Åsa Ohlsson

Background: Haemonchus contortus is a major gastrointestinal parasite of small ruminants worldwide. Its large effective population size, high fecundity, and extensive genetic variation promote rapid adaptation and the development of anthelmintic resistance. Resolving genome-wide genetic variation in H. contortus within and among populations is essential for tracking variant distribution, interpreting resistance-associated markers, and informing parasite control strategies. Although reduced representation and targeted marker approaches have provided important insights into population structure, whole genome sequencing is required to fully characterise population differentiation. This study aimed to characterise genome-wide variation within and between field-derived H. contortus populations in Sweden, using whole genome sequencing of pools of individuals from 12 sheep farms. Results: Field-derived H. contortus isolates from Swedish farms showed high genomic diversity within populations (nucleotide diversity (Pi) = 0.0211–0.0253) and relatively low genomic differentiation among most populations (fixation index (F _ST ) < 0.1). However, three population pairs displayed higher genomic differentiation (F _ST = 0.144–0.157) combined with lower genomic nucleotide diversity (Pi = 0.0211–0.0227), with divergence concentrated on chromosomes 4, 5, and X. Significantly differentiated single nucleotide polymorphisms were linked to a limited set of annotated genes involved in regulatory, signalling, and nervous system-related functions. However, the biological basis of these patterns remains unclear and will require further investigation using phenotype-linked data and farm-management metadata. Conclusions: This work establishes the first genome-wide baseline for field-derived H. contortus populations across multiple Swedish sheep farms and shows that pooled-sample whole-genome sequencing (Pool-seq) is a useful method for describing broad population-genomic patterns in this species. Our findings indicate relatively low genomic variation between Swedish H. contortus populations and high genomic variation within them. This study provides a valuable foundation for future research on parasite adaptation, resistance, surveillance, and control.

The Animal Variant Classification Guidelines (AVCG) were developed to standardize and objectify the classification of putative disease‐causing variants. These guidelines are sufficiently reproducible and are used to classify previously published and new disease‐causing variants across species. Here, the guidelines are updated (AVCG.v2), based on a three‐phase decision process. Overall, four new criteria and seven clarifying comments were added. The number of criteria has increased from 23 to 27, with three new criteria supporting pathogenicity and one new criterion supporting benign classification. Pharmacogenomic variants were determined to fall within the scope of the guidelines. These updated guidelines are being used by the Variant Pathogenicity Working Group (VPWG), part of the Animal Genetic Testing Standardization standing committee, which is a committee of elected members of the International Society for Animal Genetics (ISAG). Under the auspices of ISAG, the VPWG retrospectively classifies published putative disease‐causing variants. The pathogenicity label for a variant will be presented in the variant tables of Online Mendelian Inheritance in Animals (OMIA; https://omia.org/ ). The AVCGv.2 criteria and recommendations were developed by the expertise of the animal genetics community and the ISAG Executive Committee through the Animal Genetics Testing Standardization Committee endorses and strongly encourages their use to evaluate the evidence supporting pathogenicity of putative disease‐causing variants.

Until recently, due to the absence of standardized guidelines tailored for veterinary use, the evaluation of genetic variant pathogenicity for single‐gene diseases was based on a personal interpretation of the presented evidence, which has led to inconsistencies. With the publication of the animal variant classification guidelines (AVCG), a more objective approach became available. Variants are evaluated by the International Society of Animal Genetics (ISAG)‐endorsed Variant Pathogenicity Working Group (VPWG) based on 23 criteria and are subsequently labeled as pathogenic, likely pathogenic, variant of uncertain significance, likely benign or benign. While the accuracy was thoroughly tested in the original publication, the reproducibility of the various steps involved was only briefly checked, which is why the current analysis was performed. Each variant from a set of 150 published likely causal variants for single‐gene diseases from three species (dog, cat, horse) was independently and blindly assessed by three different VPWG reviewers, each applying the same AVCG. An overall agreement of 93% for decisions on the scope, that is, whether they fit the inclusion criteria to allow evaluation with AVCG, was found. More importantly, the reproducibility of pathogenicity label assignment was 65% and the reproducibility of clinical relevance was 83%. The reproducibility of AVCG‐pathogenicity classification is in line with reports using the human American College for Medical Genetics and Genomics and Association for Molecular Pathology guidelines for human variants. Overall, the reproducibility of the AVCG classifications as used by the ISAG‐endorsed VPWG supports the utility of these classifications in veterinary species.

Circulating microRNAs (miRNAs) are potential biomarkers for numerous diseases. Characterization of the whole blood (WB) miRNA-transcriptome (miRNome) in cats is lacking, which limits the potential use of miRNAs as biomarkers for diseases such as feline cardiovascular disease. The aims of the present study were to profile and evaluate circulating miRNAs in feline WB by high-throughput sequencing of the total miRNome in WB from twelve domestic mixed breed (DOM) and Norwegian Forest (NFO) cats stringently diagnosed with or without preclinical hypertrophic cardiomyopathy (HCM). A total of 459 mature miRNAs were identified in feline WB, of which 40 were potential novel feline miRNAs. A majority, 85.3%, of the miRNAs showed sequence similarity with human miRNAs. An effect of breed was found, with up to thirteen WB miRNAs being differentially abundant between breeds. The majority of the significant breed-specific miRNAs in feline WB could be associated with regulation of haematopoietic cells. One miRNA, miR-204-5p, was potentially associated with preclinical HCM in NFO cats, but the results need to be confirmed in a larger and sex-unbiased cohort. In conclusion, here we used miRNome-sequencing to identify hundreds of circulating miRNAs in feline WB. Breed should be considered when evaluating the miRNome in feline WB.

Background and aims: The heart is a metabolic organ rich in mitochondria. The failing heart reprograms to utilize different energy substrates, which increase its oxygen consumption. These adaptive changes contribute to increased oxidative stress. Hypertrophic cardiomyopathy (HCM) is a common heart condition, affecting approximately 15% of the general cat population. Feline HCM shares phenotypical and genotypical similarities with human HCM, but the disease mechanisms for both species are incompletely understood. Our goal was to characterize global changes in metabolome between healthy control cats and cats with different stages of HCM. Methods: Serum samples from 83 cats, the majority (70/83) of which were domestic shorthair and included 23 healthy control cats, 31 and 12 preclinical cats with American College of Veterinary Internal Medicine (ACVIM) stages B1 and B2, respectively, and 17 cats with history of clinical heart failure or arterial thromboembolism (ACVIM stage C), were collected for untargeted metabolomic analysis. Multiple linear regression adjusted for age, sex and body weight was applied to compare between control and across HCM groups. Results: Our study identified 1253 metabolites, of which 983 metabolites had known identities. Statistical analysis identified 167 metabolites that were significantly different among groups (adjusted P < 0.1). About half of the differentially identified metabolites were lipids, including glycerophospholipids, sphingolipids and cholesterol. Serum concentrations of free fatty acids, 3-hydroxy fatty acids and acylcarnitines were increased in HCM groups compared with control group. The levels of creatine phosphate and multiple Krebs cycle intermediates, including succinate, aconitate and α-ketoglutarate, also accumulated in the circulation of HCM cats. In addition, serum levels of nicotinamide and tryptophan, precursors for de novo NAD+ biosynthesis, were reduced in HCM groups versus control group. Glutathione metabolism was altered. Serum levels of cystine, the oxidized form of cysteine and cysteine-glutathione disulfide, were elevated in the HCM groups, indicative of heightened oxidative stress. Further, the level of ophthalmate, an endogenous glutathione analog and competitive inhibitor, was increased by more than twofold in HCM groups versus control group. Finally, several uremic toxins, including guanidino compounds and protein bound putrescine, accumulated in the circulation of HCM cats. Conclusions: Our study provided evidence of deranged energy metabolism, altered glutathione homeostasis and impaired renal uremic toxin excretion. Altered lipid metabolism suggested perturbed structure and function of cardiac sarcolemma membrane and lipid signalling.

Feline cardiomyopathy (FCM) is an important contributor to feline morbidity and mortality. This explorative follow-up questionnaire study was aimed at investigating the long-term outcome in cats breed-screened for FCM (BS-FCM) in three Nordic countries. Records of cats with ≥1 BS-FCM between 2004–2015 were included. Of the 1113 included cats, 104/1113 (9.3%) had developed FCM at some time-point. Fifty-nine of the 104 (56.7%) FCM cats were diagnosed within the screening program (Screen^FCM), and 33/59 (55.9%) of these were diagnosed at the first BS-FCM. Screen^FCM cats or with an owner-reported FCM diagnosis at a later time-point had a higher risk of cardiac-related death compared to cats that never developed FCM. A shorter lifespan was found in Screen^FCM cats compared to those with normal screen results (p < 0.001). Times to all-cause mortality were shorter (p < 0.001) in cats that developed FCM at any time-point compared to those that did not. Non-cardiac morbidities were similar in all screen classification groups. The large proportion of cats that developed FCM at a later time-point underscores the need for repeated screenings later in life. Cats that developed FCM at any time-point had a shorter lifespan, with a similar proportion and in line with the nature of non-cardiac morbidities, compared to those without FCM.

Circulating microRNAs (miRNAs) are potential biomarkers for numerous diseases. Characterization of the whole blood (WB) miRNA-transcriptome (miRNome) in cats is lacking, which limits the potential use of miRNAs as biomarkers for i.e. feline heart disease. The aims of the present study were to identify and evaluate circulating miRNAs in feline WB by high-throughput sequencing of the total miRNome in WB from twelve cats, representing domestic mixed breed (DOM) and Norwegian Forest (NFO) cats with or without preclinical hypertrophic cardiomyopathy (HCM). A total of 459 mature miRNAs were identified in feline WB, of which 40 were identified as potential novel miRNAs for cat. A majority, 85.3%, of the miRNAs showed sequence similarity with human miRNAs. An effect of breed was found, with up to thirteen miRNAs being differentially expressed between breeds. One miRNA, miR-204-5p, was potentially associated with preclinical HCM in NFO cats, but needs to be confirmed in a larger and sex-unbiased cohort. Most of the identified miRNAs in feline WB can be associated with regulation of haematopoietic cells. In conclusion, miRNome-sequencing can identify hundreds of circulating miRNAs in feline WB and breed should be considered when evaluating the miRNome in feline WB, especially in cohorts including preclinical patients.

Hypertrophic cardiomyopathy (HCM) is a common and potentially fatal heart disease in many cat breeds. An intronic variant in TNNT2, c.95‐108G>A, was recently reported as the cause of HCM in the Maine Coon. The aim of this study was to determine this variant’s allele frequency in different populations and its possible association with HCM. Based on 160 Maine Coon samples collected in Belgium, Italy, Sweden and the USA, the variant’s allele frequency was estimated to be 0.32. Analysis of the 99 Lives feline whole genome sequencing database showed that the TNNT2 variant also occurs in other breeds, as well as mixed‐breed cats. Comparison of 31 affected and 58 healthy cats did not reveal significantly increased odds for HCM in homozygotes. Based on the combined evidence and in agreement with the standards and guidelines for the interpretation of sequence variants, this variant is currently classified as a variant of unknown significance and should not be used for breeding decisions regarding HCM.

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a ~10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs–the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specificNEBLgene in a dachshund population and show that two of theNEBLcandidate variants have regulatory potential in heart-derived cell lines and are associated with reducedNEBLisoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity.