ABSTRACT
AIMS: To investigate trajectories of daily insulin dose requirements and glycaemic control in children, adolescents and young adults with Type 1 diabetes and to identify factors associated with changing insulin needs and deterioration in HbA1c . METHODS: The sample was a dynamic cohort of 635 children, adolescents and young adults with Type 1 diabetes from one centre. Data from clinic visits occurring over 20 years (1993-2013) were extracted from medical records. From age 7-24 years, we evaluated HbA1c and insulin dose according to sex, insulin regimen and weight status. RESULTS: Participants provided a mean ± sd of 10.7±4.3 years of insulin dose data and 12.0±4.6 years of HbA1c data. At first observation, the mean ± sd age was 10.0±2.6 years, diabetes duration was 2.8±2.1 years, insulin dose was 0.8±0.2 units/kg and HbA1c was 74±18 mmol/mol (8.9±1.6%). Insulin dose was higher in girls at ages 8-13 years (P<0.0001 to P<0.01), but higher in boys/young men at ages 16-21 years (P<0.0001 to P=0.04). HbA1c was higher in girls/young women at ages 16-24 years (P<0.0001 to P=0.01). Compared with injection therapy, pump therapy was associated with lower insulin dose at ages 8-24 years (P<0.0001 to P=0.03) and lower HbA1c at ages 8-22 years (P<0.0001 to P=0.005). HbA1c did not differ between overweight/obese and normal weight individuals, but overweight/obese individuals had higher insulin dose at ages 8-13 years (P<0.0001 to P=0.03). CONCLUSIONS: This longitudinal assessment identifies clinically meaningful modifiable (e.g. insulin regimen) and non-modifiable (e.g. sex) factors predictive of insulin requirements and HbA1c levels in young people with Type 1 diabetes; anticipatory insulin adjustments may improve glycaemic control.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 1/drug therapy , Insulin/administration & dosage , Adolescent , Adult , Blood Glucose/drug effects , Child , Cohort Studies , Diabetes Mellitus, Type 1/blood , Dose-Response Relationship, Drug , Female , Glycated Hemoglobin/drug effects , Glycated Hemoglobin/metabolism , Humans , Longitudinal Studies , Male , Prognosis , Young AdultABSTRACT
Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.
Subject(s)
Dog Diseases/genetics , Gangliosidoses, GM2/veterinary , Gene Deletion , beta-Hexosaminidase beta Chain/genetics , Animals , Dog Diseases/pathology , Dogs , Female , Gangliosidoses, GM2/genetics , Gangliosidoses, GM2/pathology , Homozygote , Microscopy, Electron/veterinaryABSTRACT
Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young-aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose-bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole-blood mitogen-stimulated T-cell proliferation was determined using a flow cytometric assay. Plasma MPA Cmax (mean ± SD, 9.33 ± 7.04 µg/ml) occurred at <1 hr. The AUC0-∞ (mean ± SD, 12.84±6.62 hr*µg/ml), MRTinf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.
Subject(s)
Immunosuppressive Agents/pharmacokinetics , Mycophenolic Acid/pharmacokinetics , Administration, Oral , Animals , CD5 Antigens/immunology , Dogs , Female , Flow Cytometry/veterinary , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Male , Mycophenolic Acid/administration & dosage , Mycophenolic Acid/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
BACKGROUND: No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. HYPOTHESIS/OBJECTIVE: Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. ANIMALS: Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. METHODS: Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. RESULTS: Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4-9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8-29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9-51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2-61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6-59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5-10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0-12.3; P < .01]). CSF pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09-90.64%) and 93.6% specific (CI 78.58-99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92-0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.
Subject(s)
Dog Diseases/cerebrospinal fluid , Neurodegenerative Diseases/veterinary , Neurofilament Proteins/cerebrospinal fluid , Spinal Cord Diseases/veterinary , Animals , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Dog Diseases/blood , Dogs , Enzyme-Linked Immunosorbent Assay/veterinary , Neurodegenerative Diseases/blood , Neurodegenerative Diseases/cerebrospinal fluid , Phosphorylation , ROC Curve , Spinal Cord Diseases/blood , Spinal Cord Diseases/cerebrospinal fluidABSTRACT
CLN2 neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease with primarily neurological signs that results from mutations in TPP1, which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Studies using a canine model for this disorder demonstrated that delivery of TPP1 enzyme to the cerebrospinal fluid (CSF) by intracerebroventricular administration of an AAV-TPP1 vector resulted in substantial delays in the onset and progression of neurological signs and prolongation of life span. We hypothesized that the treatment may not deliver therapeutic levels of this protein to tissues outside the central nervous system that also require TPP1 for normal lysosomal function. To test this hypothesis, dogs treated with CSF administration of AAV-TPP1 were evaluated for the development of non-neuronal pathology. Affected treated dogs exhibited progressive cardiac pathology reflected by elevated plasma cardiac troponin-1, impaired cardiac function and development of histopathological myocardial lesions. Progressive increases in the plasma activity levels of alanine aminotransferase and creatine kinase indicated development of pathology in the liver and muscles. The treatment also did not prevent disease-related accumulation of lysosomal storage bodies in the heart or liver. These studies indicate that optimal treatment outcomes for CLN2 disease may require delivery of TPP1 systemically as well as directly to the central nervous system.
Subject(s)
Aminopeptidases/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Genetic Therapy , Lysosomal Storage Diseases/therapy , Neuronal Ceroid-Lipofuscinoses/therapy , Serine Proteases/genetics , Aminopeptidases/therapeutic use , Animals , Dependovirus , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/therapeutic use , Disease Models, Animal , Dogs , Gene Transfer Techniques , Genetic Vectors/genetics , Genetic Vectors/therapeutic use , Humans , Infusions, Intraventricular , Lysosomal Storage Diseases/genetics , Neuronal Ceroid-Lipofuscinoses/genetics , Neurons/metabolism , Neurons/pathology , Serine Proteases/therapeutic use , Tripeptidyl-Peptidase 1ABSTRACT
A 10-month-old spayed female Cane Corso dog was evaluated after a 2-month history of progressive blindness, ataxia, and lethargy. Neurologic examination abnormalities indicated a multifocal lesion with primarily cerebral and cerebellar signs. Clinical worsening resulted in humane euthanasia. On necropsy, there was marked astrogliosis throughout white matter tracts of the cerebrum, most prominently in the corpus callosum. In the cerebral cortex and midbrain, most neurons contained large amounts of autofluorescent storage material in the perinuclear area of the cells. Cerebellar storage material was present in the Purkinje cells, granular cell layer, and perinuclear regions of neurons in the deep nuclei. Neuronal ceroid lipofuscinosis (NCL) was diagnosed. Whole genome sequencing identified a PPT1c.124 + 1G>A splice donor mutation. This nonreference assembly allele was homozygous in the affected dog, has not previously been reported in dbSNP, and was absent from the whole genome sequences of 45 control dogs and 31 unaffected Cane Corsos. Our findings indicate a novel mutation causing the CLN1 form of NCL in a previously unreported dog breed. A canine model for CLN1 disease could provide an opportunity for therapeutic advancement, benefiting both humans and dogs with this disorder.
Subject(s)
Dog Diseases/diagnosis , Neuronal Ceroid-Lipofuscinoses/veterinary , Animals , Dog Diseases/genetics , Dogs , Female , Frameshift Mutation/genetics , Magnetic Resonance Imaging/veterinary , Neuronal Ceroid-Lipofuscinoses/diagnosis , Neuronal Ceroid-Lipofuscinoses/diagnostic imaging , Neuronal Ceroid-Lipofuscinoses/geneticsABSTRACT
BACKGROUND: Neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease, has been diagnosed in young adult Australian Cattle Dogs. OBJECTIVE: Characterize the Australian Cattle Dog form of NCL and determine its molecular genetic cause. ANIMALS: Tissues from 4 Australian Cattle Dogs with NCL-like signs and buccal swabs from both parents of a fifth affected breed member. Archived DNA samples from 712 individual dogs were genotyped. METHODS: Tissues were examined by fluorescence, electron, and immunohistochemical microscopy. A whole-genome sequence was generated for 1 affected dog. A TaqMan allelic discrimination assay was used for genotyping. RESULTS: The accumulation of autofluorescent cytoplasmic storage material with characteristic ultrastructure in tissues from the 4 affected dogs supported a diagnosis of NCL. The whole-genome sequence contained a homozygous nonsense mutation: CLN5:c.619C>T. All 4 DNA samples from clinically affected dogs tested homozygous for the variant allele. Both parents of the fifth affected dog were heterozygotes. Archived DNA samples from 346 Australian Cattle Dogs, 188 Border Collies, and 177 dogs of other breeds were homozygous for the reference allele. One archived Australian Cattle Dog sample was from a heterozygote. CONCLUSIONS AND CLINICAL IMPORTANCE: The homozygous CLN5 nonsense is almost certainly causal because the same mutation previously had been reported to cause a similar form of NCL in Border Collies. Identification of the molecular genetic cause of Australian Cattle Dog NCL will allow the use of DNA tests to confirm the diagnosis of NCL in this breed.
Subject(s)
Dog Diseases/genetics , Membrane Proteins/genetics , Neuronal Ceroid-Lipofuscinoses/veterinary , Animals , Codon, Nonsense , Dogs , Female , Genetic Predisposition to Disease , Male , Neuronal Ceroid-Lipofuscinoses/genetics , PedigreeABSTRACT
We studied a recessive, progressive neurodegenerative disease occurring in Golden Retriever siblings with an onset of signs at 15 months of age. As the disease progressed these signs included ataxia, anxiety, pacing and circling, tremors, aggression, visual impairment and localized and generalized seizures. A whole genome sequence, generated with DNA from one affected dog, contained a plausibly causal homozygous mutation: CLN5:c.934_935delAG. This mutation was predicted to produce a frameshift and premature termination codon and encode a protein variant, CLN5:p.E312Vfs*6, which would lack 39 C-terminal amino acids. Eighteen DNA samples from the Golden Retriever family members were genotyped at CLN5:c.934_935delAG. Three clinically affected dogs were homozygous for the deletion allele; whereas, the clinically normal family members were either heterozygotes (n = 11) or homozygous for the reference allele (n = 4). Among archived Golden Retrievers DNA samples with incomplete clinical records that were also genotyped at the CLN5:c.934_935delAG variant, 1053 of 1062 were homozygous for the reference allele, 8 were heterozygotes and one was a deletion-allele homozygote. When contacted, the owner of this homozygote indicated that their dog had been euthanized because of a neurologic disease that progressed similarly to that of the affected Golden Retriever siblings. We have collected and stored semen from a heterozygous Golden Retriever, thereby preserving an opportunity for us or others to establish a colony of CLN5-deficient dogs.
Subject(s)
Dog Diseases/genetics , Frameshift Mutation , Membrane Proteins/genetics , Neuronal Ceroid-Lipofuscinoses/veterinary , Sequence Deletion , Animals , Base Sequence , Dogs , Homozygote , Neuronal Ceroid-Lipofuscinoses/genetics , Sequence Analysis, DNAABSTRACT
AIMS: To develop and validate the Diabetes Family Impact Scale, a scale to measure the impact of diabetes on families. METHODS: The Diabetes Family Impact Scale was developed by an iterative process, with input from multidisciplinary diabetes providers and parents of children with Type 1 diabetes. The psychometric properties of the Diabetes Family Impact Scale were assessed in parents of children with Type 1 diabetes. This assessment included internal consistency, convergent validity and exploratory factor analysis. RESULTS: Parents (n = 148) of children (mean ± sd age 12.9 ± 3.3 years) with Type 1 diabetes (mean ± sd duration 6.2 ± 3.6 years) completed the 15-item Diabetes Family Impact Scale. After eliminating one item, the 14-item measure demonstrated good internal consistency (Cronbach's α = 0.84). Correlations between the Diabetes Family Impact Scale and measures of parent diabetes burden (r = 0.48, P < 0.0001), stressful life events (r = 0.28, P = 0.0007), and child's quality of life (r = -0.52 and -0.54, P < 0.0001 for generic and diabetes-specific quality of life, respectively) supported the convergent validity of the instrument. Factor analysis identified four factors corresponding to the four survey domains (school, work, finances and family well-being). CONCLUSIONS: The Diabetes Family Impact Scale measures diabetes-specific family impacts with good internal consistency and convergent validity and may be a useful tool in clinical and research settings.
Subject(s)
Diabetes Mellitus, Type 1/psychology , Family Health , Surveys and Questionnaires/standards , Adolescent , Child , Cost of Illness , Female , Humans , Male , PsychometricsABSTRACT
BACKGROUND: Previous reports associated 2 mutant SOD1 alleles (SOD1:c.118A and SOD1:c.52T) with degenerative myelopathy in 6 canine breeds. The distribution of these alleles in other breeds has not been reported. OBJECTIVE: To describe the distribution of SOD1:c.118A and SOD1:c.52T in 222 breeds. ANIMALS: DNA from 33,747 dogs was genotyped at SOD1:c.118, SOD1:c.52, or both. Spinal cord sections from 249 of these dogs were examined. METHODS: Retrospective analysis of 35,359 previously determined genotypes at SOD1:c.118G>A or SOD1:c.52A>T and prospective survey to update the clinical status of a subset of dogs from which samples were obtained with a relatively low ascertainment bias. RESULTS: The SOD1:c.118A allele was found in cross-bred dogs and in 124 different canine breeds whereas the SOD1:c.52T allele was only found in Bernese Mountain Dogs. Most of the dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A homozygotes, but 8 dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A/G heterozygotes and had no other sequence variants in their SOD1 amino acid coding regions. The updated clinical conditions of dogs from which samples were obtained with a relatively low ascertainment bias suggest that SOD1:c.118A homozygotes are at a much higher risk of developing degenerative myelopathy than are SOD1:c.118A/G heterozygotes. CONCLUSIONS AND CLINICAL IMPORTANCE: We conclude that the SOD1:c.118A allele is widespread and common among privately owned dogs whereas the SOD1:c.52T allele is rare and appears to be limited to Bernese Mountain Dogs. We also conclude that breeding to avoid the production of SOD1:c.118A homozygotes is a rational strategy.
Subject(s)
Dog Diseases/genetics , Muscular Atrophy, Spinal/veterinary , Superoxide Dismutase/genetics , Alleles , Animals , Dogs/genetics , Gene Frequency , Genetic Predisposition to Disease/genetics , Genotype , Homozygote , Muscular Atrophy, Spinal/genetics , Mutation, Missense , Species SpecificityABSTRACT
Three young domestic shorthair cats were presented for necropsy with similar histories of slowly progressive visual dysfunction and neurologic deficits. Macroscopic examination of each cat revealed cerebral and cerebellar atrophy, dilated lateral ventricles, and slight brown discoloration of the gray matter. Histologically, there was bilateral loss of neurons within the limbic, motor, somatosensory, visual, and, to a lesser extent, vestibular systems with extensive astrogliosis in the affected regions of all 3 cases. Many remaining neurons and glial cells throughout the entire central nervous system were distended by pale yellow to eosinophilic, autofluorescent cytoplasmic inclusions with ultrastructural appearances typical of neuronal ceroid-lipofuscinoses (NCLs). Differences in clinical presentation and neurological lesions suggest that the 3 cats may have had different variants of NCL. Molecular genetic characterization in the 1 cat from which DNA was available did not reveal any plausible disease-causing mutations of the CLN1 (PPT1), CLN3, CLN5, CLN8, and CLN10 (CTSD) genes. Further investigations will be required to identify the mutations responsible for NCLs in cats.
Subject(s)
Cat Diseases/pathology , Nervous System/pathology , Neuronal Ceroid-Lipofuscinoses/veterinary , Animals , Atrophy/pathology , Atrophy/veterinary , Cats , DNA Mutational Analysis/veterinary , Fatal Outcome , Histological Techniques/veterinary , Immunohistochemistry/veterinary , Minnesota , Neuronal Ceroid-Lipofuscinoses/pathologyABSTRACT
As part of a biological control program against Spartina alterniflora Loisel. (smooth cordgrass), we simultaneously released populations of the planthopper Prokelisia marginata (van Duzee) from four geographic areas in each of five replicate field sites in the Willapa Bay estuary in Washington State. The four sources (California, Georgia, Virginia, and Rhode Island) have varying climate and seasonal regimes. We expected local adaptations would affect performance in the new environment. Using vacuum sampling, we measured population densities in spring and fall for 2 yr after release. In addition, we measured the timing of spring emergence through bi-weekly surveys of the number of nymphs residing in overwintering sites (curled leaves of senesced Spartina culms) versus on live green shoots. The observed sequence of emergence GA>CA>VA>RI was consistent with the hypothesis that this insect responds to a photoperiod cue for emergence timing. The four populations also differed in their reproductive capacity as measured by the increase in population densities over the summer months. Overall, the California and Rhode Island populations had higher population growth than those from Virginia and Georgia. Our results suggest that the climate and seasonal adaptations of biocontrol agents should be carefully considered as they can affect the performance and phenology in the new range. At the same time, it is noteworthy that all four populations were capable of establishing and growing, indicating a degree of resiliency for populations experiencing a rapid change in climate.
Subject(s)
Biological Control Agents , Environment , Hemiptera/physiology , Poaceae , Weed Control/methods , Animals , Climate Change , Introduced Species , Population Dynamics , Reproduction , Seasons , United States , WashingtonABSTRACT
AIMS: To determine incidence rates of severe hypoglycaemia and compare incidence rates by insulin regimen in a diverse sample of youth with Type 1 diabetes from two sites. METHODS: In this observational study, 255 youth (51% female) aged 9-15 years receiving varied insulin regimens provided data prospectively for a median of 1.2 years. Reported episodes of severe hypoglycaemia, defined as episodes requiring help from another person for oral treatment or episodes resulting in seizure/coma, and current insulin regimens were collected systematically. Incidence rates were calculated and compared according to insulin regimen in bivariate and multivariate analyses. RESULTS: At first encounter, participants had a median age of 12.2 years (range 9.0-15.0), median diabetes duration of 4.4 years (range 1.0-13.0) and mean HbA(1c) of 67 ± 12 mmol/mol (8.3 ± 1.1%). The incidence rate was 37.6/100 patient-years for all severe hypoglycaemia and 9.6/100 patient-years for seizure/coma. The incidence rate for severe hypoglycaemia was 31.8/100 patient-years on continuous subcutaneous insulin infusion (pump therapy), 34.4/100 patient-years on basal-bolus injections and 46.1/100 patient-years on NPH (NPH vs. pump therapy: P = 0.04). The incidence rate for seizure/coma was 4.5/100 patient-years on pump therapy, 11.1/100 patient-years on basal-bolus injections and 14.4/100 patients-years on NPH (NPH vs. pump therapy: P = 0.004). In the multivariate analysis, the rate of seizure/coma was significantly higher for those on NPH vs. pump therapy (rate ratio 2.9, P = 0.03). CONCLUSIONS: Rates of severe hypoglycaemia in youth with Type 1 diabetes remain high. Pump therapy was associated with lower rates of all severe hypoglycaemia and seizure/coma in comparison with NPH.
Subject(s)
Blood Glucose/metabolism , Cognition Disorders/epidemiology , Cognition Disorders/etiology , Diabetes Mellitus, Type 1/epidemiology , Hypoglycemia/epidemiology , Hypoglycemic Agents/therapeutic use , Insulin/therapeutic use , Adolescent , Blood Glucose Self-Monitoring , Child , Cognition Disorders/blood , Coma/epidemiology , Coma/etiology , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/complications , Drug Administration Schedule , Female , Humans , Hypoglycemia/blood , Hypoglycemia/complications , Incidence , Insulin, Long-Acting/therapeutic use , Male , Multivariate Analysis , Prognosis , Prospective Studies , Seizures/epidemiology , Seizures/etiology , Treatment OutcomeABSTRACT
The neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive lysosomal storage diseases characterized by progressive neurodegeneration and by accumulation of autofluorescent storage material in the central nervous system and other tissues. One of the most prominent clinical signs of NCL is progressive decline in cognitive function. We previously described a frame shift mutation of TPP1 in miniature long-haired Dachshunds which causes an early-onset form of NCL analogous to classical late-infantile onset NCL (CLN2) in children. Dogs homozygous for the TPP1 mutation exhibit progressive neurological signs similar to those exhibited by human patients. In order to establish biomarkers for evaluating the efficacy of ongoing therapeutic studies in this canine model, we characterized phenotypic changes in 13 dogs through 9 months of age. Cognitive function was assessed using a T-maze reversal learning (RL) task. Cognitive dysfunction was detected in affected dogs as early as 6 months of age and worsened as the disease progressed. Physical and neurological examination, funduscopy and electroretinography (ERG) were performed at regular intervals. Only the changes in ERG responses showed signs of disease progression earlier than the RL task. In the later stages of the disease clinical signs of visual and motor deficits became evident. The visual and motor deficits were not severe enough to affect the performance of dogs in the T-maze. Declining performance on the RL task is a sensitive measure of higher-order cognitive dysfunction which can serve as a useful biomarker of disease progression.
Subject(s)
Aminopeptidases/genetics , Cognition Disorders/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Maze Learning/physiology , Neuronal Ceroid-Lipofuscinoses/complications , Reversal Learning/physiology , Serine Proteases/genetics , Aminopeptidases/metabolism , Animals , Case-Control Studies , Cerebellar Ataxia/complications , Cerebellar Ataxia/genetics , Cognition Disorders/complications , Cognition Disorders/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Disease Models, Animal , Dogs , Electroretinography , Female , Frameshift Mutation , Male , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/metabolism , Serial Learning/physiology , Serine Proteases/metabolism , Tripeptidyl-Peptidase 1ABSTRACT
BACKGROUND: Bandera's neonatal ataxia (BNAt) is an autosomal recessive cerebellar ataxia that affects members of the Coton de Tulear dog breed. OBJECTIVE: To identify the mutation that causes BNAt. ANIMALS: The study involved DNA from 112 Cotons de Tulear (including 15 puppies with signs of BNAt) and 87 DNA samples from dogs of 12 other breeds. METHODS: The BNAt locus was mapped with a genome-wide association study (GWAS). The coding exons of positional candidate gene GRM1, which encodes metabotropic glutamate receptor 1, were polymerase chain reaction (PCR)-amplified and resequenced. A 3-primer PCR assay was used to genotype individual dogs for a truncated retrotransposon inserted into exon 8 of GRM1. RESULTS: The GWAS indicated that the BNAt locus was in a canine chromosome 1 region that contained candidate gene GRM1. Resequencing this gene from BNAt-affected puppies indicated that exon 8 was interrupted by the insertion of a 5'-truncated retrotransposon. All 15 BNAt-affected puppies were homozygous for the insert, whereas all other Cotons de Tulear were heterozygotes (n = 43) or homozygous (n = 54) for the ancestral allele. None of the 87 dogs from 12 other breeds had the insertion allele. CONCLUSIONS AND CLINICAL IMPORTANCE: BNAt is caused by a retrotransposon inserted into exon 8 of GRM1. A DNA test for the GRM1 retrotransposon insert can be used for genetic counseling and to confirm the diagnosis of BNAt.
Subject(s)
Cerebellar Ataxia/veterinary , Dog Diseases/genetics , Mutation , Receptors, Metabotropic Glutamate/genetics , Age of Onset , Animals , Cerebellar Ataxia/genetics , DNA Mutational Analysis/veterinary , DNA Primers/genetics , Dogs , Exons , Female , Genome-Wide Association Study , Genotype , Heterozygote , Homozygote , Male , Mutagenesis, Insertional , Open Reading Frames , Pedigree , RetroelementsABSTRACT
The neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases characterized by massive accumulation of autofluorescent storage bodies in neurons and other cells. A late-onset form of NCL occurs in Tibetan terrier dogs. Gel electrophoretic analyses of isolated storage body proteins from brains of affected dogs indicated that a protein of approximately 50 kDa was consistently prominent and a 16 kDa component was present in some brain storage body preparations. Mass spectral analysis identified the 50 kDa protein as glial fibrillary acidic protein (GFAP), isoform 2. GFAP identification was supported by immunoblot and immunohistochemical analyses. Histone H4 was the major protein in the 16 kDa component. Specific accumulation of GFAP and histone H4 in storage bodies has not been previously reported for any of the NCLs. Tibetan terrier NCL may be the canine correlate of one of the human adult-onset NCLs for which the genetic bases and storage body compositions have not yet been determined.
Subject(s)
Brain/metabolism , Gene Expression Regulation , Glial Fibrillary Acidic Protein/biosynthesis , Histones/biosynthesis , Neuronal Ceroid-Lipofuscinoses/metabolism , Amino Acid Sequence , Animals , Disease Models, Animal , Dogs , Humans , Immunohistochemistry , Mass Spectrometry/methods , Microscopy, Fluorescence/methods , Molecular Sequence Data , Protein IsoformsABSTRACT
Conventional fixation and processing of mammalian retinal tissues for transmission electron microscopic (TEM) examination is slow and produces ultrastructural artefacts in the photoreceptor cell layer. Among these artefacts are gaps between photoreceptor outer segment disc membranes and between photoreceptor cells in the region of the retina where the cell nuclei are located. A study was undertaken to determine whether a much more rapid microwave-assisted fixation and processing protocol would have an effect on the quality of ultrastructural preservation of the retina, particularly on the photoreceptor cell artefacts. The overall ultrastructural preservation of the retina was similar for the conventional and microwave-assisted techniques. However, the magnitudes of the photoreceptor artefacts were significantly reduced when microwave irradiation was used during primary fixation and processing. It is clear that, at least for the retina, employing microwave irradiation during specimen preparation for TEM results in superior ultrastructural preservation with a substantial reduction in the time required for sample preparation.