Analysis of PDE6D and PDE6G genes for generalised progressive retinal atrophy (gPRA) mutations in dogs

The δ and γ subunits of the cGMP-phosphodiesterase (PDE6D, PDE6G) genes were screened in order to identify mutations causing generalised progressive retinal atrophy (gPRA) in dogs. In the PDE6D gene, single nucleotide polymorphisms (SNP) were observed in exon 4, in introns 2 and 3 and in the 3'' untranslated region (UTR) of different dog breeds. In the coding region of the PDE6G gene, exclusively healthy Labrador Retrievers showed an A → G transition in exon 4 without amino acid exchange. SNP were also observed in introns 1 and 2 in different dog breeds. The different SNP were used as intragenic markers to investigate the involvement of both genes in gPRA. The informative substitutions allowed us to exclude mutations in the PDE6D and PDE6G genes as causing retinal degeneration in 15 of the 22 dog breeds with presumed autosomal recessively transmitted (ar) gPRA.     

Exclusion of the PDE6A gene for generalised progressive retinal atrophy in 11 breeds of dog

The cyclic guanosine monophosphate specific phosphodiesterase (cGMP-specific PDE) is a key enzyme in the phototransduction cascade of the vertebrate retina. This enzyme consists of two catalytic alpha and beta subunits, two identical inhibitory gamma subunits as well as a delta subunit. Mutations in PDE6A and the PDE6B genes lead to autosomal recessive (ar) forms of retinitis pigmentosa (RP) in human and to the homologous disease in dogs, designated generalised progressive retinal atrophy (gPRA). We investigated the PDE6A gene in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. In the coding region of PDE6A only a rare sequence variation (G103A; Asp35Asn) was found in exon 1 of two healthy Tibet Terriers and one affected Cocker Spaniel. Using single-stranded conformation polymorphism (SSCP) analyses we detected several sequence variations in eight of the PDE6A introns in different investigated breeds. Most informative for excluding the PDE6A gene as a cause for gPRA was a polymorphic microsatellite ((GT)10CG(GT)2CG(GT)12) in intron 14 and four sequence variations in intron 18 for almost all breeds investigated. The sequence variations of PDE6A did not segregate together with gPRA in 11 breeds. Since diseased animals were heterozygous for the polymorphisms, the PDE6A gene is unlikely to harbour the critical mutation causing gPRA in the following breeds: Chesapeake Bay Retriever. Entlebucher Sennenhund, Labrador Retriever. Tibet Mastiff, Dachshund (long- and wire-haired), Tibetan Terrier, Miniature Poodle. Australian Cattle Dog, Cocker Spaniel, Saarloos/Wolfshound, Sloughi.     

A 63-bp insertion in exon 2 of the porcine KIF21A gene is associated with arthrogryposis multiplex congenita

A recessive form of arthrogryposis multiplex congenita (AMC) was detected 20 years ago in the Swiss Large White (SLW) pig population. A diagnostic marker test enabled the identification of carrier animals, but the underlying causal mutation remains unknown. To identify the mutation underlying AMC, we collected SNP chip genotyping data for 11 affected piglets and 23 healthy pigs. Association testing using 47 829 SNPs confirmed that AMC maps to SSC5 (P = 9.4 × 10⁻¹³). Subsequent autozygosity mapping revealed a common 6.06 Mb region (from 66 757 970 to 72 815 151 bp) of extended homozygosity in 11 piglets affected by AMC. Using WGS data, we detected a 63-bp insertion compatible with the recessive inheritance of AMC in the second exon of KIF21A gene encoding Kinesin Family Member 21A. The 63-bp insertion is predicted to introduce a premature stop codon in KIF21A gene (p.Val41_Phe42insTer) that truncates 1614 amino acids (~97%) from the protein. We found that this deleterious allele still segregates at a frequency of 0.1% in the SLW pig population. Carrier animals can now be detected unambiguously and excluded from breeding.     

Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.     

Evaluation of ROM1 as a candidate gene in generalised progressive retinal atrophy in dogs

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and other animals. The genetic origin is unknown in most cases. We have screened the coding sequence of the ROM1 gene for disease causing mutations in Tibetan Terriers, Miniature Poodles, Dachshunds and Chesapeake Bay Retrievers by single strand conformation polymorphism analysis (SSCP). Two polymorphisms have been identified by sequencing, one in exon 1 in all examined breeds (position 210: G→A; Gly40Arg and position 252: G→T; Ala53Ser). Another polymorphism was present in exon 2 (position 1150: C→T and position 1195: C→T) segregating in Miniature Poodles. None of these polymorphisms were cosegregating with gPRA rendering a disease causing mutation in the ROM1 gene unlikely.     

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.     

Retinal dystrophy of Swedish briard/briard-beagle dogs is due to a 4-bp deletion in RPE65

The RPE65 gene encodes a 65-kDa microsomal protein expressed exclusively in retinal pigment epithelium (RPE). Mutations in the human RPE65 gene have recently been identified in patients with autosomal recessive, severe, childhood-onset retinal dystrophy. Here we report the characterization of a 2.4-kb canine Rpe65 cDNA. The longest open reading frame predicts a 533-amino-acid protein with a calculated molecular mass of about 61 kDa prior to protein modification. Sequence comparison shows that RPE65 is highly conserved throughout mammalian evolution. We have identified a homozygous 4-bp deletion (485delAAGA) in putative exon 5 of the canine Rpe65 gene in affected animals of a highly inbred kinship of Swedish briard/briard-beagle dogs, in which an autosomal recessive, early-onset, and progressive retinal dystrophy segregates. The deletion results in a frameshift and leads to a premature stop codon after inclusion of 52 canine RPE65-unrelated amino acids from residue 153 onward. More than two-thirds of the wildtype polypeptide chain will be missing, and the mutant protein is most likely nonfunctional (null allele). Clinical features of the canine disease are quite similar to those described in human. Therefore this form of canine retinal dystrophy provides an attractive animal model of the corresponding human disorder with immediate significance for various therapeutic approaches, including RPE transplantation.     

Investigation of the role of opsin gene polymorphism in generalized progressive retinal atrophies in dogs

The generalized progressive retinal atrophies (gPRAs) form a group of retinal degenerations of pedigree dogs and cats, which have a variety of genetic origins (mostly unknown). We have examined the opsin gene for polymorphisms in several breeds of pedigree dog suffering from distinct forms of gPRA, by methods including single-strand conformation polymorphism analysis, microsatellite analysis and direct sequencing. The breeds examined included the Tibetan terrier, the miniature schnauzer, the Irish setter, the miniature poodle, the Labrador retriever and the English cocker spaniel, as well as individuals from breeds in which PRA has not been described and of mixed breed. Individuals from each of the named breeds suffering from PRA were compared with clinically normal dogs. Two polymorphisms were found. One, segregating within the Tibetan terrier population, but not seen in other breeds, was a synonymous transition at nucleotide position 780 in exon 3. Inheritance of this polymorphism suggests that opsin is unlikely to contain mutations causative of gPRA in this breed. The other polymorphism occurred between all miniature schnauzers examined and dogs of other breeds. It consisted of a single base insertion in intron 2. No polymorphisms in the opsin sequence were detected in any other breed. DNA sequencing allowed rigorous exclusion of mutations in opsin as a cause of gPRA in miniature poodles, English cocker spaniels or Labrador retrievers.     

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.     

Identification of a nonframeshift 84-bp deletion in exon 13 of the cystic fibrosis gene.

Cystic fibrosis (CF) is the most frequent autosomal recessive inherited disorder in Caucasian populations. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified an 84-bp deletion in exon 13 of the CFTR gene, detected by DNA amplification and direct sequencing of 500 bp of the 5' end of exon 13. The deletion was in the maternal allele of a CF patient bearing the delta F508 deletion in the father's allele. The same 84-bp deletion could also be detected in the patient's mother. The deletion spanned from a four-A cluster in positions 1949-1952 to another four-A cluster in positions 2032-2035, including 84 bp which correspond to codons 607-634 (1949del84). The reported mutation would result in the loss of 28 amino acid residues of the R domain of the CFTR protein.     

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.     

A 971-bp insertion in the rns gene is associated with mitochondrial hypovirulence in a strain of Cryphonectria parasitica isolated from nature

In the chestnut-blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes frequently are elicited by double-stranded RNA (dsRNA) virus infections. However, some strains manifest cytoplasmically transmissible hypovirulence traits without containing any mycovirus. In this study, we describe an altered form of mtDNA that is associated with hypovirulence and senescence in a virus-free strain of C. parasitica, KFC9, which was obtained from nature and has an elevated level of cyanide-resistant respiration. In this strain, a 971-bp DNA element, named InC9, has been inserted into the first exon of the mitochondrial small-subunit ribosomal RNA (rns) gene. Sequence analysis indicates that InC9 is a type A1 group II intron that lacks a maturase-encoding ORF. RT-PCR analyses showed that the InC9 sequence is spliced inefficiently from the rRNA precursor. The KFC9 strain had very low amounts of mitochondrial ribosomes relative to virulent strains, thus most likely is deficient in mitochondrial protein synthesis and lacks at least some of the components of the cyanide-sensitive, cytochrome-mediated respiratory pathway. The attenuated-virulence trait and the splicing-defective intron are transferred asexually and concordantly by hyphal contact from hypovirulent donor strains to virulent recipients, confirming that InC9 causes hypovirulence.     

Deletion of the SELENOP gene leads to CNS atrophy with cerebellar ataxia in dogs

We investigated a hereditary cerebellar ataxia in Belgian Shepherd dogs. Affected dogs developed uncoordinated movements and intention tremor at two weeks of age. The severity of clinical signs was highly variable. Histopathology demonstrated atrophy of the CNS, particularly in the cerebellum. Combined linkage and homozygosity mapping in a family with four affected puppies delineated a 52 Mb critical interval. The comparison of whole genome sequence data of one affected dog to 735 control genomes revealed a private homozygous structural variant in the critical interval, Chr4:66,946,539_66,963,863del17,325. This deletion includes the entire protein coding sequence of SELENOP and is predicted to result in complete absence of the encoded selenoprotein P required for selenium transport into the CNS. Genotypes at the deletion showed the expected co-segregation with the phenotype in the investigated family. Total selenium levels in the blood of homozygous mutant puppies of the investigated litter were reduced to about 30% of the value of a homozygous wildtype littermate. Genotyping >600 Belgian Shepherd dogs revealed an additional homozygous mutant dog. This dog also suffered from pronounced ataxia, but reached an age of 10 years. Selenop^-/- knock-out mice were reported to develop ataxia, but their histopathological changes were less severe than in the investigated dogs. Our results demonstrate that deletion of the SELENOP gene in dogs cause a defect in selenium transport associated with CNS atrophy and cerebellar ataxia (CACA). The affected dogs represent a valuable spontaneous animal model to gain further insights into the pathophysiological consequences of CNS selenium deficiency.