Timing of standard chow exposure determines the variability of mouse phenotypic outcomes and gut microbiota profile.

Standard chow diet contributes to reproducibility in animal model experiments since chows differ in nutrient composition, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here, we measured the impact of diet (5V5M, 5V0G, 2920X, and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE), and developmental exposure (DE)) on growth & development, metabolic health indicators, and gut bacterial microbiota profiles across genetically identical C57BL6/J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on measured outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight, and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. Diet-induced beta diversity profiles were sex-dependent for LE mice. Compared to AE, LE drove 9X more diet-driven differentially abundant genera, majority of which were the result of inverse effects of 2920X and 5058. Our findings demonstrate that lifetime exposure to different chow diets has the greatest impact on reproducibility of experimental measures that are common components of preclinical mouse model studies. Importantly, weaning DE mice onto a uniform diet is likely an effective way to reduce unwanted phenotypic variability among experimental models.

A chromosome-scale fishing cat reference genome for the evaluation of potential germline risk variants.

The fishing cat, Prionailurus viverrinus, faces a population decline, increasing the importance of maintaining healthy zoo populations. Unfortunately, zoo-managed individuals currently face a high prevalence of transitional cell carcinoma (TCC), a form of bladder cancer. To investigate the genetics of inherited diseases among captive fishing cats, we present a chromosome-scale assembly, generate the pedigree of the zoo-managed population, reaffirm the close genetic relationship with the Asian leopard cat (Prionailurus bengalensis), and identify 7.4 million single nucleotide variants (SNVs) and 23,432 structural variants (SVs) from whole genome sequencing (WGS) data of healthy and TCC cats. Only BRCA2 was found to have a high recurrent number of missense mutations in fishing cats diagnosed with TCC when compared to inherited human cancer risk variants. These new fishing cat genomic resources will aid conservation efforts to improve their genetic fitness and enhance the comparative study of feline genomes.

The Expected Behaviors of Posterior Predictive Tests and Their Unexpected Interpretation.

Poor fit between models of sequence or trait evolution and empirical data is known to cause biases and lead to spurious conclusions about evolutionary patterns and processes. Bayesian posterior prediction is a flexible and intuitive approach for detecting such cases of poor fit. However, the expected behavior of posterior predictive tests has never been characterized for evolutionary models, which is critical for their proper interpretation. Here, we show that the expected distribution of posterior predictive P-values is generally not uniform, in contrast to frequentist P-values used for hypothesis testing, and extreme posterior predictive P-values often provide more evidence of poor fit than typically appreciated. Posterior prediction assesses model adequacy under highly favorable circumstances, because the model is fitted to the data, which leads to expected distributions that are often concentrated around intermediate values. Nonuniform expected distributions of P-values do not pose a problem for the application of these tests, however, and posterior predictive P-values can be interpreted as the posterior probability that the fitted model would predict a dataset with a test statistic value as extreme as the value calculated from the observed data.

Association of a novel dystrophin (DMD) genetic nonsense variant in a cat with X-linked muscular dystrophy with a mild clinical course.

X-linked muscular dystrophy in cats (FXMD) is an uncommon disease, with few reports describing its pathogenic genetic variants. A 9-year-old castrated male domestic shorthair cat was presented with persistent muscle swelling and breathing difficulty from 3 years of age. Serum activity of alanine aminotransferase, aspartate transaminase, and creatine kinase were abnormally high. Physical and neurological examinations showed muscle swelling in the neck and proximal limb, slow gait, and occasional breathing difficulties. Electromyography showed pseudomyotonic discharges and complex repetitive discharges with a "dive-bomber" sound. Histopathology revealed muscle necrosis and regeneration. Whole-genome sequencing identified a novel and unique hemizygous nonsense genetic variant, c.8333G > A in dystrophin (DMD), potentially causing a premature termination codon (p.Trp2778Ter). Based on a combination of clinical and histological findings and the presence of the DMD nonsense genetic variant, this case was considered FXMD, which showed mild clinical signs and long-term survival, even though immunohistochemical characterization was lacking.

ARG1 Is a Potential Prognostic Marker in Metastatic Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy. While the majority of patients present with early-stage and low-grade EC and have an excellent prognosis, a subset has metastatic disease at presentation or develops distant recurrence after initial treatment of the primary. However, the lack of prognostic biomarkers for metastatic EC is a critical barrier. Arginase 1 (ARG1) regulates the last step of the urea cycle, and an increase in ARG1 has been correlated as a poor prognostic factor in a variety of cancers. In the present study, ARG1 expression was evaluated as a potential prognostic marker for metastatic EC in endometrial hyperplasia and cancer of mice with Pten mutation as well as Pten and Mig-6 double mutations. While Pten mutation in the uterus is not sufficient for distant metastasis, mice with concurrent ablation of Mig-6 and Pten develop distant metastasis. Our immunostaining and RT-qPCR analysis revealed that the expression of ARG1 in early stage of EC as well as endometrial hyperplasia from mice deficient in Mig-6 and Pten mutations significantly increased compared to Pten mutation in the uterus. The results suggest that a high level of ARG1 is associated with poor prognosis in association with EC of mouse.

Precision medicine using whole genome sequencing identifies a novel dystrophin (DMD) variant for X-linked muscular dystrophy in a cat.

BACKGROUND: Muscular dystrophies (MDs) are a large, heterogeneous group of degenerative muscle diseases. X-linked dystrophin-deficient MD in cats is the first genetically characterized cat model for a human disease and a few novel forms have been identified. HYPOTHESIS/OBJECTIVES: Muscular dystrophy was suspected in a young male domestic shorthair cat. Clinical, molecular, and genetic techniques could provide a definitive diagnosis. ANIMALS: A 1-year-old male domestic shorthair cat presented for progressive difficulty walking, macroglossia and dysphagia beginning at 6 months of age. The tongue was thickened, protruded with constant ptyalism, and thickening and rigidity of the neck and shoulders were observed. METHODS: A complete neurological examination, baseline laboratory evaluation and biopsies of the trapezius muscle were performed with owner consent. Indirect immunofluorescence staining of muscle cryosections was performed using several monoclonal and polyclonal antibodies against dystrophy-associated proteins. DNA was isolated for genomic analyses by whole genome sequencing and comparison to DNA variants in the 99 Lives Cat Genome Sequencing dataset. RESULTS AND CLINICAL IMPORTANCE: Aspartate aminotransferase (687 IU/L) and creatine kinase (24 830 IU/L) activities were increased and mild hypokalemia (3.7 mmol/L) was present. Biopsy samples from the trapezius muscle confirmed a degenerative and regenerative myopathy and protein alterations identified by immunohistochemistry resulted in a diagnosis of a in dystrophin-deficient form of X-linked MD. A stop gain variant (c.4849C>T; p.Gln1617Ter) dystrophin was identified by genome sequencing. Precision/genomic medicine efforts for the domestic cat and in veterinary medicine support disease variant and animal model discovery and provide opportunities for targeted treatments for companion animals.

Beta-mannosidosis in a domestic cat associated with a missense variant in MANBA.

A 6-month-old cat of unknown ancestry presented for a neurologic evaluation due to progressive motor impairment. Complete physical and neurologic examinations suggested the disorder was likely to be hereditary, although the signs were not consistent with any previously described inherited disorders in cats. Due to the progression of disease signs including severely impaired motor function and cognitive decline, the cat was euthanized at approximately 10.5 months of age. Whole genome sequence analysis identified a homozygous missense variant c.2506G > A in MANBA that predicts a p.Gly836Arg alteration in the encoded lysosomal enzyme ß -mannosidase. This variant was not present in the whole genome or whole exome sequences of any of the 424 cats represented in the 99 Lives Cat Genome dataset. ß -Mannosidase enzyme activity was undetectable in brain tissue homogenates from the affected cat, whereas α-mannosidase enzyme activities were elevated compared to an unaffected cat. Postmortem examination of brain and retinal tissues revealed massive accumulations of vacuolar inclusions in most cells, similar to those reported in animals of other species with hereditary ß -mannosidosis. Based on these findings, the cat likely suffered from ß -mannosidosis due to the abolition of ß -mannosidase activity associated with the p.Gly836Arg amino acid substitution. p.Gly836 is located in the C-terminal region of the protein and was not previously known to be involved in modulating enzyme activity. In addition to the vacuolar inclusions, some cells in the brain of the affected cat contained inclusions that exhibited lipofuscin-like autofluorescence. Electron microscopic examinations suggested these inclusions formed via an autophagy-like process.

Cardiomyopathy associated 5 (CMYA5) implicated as a genetic risk factor for radial hemimelia in Siamese cats.

OBJECTIVES: The present study aimed to determine the inheritance pattern and genetic cause of congenital radial hemimelia (RH) in cats. METHODS: Clinical and genetic analyses were conducted on a Siamese cat family (n = 18), including two siblings with RH. Radiographs were obtained for the affected kittens and echocardiograms of an affected kitten and sire. Whole genome sequencing was completed on the two cases and the parents. Genomic data were compared with the 99 Lives Cat Genome data set of 420 additional domestic cats with whole genome and whole exome sequencing data. Variants were considered as homozygous in the two cases of the siblings with RH and heterozygous in the parents. Candidate variants were genotyped by Sanger sequencing in the extended pedigree. RESULTS: Radiographs of the female kitten revealed bilateral absence of the radii and bowing of the humeri, while the male kitten showed a dysplastic right radius. Echocardiography suggested the female kitten had restrictive cardiomyopathy with a positive left atrial-to-aortic root ratio (LA:Ao = 1.83 cm), whereas hypertrophic cardiomyopathy was more likely in the sire, showing diastolic dysfunction using tissue Doppler imaging (59.06 cm/s). Twenty-two DNA variants were unique and homozygous in the affected kittens and heterozygous in the parents. Seven variants clustered in one chromosomal region, including two frameshift variants in cardiomyopathy associated 5 (CMYA5) and five variants in junction mediating and regulatory protein, P53 cofactor (JMY ), including a missense and an in-frame deletion. CONCLUSIONS AND RELEVANCE: The present study suggested an autosomal recessive mode of inheritance with variable expression for RH in the Siamese cat family. Candidate variants for the phenotype were identified, implicating their roles in bone development. These genes should be considered as potentially causal for other cats with RH. Siamese cat breeders should consider genetically testing their cats for these variants to prevent further dissemination of the suspected variants within the breed.

Precision medicine using whole genome sequencing in a cat identifies a novel COL5A1 variant for classical Ehlers-Danlos syndrome.

BACKGROUND: Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable connective tissue disorders occurring in both human and veterinary patients. The genetics of these disorders are poorly described in small animal patients. HYPOTHESIS/OBJECTIVES: Define the clinical manifestations and genetic cause of a suspected form of EDS in a cat. ANIMALS: A 14-week-old male domestic medium hair cat was presented with skin hyperextensibility and fragility. The classic tragic facial expression was observed as well as chronic pruritus and mild hyperesthesia. METHODS: Blood samples and a skin biopsy sample were collected from the affected cat. Clinical examinations, histology, electron microscopy and whole genome sequencing were conducted to characterize the clinical presentation and identify possible pathogenic DNA variants to support a diagnosis. Criteria defining variant pathogenicity were examined including human disease variant databases. RESULTS: Histology showed sparse, disorganized collagen and an increase in cutaneous mast cells. Electron microscopy identified ultrastructural defects commonly seen in collagen type V alpha 1 chain (COL5A1) variants including flower-like collagen fibrils in cross-section. Whole genome sequencing and comparison with 413 cats in the 99 Lives Cat Genome Sequencing Consortium database identified a novel splice acceptor site variant at exon 4 in COL5A1 (c.501-2A>C). CONCLUSIONS AND CLINICAL IMPORTANCE: Our report broadens the current understanding of EDS in veterinary patients and supports the use of precision medicine techniques in clinical veterinary practice. The classification of variants for pathogenicity should be considered in companion animals.

Feline precision medicine using whole-exome sequencing identifies a novel frameshift mutation for vitamin D-dependent rickets type 2.

OBJECTIVES: A 14-week-old female domestic longhair kitten presented with shifting lameness and disproportionately smaller size compared with a co-housed littermate. METHODS: Hematology and serum biochemical testing were conducted to investigate causes for delayed growth, and radiographs of the appendicular skeleton were obtained. RESULTS: The afflicted kitten had marked hypocalcemia, mild hypophosphatemia and substantial elevations in alkaline phosphatase activity, as well as pathognomonic radiographic findings consistent with rickets. Skeletal changes and hypocalcemia prompted testing of concentrations of parathyroid hormone (PTH) and vitamin D metabolites. Endocrine testing demonstrated significant increases in serum concentrations of PTH and 1,25-dihydroxycholecalciferol (calcitriol), supporting a diagnosis of vitamin D-dependent rickets type 2. Provision of analgesia, supraphysiologic doses of calcitriol and calcium carbonate supplementation achieved normalization of the serum calcium concentration and restoration of normal growth, although some skeletal abnormalities persisted. Once skeletally mature, ongoing calcitriol supplementation was not required. Whole-exome sequencing (WES) was conducted to identify the underlying DNA variant. A cytosine deletion at cat chromosome position B4:76777621 in VDR (ENSFCAT00000029466:c.106delC) was identified and predicted to cause a stop codon in exon 2 (p.Arg36Glufs*18), disrupting >90% of the receptor. The variant was unique and homozygous in this patient and absent in the sibling and approximately 400 other cats for which whole-genome and whole-exome data were available. CONCLUSIONS AND RELEVANCE: A unique, heritable form of rickets was diagnosed in a domestic longhair cat. WES identified a novel frameshift mutation affecting the gene coding for the vitamin D3 receptor, determining the likely causal genetic variant. Precision medicine techniques, including whole-exome and whole-genome sequencing, can be a standard of care in cats to identify disease etiologies, and to target therapeutics and personalize treatment.

Full Viral Genome Sequencing and Phylogenomic Analysis of Feline Herpesvirus Type 1 (FHV-1) in Cheetahs (Acinonyx jubatus).

Feline herpesvirus type 1 (FHV-1) is endemic in captive cheetahs and sporadically causes devastating disease. Modified live vaccines (MLV), intended for use in domestic cats, are used in some captive cheetah populations and have been anecdotally linked to disease in certain subpopulations. Ten FHV-1 isolates from ten captive cheetahs and one isolate from an MLV used to inoculate four of the host animals were analyzed. Viral DNA was extracted for full-genome sequencing by Illumina MiSeq with viral genomes then used for phylogenomic and recombinational analyses. The FHV-1 shed by vaccinated cheetahs were almost identical to the MLV, with few variants among viral genomes. Eight cheetah FHV-1 isolates and the MLV were grouped in a clade along with FHV-1 isolates from domestic cats in the USA. The remaining two cheetah FHV-1 isolates (unknown host vaccine status) were not associated with a clade. The likely ancestral origin of these two isolates involves recombination events between Australian domestic cat and cheetah FHV-1 isolates. Collectively, these data suggest that the MLV is capable of causing clinical disease and viral shedding in some cheetahs and represents evidence of interspecies transmission of virus between domestic and wild cats.

Phylogenomic Analysis of Global Isolates of Canid Alphaherpesvirus 1.

Canid alphaherpesvirus 1 (CHV-1) is a widespread pathogen of dogs with multiple associated clinical signs. There has been limited prior investigation into the genomics and phylogeny of this virus using whole viral genome analysis. Fifteen CHV-1 isolates were collected from animals with ocular disease based in the USA. Viral DNA was extracted for Illumina MiSeq full genome sequencing from each isolate. These data were combined with genomes of previously sequenced CHV-1 isolates obtained from hosts in the UK, Australia and Brazil. Genomic, recombinational and phylogenetic analysis were performed using multiple programs. Two isolates were separated into a clade apart from the remaining isolates and accounted for the majority of genomic distance (0.09%): one was obtained in 2019 from a USA-based host (ELAL-1) and the other in 2012 from a host in Brazil (BTU-1). ELAL-1 was found to contain variants previously reported in BTU-1 but also novel variants in the V57 gene region. Multiple non-synonymous variants were found in USA-based isolates in regions associated with antiviral resistance. Evidence of recombination was detected between ELAL-1 and BTU-1. Collectively, this represents evidence of trans-boundary transmission of a novel form of CHV-1, which highlights the importance of surveillance for this pathogen in domestic dog populations.

Genomic analysis for virulence determinants in feline herpesvirus type-1 isolates.

Feline herpesvirus type 1 (FHV-1) is a widespread cause of respiratory and ocular disease in domestic cats. A spectrum of disease severity is observed in host animals, but there has been limited prior investigation into viral genome factors which could be responsible. Stocks of FHV-1 were established from oropharyngeal swabs obtained from twenty-five cats with signs of infection housed in eight animal shelters around the USA. A standardized numerical host clinical disease severity scoring scheme was used for each cat from which an isolate was obtained. Illumina MiSeq was used to sequence the genome of each isolate. Genomic homogeneity among isolates was relatively high. A general linear model for fixed effects determined that only two synonymous single nucleotide polymorphisms across two genes (UL37/39) in the same isolate (from one host animal with a low disease severity score) were significantly associated (p ≤ 0.05) with assigned host respiratory and total disease severity score. No variants in any isolate were found to be significantly associated with assigned host ocular disease severity score. A concurrent analysis of missense mutations among the viral isolates identified three genes as being primarily involved in the observed genomic variation, but none were significantly associated with host disease severity scores. An ancestral state likelihood reconstruction was performed and determined that there was no evidence of a connection between host disease severity score and viral evolutionary state. We conclude from our results that the spectrum of host disease severity observed with FHV-1 is unlikely to be primarily related to viral genomic variations, and is instead due to host response and/or other factors.

Hinge Region in DNA Packaging Terminase pUL15 of Herpes Simplex Virus: A Potential Allosteric Target for Antiviral Drugs.

Approximately 80% of adults are infected with a member of the herpesviridae family. Herpesviruses establish life-long latent infections within neurons, which may reactivate into lytic infections due to stress or immune suppression. There are nine human herpesviruses (HHV) posing health concerns from benign conditions to life threatening encephalitis, including cancers associated with viral infections. The current treatment options for most HHV conditions mainly include several nucleoside and nucleotide analogs targeting viral DNA polymerase. Although these drugs help manage infections, their common mechanism of action may lead to the development of drug resistance, which is particularly devastating in immunocompromised patients. Therefore, new classes of drugs directed against novel targets in HHVs are necessary to alleviate this issue. We analyzed the conservation rates of all proteins in herpes simplex virus 1 (HHV-1), a representative of the HHV family and one of the most common viruses infecting the human population. Furthermore, we generated a full-length structure model of the most conserved HHV-1 protein, the DNA packaging terminase pUL15. A series of computational analyses were performed on the model to identify ATP and DNA binding sites and characterize the dynamics of the protein. Our study indicates that proteins involved in HHV-1 DNA packaging and cleavage are amongst the most conserved gene products of HHVs. Since the packaging protein pUL15 is the most conserved among all HHV-1 gene products, the virus will have a lower chance of developing resistance to small molecules targeting pUL15. A subsequent analysis of the structure of pUL15 revealed distinct ATP and DNA binding domains and the elastic network model identifies a functionally important hinge region between the two domains of pUL15. The atomic information on the active and allosteric sites in the ATP- and DNA-bound model of pUL15 presented in this study can inform the structure-based drug discovery of a new class of drugs to treat a wide range of HHVs.

Identification and Visualization of Functionally Important Domains and Residues in Herpes Simplex Virus Glycoprotein K(gK) Using a Combination of Phylogenetics and Protein Modeling.

Alphaherpesviruses are a subfamily of herpesviruses that include the significant human pathogens herpes simplex viruses (HSV) and varicella zoster virus (VZV). Glycoprotein K (gK), conserved in all alphaherpesviruses, is a multi-membrane spanning virion glycoprotein essential for virus entry into neuronal axons, virion assembly, and pathogenesis. Despite these critical functions, little is known about which gK domains and residues are most important for maintaining these functions across all alphaherpesviruses. Herein, we employed phylogenetic and structural analyses including the use of a novel model for evolutionary rate variation across residues to predict conserved gK functional domains. We found marked heterogeneity in the evolutionary rate at the level of both individual residues and domains, presumably as a result of varying selective constraints. To clarify the potential role of conserved sequence features, we predicted the structures of several gK orthologs. Congruent with our phylogenetic analysis, slowly evolving residues were identified at potentially structurally significant positions across domains. We found that using a quantitative measure of amino acid rate variation combined with molecular modeling we were able to identify amino acids predicted to be critical for gK protein structure/function. This analysis yields targets for the design of anti-herpesvirus therapeutic strategies across all alphaherpesvirus species that would be absent from more traditional analyses of conservation.

P3: Phylogenetic Posterior Prediction in RevBayes.

Tests of absolute model fit are crucial in model-based inference because poorly structured models can lead to biased parameter estimates. In Bayesian inference, posterior predictive simulations can be used to test absolute model fit. However, such tests have not been commonly practiced in phylogenetic inference due to a lack of convenient and flexible software. Here, we describe our newly implemented tests of model fit using posterior predictive testing, based on both data- and inference-based test statistics, in the phylogenetics software RevBayes. This new implementation makes a large spectrum of models available for use through a user-friendly and flexible interface.

Short Communication: Lack of Support for Socially Connected HIV-1 Transmission Among Young Adult Black Men Who Have Sex with Men.

We explore the phylogenetic relationships among HIV sequences sampled from young adult black men who have sex with men (YAB-MSM), who are connected through peer referral/social ties and who attend common venues. Using 196 viral sequences sampled from the peripheral blood mononuclear cells of 10 individuals, our preliminary phylogenetic results indicate that these socially connected YAB-MSM are infected with distantly related viruses and provide no evidence for viral transmission between network members. Our results suggest that HIV-prevention strategies that target young adult MSM should extend beyond their network members and local community.

Synthesis of phylogeny and taxonomy into a comprehensive tree of life.

Reconstructing the phylogenetic relationships that unite all lineages (the tree of life) is a grand challenge. The paucity of homologous character data across disparately related lineages currently renders direct phylogenetic inference untenable. To reconstruct a comprehensive tree of life, we therefore synthesized published phylogenies, together with taxonomic classifications for taxa never incorporated into a phylogeny. We present a draft tree containing 2.3 million tips-the Open Tree of Life. Realization of this tree required the assembly of two additional community resources: (i) a comprehensive global reference taxonomy and (ii) a database of published phylogenetic trees mapped to this taxonomy. Our open source framework facilitates community comment and contribution, enabling the tree to be continuously updated when new phylogenetic and taxonomic data become digitally available. Although data coverage and phylogenetic conflict across the Open Tree of Life illuminate gaps in both the underlying data available for phylogenetic reconstruction and the publication of trees as digital objects, the tree provides a compelling starting point for community contribution. This comprehensive tree will fuel fundamental research on the nature of biological diversity, ultimately providing up-to-date phylogenies for downstream applications in comparative biology, ecology, conservation biology, climate change, agriculture, and genomics.

Next generation phylogeography of cave and surface Astyanax mexicanus.

The loss of traits is a commonly observed evolutionary pattern in cave organisms, but due to extensive morphological convergence, inferring relationships between cave and surface populations can be difficult. For instance, Astyanax mexicanus (the blind Mexican cavefish) is thought to have repeatedly lost its eyes following colonization of cave environments, but the number of evolutionarily independent invasions of this species into caves remains unclear. Because of these repeated losses, it has become a model organism for studying the genetic basis of phenotypic trait loss. Here we reconstruct a high-resolution phylogeography for A. mexicanus inferred from both mitochondrial DNA and several thousand single nucleotide polymorphisms. We provide novel insight into the origin of cave populations from the Sabinos and Río Subterráneo caves and present evidence that the Sabinos cave population is part of a unique cave lineage unrelated to other A. mexicanus cave populations. Our results indicate A. mexicanus cave populations have at least four independent origins.