Mohammad Mejbah Uddin

Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan

Neuronal ceroid lipofuscinosis (NCL) constitutes a group of recessively inherited lysosomal storage diseases that primarily affect neuronal cells. Such diseases share certain clinical and pathologic features in human beings and animals. Neuronal ceroid lipofuscinosis in Border Collie dogs was first detected in Australia in the 1980s, and the pathogenic mutation was shown to be a nonsense mutation (c.619C>T) in exon 4 in canine CLN5 gene. In the present study, novel rapid genotyping assays including polymerase chain reaction (PCR)–restriction fragment length polymorphism, PCR primer–induced restriction analysis, mutagenically separated PCR, and real-time PCR with TaqMan minor groove binder probes, were developed. The utility of microchip electrophoresis was also evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies in Japan using these assays to determine the current allele frequency in Japan, providing information to control and prevent this disease in the next stage. All assays developed in the current study are available to discriminate these genotypes, and microchip electrophoresis showed a timesaving advantage over agarose gel electrophoresis. Of all assays, real-time PCR was the most suitable for large-scale examination because of its high throughput. The genotyping survey demonstrated that the carrier frequency was 8.1%. This finding suggested that the mutant allele frequency of NCL in Border Collies is high enough in Japan that measures to control and prevent the disease would be warranted. The genotyping assays developed in the present study could contribute to the prevention of NCL in Border Collies.

Neuronal Loss and Decreased GLT-1 Expression Observed in the Spinal Cord of Pembroke Welsh Corgi Dogs With Canine Degenerative Myelopathy:

Canine degenerative myelopathy (DM) is a progressive neurodegenerative disease that is frequently found in Pembroke Welsh Corgi (PWC) dogs. Canine DM is potentially a spontaneous animal model for human amyotrophic lateral sclerosis (ALS) because of similar lesions and the involvement of superoxide dismutase 1 (SOD1) mutation. However, the ventral horn lesion in DM has not been characterized in detail. Glutamate excitotoxicity due to deficiency of the glutamine-glutamate cycle has been implicated in neuron death in ALS. Thus, we examined 5 PWC dogs with an SOD1 mutation that were affected by DM, 5 non-DM PWC dogs, and 5 Beagle dogs without neurologic signs to assess the neuronal changes and the expression levels of 2 glial excitatory amino acid transporters (glutamate transporter 1 [GLT-1] and glutamate/aspartate transporter [GLAST]). The number of neurons in the spinal ventral horns of the DM dogs was significantly decreased, whereas no change was found in the cell size. Chromatolysis, lipofuscin-laden neurons, and marked synapse loss were also observed. GLT-1 expression was strikingly decreased in DM dogs, whereas GLAST expression showed no significant change. The results indicate that excitotoxicity related to the reduced expression of GLT-1, but not GLAST, may be involved in neuron loss in DM, as in human ALS, whereas intraneuronal events may differ between the 2 diseases.

A frameshift mutation in the canine HEXB gene in toy poodles with GM2 gangliosidosis variant 0 (Sandhoff disease)

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene. Toy poodles recently were reported as the second breed of dog with SD. The present paper describes the molecular defect of this canine SD as the first identification of a pathogenic mutation in the canine HEXB gene. Genomic and complementary DNA sequences covering exonic regions of the canine HEXB gene, except exon 1, were analysed using DNA and RNA in an affected dog. A homozygous single base pair deletion of guanine in exon 3 was identified at nucleotide position 283 of the putative open reading frame (c.283delG). This mutation has the potential to cause a frameshift resulting in the alteration of valine at amino acid position 59 to a stop codon (p.V59fsX). Genotyping using the mutagenically separated PCR method demonstrated a correlation between phenotype and genotype in dogs with a pedigree related to the disease and that the mutation was rare in a randomly-selected population of toy poodles. These results strongly suggest that the deletion is pathogenic.

Rapid genotyping assays for the 4–base pair deletion of canine MDR1/ABCB1 gene and low frequency of the mutant allele in Border Collie dogs

P-glycoprotein, encoded by the MDR1 or ABCB1 gene, is an integral component of the blood–brain barrier as an efflux pump for xenobiotics crucial in limiting drug uptake into the central nervous system. Dogs homozygous for a 4–base pair deletion of the canine MDR1 gene show altered expression or function of P-glycoprotein, resulting in neurotoxicosis after administration of the substrate drugs. In the present study, the usefulness of microchip electrophoresis for genotyping assays detecting this deletion mutation was evaluated. Mutagenically separated polymerase chain reaction (MS-PCR) and real-time PCR assays were newly developed and evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies dogs in Japan to determine the allele frequency in this breed. Microchip electrophoresis showed advantages in detection sensitivity and time saving over other modes of electrophoresis. The MS-PCR assay clearly discriminated all genotypes. Real-time PCR assay was most suitable for a large-scale survey due to its high throughput and rapidity. The genotyping survey demonstrated that the carrier and mutant allele frequencies were 0.49% and 0.25%, respectively, suggesting that the mutant allele frequency in Border Collies is markedly low compared to that in the susceptible dog breeds such as rough and smooth Collies.

A Novel Rapid Genotyping Technique for Collie Eye Anomaly: SYBR Green–Based Real-Time Polymerase Chain Reaction Method Applicable to Blood and Saliva Specimens on Flinders Technology Associates Filter Paper

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green–based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green–based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green–based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

Clinical Anatomy of the Head Region of the Black Bengal Goat in Bangladesh

SUMMARY: The study involved the measurements of some clinically important landmarks for regional anesthesia in the maxillaryand mandibular regions of head of fifty Black Bengal (BB) goats. The distance from facial tuberosity to the infra-orbital canal and fromthe latter to the root of the alveolar tooth directly ventral to it were 1.85 cm and 1.75 cm respectively. Both the distances from facialtuberosity to infra-orbital canal and from infra-orbital canal to the root of the alveolar tooth are of clinical important even in live animalas a guide for tracking infra-orbital nerve because of facial tuberosity. The distance between the lateral end of the alveolus of the incisortooth to the mental foramen was 2.11 cm and are from the mental foramen to the caudal mandibular border was 11.69 cm and import antfor the regional anesthesia of mental nerve. The length and maximum height of the mandibles were 14.21 cm and 8.83 cm respectiv ely.The distance from Caudal border of mandible to below mandibular foramen was 1.47 cm, while the distance from the mandibularforamen to the base of the mandible, Caudal border of mandibular to the level of mandibular foramen and mandibular foramen to b orderof mandibular angle were 3.64cm, 0.90 cm and 2.65 cm respectively. Parameters regarding mandible are of c linical important lan dmarksand will be guide for the regional anesthesia of mandibular nerve block. However, these data was discussed with regard to their applicationin proper tracking of infra-orbital, mental and mandibular nerves in aid of regional anesthetic procedures for the clinical aff ection aroundthe head of the Black Bengal goat.

Mutational analysis of the feline CLN3 gene and an ultrastructural evaluation of lysosomal storage materials in a cat with neuronal ceroid lipofuscinosis: An investigation into the molecular basis of the disease

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL.

High Frequency of a Single Nucleotide Substitution (c.-6-180T>G) of the Canine MDR1/ABCB1 Gene Associated with Phenobarbital-Resistant Idiopathic Epilepsy in Border Collie Dogs

A single nucleotide substitution (c.-6-180T>G) associated with resistance to phenobarbital therapy has been found in the canine MDR1/ABCB1 gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9%) in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies.

Real-time PCR genotyping assay for canine trapped neutrophil syndrome and high frequency of the mutant allele in Border collies.

Trapped neutrophil syndrome is an autosomal recessive inherited neutropenia in Border collies. The causative mutation is a 4base pair deletion in exon 19 of the canine VPS13B gene. In this study, a real-time PCR assay was developed and a genotyping survey was carried out in Border collies in Japan. The carrier frequency was 11.1%, suggesting that the mutant allele frequency is high enough to warrant measures to control and prevent the disease.

Collie eye anomaly in Hokkaido dogs: case study

OBJECTIVE To describe a Hokkaido dog, one of the traditional Japanese breeds that was affected by Collie eye anomaly (CEA), and to report the genotype of this dog and the Hokkaido dog allelic frequency of the CEA-associated mutation. CASE A nine-month-old intact female Hokkaido dog without any obvious visual disturbance was diagnosed ophthalmoscopically with CEA. Severe choroidal hypoplasia was observed in the bilateral temporal area adjacent to the optic nerve head, appearing as whitish areas. Therefore, the dog was suspected of possessing the CEA-associated mutation that was previously reported as an intronic 7.8-kilo base deletion in the canine NHEJ1 gene. PROCEDURES SYBR Green-based real-time PCR with a melting curve analysis, conventional PCR with agarose gel electrophoresis, and direct DNA sequencing were carried out to determine the genotype of the dog. Furthermore, a preliminary genotyping survey was carried out in 17 Hokkaido dogs from three kennels using the real-time PCR method, and the pedigree relationships were analyzed using their pedigree papers. RESULTS The Hokkaido dog affected by CEA was proven to possess the CEA-associated mutation. Of these 17 Hokkaido dogs, 12 dogs were heterozygous carriers and five dogs were affected by this mutation. The preliminary genotyping survey and pedigree analysis demonstrated that the allelic frequency of the CEA-associated mutation is very high in Hokkaido dogs. CONCLUSION These data suggest that the Hokkaido breed is highly susceptible to CEA because of the known CEA-associated mutation much like the Collie-related breeds.