A SACS deletion variant in Great Pyrenees dogs causes autosomal recessive neuronal degeneration.

ARSACS (autosomal recessive spastic ataxia of Charlevoix-Saguenay) is a human neurological disorder characterized by progressive cerebellar ataxia and peripheral neuropathy. A recently recognized disorder in Great Pyrenees dogs is similarly characterized by widespread central nervous system degeneration leading to progressive cerebellar ataxia and spasticity, combined with peripheral neuropathy. Onset of clinical signs occurred in puppies as young as 4 months of age, with slow progression over several years. A multi-generation pedigree suggested an autosomal recessive mode of inheritance. Histopathology revealed consistent cerebellar Purkinje cell degeneration, neuronal degeneration in brainstem nuclei, widespread spinal cord white matter degeneration, ganglion cell degeneration, inappropriately thin myelin sheaths or fully demyelinated peripheral nerve fibers, and normal or only mild patterns of denervation atrophy in skeletal muscles. Genome-wide single nucleotide polymorphism (SNP) genotype data was collected from 6 cases and 26 controls, where homozygosity mapping identified a 3.3 Mb region on CFA25 in which all cases were homozygous and all controls were either heterozygous or homozygous for alternate haplotypes. This region tagged the SACS gene where variants are known to cause ARSACS. Sanger sequencing of SACS in affected dogs identified a 4 bp deletion that causes a frame shift and truncates 343 amino acids from the C terminus of the encoded sacsin protein (p.Val4244AlafsTer32). Our clinical and histopathological descriptions of this canine disorder contribute to the description of human ARSACS and represents the first naturally occurring large animal model of this disorder.

Exploration of fine-scale recombination rate variation in the domestic horse.

Total genetic map length and local recombination landscapes typically vary within and across populations. As a first step to understanding the recombination landscape in the domestic horse, we calculated population recombination rates and identified likely recombination hotspots using approximately 1.8 million SNP genotypes for 485 horses from 32 distinct breeds. The resulting breed-averaged recombination map spans 2.36 Gb and accounts for 2939.07 cM. Recombination hotspots occur once per 23.8 Mb on average and account for ∼9% of the physical map length. Regions with elevated recombination rates in the entire cohort were enriched for genes in pathways involving interaction with the environment: immune system processes (specifically, MHC class I and class II genes), responses to stimuli, and serotonin receptor pathways. We found significant correlations between differences in local recombination rates and population differentiation quantified by F ST Analysis of breed-specific maps revealed thousands of hotspot regions unique to particular breeds, as well as unique "coldspots," regions where a particular breed showed below-average recombination, whereas all other breeds had evidence of a hotspot. Finally, we identified relative enrichment (P = 5.88 × 10-27) for the in silico-predicted recognition motif for equine PR/SET domain 9 (PRDM9) in recombination hotspots. These results indicate that selective pressures and PRDM9 function contribute to variation in recombination rates across the domestic horse genome.

Identification and validation of genetic variants predictive of gait in standardbred horses.

Several horse breeds have been specifically selected for the ability to exhibit alternative patterns of locomotion, or gaits. A premature stop codon in the gene DMRT3 is permissive for "gaitedness" across breeds. However, this mutation is nearly fixed in both American Standardbred trotters and pacers, which perform a diagonal and lateral gait, respectively, during harness racing. This suggests that modifying alleles must influence the preferred gait at racing speeds in these populations. A genome-wide association analysis for the ability to pace was performed in 542 Standardbred horses (n = 176 pacers, n = 366 trotters) with genotype data imputed to ~74,000 single nucleotide polymorphisms (SNPs). Nineteen SNPs on nine chromosomes (ECA1, 2, 6, 9, 17, 19, 23, 25, 31) reached genome-wide significance (p < 1.44 x 10-6). Variant discovery in regions of interest was carried out via whole-genome sequencing. A set of 303 variants from 22 chromosomes with putative modifying effects on gait was genotyped in 659 Standardbreds (n = 231 pacers, n = 428 trotters) using a high-throughput assay. Random forest classification analysis resulted in an out-of-box error rate of 0.61%. A conditional inference tree algorithm containing seven SNPs predicted status as a pacer or trotter with 99.1% accuracy and subsequently performed with 99.4% accuracy in an independently sampled population of 166 Standardbreds (n = 83 pacers, n = 83 trotters). This highly accurate algorithm could be used by owners/trainers to identify Standardbred horses with the potential to race as pacers or as trotters, according to the genotype identified, prior to initiating training and would enable fine-tuning of breeding programs with designed matings. Additional work is needed to determine both the algorithm's utility in other gaited breeds and whether any of the predictive SNPs play a physiologically functional role in the tendency to pace or tag true functional alleles.

NME5 frameshift variant in Alaskan Malamutes with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a hereditary defect of motile cilia in humans and several domestic animal species. Typical clinical findings are chronic recurrent infections of the respiratory tract and fertility problems. We analyzed an Alaskan Malamute family, in which two out of six puppies were affected by PCD. The parents were unaffected suggesting autosomal recessive inheritance. Linkage and homozygosity mapping defined critical intervals comprising ~118 Mb. Whole genome sequencing of one case and comparison to 601 control genomes identified a disease associated frameshift variant, c.43delA, in the NME5 gene encoding a sparsely characterized protein associated with ciliary function. Nme5-/- knockout mice exhibit doming of the skull, hydrocephalus and sperm flagellar defects. The genotypes at NME5:c.43delA showed the expected co-segregation with the phenotype in the Alaskan Malamute family. An additional unrelated Alaskan Malamute with PCD and hydrocephalus that became available later in the study was also homozygous mutant at the NME5:c.43delA variant. The mutant allele was not present in more than 1000 control dogs from different breeds. Immunohistochemistry demonstrated absence of the NME5 protein from nasal epithelia of an affected dog. We therefore propose NME5:c.43delA as the most likely candidate causative variant for PCD in Alaskan Malamutes. These findings enable genetic testing to avoid the unintentional breeding of affected dogs in the future. Furthermore, the results of this study identify NME5 as a novel candidate gene for unsolved human PCD and/or hydrocephalus cases.

Correction to: Genomic diversity and population structure of the Leonberger dog breed.

An amendment to this paper has been published and can be accessed via the original article.

Genomic diversity and population structure of the Leonberger dog breed.

BACKGROUND: Leonberger is a giant dog breed formed in the 1850s in Germany. Its post-World War II popularity has resulted in a current global population of ~ 30,000 dogs. The breed has predispositions to neurodegenerative disorders and cancer, which is likely due in large part to limited genetic diversity. However, to date there is no scientific literature on the overall demography and genomic architecture of this breed. RESULTS: We assessed extensive pedigree records, SNP array genotype data, and whole-genome sequences (WGS) on 142,072, 1203 and 39 Leonberger dogs, respectively. Pedigree analyses identified 22 founder animals and revealed an apparent popular sire effect. The average pedigree-based inbreeding coefficient of 0.29 and average kinship of 0.31 show a dramatic loss of genetic diversity. The observed average life span decreased over time from 9.4 years in 1989 to 7.7 years in 2004. A global health survey confirmed a high prevalence of cancer and neurological disorders. Analysis of SNP-based runs of homozygosity (ROH) identified 125,653 ROH with an average length of 5.88 Mb, and confirmed an average inbreeding coefficient of 0.28. Genome-wide filtering of the WGS data revealed 28 non-protein-changing variants that were present in all Leonberger individuals and a list of 22 potentially pathogenic variants for neurological disorders of which 50% occurred only in Leonbergers and 50% occurred rarely in other breeds. Furthermore, one of the two mtDNA haplogroups detected was present in one dog only. CONCLUSIONS: The increasing size of the Leonberger population has been accompanied by a considerable loss of genetic diversity after the bottleneck that occurred in the 1940s due to the intensive use of popular sires resulting in high levels of inbreeding. This might explain the high prevalence of certain disorders; however, genomic data provide no evidence for fixed coding variants that explain these predispositions. The list of candidate causative variants for polyneuropathy needs to be further evaluated. Preserving the current genetic diversity is possible by increasing the number of individuals for breeding while restricting the number of litters per sire/dam. In addition, outcrossing would help optimize long-term genetic diversity and contribute to the sustainability and health of the population.

Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice.

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

A GJA9 frameshift variant is associated with polyneuropathy in Leonberger dogs.

BACKGROUND: Many inherited polyneuropathies (PN) observed in dogs have clinical similarities to the genetically heterogeneous group of Charcot-Marie-Tooth (CMT) peripheral neuropathies in humans. The canine disorders collectively show a variable expression of progressive clinical signs and ages of onset, and different breed prevalences. Previously in the Leonberger breed, a variant highly associated with a juvenile-onset PN was identified in the canine orthologue of a CMT-associated gene. As this deletion in ARHGEF10 (termed LPN1) does not explain all cases, PN in this breed may encompass variants in several genes with similar clinical and histopathological features. RESULTS: A genome-wide comparison of 173 k SNP genotypes of 176 cases, excluding dogs homozygous for the ARHGEF10 variant, and 138 controls, was carried out to detect further PN-associated variants. A single suggestive significant association signal on CFA15 was found. The genome of a PN-affected Leonberger homozygous for the associated haplotype was sequenced and variants in the 7.7 Mb sized critical interval were identified. These variants were filtered against a database of variants observed in 202 genomes of various dog breeds and 3 wolves, and 6 private variants in protein-coding genes, all in complete linkage disequilibrium, plus 92 non-coding variants were revealed. Five of the coding variants were predicted to have low or moderate effect on the encoded protein, whereas a 2 bp deletion in GJA9 results in a frameshift of high impact. GJA9 encodes connexin 59, a connexin gap junction family protein, and belongs to a group of CMT-associated genes that have emerged as important components of peripheral myelinated nerve fibers. The association between the GJA9 variant and PN was confirmed in an independent cohort of 296 cases and 312 controls. Population studies showed a dominant mode of inheritance, an average age of onset of approximately 6 years, and incomplete penetrance. CONCLUSIONS: This GJA9 variant represents a highly probable candidate variant for another form of PN in Leonberger dogs, which we have designated LPN2, and a new candidate gene for CMT disease. To date, approximately every third PN-diagnosed Leonberger dog can be explained by the ARHGEF10 or GJA9 variants, and we assume that additional genetic heterogeneity in this condition exists in the breed.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

An ARHGEF10 deletion is highly associated with a juvenile-onset inherited polyneuropathy in Leonberger and Saint Bernard dogs.

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10-10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3'-end of exon 17 and six nucleotides from the 5'-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3'-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.

Identification and validation of risk loci for osteochondrosis in standardbreds.

BACKGROUND: Osteochondrosis (OC), simply defined as a failure of endochondral ossification, is a complex disease with both genetic and environmental risk factors that is commonly diagnosed in young horses, as well as other domestic species. Although up to 50 % of the risk for developing OC is reportedly inherited, specific genes and alleles underlying risk are thus far completely unknown. Regions of the genome identified as associated with OC vary across studies in different populations of horses. In this study, we used a cohort of Standardbred horses from the U.S. (n = 182) specifically selected for a shared early environment (to reduce confounding factors) to identify regions of the genome associated with tarsal OC. Subsequently, putative risk variants within these regions were evaluated in both the discovery population and an independently sampled validation population of Norwegian Standardbreds (n = 139) with tarsal OC. RESULTS: After genome-wide association analysis of imputed data with information from >200,000 single nucleotide polymorphisms, two regions on equine chromosome 14 were associated with OC in the discovery cohort. Variant discovery in these and 30 additional regions of interest (including 11 from other published studies) was performed via whole-genome sequencing. 240 putative risk variants from 10 chromosomes were subsequently genotyped in both the discovery and validation cohorts. After correction for population structure, gait (trot or pace) and sex, the variants most highly associated with OC status in both populations were located within the chromosome 14 regions of association. CONCLUSIONS: The association of putative risk alleles from within the same regions with disease status in two independent populations of Standardbreds suggest that these are true risk loci in this breed, although population-specific risk factors may still exist. Evaluation of these loci in other populations will help determine if they are specific to the Standardbred breed, or to tarsal OC or are universal risk loci for OC. Further work is needed to identify the specific variants underlying OC risk within these loci. This is the first step towards the long-term goal of constructing a genetic risk model for OC that allows for genetic testing and quantification of risk in individuals.

Heritability of Recurrent Exertional Rhabdomyolysis in Standardbred and Thoroughbred Racehorses Derived From SNP Genotyping Data.

Recurrent exertional rhabdomyolysis (RER) in Thoroughbred and Standardbred racehorses is characterized by episodes of muscle rigidity and cell damage that often recur upon strenuous exercise. The objective was to evaluate the importance of genetic factors in RER by obtaining an unbiased estimate of heritability in cohorts of unrelated Thoroughbred and Standardbred racehorses. Four hundred ninety-one Thoroughbred and 196 Standardbred racehorses were genotyped with the 54K or 74K SNP genotyping arrays. Heritability was calculated from genome-wide SNP data with a mixed linear and Bayesian model, utilizing the standard genetic relationship matrix (GRM). Both the mixed linear and Bayesian models estimated heritability of RER in Thoroughbreds to be approximately 0.34 and in Standardbred racehorses to be approximately 0.45 after adjusting for disease prevalence and sex. To account for potential differences in the genetic architecture of the underlying causal variants, heritability estimates were adjusted based on linkage disequilibrium weighted kinship matrix, minor allele frequency and variant effect size, yielding heritability estimates that ranged between 0.41-0.46 (Thoroughbreds) and 0.39-0.49 (Standardbreds). In conclusion, between 34-46% and 39-49% of the variance in RER susceptibility in Thoroughbred and Standardbred racehorses, respectively, can be explained by the SNPs present on these 2 genotyping arrays, indicating that RER is moderately heritable. These data provide further rationale for the investigation of genetic mutations associated with RER susceptibility.

Genome-wide analysis reveals selection for important traits in domestic horse breeds.

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an F(ST)-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.

A high density SNP array for the domestic horse and extant Perissodactyla: utility for association mapping, genetic diversity, and phylogeny studies.

An equine SNP genotyping array was developed and evaluated on a panel of samples representing 14 domestic horse breeds and 18 evolutionarily related species. More than 54,000 polymorphic SNPs provided an average inter-SNP spacing of ∼43 kb. The mean minor allele frequency across domestic horse breeds was 0.23, and the number of polymorphic SNPs within breeds ranged from 43,287 to 52,085. Genome-wide linkage disequilibrium (LD) in most breeds declined rapidly over the first 50-100 kb and reached background levels within 1-2 Mb. The extent of LD and the level of inbreeding were highest in the Thoroughbred and lowest in the Mongolian and Quarter Horse. Multidimensional scaling (MDS) analyses demonstrated the tight grouping of individuals within most breeds, close proximity of related breeds, and less tight grouping in admixed breeds. The close relationship between the Przewalski's Horse and the domestic horse was demonstrated by pair-wise genetic distance and MDS. Genotyping of other Perissodactyla (zebras, asses, tapirs, and rhinoceros) was variably successful, with call rates and the number of polymorphic loci varying across taxa. Parsimony analysis placed the modern horse as sister taxa to Equus przewalski. The utility of the SNP array in genome-wide association was confirmed by mapping the known recessive chestnut coat color locus (MC1R) and defining a conserved haplotype of ∼750 kb across all breeds. These results demonstrate the high quality of this SNP genotyping resource, its usefulness in diverse genome analyses of the horse, and potential use in related species.

The American Quarter Horse: population structure and relationship to the thoroughbred.

A breed known for its versatility, the American Quarter Horse (QH), is increasingly bred for performance in specific disciplines. The impact of selective breeding on the diversity and structure of the QH breed was evaluated using pedigree analysis and genome-wide SNP data from horses representing 6 performance groups (halter, western pleasure, reining, working cow, cutting, and racing). Genotype data (36 037 single nucleotide polymorphisms [SNPs]) from 36 Thoroughbreds were also evaluated with those from the 132 performing QHs to evaluate the Thoroughbred's influence on QH diversity. Results showed significant population structure among all QH performance groups excepting the comparison between the cutting and working cow horses; divergence was greatest between the cutting and racing QHs, the latter of which had a large contribution of Thoroughbred ancestry. Significant coancestry and the potential for inbreeding exist within performance groups, especially when considering the elite performers. Relatedness within performance groups is increasing with popular sires contributing disproportionate levels of variation to each discipline. Expected heterozygosity, inbreeding, F ST, cluster, and haplotype analyses suggest these QHs can be broadly classified into 3 categories: stock, racing, and pleasure/halter. Although the QH breed as a whole contains substantial genetic diversity, current breeding practices have resulted in this variation being sequestered into subpopulations.

Evidence of positive selection for a glycogen synthase (GYS1) mutation in domestic horse populations.

A dominantly inherited gain-of-function mutation in the glycogen synthase (GYS1) gene, resulting in excess skeletal muscle glycogen, has been identified in more than 30 horse breeds. This mutation is associated with the disease Equine Polysaccharide Storage Myopathy Type 1, yet persists at high frequency in some breeds. Under historical conditions of daily work and limited feed, excess muscle glycogen may have been advantageous, driving the increase in frequency of this allele. Fine-scale DNA sequencing in 80 horses and genotype assays in 279 horses revealed a paucity of haplotypes carrying the mutant allele when compared with the wild-type allele. Additionally, we found increased linkage disequilibrium, measured by relative extended haplotype homozygosity, in haplotypes carrying the mutation compared with haplotypes carrying the wild-type allele. Coalescent simulations of Belgian horse populations demonstrated that the high frequency and extended haplotype associated with the GYS1 mutation were unlikely to have arisen under neutrality or due to population demography. In contrast, in Quarter Horses, elevated relative extended haplotype homozygosity was associated with multiple haplotypes and may be the result of recent population expansion or a popular sire effect. These data suggest that the GYS1 mutation underwent historical selection in the Belgian, but not in the Quarter Horse.

Haplotype diversity in the equine myostatin gene with focus on variants associated with race distance propensity and muscle fiber type proportions.

Two variants in the equine myostatin gene (MSTN), including a T/C SNP in the first intron and a 227-bp SINE insertion in the promoter, are associated with muscle fiber type proportions in the Quarter Horse (QH) and with the prediction of race distance propensity in the Thoroughbred (TB). Genotypes from these loci, along with 18 additional variants surrounding MSTN, were examined in 301 horses of 14 breeds to evaluate haplotype relationships and diversity. The C allele of intron 1 was found in 12 of 14 breeds at a frequency of 0.27; the SINE was observed in five breeds, but common in only the TB and QH (0.73 and 0.48 respectively). Haplotype data suggest the SINE insertion is contemporary to and arose upon a haplotype containing the intron 1 C allele. Gluteal muscle biopsies of TBs showed a significant association of the intron 1 C allele and SINE with a higher proportion of Type 2B and lower proportion of Type 1 fibers. However, in the Belgian horse, in which the SINE is not present, the intron 1 SNP was not associated with fiber type proportions, and evaluation of fiber type proportions across the Belgian, TB and QH breeds shows the significant effect of breed on fiber type proportions is negated when evaluating horses without the SINE variant. These data suggest the SINE, rather than the intron 1 SNP, is driving the observed muscle fiber type characteristics and is the variant targeted by selection for short-distance racing.

Multi-Allelic Mitochondrial DNA Deletions in an Adult Dog with Chronic Weakness, Exercise Intolerance and Lactic Acidemia.

(1) Background: An adult dog was presented to a board-certified veterinary neurologist for evaluation of chronic weakness, exercise intolerance and lactic acidemia. (2) Methods: A mitochondrial myopathy was diagnosed based on the histological and histochemical phenotype of numerous COX-negative muscle fibers. Whole-genome sequencing established the presence of multiple extended deletions in the mitochondrial DNA (mtDNA), with the highest prevalence between the 1-11 kb positions of the approximately 16 kb mitochondrial chromosome. Such findings are typically suggestive of an underlying nuclear genome variant affecting mitochondrial replication, repair, or metabolism. (3) Results: Numerous variants in the nuclear genome unique to the case were identified in the whole-genome sequence data, and one, the insertion of a DYNLT1 retrogene, whose parent gene is a regulator of the mitochondrial voltage-dependent anion channel (VDAC), was considered a plausible causal variant. (4) Conclusions: Here, we add mitochondrial deletion disorders to the spectrum of myopathies affecting adult dogs.

Variants in CLCN1 and PDE4C Associated with Muscle Hypertrophy, Dysphagia, and Gait Abnormalities in Young French Bulldogs.

(1) Background: Muscle hypertrophy, swallowing disorders, and gait abnormalities are clinical signs common to many muscle diseases, including muscular dystrophies, non-dystrophic myotonias, genetic myopathies associated with deficiency of myostatin, and acquired inflammatory myopathies. Here, we investigated underlying causes of this triad of clinical signs in four young French bulldogs via muscle histopathology coupled with whole genome and Sanger sequencing. (2) Methods: Dogs were evaluated by veterinary clinical internists and neurologists, and biopsies were obtained for histopathological diagnosis. DNA was submitted for whole genome sequencing, followed by bioinformatics evaluation and confirmation of variants via Sanger sequencing in two cases. (3) Results: Two novel variants were identified. The first, found in two related French bulldogs, was a homozygous variant in the chloride channel gene CLCN1 known to cause non-dystrophic congenital myotonia, and the second, found in an unrelated French bulldog, was a heterozygous variant in the cAMP phosphodiesterase gene PDE4C, which is the major phosphodiesterase expressed in skeletal muscle and may play a role in decreasing muscle atrophy. An underlying molecular basis in one other case has not yet been identified. (4) Conclusions: Here, we identified two novel variants, one in the CLCN1 and one in the PDE4C gene, associated with clinical signs of muscle hypertrophy, dysphagia, and gait abnormalities, and we suggested other bases of these phenotypes in French bulldogs that are yet to be discovered. Identification of genes and deleterious variants associated with these clinical signs may assist breeders in improving the overall health of this very popular breed and may lead to the identification of new therapies to reverse muscle atrophy in people and animals with neuromuscular diseases.