Identification of the mutation causing progressive retinal atrophy in Old Danish Pointing Dog

Progressive retinal atrophy (PRA) is a common cause of blindness in many dog breeds. It is most often inherited as a simple Mendelian trait, but great genetic heterogeneity has been demonstrated both within and between breeds. In many breeds the genetic cause of the disease is not known, and until now, the Old Danish Pointing Dog (ODP) has been one of those breeds. ODP is one of the oldest dog breeds in Europe. Seventy years ago the breed almost vanished, but today a population still exists, primarily in Denmark but with some dogs in Germany and Sweden. PRA has been diagnosed in ODP since the late 1990s. It resembles late onset PRA in other dog breeds, and it is inherited as an autosomal recessive trait. In the present study, we performed whole‐genome sequencing and identified a single base insertion (c.3149_3150insC) in exon 1 of C17H2orf71. This is the same mutation previously found to cause PRA in Gordon Setters and Irish Setters, and it was later found in Tibetan Terrier, Standard Poodle and the Polski Owczarek Nizinny. The presence of the mutation in such a diverse range of breeds indicates an origin preceding creation of modern dog breeds. Hence, we screened 262 dogs from 44 different breeds plus four crossbred dogs, and can subsequently add Miniature Poodle and another polish sheepdog, the Polski Owczarek Podhalanski, to the list of affected breeds.     

Progressive retinal atrophy in Shetland sheepdog is associated with a mutation in the CNGA1 gene

Progressive retinal atrophy (PRA) is the collective name of a class of hereditary retinal dystrophies in the dog and is often described as the equivalent of retinitis pigmentosa in humans. PRA is characterized by visual impairment due to degeneration of the photoreceptors in the retina, usually leading to blindness. PRA has been reported in dogs from more than 100 breeds and can be genetically heterogeneous both between and within breeds. The disease can be subdivided by age at onset and rate of progression. Using genome‐wide association with 15 Shetland Sheepdog (Sheltie) cases and 14 controls, we identified a novel PRA locus on CFA13 (P_raw = 8.55 × 10^?7, P_genome = 1.7 × 10^?4). CNGA1, which is known to be involved in human cases of retinitis pigmentosa, was located within the associated region and was considered a likely candidate gene. Sequencing of this gene identified a 4‐bp deletion in exon 9 (c.1752_1755delAACT), leading to a frameshift and a premature stop codon. The study indicated genetic heterogeneity as the mutation was present in all PRA‐affected individuals in one large family of Shelties, whereas some other cases in the studied Sheltie population were not associated with this CNGA1 mutation. To our knowledge, this is the first report of a mutation in CNGA1 causing PRA in dogs.     

An Intronic SINE Insertion in FAM161A that Causes Exon-Skipping Is Associated with Progressive Retinal Atrophy in Tibetan Spaniels and Tibetan Terriers

Progressive retinal atrophy (PRA) in dogs is characterised by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds and is known to be genetically heterogeneous between breeds. Around 19 mutations have now been identified that are associated with PRA in around 49 breeds, but for the majority of breeds the mutation(s) responsible have yet to be identified. Using genome-wide association with 22 Tibetan Spaniel PRA cases and 10 controls, we identified a novel PRA locus, PRA3, on CFA10 (p_raw = 2.01×10⁻⁵, p_genome = 0.014), where a 3.8 Mb region was homozygous within 12 cases. Using targeted next generation sequencing, a short interspersed nuclear element insertion was identified near a splice acceptor site in an intron of a provocative gene, FAM161A. Analysis of mRNA from an affected dog revealed that the SINE causes exon skipping, resulting in a frame shift, leading to a downstream premature termination codon and possibly a truncated protein product. This mutation segregates with the disease in 22 out of 35 cases tested (63%). Of the PRA controls, none are homozygous for the mutation, 15% carry the mutation and 85% are homozygous wildtype. This mutation was also identified in Tibetan Terriers, although our results indicate that PRA is genetically heterogeneous in both Tibetan Spaniels and Tibetan Terriers.     

A CNGB1 Frameshift Mutation in Papillon and Phalène Dogs with Progressive Retinal Atrophy

Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA) or degeneration resembles human retinitis pigmentosa (RP) and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD) of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (P_Fisher = 1.4×10⁻⁸) with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes.     

A frameshift mutation in golden retriever dogs with progressive retinal atrophy endorses SLC4A3 as a candidate gene for human retinal degenerations

Progressive retinal atrophy (PRA) in dogs, the canine equivalent of retinitis pigmentosa (RP) in humans, is characterised by vision loss due to degeneration of the photoreceptor cells in the retina, eventually leading to complete blindness. It affects more than 100 dog breeds, and is caused by numerous mutations. RP affects 1 in 4000 people in the Western world and 70% of causal mutations remain unknown. Canine diseases are natural models for the study of human diseases and are becoming increasingly useful for the development of therapies in humans. One variant, prcd-PRA, only accounts for a small proportion of PRA cases in the Golden Retriever (GR) breed. Using genome-wide association with 27 cases and 19 controls we identified a novel PRA locus on CFA37 (p_raw = 1.94×10⁻¹⁰, p_genome = 1.0×10⁻⁵), where a 644 kb region was homozygous within cases. A frameshift mutation was identified in a solute carrier anion exchanger gene (SLC4A3) located within this region. This variant was present in 56% of PRA cases and 87% of obligate carriers, and displayed a recessive mode of inheritance with full penetrance within those lineages in which it segregated. Allele frequencies are approximately 4% in the UK, 6% in Sweden and 2% in France, but the variant has not been found in GRs from the US. A large proportion of cases (approximately 44%) remain unexplained, indicating that PRA in this breed is genetically heterogeneous and caused by at least three mutations. SLC4A3 is important for retinal function and has not previously been associated with spontaneously occurring retinal degenerations in any other species, including humans.     

Identical mutation in a novel retinal gene causes progressive rod-cone degeneration in dogs and retinitis pigmentosa in humans

Progressive rod-cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and a 53-aa protein in the mouse; the first 24 aa, coded for by exon 1, are highly conserved in 14 vertebrate species. A homozygous mutation (TGC --> TAC) in the second codon shows complete concordance with the disorder in 18 different dog breeds/breed varieties tested. The same homozygous mutation was identified in a human patient from Bangladesh with autosomal recessive RP. Expression studies support the predominant expression of this gene in the retina, with equal expression in the retinal pigment epithelium, photoreceptor, and ganglion cell layers. This study provides strong evidence that a mutation in the novel gene PRCD is the cause of autosomal recessive retinal degeneration in both dogs and humans.     

A second KRT71 allele in curly coated dogs

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant ¹⁵¹Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein‐changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C‐terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs.     

Canine models of ocular disease: outcross breedings define a dominant disorder present in the English mastiff and bull mastiff dog breeds

Progressive retinal atrophies (PRA) are a heterogeneous group of inherited eye diseases common to both dogs and man. Over 100 individual canine breeds display some sort of retinal degeneration, making the dog an extremely valuable resource both for finding the genetic determinants of inherited blindness and for developing naturally occurring animal models that mimic human disease. Progressive retinal atrophies within the English mastiff displayed an ambiguous mode of inheritance. By conducting outcross matings between affected English mastiffs and normal animals from other breeds, the mode of inheritance was confirmed as dominant. This directed candidate gene analysis and led to identification of two synonymous mutations and one nonsynonymous mutation within the canine rhodopsin gene. The nonsynonymous mutation (T4R) is the cause of PRA in the English mastiff, and a test was developed to investigate its presence in 17 additional breeds. Testing of PRA-affected animals from 16 breeds revealed that none carry the T4R mutation, indicating a different cause of PRA. Analysis of two affected bull mastiffs revealed one heterozygote (+/T4R) and one homozygous normal individual (+/+). These findings suggest that the genetic origin of PRA is often breed specific and underline the value of outcross mating to circumvent problems that act to mask the mode of inheritance.     

Reverse genetic screen for loss‐of‐function mutations uncovers a frameshifting deletion in the melanophilin gene accountable for a distinctive coat color in Belgian Blue cattle

In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10‐bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large‐scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild‐type allele at the co‐dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named ‘cool gray’. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.     

Allelic heterogeneity of FGF5 mutations causes the long‐hair phenotype in dogs

Hitherto, the only known mutant gene leading to the long‐hair phenotype in mammals is the fibroblast growth factor 5 (FGF5). In many dog breeds, the previously discovered FGF5:p.Cys95Phe mutation appeared completely concordant with the long‐hair phenotype, but for some breeds, the long‐hair phenotype could not be resolved. First, we studied the role of the FGF5:p.Cys95Phe and FGF5:g.145_150dupACCAGC mutations in 268 dogs descending from 27 breeds and seven wolves. As these mutations did not explain all the long‐hair phenotypes, all exons and their neighbouring regions of FGF5 were re‐sequenced. We detected three novel mutations in the coding sequence and one novel non‐coding splice‐site mutation in FGF5 associated with the long‐hair phenotype. The FGF5:p.Ala193Val polymorphism was perfectly consistent with long hair in Akitas and probably in Siberian huskies, too. Dogs of the long‐hair breed Samoyed were either homozygous or compound heterozygous for the FGF5:p.Ala193Val or the FGF5:p.Cys95Phe polymorphisms respectively. The two newly detected polymorphisms FGF5:c.559_560dupGG and FGF5:g.8193T>A and the known mutation FGF5:p.Cys95Phe explained the long‐hair phenotype of all Afghan hounds analysed. An FGF5:c.556_571del16 mutation was found in one longhaired Eurasier. All long‐hair‐associated mutations follow a recessive mode of inheritance, and allelic heterogeneity was a common finding in breeds other than Akita.     

Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166^∗]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156−2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156−2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.     

Explicit evidence for a missense mutation in exon 4 of SLC45A2 gene causing the pearl coat dilution in horses

Four loci seem responsible for the dilution of the basic coat colours in horse: Dun (D), Silver Dapple (Z), Champagne (CH) and Cream (C). Apart from the current phenotypes ascribed to these loci, pearl has been described as yet another diluted coat colour in this species. To date, this coat colour seems to segregate only in the Iberian breeds Purebred Spanish horse and Lusitano and has also been described in breeds of Iberian origin, such as Quarter Horses and Paint Horse, where it is referred to as the ‘Barlink Factor’. This phenotype segregates in an autosomal recessive manner and resembles some of the coat colours produced by the champagne CH^CH and cream C^Cr alleles, sometimes being difficult to distinguish among them. The interaction between compound heterozygous for the pearl C^prl and cream C^Cr alleles makes SLC45A2 the most plausible candidate gene for the pearl phenotype in horses. Our results provide documented evidence for the missense variation in exon 4 [SLC45A2:c.985G>A; SLC45A2:p.(Ala329Thr)] as the causative mutation for the pearl coat colour. In addition, it is most likely involved as well in the cremello, perlino and smoky cream like phenotypes associated with the compound C^Cr and C^prl heterozygous genotypes (known as cream pearl in the Purebred Spanish horse breed). The characterization of the pearl mutation allows breeders to identify carriers of the C^prl allele and to select this specific coat colour according to personal preferences, market demands or studbook requirements as well as to verify segregation within particular pedigrees.     

DRD4 and TH gene polymorphisms are associated with activity,impulsivity and inattention in Siberian Husky dogs

Both dopamine receptor D4 (DRD4) exon 3 and tyrosine hydroxylase (TH) intron 4 repeat polymorphisms have been linked to activity and impulsivity in German Shepherd dogs (GSDs). However, the results in GSDs may not be generalisable to other breeds, as allelic frequencies vary markedly among breeds. We selected the Siberian Husky for further study, because it is highly divergent from most dog breeds, including the GSD. The study sample consisted of 145 racing Siberian Huskies from Europe and North America. We found that this breed possesses seven DRD4 length variants, two to five more variants than found in other breeds. Among them was the longest known allele, previously described only in wolves. Short alleles of the DRD4 and TH repeat polymorphisms were associated with higher levels of activity, impulsivity and inattention. Siberian Huskies possessing at least one short allele of the DRD4 polymorphism displayed greater activity in a behavioural test battery than did those with two long alleles. However, the behavioural test was brief and may not have registered variation in behaviour across time and situations. Owners also completed the Dog‐Attention Deficit Hyperactivity Disorder Rating Scale (Dog‐ADHD RS), a more general measure of activity and attention. Siberian Huskies from Europe with two short alleles of the TH polymorphism received higher ratings of inattention on the Dog‐ADHD RS than did those with the long allele. Investigation of the joint effect of DRD4 and TH showed that dogs possessing long alleles at both sites were scored as less active–impulsive than were others. Our results are aligned with previous studies showing that DRD4 and TH polymorphisms are associated with activity–impulsivity related traits in dogs. However, the prevalence of variants of these genes differs across breeds, and the functional role of specific variants is unclear.     

Ultrasonographic evaluation of the supraspinous ligament in a series of ridden and unridden horses and horses with unrelated back pathology

Background

Dogs have the second largest number of genetic diseases, after humans. Among the diseases present in dogs, progressive retinal atrophy has been reported in more than a hundred breeds. In some of them, the mutation has been identified and genetic tests have allowed the identification of carriers, thus enabling a drastic reduction in the incidence of the disease. The Finnish lapphund is a dog breed presenting late-onset progressive retinal atrophy for which the disease locus remains unknown.

Results

In this study we mapped the progressive retinal atrophy locus in the Finnish lapphund using a DNA pooling approach, assuming that all affected dogs within the breed share the same identical-by descent-mutation as the cause of the disease (genetic homogeneity). Autosomal recessive inheritance was also assumed, after ruling out, from pedigree analysis, dominant and X-linked inheritance. DNA from 12 Finnish lapphund cases was mixed in one pool, and DNA from 12 first-degree relatives of these cases was mixed to serve as the control pool. The 2 pools were tested with 133 microsatellite markers, 3 of which showed a shift towards homozygosity in the cases. Individual genotyping with these 3 markers confirmed homozygosity for the GALK1 microsatellite only (chromosome 9). Further individual genotyping with additional samples (4 cases and 59 controls) confirmed the association between this marker and the disease locus (p < 0.001). Closely related to this breed are the Swedish lapphund and the Lapponian herder for which a small number of retinal atrophy cases have been reported. Swedish lapphund cases, but not Lapponian herder cases, had the same GALK1 microsatellite genotype as Finnish lapphund cases.

Conclusion

The locus for progressive rod-cone degeneration is known to be close to the GALK1 locus, on the telomeric region of chromosome 9, where the retinal atrophy locus of the Finnish lapphund has been mapped. This suggests that the disease in this breed, as well as in the Swedish lapphund, may correspond to progressive rod-cone degeneration. This would increase the number of known dog breeds having this particular form of progressive retinal atrophy.     

Not another type of potato: MC1R and the russet coloration of Burmese cats

The Burmese is a breed of domestic cat that originated in Southeast Asia and was further developed in the United States. Variants in melanocortin 1 receptor (MC1R) causes common coat colour phenotypes in a variety of mammalian species but only limited colour variation in the domestic cat. Known as the extension (E) locus, melanocortin 1 receptor (MC1R) interacts with the agouti locus to produce the eumelanin and pheomelanin pigments. Recently, a novel reddish coloration, which is termed russet, was identified in the Burmese cat breed. Because this russet Burmese coloration changes with aging, MC1R was suggested as candidate gene. The similar colouration in specific lineages of Norwegian Forest cat known as amber (e) (c.250G>A; p.Asp84Asn) was excluded for this Burmese phenotype. The complete 954‐bp coding region of MC1R was directly sequenced in russet Burmese and suspected carriers. A 3‐bp deletion (c.439_441del) associated with the deletion of a phenyalanine (p.Phe146del) in the protein sequence was identified. All russet coloured cats were homozygous for the variant, and all obligate carriers were heterozygous, confirming that the deletion segregated concordantly with colouring in Burmese cats from the New Zealand foundation lineage. The variant was not identified in 442 cats from 26 different breeds and random‐bred cats. Twenty‐six Burmese from the USA did not have the variant. This MC1R variant defines a unique coloration and the second breed‐specific MC1R variant in cats. The interactions of the two recessive feline MC1R alleles (E  >  e, e^r) is unknown.     

Invasive submerged freshwater macrophytes are more plastic in their response to light intensity than to the availability of free CO2 in air‐equilibrated water

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The oxalyl‐CoA synthetase‐regulated oxalate and its distinct effects on resistance to bacterial blight and aluminium toxicity in rice

• Oxalic acid is widely distributed in biological systems and known to play functional roles in plants. The gene AAE3 was recently identified to encode an oxalyl‐CoA synthetase (OCS) in Arabidopsis that catalyses the conversion of oxalate and CoA into oxalyl‐CoA. It will be particularly important to characterise the homologous gene in rice since rice is not only a monocotyledonous model plant, but also a staple food crop.
• Various enzymatic and biological methods have been used to characterise the homologous gene.
• We first defined that AAE3 in the rice genome (OsAAE3) also encodes an OCS enzyme. Its K_m for oxalate is 1.73 ± 0.12 mm , and V_m is 6824.9 ± 410.29 U·min^?1·mg protein^?1. Chemical modification and site‐directed mutagenesis analyses identified thiols as the active site residues for rice OCS catalysis, suggesting that the enzyme might be regulated by redox state. Subcellular localisation assay showed that the enzyme is located in the cytosol and predominantly distributed in leaf epidermal cells. As expected, oxalate levels increased when OCS was suppressed in RNAi transgenic plants. More interestingly, OCS‐suppressed plants were more susceptible to bacterial blight but more resistant to Al toxicity.
• The results demonstrate that the OsAAE3‐encoded protein also acts as an OCS in rice, and may play different roles in coping with stresses. These molecular, enzymatic and functional data provide first‐hand information to further clarify the function and mechanism of OCS in rice plants.