Ocorrência do SNP c.767G>T no gene DNM1responsável pelo colapso induzido pelo exercício em cães da raça Labrador Retriever no estado de São Paulo / Occurrence of DNM1 SNP c.767G>T responsible for exercise-induced collapse in Labrador Retriever in the Sao Paulo state

O colapso induzido pelo exercício (EIC) é considerado uma síndrome autossômica recessiva que afeta principalmente cães da raça Labrador Retriever. A doença é caracterizada por fraqueza muscular e colapso após exercício intenso. Usualmente, ocorre recuperação clínica após o episódio, mas alguns animais podem vir a óbito. Os sinais clínicos são decorrentes do polimorfismo de base única (SNP) c.767G>T no gene Dynamin 1 (DNM1). O objetivo deste trabalho foi determinar a ocorrência deste SNP em 321 cães da raça Labrador Retriever do Estado de São Paulo. Primers específicos para a amplificação de todo o exon 6 do gene DNM1 foram usados nas PCRs utilizando DNA a partir de amostras de sangue ou swab bucal, a avaliação final foi realizada com sequenciamento direto dos produtos da PCR. Dentre os 321 animais estudados, 3,4 % (11/321) eram homozigotos para o SNP c.767G>T no gene DNM1 e 24,6% (79/321) eram heterozigotos. Somente um dos 11 animais homozigotos apresentavam sinais clínicos compatíveis com a EIC. Este é o primeiro estudo sobre a ocorrência deste SNP no Brasil e considerando que quase 25% dos animais estudados eram heterozigotos, a genotipagem dos animais para este SNP pode ser importante antes dos acasalamentos para cães desta raça. A EIC deve ser considerada nos diagnósticos diferenciais de enfermidades neuromusculares em cães da raça Labrador Retriever.

The exercise-induced collapse (EIC) is considered an autosomal recessive syndrome that mainly affects Labrador Retriever dogs. The disease is characterized by muscle weakness and collapse after intense exercise. Recovery usually occurs after exercise but some animals may die. The clinical signs occurs due to the single-nucleotide polymorphism (SNP) c.767G>T in Dynamin 1 (DNM1) gene. The aim of this study was to evaluate the occurrence of this SNP in 321 Labrador Retriever dogs from São Paulo state. Specific primers for amplification of the entire exon 6 of the DNM1 gene were used in a PCR performed with DNA from blood or buccal swab samples, direct sequencing was performed for the final evaluation. Among 321 animals studied, 3.4% (11/321) of animals were homozygous for the DNM1 SNP (c.767G>T) and 24.6% (79/321) were heterozygous. Only one of the 11 homozygous animals in this study had previous clinical signs compatible with this disease. This is the first study that evaluated the occurrence of DNM1 SNP (c.767G>T) gene in Brazil and considering that almost 25% of the studied animals were heterozygous, the routinely evaluation of this SNP may be important before this breed mating The EIC should be include in the differential diagnosis of neuromuscular diseases in Labrador Retriever dogs.

Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR.

A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations.

Identification of risk factors for exertional heat illness: a brief commentary on genetic testing.

OBJECTIVE: This commentary discusses known links between Exertional Heat Illness (EHI), Malignant Hyperthermia (MH), and other hereditary diseases of muscle. Genetic and functional testing is also evaluated as measures of fitness to return to duty/play. DATA SOURCES: Reviews and research articles from Sports Medicine, Applied Physiology, and Anesthesiology. DATA EXTRACTION: Detailed comparisons of existing literature regarding clinical cases of EHI and MH and the potential utility of genetic testing, specifically the ryanodine receptor (RYR1) gene and other genes related to disorders of skeletal muscle. DATA SYNTHESIS: EHI is a complex disorder wherein physiological, environmental, and hereditary factors interact to endanger an individual's ability to maintain thermal homeostasis. CONCLUSIONS: Individuals' genetic background is likely to play an important role, particularly when EHI recurs. Recurrent EHI has been associated with MH and other genetic disorders, highlighting the importance of identification and exclusion of individuals with known high risk factors.

Is there a link between malignant hyperthermia and exertional heat illness?

Exertional heat illness (EHI) and malignant hyperthermia (MH) are two potentially lethal conditions. It has been suggested that a subset of MH susceptible persons may be predisposed to EHI. We examine the current understanding of these disorders and explore evidence of a relationship. Screening for the muscle type I ryanodine receptor gene should help clarify the relationship between MH and EHI.

[Two familial cases with exertion-induced heat stroke--relationship to malignant hyperthermia].

Two patients in a family of exertion-induced heat stroke were reported. Case 1: A 23-year-old male, paternal cousin of case 2, was admitted to our hospital because of loss of consciousness during running under a burning sun. On physical and neurological examinations, he was deeply comatose with high fever, tachycardia, and increased deep tendon reflexes. Laboratory findings disclosed rhabdomyolysis, acute renal failure, disseminated intravascular coagulation, liver injury, and brain edema. He recovered after intensive cooling, some antibiotics, glycerol and sodium dantrolene administration. Case 2: A 19-year-old male experienced loss of consciousness and high fever during playing soccer at 15 years of age, and was admitted to a hospital. On admission, he had high fever of 38.7 degrees C, and increased serum CK level. He recovered two weeks after admission. He was readmitted to our hospital to evaluate the predisposition for malignant hyperthermia. His physical and neurological examinations showed no abnormalities. Routine laboratory findings were within normal limits. Muscle biopsy findings of cases 1 and 2 were mildly increased number of fibers with centrally placed nuclei. Caffeine test on skinned muscle fibers from the biopsies showed normal response in both type 1 and 2 fibers. The present patients were diagnosed as having exertion-induced heat stroke, but with no increased muscle fiber sensitivity to caffeine, suggesting that the pathomechanism differs from that of malignant hyperthermia induced by malfunction of sarcoplasmic reticulum.