An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease.

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.

Abnormal Appearance of the Area Centralis in Labrador Retrievers With an ABCA4 Loss-of-function Mutation.

PURPOSE: To study retinal appearance and morphology in Labrador retrievers (LRs) heterozygous and homozygous for an ABCA4 loss-of-function mutation. METHODS: Ophthalmic examination, including ophthalmoscopy and simple testing of vision, was performed in five ABCA4wt/wt, four ABCA4wt/InsC, and six ABCA4InsC/InsC LRs. Retinas were also examined with confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT). Infrared and fundus autofluorescence (FAF) images were studied, and outer nuclear layer (ONL) and neuroretinal thickness were measured in the central and peripheral area centralis. RESULTS: Clinical signs in young ABCA4InsC/InsC LRs were subtle, whereas ophthalmoscopic findings and signs of visual impairment were obvious in old ABCA4InsC/InsC LRs. Retinal appearance and vision testing was unremarkable in heterozygous LRs regardless of age. The cSLO/OCT showed abnormal morphology including ONL thinning, abnormal outer retinal layer segmentation, and focal loss of retinal pigment epithelium in the fovea equivalent in juvenile ABCA4InsC/InsC LRs. The abnormal appearance extended into the area centralis and visual streak in middle-aged ABCA4InsC/InsC and then spread more peripherally. A mild phenotype was seen on cSLO/OCT and FAF in middle-aged to old ABCA4wt/InsC LRs. CONCLUSIONS: Abnormal appearance and morphology in the fovea equivalent are present in juvenile ABCA4InsC/InsC. In the older affected LRs, the visual streak and then the peripheral retina also develop an abnormal appearance. Vision deteriorates slowly, but some vision is retained throughout life. Older heterozygotes may show a mild retinal phenotype but no obvious visual impairment. The ABCA4InsC/InsC LR is a potential model for ABCA4-mediated retinopathies/juvenile-onset Stargardt disease in a species with human-sized eyes. TRANSLATIONAL RELEVANCE: The ABCA4InsC mutation causes juvenile-onset abnormal appearance of the fovea equivalent in affected dogs that slowly spreads in the retina, while only a mild phenotype is seen in older carriers. This is the first non-primate, large-animal model for ABCA4-related/STGD1 retinopathies in a species with a fovea equivalent.

A novel canine reference genome resolves genomic architecture and uncovers transcript complexity.

We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. Annotation with generated and existing long and short read RNA-seq, miRNA-seq and ATAC-seq, revealed that 32.1% of lifted over CanFam3.1 gaps harboured previously hidden functional elements, including promoters, genes and miRNAs in GSD_1.0. A catalogue of canine "dark" regions was made to facilitate mapping rescue. Alignment in these regions is difficult, but we demonstrate that they harbour trait-associated variation. Key genomic regions were completed, including the Dog Leucocyte Antigen (DLA), T Cell Receptor (TCR) and 366 COSMIC cancer genes. 10x linked-read sequencing of 27 dogs (19 breeds) uncovered 22.1 million SNPs, indels and larger structural variants. Subsequent intersection with protein coding genes showed that 1.4% of these could directly influence gene products, and so provide a source of normal or aberrant phenotypic modifications.

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole.

BACKGROUND: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. METHODS: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. RESULTS: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. CONCLUSIONS: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

Deletion in the Bardet-Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs.

In golden retriever dogs, a 1 bp deletion in the canine TTC8 gene has been shown to cause progressive retinal atrophy (PRA), the canine equivalent of retinitis pigmentosa. In humans, TTC8 is also implicated in Bardet-Biedl syndrome (BBS). To investigate if the affected dogs only exhibit a non-syndromic PRA or develop a syndromic ciliopathy similar to human BBS, we recruited 10 affected dogs to the study. The progression of PRA for two of the dogs was followed for 2 years, and a rigorous clinical characterization allowed a careful comparison with primary and secondary characteristics of human BBS. In addition to PRA, the dogs showed a spectrum of clinical and morphological signs similar to primary and secondary characteristics of human BBS patients, such as obesity, renal anomalies, sperm defects, and anosmia. We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-length TTC8 transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet-Biedl syndrome with heterogeneous clinical signs.

Genome-wide analysis of chimpanzee genes with premature termination codons.

BACKGROUND: Premature termination codons (PTCs) cause mRNA degradation or a truncated protein and thereby contribute to the transcriptome and proteome divergence between species. Here we present the first genome-wide study of PTCs in the chimpanzee. By comparing the human and chimpanzee genome sequences we identify and characterize genes with PTCs, in order to understand the contribution of these mutations to the transcriptome diversity between the species. RESULTS: We have studied a total of 13,487 human-chimpanzee gene pairs and found that ~8% were affected by PTCs in the chimpanzee. A majority (764/1,109) of PTCs were caused by insertions or deletions and the remaining part was caused by substitutions. The distribution of PTC genes varied between chromosomes, with Y having the highest proportion. Furthermore, the density of PTC genes varied on a megabasepair scale within chromosomes and we found the density to be correlated both with indel divergence and proximity to the telomere. Within genes, PTCs were more common close to the 5' and 3' ends of the amino acid sequence. Gene Ontology classification revealed that olfactory receptor genes were over represented among the PTC genes. CONCLUSION: Our results showed that the density of PTC genes fluctuated across the genome depending on the local genomic context. PTCs were preferentially located in the terminal parts of the transcript, which generally have a lower frequency of functional domains, indicating that selection was operating against PTCs at sites central to protein function. The enrichment of GO terms associated with olfaction suggests that PTCs may have influenced the difference in the repertoire of olfactory genes between humans and chimpanzees. In summary, 8% of the chimpanzee genes were affected by PTCs and this type of variation is likely to have an important effect on the transcript and proteomic divergence between humans and chimpanzees.

Resistance to pyrantel embonate and efficacy of fenbendazole in Parascaris univalens on Swedish stud farms.

The aims of this study were to determine the species of Parascaris present in foals in Sweden and to establish whether anthelmintic resistance to pyrantel and fenbendazole is present on Swedish stud farms. Ascarid eggs collected from different regions in Sweden were karyotyped and were all identified as Parascaris univalens, characterized by one chromosomal pair. Faecal egg count reduction tests were performed on a total of 142 foals on 9 farms between September 2016 and May 2017. Healthy foals with at least 150 eggs per gram faeces (EPG) were included in the study and treated with oral pastes of pyrantel embonate or fenbendazole according to manufacturer instructions. The efficacy of the drugs was calculated by a Bayesian model using the R package "eggCounts". In accordance with the American Association of Equine Practitioners, parasites were classified as resistant to pyrantel if the reduction in EPG was ≤ 85% and to fenbendazole if the observed efficacy was ≤ 90%. Four of eleven groups treated with pyrantel had an observed efficacy of ≤ 85%, and as many as 43% of the foals treated with pyrantel excreted eggs 10-16 days after treatment. In contrast, one of the six groups treated with fenbendazole had an observed efficacy of ≤ 90%, and only 6% of all foals were excreting eggs 10-16 days after treatment. Since resistance to ivermectin has earlier been shown to be widespread in Parascaris spp. in Sweden it is likely that multiresistant populations are present on Swedish stud farms. This is the first study showing the existence of pyrantel-resistant Parascaris spp. in Europe, and the first ever study where anthelmintic resistance has been shown in P. univalens.

Genomic structure of the horse major histocompatibility complex class II region resolved using PacBio long-read sequencing technology.

The mammalian Major Histocompatibility Complex (MHC) region contains several gene families characterized by highly polymorphic loci with extensive nucleotide diversity, copy number variation of paralogous genes, and long repetitive sequences. This structural complexity has made it difficult to construct a reliable reference sequence of the horse MHC region. In this study, we used long-read single molecule, real-time (SMRT) sequencing technology from Pacific Biosciences (PacBio) to sequence eight Bacterial Artificial Chromosome (BAC) clones spanning the horse MHC class II region. The final assembly resulted in a 1,165,328 bp continuous gap free sequence with 35 manually curated genomic loci of which 23 were considered to be functional and 12 to be pseudogenes. In comparison to the MHC class II region in other mammals, the corresponding region in horse shows extraordinary copy number variation and different relative location and directionality of the Eqca-DRB, -DQA, -DQB and -DOB loci. This is the first long-read sequence assembly of the horse MHC class II region with rigorous manual gene annotation, and it will serve as an important resource for association studies of immune-mediated equine diseases and for evolutionary analysis of genetic diversity in this region.

Whole-Genome Sequencing of a Canine Family Trio Reveals a FAM83G Variant Associated with Hereditary Footpad Hyperkeratosis.

Over 250 Mendelian traits and disorders, caused by rare alleles have been mapped in the canine genome. Although each disease is rare in the dog as a species, they are collectively common and have major impact on canine health. With SNP-based genotyping arrays, genome-wide association studies (GWAS) have proven to be a powerful method to map the genomic region of interest when 10-20 cases and 10-20 controls are available. However, to identify the genetic variant in associated regions, fine-mapping and targeted resequencing is required. Here we present a new approach using whole-genome sequencing (WGS) of a family trio without prior GWAS. As a proof-of-concept, we chose an autosomal recessive disease known as hereditary footpad hyperkeratosis (HFH) in Kromfohrländer dogs. To our knowledge, this is the first time this family trio WGS-approach has been used successfully to identify a genetic variant that perfectly segregates with a canine disorder. The sequencing of three Kromfohrländer dogs from a family trio (an affected offspring and both its healthy parents) resulted in an average genome coverage of 9.2X per individual. After applying stringent filtering criteria for candidate causative coding variants, 527 single nucleotide variants (SNVs) and 15 indels were found to be homozygous in the affected offspring and heterozygous in the parents. Using the computer software packages ANNOVAR and SIFT to functionally annotate coding sequence differences, and to predict their functional effect, resulted in seven candidate variants located in six different genes. Of these, only FAM83G:c155G > C (p.R52P) was found to be concordant in eight additional cases, and 16 healthy Kromfohrländer dogs.

Evaluation of whole-genome sequencing of four Chinese crested dogs for variant detection using the ion proton system.

BACKGROUND: Next generation sequencing (NGS) has traditionally been performed by large genome centers, but in recent years, the costs for whole-genome sequencing (WGS) have decreased substantially. With the introduction of smaller and less expensive "desktop" systems, NGS is now moving into the general laboratory. To evaluate the Ion Proton system for WGS we sequenced four Chinese Crested dogs and analyzed the data quality in terms of genome and exome coverage, the number of detected single nucleotide variants (SNVs) and insertions and deletions (INDELs) and the genotype concordance with the Illumina HD canine SNP array. For each of the four dogs, a 200 bp fragment library was constructed from genomic DNA and sequenced on two Ion PI chips per dog to reach mean coverage of 6-8x of the canine genome (genome size ≈ 2.4 Gb). RESULTS: On average, each Ion PI chip yielded approximately 73.3 million reads with a mean read length of 130 bp (~9.5 Gb sequence data) of which 98.5 % could be aligned to the canine reference genome (CanFam3.1). By sequencing a single dog using one fragment library and two Ion PI chips, on average 80 % of the genome and 77 % exome was covered by at least four reads. After removing duplicate reads (20.7 %) the mean coverage across the whole genome was 6x. Using sequence data from all four individuals (four fragment libraries and eight Ion PI chips) the genome and exome coverage could be further increased to 97.2 and 94.3 %, respectively. We detected 4.83 million unique SNPs and 6.10 million unique INDEL positions across all individuals. A comparison between SNP genotypes detected with the WGS and the 170 K Illumina HD canine SNP array showed 90 % concordance. CONCLUSIONS: We have evaluated whole-genome sequencing on the Ion Proton system for genetic variant detection in four Chinese crested dogs. Even though INDEL calling with Ion Proton data is challenging due to specific platform errors, in case of SNP calling it can serve as an alternative to other next-generation sequencing platforms and SNP genotyping arrays, in studies aiming to identify causative mutations for rare monogenic diseases. In addition, we have identified new genetic variants of the Chinese Crested dog that will contribute to further whole-genome sequencing studies aimed to identify mutations associated with monogenic diseases with autosomal recessive inheritance.