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 novel insertion-induced frameshift mutation of the SLC26A4 gene in a Korean family with Pendred syndrome

Pendred syndrome (PS) is an autosomal recessive disorder characterized by congenital bilateral sensorineural hearing loss, goiter, and incomplete iodide organification. Patients with PS also have structural anomalies of the inner ear such as enlarged vestibular aqueducts (EVA) and Mondini's malformation. The goiter, which is a major clinical manifestation of PS, usually develops around adolescence. PS is caused by biallelic mutations of the SLC26A4 gene, while nonsyndromic bilateral EVA is associated with zero or one SLC26A4 mutant allele. We report here a Korean family including a young female with PS who had goiter and progressive, fluctuating sensorineural hearing loss that could be partially recovered by oral steroid treatment. Genetic investigation revealed compound heterozygous mutations for p.R677AfsX11, a novel frameshift mutation, and p.H723R in the SLC26A4 gene. Our findings provide detailed information regarding the distribution of mutant alleles for PS and may serve as a foundation for studies to comprehend the genetic portion of syndromic hearing loss.     

Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs

Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1 , a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.     

Expression pattern, genomic structure and evaluation of the human SLC30A4 gene as a candidate for acrodermatitis enteropathica

Slc30a4 is the fourth and last identified member of a mammalian proteins family presumably involved in the cellular transport of zinc, solute carrier family 30. The murine homologue of the human SLC30A4 gene has previously been investigated and found responsible for the lm, a phenotype due to zinc deficiency. According to the strong homology between mouse and human SLC30A4 coding sequences, and to the very similar clinical features encountered in the murine lm and in human acrodermatitis enteropathica, SLC30A4 has appeared to us to be a good candidate for acrodermatitis enteropathica. Here we detail the genomic structure of human SLC30A4 together with its localization on chromosome 15q15-q21. We also report the mutational analysis of human SLC30A4 in ten families with acrodermatitis enteropathica, which enabled us to exclude this gene from any involvement in the disorder of the patients examined.     

Late‐onset progressive retinal atrophy in the Gordon and Irish Setter breeds is associated with a frameshift mutation in C2orf71

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 14 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 16 Gordon Setter PRA cases and 22 controls, we identified a novel PRA locus, termed rod–cone degeneration 4 (rcd4), on CFA17 (P_raw = 2.22 × 10^?8, P_genome = 2.00 × 10^?5), where a 3.2‐Mb region was homozygous within cases. A frameshift mutation was identified in C2orf71, a gene located within this region. This variant was homozygous in 19 of 21 PRA cases and was at a frequency of approximately 0.37 in the Gordon Setter population. Approximately 10% of cases in our study (2 of 21) are not associated with this C2orf71 mutation, indicating that PRA in this breed is genetically heterogeneous and caused by at least two mutations. This variant is also present in a number of Irish Setter dogs with PRA and has an estimated allele frequency of 0.26 in the breed. The function of C2orf71 remains unknown, but it is important for retinal development and function and has previously been associated with autosomal recessive retinitis pigmentosa in 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.     

Intranasal oxytocin and a polymorphism in the oxytocin receptor gene are associated with human-directed social behavior in golden retriever dogs

The oxytocin system may play an important role in dog domestication from the wolf. Dogs have evolved unique human analogue social skills enabling them to communicate and cooperate efficiently with people. Genomic differences in the region surrounding the oxytocin receptor (OXTR) gene have previously been associated with variation in dogs' communicative skills. Here we have utilized the unsolvable problem paradigm to investigate the effects of oxytocin and OXTR polymorphisms on human-directed contact seeking behavior in 60 golden retriever dogs. Human-oriented behavior was quantified employing a previously defined unsolvable problem paradigm. Behaviors were tested twice in a repeated, counterbalanced design, where dogs received a nasal dose of either oxytocin or saline 45 min before each test occasion. Buccal DNA was analysed for genotype on three previously identified SNP-markers associated with OXTR. The same polymorphisms were also genotyped in 21 wolf blood samples to explore potential genomic differences between the species. Results showed that oxytocin treatment decreased physical contact seeking with the experimenter and one of the three polymorphisms was associated with degree of physical contact seeking with the owner. Dogs with the AA-genotype at this locus increased owner physical contact seeking in response to oxytocin while the opposite effect was found in GG-genotype individuals. Hence, intranasal oxytocin treatment, an OXTR polymorphism and their interaction are associated with dogs' human-directed social skills, which can explain previously described breed differences in oxytocin response. Genotypic variation at the studied locus was also found in wolves indicating that it was present even at the start of dog domestication.     

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.     

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.     

Evaluation of RDS/Peripherin and ROM1 as candidate genes in generalised progressive retinal atrophy and exclusion of digenic inheritance

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in the RDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate gene RDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.     

A simplified explanation for the frameshift mutation that created a novel C-terminal motif in the APETALA3 gene lineage

Background  

The evolution of type II MADS box genes has been extensively studied in angiosperms. One of the best-understood subfamilies is that of the Arabidopsis gene APETALA3 (AP3). Previous work has demonstrated that the ancestral paleo AP3 lineage was duplicated at some point within the basal eudicots to give rise to the paralogous TM6 and eu AP3 lineages. This event was followed in eu AP3 orthologs by the replacement of the C-terminal paleoAP3 motif with the derived euAP3 motif. It has been suggested that the new motif was created by an eight-nucleotide insertion that produced a translational frameshift.     

Identification of a doublet missense substitution in the bovine LRP4 gene as a candidate causal mutation for syndactyly in Holstein cattle

Syndactyly in Holstein cattle is an autosomal recessive abnormality characterized by the fusion of the functional digits. This disorder has been previously mapped to the telomeric part of bovine chromosome 15. Here, we describe the fine-mapping of syndactyly in Holstein cattle to a 3.5-Mb critical interval using a comparative mapping approach and an extended pedigree generated by embryo transfer. We report genetic evidence for the exclusion of two genes previously suggested as candidates (EXT2 and ALX4) and describe the identification of a doublet mutation in complete linkage disequilibrium with syndactyly in one gene of the critical interval: LRP4. Finally, based on recent discoveries concerning the mouse mutants dan and mdig and a mouse knockout for Lrp4, we present solid evidence that the subsequent substitution in LRP4 exon 33 is a strong candidate causal mutation for syndactyly in Holstein cattle.     

Cloning and characterization of human homologue of Drosophila retinal degeneration B: A candidate gene for degenerative retinal diseases

Mutations in the Drosophila retinal degeneration B (D-rdgB) gene cause light-enhanced retinal degeneration. Here, we report the isolation of the cDNA encoding human homologue of the D-rdgB and initial characterization of the gene products. Like D-rdgB, the human rdgB homologue (H-rdgB) is a transmembrane protein with the N-terminus sharing high homology to two closely related cytosolic proteins, phosphatidylinositol transfer protein (PITP) α and β, indicating that rdgB like proteins belong to the family of PITP proteins. Using Northern and Western blotting, we demonstrated that the rdgB homologue is expressed in rat retina, olfactory bulb, and brain, but not in nonneuronal tissues. In the rat retina, immunoreactivity of the rdgB homologue was observed in photoreceptors and throughout the inner nuclear and plexiform layers; the strongest staining was in the inner plexiform layer. In the photoreceptor cells, the rdgB homologue was located primarily in the inner segment where sorting and traffic of membranes required for outer segment assembly take place. These data, together with recent findings showing PITPs as an important component of intracellular membrane traffic apparatus in mammalian cells, suggest that rdgB homologue may play a role in photoreceptor membrane renewal and in neurotransmitter release. Furthermore, using somatic hybrid cell hybridization and fluorescence in situ hybridization H-rdgB gene was mapped to human chromosome 11q13, a region known to contain several retinopathy loci, including Best disease and Bardet-Biedl syndrome I. Therefore, H-rdgB gene is an attractive candidate for several inherited retinal degenerative diseases. Dev. Genet. 20:235–245, 1997. © 1997 Wiley-Liss, Inc.     

Generalized progressive retinal atrophy of Sloughi dogs is due to an 8-bp insertion in exon 21 of the PDE6B gene

We investigated the gene encoding the beta subunit of cGMP phosphodiesterase (PDE6B) as a candidate for generalized progressive retinal atrophy (gPRA), an autosomal recessively transmitted eye disease in dogs. The PDE6B gene was isolated from a genomic library. Single-strand conformation polymorphism analysis revealed eight intronic variations in different subsets of the 14 dog breeds investigated. In addition, we identified an 8-bp insertion after codon 816 in certain Sloughi dogs. Analysis of PRA-affected and obligatory carrier Sloughis showed that this mutation cosegregates with disease status in a large pedigree. All other exchanges identified were not located in functionally relevant parts of the gene (e.g., in the splice signal consensus sites). In most dog breeds (Labrador retriever, Tibetan mastiff, dachshund, Tibetan terrier, miniature poodle, Australian cattle dog, cocker spaniel, collie, Saarloos wolfhound, Chesapeake Bay retriever, and Yorkshire terrier), PDE6B was excluded as a candidate gene for gPRA because heterozygous allele constellations were detected in diseased animals. Therefore, the PDE6B sequence variations did not segregate together with the mutation(s) causing gPRA. Direct and indirect DNA tests concerning gPRA can be offered now for a variety of different dog breeds.     

Analysis of the SLC4A1 gene in three Mexican patients with hereditary spherocytosis: Report of a novel mutation

We analyzed the SLC4A1 gene in three Mexican patients with Hereditary Spherocytosis (HS). The promoter and all 20 exons were investigated through heteroduplex analysis and DNA sequencing. No DNA changes were detected in one of the three patients. Two well-known polymorphisms, Memphis I and the Diego-a blood group, were detected in another one. In the third, the HS phenotype could be explained by the novel 1885_1888dupCCGG mutation found in heterozygosis. This frameshift mutation is predicted to result in a truncated and unstable protein lacking normal functions.