Canine noninflammatory alopecia: An approach to its classification and a diagnostic aid.

Noninflammatory alopecia is common in dogs and is a frequent cause to consult a veterinarian. It is also a common reason to take biopsies. Noninflammatory alopecia can be attributed to a decreased formation or cytodifferentiation of the hair follicle or the hair shaft in utero, resulting in congenital alopecia. Congenital alopecia often has a hereditary cause, and examples of such disorders are ectodermal dysplasias associated with gene variants of the ectodysplasin A gene. Noninflammatory alopecia may also be caused by impaired postnatal regeneration of hair follicles or shafts. Such disorders may have a clear breed predilection, and alopecia starts early in life. A hereditary background is suspected in those cases but has not been proven. They are referred to as follicular dysplasia although some of these disorders present histologically like a hair cycle disturbance. Late-onset alopecia is usually acquired and may be associated with endocrinopathies. Other possible causes are impaired vascular perfusion or stress. As the hair follicle has limited possible responses to altered regulation, and histopathology may change during the course of a disease, a detailed clinical history, thorough clinical examination including blood work, appropriate biopsy site selection, and detailed histological findings need to be combined to achieve a final diagnosis. This review aims to provide an overview about the known noninflammatory alopecic disorders in dogs. As the pathogenesis of most disorders is unknown, some statements are based on comparative aspects or reflect the authors' opinion.

Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy.

Dogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

Critical Decrease in the Level of Axon Guidance Receptor ROBO1 in Rod Synaptic Terminals Is Followed by Axon Retraction.

Purpose: To define remodeling of photoreceptor synaptic terminals and second-order retinal neurons in canine X-linked progressive retinal atrophy 1 caused by a five-nucleotide deletion in the RPGR exon ORF15. Methods: Retinas of normal and mutant dogs were used for gene expression, Western blot, and immunohistochemistry. Cell-specific markers were used to examine disease-dependent retinal remodeling. Results: In mutant retinas, a number of rod axon terminals retract into the outer nuclear layer. This neuritic atrophy preceded significant loss of rods and was evident early in disease. Rod bipolar and horizontal cell processes were found to extend into the outer nuclear layer, where they seemed to form contacts with the spherules of rod photoreceptors. No ectopic rewiring was observed. Because cytoskeletal reorganization was previously shown to underlie photoreceptor axon retraction, we examined normal and mutant retinas for expression of axon guidance receptors ROBO1 and ROBO2, which are known to regulate actin cytoskeleton dynamics. We found that the overall expression of both ROBO1 and ROBO2 is retained at the same level in premature and fully developed normal retinas. However, analysis of predisease and early disease retinas identified markedly decreased levels of ROBO1 in rod spherules compared with controls. In contrast, no differences in ROBO1 signals were noted in cone pedicles in normal and mutant retinas, where ROBO1 levels remained similarly low. Conclusions: Depletion of ROBO1 in rod synaptic terminals correlates with the remodeling of axonal and dendritic processes in the outer retina of dogs with X-linked progressive retinal atrophy 1 and may play a role in the retraction of rod axons.

Genome-wide association study and whole-genome sequencing identify a deletion in LRIT3 associated with canine congenital stationary night blindness.

Congenital stationary night blindness (CSNB), in the complete form, is caused by dysfunctions in ON-bipolar cells (ON-BCs) which are secondary neurons of the retina. We describe the first disease causative variant associated with CSNB in the dog. A genome-wide association study using 12 cases and 11 controls from a research colony determined a 4.6 Mb locus on canine chromosome 32. Subsequent whole-genome sequencing identified a 1 bp deletion in LRIT3 segregating with CSNB. The canine mutant LRIT3 gives rise to a truncated protein with unaltered subcellular expression in vitro. Genetic variants in LRIT3 have been associated with CSNB in patients although there is limited evidence regarding its apparently critical function in the mGluR6 pathway in ON-BCs. We determine that in the canine CSNB retina, the mutant LRIT3 is correctly localized to the region correlating with the ON-BC dendritic tips, albeit with reduced immunolabelling. The LRIT3-CSNB canine model has direct translational potential enabling studies to help understand the CSNB pathogenesis as well as to develop new therapies targeting the secondary neurons of the retina.

Nonketotic hyperglycinemia in captive-bred Vervet monkeys (Chlorocebus aethiops) with cataracts.

BACKGROUND: Nonketotic hyperglycinemia (NKH) is a rare metabolic disorder that is characterized by high levels of glycine in plasma and cerebrospinal fluid in humans. In this study, total congenital cataract captive-bred Vervet monkeys (Chlorocebus aethiops) that are hyperglycinemic were screened to identify mutations in Bola type 3 (BOLA3), glutaredoxin 5 (GLRX5), and lipoate synthase (LIAS) genes. METHODS: Twenty-four Vervet monkeys (12 hyperglycinemic and 12 healthy controls) were selected for mutation analysis using polymerase chain reaction (PCR), Sanger sequencing, and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Novel sequence variants were identified in BOLA3 (R23H and Q38R) and LIAS (R369I and A371A), and gene expression in the control group was significantly lower compared to the hyperglycinemic group (P < 0.05). CONCLUSION: The data obtained from this study will contribute to generation of new knowledge regarding the involvement of these genes in NKH development.

Abnormal Expression of miR-21 in Kidney Tissue of Dogs With X-Linked Hereditary Nephropathy: A Canine Model of Chronic Kidney Disease.

MicroRNAs (miRNAs) are a group of small noncoding RNAs that act as regulators of posttranslational gene/protein expression and are known to play a key role in physiological and pathological processes. The objective of our study was to compare expression of miR-21 in renal tissue from dogs affected with chronic kidney disease (CKD) caused by X-linked hereditary nephropathy (XLHN), a disease equivalent to human Alport syndrome, to that from unaffected dogs. Additionally, we sought to characterize changes in relative mRNA expression of various genes associated with miR-21 function. miRNA was isolated from kidney tissue collected from both affected dogs and unaffected, age-matched littermates at defined milestones of disease progression, including end-stage renal disease (ESRD). Additionally, autopsy samples from affected dogs at ESRD and corresponding unaffected dogs were evaluated. Samples were scored based on histological changes, and relative expression of miR-21 and kidney disease-related genes was determined using quantitative real-time polymerase chain reaction. In affected dogs, significant upregulation of kidney miR-21 was first detected at the milestone corresponding with increased serum creatinine. Furthermore, miR-21 expression correlated significantly with urine protein: urine creatinine ratio, serum creatinine concentration, glomerular filtration rate, and histologic lesions (glomerular damage, tubular damage, chronic inflammation, and fibrosis). At end-stage disease, COL1A1, TGFB1 and its receptor, TGFB2, and Serpine1 were upregulated, while PPARA, PPARGC1A, ACADM, SOD1, and EGF were downregulated. In conclusion, miR-21 is abnormally upregulated in the kidneys of dogs with CKD caused by XLHN, which may play an important pathologic role in the progression of disease by dysregulating multiple pathways.

Autosomal recessive congenital cataract in captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: The aim of the study was to evaluate the genetic predisposition of congenital cataract in a colony of captive-bred vervet monkeys. METHODS: Four congenital cataract genes: glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat shock transcription factor 4 (HSF4), crystallin alpha A (CRYAA) and lens intrinsic membrane protein-2 (LIM2) were screened, sequenced and analysed for possible genetic variants in 36 monkeys. Gene expression was also evaluated in these genes. RESULTS: Fifteen sequence variants were identified in the coding regions of three genes (GCNT2, HSF4 and CRYAA). Of these variations, only three were missense mutations (M258V, V16I and S24N) and identified in the GCNT2 transcripts A, B and C, respectively, which resulted in a downregulated gene expression. CONCLUSION: Although the three missense mutations in GCNT2 have a benign effect, a possibility exists that the candidate genes (GCNT2, HSF4 and CRYAA) might harbour mutations that are responsible for total congenital cataract.

RNA-seq of serial kidney biopsies obtained during progression of chronic kidney disease from dogs with X-linked hereditary nephropathy.

Dogs with X-linked hereditary nephropathy (XLHN) have a glomerular basement membrane defect that leads to progressive juvenile-onset renal failure. Their disease is analogous to Alport syndrome in humans, and they also serve as a good model of progressive chronic kidney disease (CKD). However, the gene expression profile that affects progression in this disease has only been partially characterized. To help fill this gap, we used RNA sequencing to identify differentially expressed genes (DEGs), over-represented pathways, and upstream regulators that contribute to kidney disease progression. Total RNA from kidney biopsies was isolated at 3 clinical time points from 3 males with rapidly-progressing CKD, 3 males with slowly-progressing CKD, and 2 age-matched controls. We identified 70 DEGs by comparing rapid and slow groups at specific time points. Based on time course analysis, 1,947 DEGs were identified over the 3 time points revealing upregulation of inflammatory pathways: integrin signaling, T cell activation, and chemokine and cytokine signaling pathways. T cell infiltration was verified by immunohistochemistry. TGF-ß1 was identified as the primary upstream regulator. These results provide new insights into the underlying molecular mechanisms of disease progression in XLHN, and the identified DEGs can be potential biomarkers and therapeutic targets translatable to all CKDs.

Redundant contribution of a Transient Receptor Potential cation channel Member 1 exon 11 single nucleotide polymorphism to equine congenital stationary night blindness.

BACKGROUND: Congenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts. RESULTS: Retinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression. CONCLUSIONS: These finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.

Molecular studies of phenotype variation in canine RPGR-XLPRA1.

PURPOSE: Canine X-linked progressive retinal atrophy 1 (XLPRA1) caused by a mutation in retinitis pigmentosa (RP) GTPase regulator (RPGR) exon ORF15 showed significant variability in disease onset in a colony of dogs that all inherited the same mutant X chromosome. Defective protein trafficking has been detected in XLPRA1 before any discernible degeneration of the photoreceptors. We hypothesized that the severity of the photoreceptor degeneration in affected dogs may be associated with defects in genes involved in ciliary trafficking. To this end, we examined six genes as potential disease modifiers. We also examined the expression levels of 24 genes involved in ciliary trafficking (seven), visual pathway (five), neuronal maintenance genes (six), and cellular stress response (six) to evaluate their possible involvement in early stages of the disease. METHODS: Samples from a pedigree derived from a single XLPRA1-affected male dog outcrossed to unrelated healthy mix-bred or purebred females were used for immunohistochemistry (IHC), western blot, mutational and haplotype analysis, and gene expression (GE). Cell-specific markers were used to examine retinal remodeling in the disease. Single nucleotide polymorphisms (SNPs) spanning the entire RPGR interacting and protein trafficking genes (RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B) were genotyped in the pedigree. Quantitative real-time PCR (qRT-PCR) was used to examine the expression of a total of 24 genes, including the six genes listed. RESULTS: Examination of cryosections from XLPRA1-affected animals of similar age (3-4 years) with different disease severity phenotype revealed mislocalization of opsins and upregulation of the Müller cell gliosis marker GFAP. Four to ten haplotypes per gene were identified in RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B for further assessment as potential genetic modifiers of XLPRA1. No correlation was found between the haplotypes and disease severity. During mutational analysis, several new variants, including a single intronic mutation in RAB8A and three mutations in exon 3 of DFNB31 were described (c.970G>A (V324I), c.978T>C (G326=), and c.985G>A (A329T)). Expression analysis of stress response genes in 16-week-old predisease XLPRA1 retinas revealed upregulation of GFAP but not HSPA5, DDIT3, HSPA4, HSP90B1, or HIF1A. Western blot analysis confirmed GFAP upregulation. In the same predisease group, no significant differences were found in the expression of 18 selected genes (RHO, OPN1LW, OPN1MW, RLBP1, RPGRORF15, RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, RAB11B, CRX, RCVRN, PVALB, CALB1, FGFR1, NTRK2, and NTRK3) involved in neuronal function. CONCLUSIONS: Lack of association between haplotypes of RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B and the disease phenotype suggests that these genes are not genetic modifiers of XLPRA1. Upregulation of GFAP, an established indicator of the Müller cell gliosis, manifests as an important early feature of the disease.

X-Linked Hereditary Nephropathy in Navasota Dogs: Clinical Pathology, Morphology, and Gene Expression During Disease Progression.

X-linked hereditary nephropathy (XLHN) in Navasota dogs is a spontaneously occurring disease caused by a mutation resulting in defective production of type IV collagen and juvenile-onset renal failure. The study was aimed at examining the evolution of renal damage and the expression of selected molecules potentially involved in the pathogenesis of XLHN. Clinical data and renal samples were obtained in 10 XLHN male dogs and 5 controls at 4 (T0), 6 (T1), and 9 (T2) months of age. Glomerular and tubulointerstitial lesions were scored by light microscopy, and the expression of 21 molecules was investigated by quantitative real-time polymerase chain reaction with selected proteins evaluated by immunohistochemistry. No significant histologic lesions or clinicopathologic abnormalities were identified in controls at any time-point. XLHN dogs had progressive proteinuria starting at T0. At T1, XLHN dogs had a mesangioproliferative glomerulopathy with glomerular loss, tubular necrosis, and interstitial fibrosis. At T2, glomerular and tubulointerstitial lesions were more severe, particularly glomerular loss, interstitial fibrosis, and inflammation. At T0, transforming growth factor ß, connective tissue growth factor, and platelet-derived growth factor α mRNA were overexpressed in XLHN dogs compared with controls. Clusterin and TIMP1 transcripts were upregulated in later stages of the disease. Transforming growth factor ß, connective tissue growth factor, and platelet-derived growth factor α should be considered as key players in the initial events of XHLN. Clusterin and TIMP1 appear to be more associated with the progression rather than initiation of tubulointerstitial damage in chronic renal disease.

Twenty-five thousand years of fluctuating selection on leopard complex spotting and congenital night blindness in horses.

Leopard complex spotting is inherited by the incompletely dominant locus, LP, which also causes congenital stationary night blindness in homozygous horses. We investigated an associated single nucleotide polymorphism in the TRPM1 gene in 96 archaeological bones from 31 localities from Late Pleistocene (approx. 17 000 YBP) to medieval times. The first genetic evidence of LP spotting in Europe dates back to the Pleistocene. We tested for temporal changes in the LP associated allele frequency and estimated coefficients of selection by means of approximate Bayesian computation analyses. Our results show that at least some of the observed frequency changes are congruent with shifts in artificial selection pressure for the leopard complex spotting phenotype. In early domestic horses from Kirklareli-Kanligecit (Turkey) dating to 2700-2200 BC, a remarkably high number of leopard spotted horses (six of 10 individuals) was detected including one adult homozygote. However, LP seems to have largely disappeared during the late Bronze Age, suggesting selection against this phenotype in early domestic horses. During the Iron Age, LP reappeared, probably by reintroduction into the domestic gene pool from wild animals. This picture of alternating selective regimes might explain how genetic diversity was maintained in domestic animals despite selection for specific traits at different times.

Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease.

OBJECTIVES: To evaluate the predictive value of plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) and nitric oxide end-products (NOx) as markers for progression of mitral regurgitation caused by myxomatous mitral valve disease. ANIMALS: Seventy-eight privately owned Cavalier King Charles spaniels with naturally occurring myxomatous mitral valve disease. METHODS: Prospective longitudinal study comprising 312 measurements over a 4.5 year period. Clinical values were recorded, NT-proANP concentrations were measured by radioimmunoassay, and NOx were analyzed colorimetrically. To predict congestive heart failure (CHF), Cox proportional hazards models with time-varying covariates were constructed. RESULTS: The hazard ratio for NT-proANP (per 1000 pmol/l increase) to predict future CHF was 6.7 (95% confidence interval, 3.6-12.5; p < 0.001). The median time to CHF for dogs with NT-proANP levels >1000 pmol/l was 11 months (95% confidence interval, 5.6-12.6 months), compared to 54 months (46 - infinity) for dogs with concentrations ≤ 1000 pmol/l (p < 0.001). Due to intra- and inter-individual variability, most corresponding analyses for NOx were insignificant but dogs reaching CHF had a lower mean NOx concentration than dogs not reaching CHF (23 vs. 28 µmol/l, p = 0.016). Risk of CHF increased with increase in heart rate (>130 beats per minute) and grade of murmur (≥ 3/6). CONCLUSIONS: The risk of CHF due to mitral regurgitation is increased in dogs with blood NT-proANP concentrations above 1000 pmol/l. Measurement of NT-proANP can be a valuable tool to identify dogs that may develop CHF within months.

Urinary biomarkers of renal disease in dogs with X-linked hereditary nephropathy.

BACKGROUND: Sensitive and specific biomarkers for early tubulointerstitial injury are lacking. HYPOTHESIS: The excretion of certain urinary proteins will correlate with the state of renal injury in dogs with chronic kidney disease. ANIMALS: Twenty-five male colony dogs affected with X-linked hereditary nephropathy (XLHN) and 19 unaffected male littermates were evaluated. METHODS: Retrospective analysis of urine samples collected every 2-4 weeks was performed. Urine proteins evaluated were retinol binding protein (uRBP/c), ß2-microglobulin (uB2M), N-acetyl-ß-D-glucosaminidase (uNAG/c), neutrophil gelatinase-associated lipocalin (uNGAL/c), and immunoglobulin G (uIgG/c). Results were correlated with serum creatinine concentration (sCr), glomerular filtration rate (GFR), urine protein : creatinine ratio, and histopathologic analysis of serial renal biopsies. Analytical validation was performed for all assays; uNAG stability was evaluated. RESULTS: All urinary biomarkers distinguished affected dogs from unaffected dogs early in their disease process, increasing during early and midstages of disease. uRBP/c correlated most strongly with conventional measures of disease severity, including increasing sCr (r = 0.89), decreasing GFR (r = -0.77), and interstitial fibrosis (r = 0.80), P < .001. However, multivariate analysis revealed age, sCr, uIgG/c, and uB2M, but not uRBP/c, as significant independent predictors of GFR (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: All urinary biomarkers were elevated before sCr increased, but typically after proteinuria developed in dogs with progressive glomerular disease because of XLHN. uRBP/c measurement might be promising as a noninvasive tool for diagnosis and monitoring of tubular injury and dysfunction in dogs.

Inherited polyneuropathy in Leonberger dogs.

BACKGROUND: Although reporting the same clinical phenotype, inherited polyneuropathy in Leonberger dogs (ILPN) has been attributed to various modes of inheritance. HYPOTHESIS: The ILPN is one disease with a major risk factor on chromosome X. ANIMALS: Dogs affected by ILPN (n = 104). METHODS: Pedigree analyses were performed by means of a case-control approach. Data were retrieved either from medical records of cases diagnosed by the first author (n = 13), from breeders (n = 18) or from different registries publishing data on affected dogs (n = 73). A comparison was made between the X-chromosome ancestry of fathers of affected male dogs and the ancestry of the X-chromosomes of mothers of affected dogs of either sex. A systematic random sample, obtained from an international database of registered Leonberger dogs, served as a reference population regarding ancestry. RESULTS: Having one particular female, born 1943, in the X-chromosomal lineage is a major risk factor for developing ILPN. Sex distribution among affected dogs is in favor of a risk factor on the X-chromosome and contradicts a monogenic autosomal or mitochondrial inheritance. CONCLUSIONS AND CLINICAL IMPORTANCE: The ILPN is considered most likely to be one disease, and the inheritance of ILPN is best explained by an underlying X-linked mode of transmission for the phenotype. However, age at onset and severity of signs might be determined by contributing loci. This has consequences in molecular genetic studies and for breeding strategies aimed at eliminating this disease.

A novel movement disorder in related male Labrador Retrievers characterized by extreme generalized muscular stiffness.

OBJECTIVES: To describe the clinical phenotype of a new motor disorder in Labrador Retrievers. ANIMALS AND METHODS: Case series study. Seven young male Labrador Retrievers presented for evaluation of stiff gait. RESULTS: All affected dogs had generalized muscular stiffness, persistent at rest and resulting in restricted joint movements. They showed a forward flexed posture, festinating gait, and bradykinesia. Signs developed between 2 and 16 months of age and tended to stabilize in adulthood. Needle electromyogram in the conscious state showed continuous motor unit activity in resting epaxial and proximal limb muscles. This activity was abolished by general anesthesia. Muscle and nerve histopathology was normal. In 2 dogs necropsied, astrocytosis was evident throughout the spinal cord gray matter, reticular formation and caudate nuclei. Decreased neuronal counts were selectively found in the spinal cord Rexed's lamina VII, but not in VIII and IX. Pedigree analysis showed that the affected dogs were from 5 related litters. CONCLUSIONS AND CLINICAL IMPORTANCE: This new hypertonicity syndrome in Labrador Retrievers is unique because of the selective distribution of the histological lesions, the lack of progression in adulthood, and its exclusive occurrence in male dogs. Pedigree analysis suggests an X-linked hereditary disease, although other modes of inheritance cannot be ruled out with certainty. We hypothesize that altered output from basal nuclei and reticular formation together with motor neuron disinhibition caused by a decreased number of spinal cord interneurons leads to the muscular stiffness.

Age-dependent disease expression determines remodeling of the retinal mosaic in carriers of RPGR exon ORF15 mutations.

PURPOSE: To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 and XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15. METHODS: Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events. RESULTS: A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches an increased density of cones was found to persist over time. Patches of disease gradually disappeared so that by 39 weeks of age the overall retinal morphology, albeit thinner, had improved lamination. In older XLPRA2 carriers (>or=8.8 years), extended regions of severe degeneration occurred in the peripheral/mid-peripheral retina. In XLPRA1 carriers, opsin mislocalization and rare events of rod death were detected by TUNEL assay at 20 weeks of age; however, only patchy degeneration was seen by 1.4 years and was still apparent at 7.8 years. CONCLUSIONS: The time of onset and the progression of the disease differed between the two models. In the early-onset form (XLPRA2) the morphologic appearance of the retinal mosaic changed as a function of age, suggesting that structural plasticity persists in the early postnatal canine retina as mutant photoreceptors die. In the late-onset form (XLPRA1), patches of disease persisted until later ages.

X chromosome inactivation patterns in normal and X-linked hereditary nephropathy carrier dogs.

Alport syndrome (AS) and hereditary nephropathy (HN) are glomerular nephropathies caused by mutations in the genes encoding the type IV collagens. In a mixed breed of dog, termed Navasota (NAV) dogs, X-linked hereditary nephropathy (XLHN) is caused by a 10-bp deletion in exon 9 of COL4A5. Males harboring this mutation succumb to end-stage renal disease before 18 months of age. In contrast, female carriers of this disease survive much longer, most have a normal life-span, and vary in disease progression as compared with XLHN-affected males. X chromosome inactivation (XCI) patterns have been studied in human X-linked AS carriers and some have been shown to have a high degree of skewed XCI. However, similar studies have never been reported in an animal model of this disease. Therefore, patterns of XCI were examined in XLHN-carrier NAV dogs. The variation in XCI among the 26 XLHN-carrier and seven normal female NAV dogs studied was low and only three were found to preferentially inactivate one X chromosome, all of which were XLHN-carriers. The average skewedness among all dogs was 59% and 57% among the XLHN-carriers. No significant difference in XCI was found between the two groups (P = 0.477). It is clear from these data that genotype does not seem to have an effect on inactivation; the majority of these dogs have random patterns of XCI. Highly skewed X chromosome inactivation also appears to be random, given that no difference was observed between the XLHN-carriers and normal females. Because of the apparent rarity of skewed XCI, these dogs may not be a suitable model for studying a potential correlation between this phenomenon and disease progression.

Genetic cause of autosomal recessive hereditary nephropathy in the English Cocker Spaniel.

BACKGROUND: Autosomal recessive hereditary nephropathy (ARHN) in the English Cocker Spaniel is caused by a type IV collagen defect, but the underlying mutation is unknown. ANIMALS: One hundred thirty-four English Cocker Spaniels (12 with ARHN, 8 obligate carriers, and 114 others), 3 mixed breed dogs with X-linked hereditary nephropathy (XLHN), and 7 other dogs without hereditary nephropathy were included. METHODS: Diagnosis of ARHN was based on transmission electron microscopy and immunostaining of kidney. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to compare COL4A3, COL4A4, and COL4A5 mRNA concentrations in the renal cortex from ARHN-affected English Cocker Spaniels, XLHN-affected dogs, and dogs without hereditary nephropathy. The entire coding region of COL4A4 was sequenced in 2 ARHN-affected dogs, 2 obligate carriers, 2 English Cocker Spaniels of unknown status, and 2 healthy mixed breed dogs. The exon containing the mutation was sequenced for all 134 English Cocker Spaniels. RESULTS: Quantitative real time RT-PCR implicated COL4A4 as the gene harboring the mutation, and sequencing identified a single nucleotide substitution at base 115 as the cause of ARHN in English Cocker Spaniels. This mutation, which causes a premature stop codon in exon 3 of COL4A4, was segregated with clinical status in all affected dogs and obligate carriers. The mutation also was identified in 39 of 114 other English Cocker Spaniels with previously unknown status. CONCLUSIONS AND CLINICAL IMPORTANCE: The cause of this disease has been identified, and use of a test for the mutation will permit eradication of ARHN in the English Cocker Spaniel.