either copy number variable regions or fixed duplications. Comparison with human genomic copy number maps based on previously published data shows that, despite overall similarities in the genome-wide distribution of these regions, there are specific differences at the chromosome level. Some of these create differences in the copy number profile between human disease genes and their rhesus macaque orthologs. Our results highlight the importance of addressing the number of copies of target genes in the design of experiments and cautions against human-centered assumptions in research conducted with model organisms. Overall, we present a genome-wide copy number map from a large sample of rhesus macaque individuals representing an important novel contribution concerning the evolution of copy number in primate genomes.

Origins and Long-Term Patterns of Copy-Number Variation in Rhesus Macaques.

Mutations play a key role in the development of disease in an individual and the evolution of traits within species. Recent work in humans and other primates has clarified the origins and patterns of single-nucleotide variants, showing that most arise in the father's germline during spermatogenesis. It remains unknown whether larger mutations, such as deletions and duplications of hundreds or thousands of nucleotides, follow similar patterns. Such mutations lead to copy-number variation (CNV) within and between species, and can have profound effects by deleting or duplicating genes. Here, we analyze patterns of CNV mutations in 32 rhesus macaque individuals from 14 parent-offspring trios. We find the rate of CNV mutations per generation is low (less than one per genome) and we observe no correlation between parental age and the number of CNVs that are passed on to offspring. We also examine segregating CNVs within the rhesus macaque sample and compare them to a similar data set from humans, finding that both species have far more segregating deletions than duplications. We contrast this with long-term patterns of gene copy-number evolution between 17 mammals, where the proportion of deletions that become fixed along the macaque lineage is much smaller than the proportion of segregating deletions. These results suggest purifying selection acting on deletions, such that the majority of them are removed from the population over time. Rhesus macaques are an important biomedical model organism, so these results will aid in our understanding of this species and the disease models it supports.

Infant inhibited temperament in primates predicts adult behavior, is heritable, and is associated with anxiety-relevant genetic variation.

An anxious or inhibited temperament (IT) early in life is a major risk factor for the later development of stress-related psychopathology. Starting in infancy, nonhuman primates, like humans, begin to reveal their temperament when exposed to novel situations. Here, in Study 1 we demonstrate this infant IT predicts adult behavior. Specifically, in over 600 monkeys, we found that individuals scored as inhibited during infancy were more likely to refuse treats offered by potentially-threatening human experimenters as adults. In Study 2, using a sample of over 4000 monkeys from a large multi-generational family pedigree, we demonstrate that infant IT is partially heritable. The data revealed infant IT to reflect a co-inherited substrate that manifests across multiple latent variables. Finally, in Study 3 we performed whole-genome sequencing in 106 monkeys to identify IT-associated single-nucleotide variations (SNVs). Results demonstrated a genome-wide significant SNV near CTNNA2, suggesting a molecular target worthy of additional investigation. Moreover, we observed lower p values in genes implicated in human association studies of neuroticism and depression. Together, these data demonstrate the utility of our model of infant inhibited temperament in the rhesus monkey to facilitate discovery of genes that are relevant to the long-term inherited risk to develop anxiety and depressive disorders.

Clinical presentation, treatment, and genetic and histopathological analysis of juvenile cataracts and secondary glaucoma in a rhesus macaque (Macaca mulatta).

This report describes the clinical and histological findings, genetic study, and treatment in a 1.3-year-old rhesus macaque with bilateral cataracts and unilateral secondary glaucoma. Intravitreal injection of gentamicin decreased the intraocular pressure from 56 to <2 mm Hg. A putative genetic cause of the cataracts was not identified.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 2 million fatalities since it first emerged in late 2019. As we write, infection rates are at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary target of SARS-CoV-2 is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predict that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results while finding additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques.

Over the past two decades, 33 cases of colonic adenocarcinomas have been diagnosed in rhesus macaques (Macaca mulatta) at the nonhuman primate colony of the Keeling Center for Comparative Medicine and Research at The University of Texas MD Anderson Cancer Center. The distinctive feature in these cases, based on PET/computed tomography (CT) imaging, was the presence of two or three tumor lesions in different locations, including proximal to the ileocecal juncture, proximal to the hepatic flexure, and/or in the sigmoid colon. These colon carcinoma lesions selectively accumulated [18F]fluorodeoxyglucose ([18F]FDG) and [18F]fluoroacetate ([18F]FACE) at high levels, reflecting elevated carbohydrate and fatty acid metabolism in these tumors. In contrast, the accumulation of [18F]fluorothymidine ([18F]FLT) was less significant, reflecting slow proliferative activity in these tumors. The diagnoses of colon carcinomas were confirmed by endoscopy. The expression of MLH1, MSH2, and MSH6 proteins and the degree of microsatellite instability (MSI) was assessed in colon carcinomas. The loss of MLH1 protein expression was observed in all tumors and was associated with a deletion mutation in the MLH1 promoter region and/or multiple single-nucleotide polymorphism (SNP) mutations in the MLH1 gene. All tumors exhibited various degrees of MSI. The pedigree analysis of this rhesus macaque population revealed several clusters of affected animals related to each other over several generations, suggesting an autosomal dominant transmission of susceptibility for colon cancer. The newly discovered hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques, termed MLH1-rheMac, may serve as a model for development of novel approaches to diagnosis and therapy of Lynch syndrome in humans.

Unusual sequence characteristics of human chromosome 19 are conserved across 11 nonhuman primates.

BACKGROUND: Human chromosome 19 has many unique characteristics including gene density more than double the genome-wide average and 20 large tandemly clustered gene families. It also has the highest GC content of any chromosome, especially outside gene clusters. The high GC content and concomitant high content of hypermutable CpG sites raises the possibility chromosome 19 exhibits higher levels of nucleotide diversity both within and between species, and may possess greater variation in DNA methylation that regulates gene expression. RESULTS: We examined GC and CpG content of chromosome 19 orthologs across representatives of the primate order. In all 12 primate species with suitable genome assemblies, chromosome 19 orthologs have the highest GC content of any chromosome. CpG dinucleotides and CpG islands are also more prevalent in chromosome 19 orthologs than other chromosomes. GC and CpG content are generally higher outside the gene clusters. Intra-species variation based on SNPs in human common dbSNP, rhesus, crab eating macaque, baboon and marmoset datasets is most prevalent on chromosome 19 and its orthologs. Inter-species comparisons based on phyloP conservation show accelerated nucleotide evolution for chromosome 19 promoter flanking and enhancer regions. These same regulatory regions show the highest CpG density of any chromosome suggesting they possess considerable methylome regulatory potential. CONCLUSIONS: The pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur representing 74 million years of primate evolution. Much CpG variation exists both within and between primate species with a portion of this variation occurring in regulatory regions.

MHC genotyping from rhesus macaque exome sequences.

Indian rhesus macaque major histocompatibility complex (MHC) variation can influence the outcomes of transplantation and infectious disease studies. Frequently, rhesus macaques are MHC genotyped to identify variants that could account for unexpected results. Since the MHC is only one region in the genome where variation could impact experimental outcomes, strategies for simultaneously profiling variation in the macaque MHC and the remainder of the protein coding genome would be useful. Here we determine MHC class I and class II genotypes using target-capture probes enriched for MHC sequences, a method we term macaque exome sequence (MES) genotyping. For a cohort of 27 Indian rhesus macaques, we describe two methods for obtaining MHC genotypes from MES data and demonstrate that the MHC class I and class II genotyping results obtained with these methods are 98.1% and 98.7% concordant, respectively, with expected MHC genotypes. In contrast, conventional MHC genotyping results obtained by deep sequencing of short multiplex PCR amplicons were only 92.6% concordant with expectations for this cohort.

The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences.

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease.

Intergenerational neural mediators of early-life anxious temperament.

Understanding the heritability of neural systems linked to psychopathology is not sufficient to implicate them as intergenerational neural mediators. By closely examining how individual differences in neural phenotypes and psychopathology cosegregate as they fall through the family tree, we can identify the brain systems that underlie the parent-to-child transmission of psychopathology. Although research has identified genes and neural circuits that contribute to the risk of developing anxiety and depression, the specific neural systems that mediate the inborn risk for these debilitating disorders remain unknown. In a sample of 592 young rhesus monkeys that are part of an extended multigenerational pedigree, we demonstrate that metabolism within a tripartite prefrontal-limbic-midbrain circuit mediates some of the inborn risk for developing anxiety and depression. Importantly, although brain volume is highly heritable early in life, it is brain metabolism-not brain structure-that is the critical intermediary between genetics and the childhood risk to develop stress-related psychopathology.

Hybrid de novo genome assembly and centromere characterization of the gray mouse lemur (Microcebus murinus).

BACKGROUND: The de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly. METHODS: We used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome. RESULTS: We improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies. CONCLUSIONS: We describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes.