Novel genotyping assay for the nt230 (del4) ABCB1 gene mutation and its allele frequency in Border Collie dogs in Mexico

A 4-bp deletion in the ATP-binding cassette subfamily B member 1 (ABCB1) gene, also referred to as the multidrug resistance gene (MDR1), produces stop codons that cause premature termination of P-glycoprotein 1 (P-gp) synthesis. Dogs with the homozygous mutation do not express functional P-gp, which increases their sensitivity markedly to many common veterinary drugs. We detected the nt230 (del4) ABCB1 mutation in Border Collie dogs in western Mexico with a simple and affordable primer-introduced restriction analysis PCR (PIRA-PCR). PIRA-PCR clearly identified all genotypes in our sample of 104 dogs. Genotype frequencies were 0.952 (wild/wild), 0.029 (wild/mut) and 0.019 (mut/mut). Allele frequencies were 0.033 (mutant alleles) and 0.966 (wild-type alleles). In this small subset of the Mexican dog population, we found a higher prevalence of the nt230 (del4) MDR1/ABCB1 gene mutation than reported in other countries.     

Frequency of pulmonary mineralization and hypoxemia in 21 dogs with pituitary-dependent hyperadrenocorticism

The purpose of this study was to determine the frequency of hypoxemia and pulmonary mineralization using ^99mTc-methylene diphosphonate (^99mTc-MDP) in dogs with pituitary-dependent hyperadrenocorticism (PDH). Twenty-one dogs with PDH were pro-spectively evaluated using thoracic radiography, arterial blood gas analysis, and bone phase and pulmonary perfusion scintigraphy (using ^99mTc-macro-aggregated albumin [^99mTc-MAA]). The radiographs and bone and perfusion studies were evaluated subjectively. An averaged quantitative count density ratio was calculated between the thorax and cranial thoraco-lumbar vertebrae from lateral thoracic ^99mTc-MDP images. Thoracic: vertebral ratios were calculated using ^99mTc-MDP studies from 21 control dogs. The thoracic: vertebral ratios were compared between the 2 groups (PDH and control). The mean age (±SD) of the 21 PDH dogs was 10.2 (±3) years, whereas the mean age of the control group was 9.8 (±3) years. Seven of the 21 dogs with PDH were hypoxemic (denned as an arterial partial pressure of oxygen [PaO₂] <80 mm Hg) with an average PaO₂ (±SD) of 62 (±15) mm Hg. Of the 7 hypoxemic dogs, 2 were found to have pulmonary mineralization based on bone scintigraphic images. Pulmonary perfusion abnormalities were not identified using ^99mTc-MAA in any of the 21 PDH dogs. Six PDH dogs had an abnormal interstitial pulmonary pattern and 5 of these dogs were hypoxemic. The average quantitative thoracic: vertebral ratio was not significantly different between the PDH and control dogs (0.5 ± 0.4 versus 0.4 ± 0.1, P = .16). Causes of hypoxemia other than pulmonary thromboembolism should be considered in dogs with PDH. Pulmonary mineralization may contribute to hypoxemia in dogs with PDH.     

QUANTITATIVE HEPATOBILIARY SCINTIGRAPHY USING 99mTC-DISIDA IN THE DOG

Quantitative hepatobiliary scintigraphy using ^99mTc-2,6 diisopropylphenylcarbamoylmethyl iminodiacetic acid (DISIDA) was performed in normal dogs. ^99mTc-DISIDA is excreted by the hepatocyte into the biliary tract. Hepatic blood flow from arterial and portal venous sources was measured as the hepatic perfusion index. The hepatic clearance of the radiopharmaceutical was used to assess hepatocyte function and parenchymal biliary transport. Biliary ejection was evaluated using synthetic cholecystokinin infusion. The procedure should be useful in evaluating dogs with hepatic and biliary disease.     

VALIDATION OF DECONVOLUTIONAL ANALYSIS FOR THE MEASUREMENT OF HEPATIC FUNCTION IN DOGS WITH TOXIC-INDUCED LIVER DISEASE

The extraction of the hepatobiliary radiopharmaceutical ^99mTc-mebrofenin ( Choletec ) by the liver can be used to evaluate the severity of hepatocellular disease. The hepatic parenchymal cells extract mebrofenin from the blood by the same active transport mechanism as bilirubin. The ability of the liver to extract ^99mTc-membrofenin is a measure of hepatic parenchymal cell function. In this study, we induced hepatocellular disease by administration of a hepatotoxic drug and compared a direct method of determining the hepatic extraction of ^99mTc-membrofenin to hepatic extraction fraction derived from deconvolutional analysis. We also compared both methods of calculating the hepatic extraction of ^99mTc-membrofenin to liver histopathology. Hepatic extraction fraction derived from deconvolutional analysis correlated very well to the direct measurement technique (R=0.922, p<0.001). Both methods of determining hepatic extraction correlated well to quantitative histopathology, having the same correlation coefficient and p values. (R=-0.833, p=0.003). As the hepatic extraction ^99mTc-membrofenin decreased, the severity of the histopathologic lesions of the liver increased in a linear fashion. There was a significant correlation of the hepatic excretion T_1/2 to quantititative histopathology (R=0.949, p<0.001). The hepatic excretion T_1/2 increased as the severity of the histopathologic lesions of the liver increased. Hepatic extraction (HEF) and excretion of ^99mTc-membrofenin are good predictors of the severity of hepatocellular damage in toxic induced liver disease. This study helps validate the premise that HEF derived from deconvolutional analysis ois a good predictor of the actual first pass hepatic extraction of ^99mTc-membrofenin.     

COMPARISON BETWEEN THE SCINTIGRAPHIC UPTAKE AND PLASMA CLEARANCE OF 99mTc-DIETHYLENETRIAMINEPENTACETIC ACID (DTPA) FOR THE EVALUATION OF THE GLOMERULAR FILTRATION RATE IN DOGS

The scintigraphically measured percentage dose uptake of ^99mTc-DTPA by the kidneys and the plasma clearance of ^99mTc-DTPA have been reported to correlate well with inulin clearance. These two parameters were evaluated in seven dogs with known or suspected naturally occurring renal disease and compared to simultaneously measured renal inulin clearance. Correlation between inulin clearance and the ^99mTc-DTPA plasma clearance was better ( p =.0016) than the correlation between the percentage DTPA uptake by the kidney. It was concluded that measurement of ^99mTc-DTPA plasma clearance is a more accurate method to estimate global glomerular filtration rate (GFR) than the percentage kidney uptake.     

Effect of short-term treatment with meloxicam and pimobendan on the renal function in healthy beagle dogs

The aim of the study was to investigate the renal function in clinically normal dogs receiving meloxicam and pimobendan alone or in combination. Ten adult female beagle dogs were administered the treatment for 7 days in a randomized crossover trial (control/meloxicam/pimobendan/meloxicam and pimobendan). Renal function was assessed by blood urea, creatinine, sodium, potassium and chloride concentrations and by glomerular filtration rate, measured by means of renal scintigraphy [renal uptake of ^99mTc-diethylenetriaminepentacetic acid (DTPA)] and plasma clearance of ^99mTc-DTPA. As compared with the control group, renal uptake and plasma clearance of ^99mTc-DTPA were not significantly modified after a 7-day period of treatment with meloxicam or pimobendan alone, or meloxicam and pimobendan in combination. Furthermore, urea, creatinine, sodium, potassium and chloride levels in the serum of the dogs during the 7-day period treatment were not significantly modified in relation to the treatments. It was therefore concluded that meloxicam and pimobendan alone or in combination did not alter renal function in healthy dogs.     

Canine ABCB1 and macrocyclic lactones: heartworm prevention and pharmacogenetics

The impact of drug transporters on drug pharmacokinetics and pharmacodynamics has been increasingly recognized in recent years. P-glycoprotein (P-gp), the product of the ABCB1 (formerly MDR1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. P-gp is expressed by a variety of normal tissues, including the intestines, brain capillary endothelial cells, renal tubular cells, and biliary canalicular cells, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs and enhances their excretion from the body. Many drugs used in veterinary medicine are substrates for P-gp, including many chemotherapeutic agents and macrocyclic lactones (avermectins and milbemycin). A 4-base pair deletion mutation in the ABCB1 gene occurs in many herding breed dogs, including collies, Australian shepherds, and Shetland sheepdogs. The mutation (ABCB1-1Delta) renders affected animals extremely susceptible to toxicosis induced by substrate drugs, such as the macrocyclic lactones at doses well below those tolerated by dogs with the wild-type ABCB1 gene. However, at the manufacturer's recommended dose, all FDA-approved heartworm preventive products marketed in the United States are safe, even for dogs with the ABCB1 mutant/mutant genotype.     

99mTc-METHOXY-ISOBUTYL-ISONITRILE (SESTAMIBI) IMAGING OF MALIGNANT CANINE LYMPHOMA

Technetium-99m methoxy-isobutyl-isonitrile (sestamibi) imaging of malignant canine lymphoma was performed in thirteen dogs 1 hour after intravenous injection of ^99mTc-sestamibi at 13 MBq (0.35 mCi) per kilogram body weight. Abnormal tracer uptake was visualized in the liver, spleen, bone marrow, and mesenteric, inguinal, popliteal, sternal, cranial cervical and mandibular lymph nodes. Radiopharmaceutical uptake was also noted in a nasal mass. One large neoplastic renal mass did not have demonstrable sestamibi uptake. Other regions had no significant difference in the target:background ratios when compared with values from normal dogs ( P > 0.05). ^99mTc-sestamibi can be used to image malignant lymphoma, and has potential applications in the management of patients to document response to treatment and to stage of extent of disease.     

LYMPH NODE UPTAKE OF 99MTc-MDP DURING BONE SCINTIGRAPHY IN DOGS

Localization of ^99mTc-MDP in lymph nodes was apparent on the three-hour bone-scan image in seven dogs. In six dogs injection or leakage of the radiopharmaceutical into the perivascular tissues was associated with subsequent uptake in an ipsilateral lymph node. In the remaining dog, ^99mTc-MDP localized in a lymph node infiltrated by metastatic osteosarcoma. This aided staging of the tumor. Possible mechanisms of ^99mTc-MDP localization in soft tissues are briefly reviewed.     

IN-VIVO TESTING OF TECHNETIUM-99m-LABELED MONOCLONAL ANTIBODIES SPECIFIC FOR DIROFILARIA IMMITIS

Monoclonal antibodies specific for Dirofilaria immitis were labeled with technetium-99m (^99mTc) and tested in vivo in five heartworm positive and five heartworm negative dogs. Diethlyenetriaminepentaactic acid (DTPA) was used to label the ^99mTc to the antibody. Radiochemical purity of the ^99mTc-labeled antibodies (^99mTc-MoAb) was determined by gel filtration and by instant thin-layer chromatography. The uptake percentages in the blood, heart, lung, liver, and kidneys were determined from tissues obtained at necropsy and by computer analysis of images obtained at two and 24 hours. The percent uptake in the heart region of interest (ROI) at two hours was significantly higher in the heartworm-positive dogs. There was significant correlation between the uptake in the heart at two hours to the number and activity of the worms at necropsy.     

QUANTITATIVE THYROID SCINTIGRAPHY IN GREYHOUNDS SUSPECTED OF PRIMARY HYPOTHYROIDISM

The existence of hypothyroidism in greyhounds remains controversial and its investigation is complicated by the low circulating thyroid hormone concentrations typically found in healthy dogs of this breed. Quantitative measurement of thyroidal technetium-99m pertechnetate (^99mTcO₄⁻) uptake is known to be useful in assessing thyroid function in other breeds. The aim of this study was to evaluate thyroid scintigraphy as a method of assessing thyroid function in greyhounds suspected of primary hypothyroidism. Twenty greyhounds (eight females, 12 males) were studied. Thirteen had bald thigh syndrome and seven poor performance and low total T4. Total T4 concentrations were decreased in 18 (90%), and free T4 in two (10%) dogs. All canine thyroid stimulating hormone concentrations were within the reference interval. Thyroidal ^99mTcO₄⁻ uptake values (mean ± SD, 0.76 ± 0.26%) were within the reference limits published for euthyroid dogs (0.39–1.86%) making hypothyroidism highly unlikely. There were no significant differences ( P <0.05) when comparing data between dogs with bald thigh syndrome (13 dogs) and the remaining dogs (seven dogs). Seventeen (85%) dogs had higher uptake in the left thyroid gland than in the right that might reflect an anatomic feature of the greyhound breed. Calculation of percent thyroidal uptake of ^99mTcO₄⁻ is more accurate than thyroid:salivary gland ratios because of high variability in salivary gland uptake. Percent thyroidal uptake of ^99mTcO₄⁻ should be used when assessing thyroid function scintigraphically in the greyhound breed.     

Nuclear Medical Bone Imaging and Targeted Radiography for Evaluation of Skeletal Neoplasms in 23 Dogs

Nuclear medical bone imaging was used in combination with targeted radiography to detect metastatic or multicentric lesions in 23 dogs with skeletal neoplasms. Each dog with a radio-graphically diagnosed skeletal neoplasm was injected with Technetium-99m labeled methylene diphosphonate (^99mTc-MDP) (15.0 mCi intravenously) for whole body imaging. All areas with increased uptake of ^99mTc-MDP were radiographed. In 19 dogs, the amount of bone shaft involvement in primary sites indicated by bone imaging was larger than the amount indicated by radiography. Eighteen dogs had secondary areas of increased ^99mTc-MDP uptake, six of which had secondary areas that were suspected radiographically to be neoplastic. Four dogs had lesions characterized histologically as neoplasia, one as bone infarction and one as normal cortical bone. Positive predictive value for this strategy was 66.7%. Results of this study suggest that nuclear medical bone imaging with targeted radiography is a quick, noninvasive technique with a good positive predictive value for evaluation of the skeleton for metastatic or multicentric sites of neoplasia.     

TECHNETIUM-99M AND IODINE-131 THYROID SCINTIGRAPHY IN NORMAL AND RADIOTHYROIDECTOMIZED COCKATIELS (NYMPHICUS HOLLANDICUS)

Thyroid scintigraphy using sodium ^99mTechnetium pertechnetate (⁹⁹TcO₄-) was performed in normal and radiothyroidectomized cockatiels ( Nymphicus hollandicus ). ¹³¹I scintigraphy was performed in the course of ablating the thyroid glands. The thyroid glands in normal birds were clearly visualized, but were not individually resolvable. Thyroid glands were not visualized in thyroid-ablated birds. With ^99mTcO₄ scans, thyroid (or other region of interest)/body count density ratios were used for comparisons of normal and thyroid-ablated birds. Normal ^99mTcO₄- thyroid/body ratios (mean +/− SD) for the dorsal and lateral views were 1.83 +/− 0.31 and 1.70 +/− 0.34 respectively. Pertechnetate thyroid/body count density ratios decreased (to 0.93 -/−0.14 and 0.88 +/− 0.12 for dorsal and lateral views respectively) after thyroid ablation, while crop/body ratios increased. ^99mTcO₄- thyroid scintigraphy, therefore, was capable of detecting hypofunctional thyroid abnormalities in ¹³¹I radiothyroidectomized cockatiels.     

ABCB1-1Δ (MDR1-1Δ) genotype is associated with adverse reactions in dogs treated with milbemycin oxime for generalized demodicosis

Twenty-two dogs diagnosed with generalized demodicosis were treated with milbemycin oxime (MO) because of poor response to previous therapies or because the dog was a breed known to be susceptible to ivermectin toxicosis. Fifteen of the 22 dogs were herding breeds. Doses of MO ranged from 1.0 to 2.2 mg kg⁻¹ day⁻¹ per os. Cheek swab samples were obtained in order to determine each dog's ABCB 1 genotype. Adverse drug reactions were recorded for each dog by the owners and/or veterinarians. The ABCB 1-1Δ genotype was significantly associated with the development of an adverse reaction (neurological toxicity) after treatment with MO. None of the 19 dogs with the wild-type ABCB1 allele experienced adverse reactions, whereas two dogs homozygous for the ABCB1-1Δ mutation developed ataxia. Assessing the ABCB1-1Δ genotype prior to MO administration may prevent neurological toxicity in these patients.     

PULMONARY MINERALIZATION IN FOUR DOGS WITH CUSHING'S SYNDROME

The clinical and imaging features of four dogs with Cushing's syndrome and pulmonary mineralization are reviewed. Three dogs presented with a primary complaint of respiratory distress/dyspnea. Three dogs had pituitary dependent Cushing's syndrome, while the remaining one dog had iatrogenic Cushing's syndrome. Each dog had clinical features typical for Cushing's syndrome. Two of the dogs were euthanized due to progressive hypoxemia. In each dog, the serum calcium, phosphorous, blood urea nitrogen and creatinine were normal.
A generalized increase in unstructured interstitial pulmonary opacity with diffuse mineralization was noted on thoracic radiographs of all dogs. In one dog, an ill-defined nodular interstitial pattern of mineralization was present. Delayed bone phase scintigraphy using ^99mTechnetium methylene diphosphonate documented generalized pulmonary uptake in two dogs. ^99mTechnetium labeled microaggregated albumin lung perfusion scans were normal in these two dogs. ^99mTc-MDP scintigraphy can provide useful information in diagnosing pulmonary mineralization in Cushingoid dogs.     

CANINE VENTRAL BODY WALL LYMPHOSCINTIGRAPHY

The scintigraphic characteristics of lymph nodes draining the ventral body wall were analyzed in 12 dogs as to their pattern of appearance and image contrast. Each dog was scanned using ^99mTc-antimony sulfide colloid and ^99mTc-dextran, and the imaging characteristics of these agents were compared. A large degree of variability exists in the number and pattern of lymph node appearance with either agent. No significant difference in average numbers of lymph nodes per dog was shown between agents. However, ^99mTc-antimony sulfide colloid provided a significantly (p<.05) better target-to-background ratio, which determines the contrast with which lymph nodes stand out from background activity. From these findings, it is suggested that too great a variability in lymph node appearance exists using this procedure to allow a high degree of confidence in the diagnosis of metastasis to lymph nodes of the ventral body wall. However, for other purposes, ^99mTc-antimony sulfide colloid is the lymphoscintigraphic agent of choice.     

The ABCB1-1Δ mutation is not responsible for subchronic neurotoxicity seen in dogs of non-collie breeds following macrocyclic lactone treatment for generalized demodicosis

P-glycoprotein (P-gp), encoded by the multiple drug resistance gene ABCB1 (also known as MDR1 ), is an integral component of the blood brain barrier crucial in limiting drug uptake into the central nervous system. Altered expression or function of P-gp, as seen in dogs of the collie lineage homozygous for the nt228(del4) mutation of the ABCB1 gene ( ABCB1-1Δ ), can result in potentially fatal neurotoxicosis, especially following administration of systemic macrocyclic lactones (SML). Occasionally, dogs from unrelated breeds develop subchronic signs of neurotoxicity when receiving SML to treat generalized demodicosis. It is possible that these dogs are heterozygous carriers of the ABCB1-1Δ mutation, resulting in decreased P-gp activity and central neurotoxicosis. Cheek swabs were collected from 28 dogs with generalized demodicosis that had shown subchronic signs of neurotoxicity following daily oral administration of ivermectin or other SML. Ten of these animals received concurrent systemic treatment with other confirmed or putative P-gp substrates. After DNA extraction, the relevant portion of the ABCB1 gene was amplified by polymerase chain reaction, and sequenced. Twenty-seven dogs were homozygous normal while one dog was heterozygous for the ABCB1-1Δ mutation. Therefore, with the exception of one dog, the observed neurotoxicity could not be attributed to the ABCB1-1Δ mutation. Possible explanations for the adverse reactions observed include pharmacological interactions (administration of SML with other P-gp substrates or inhibitors), excessively high doses, polymorphisms in P-gp expression, uncharacterized mutations in the ABCB1 gene or in another gene, or phenomena unrelated to the SML–P-gp interaction.     

ABCB1-1Δ Polymorphism Can Predict Hematologic Toxicity in Dogs Treated with Vincristine

Background: Dogs that harbor the naturally occurring ABCB1-1Δ polymorphism experience increased susceptibility to avermectin-induced neurological toxicosis as a result of deficient P-glycoprotein function. Whether or not the ABCB1-1Δ polymorphism affects susceptibility to toxicity of other P-glycoprotein substrate drugs has not been studied.
Hypothesis: Dogs that possess the ABCB1-1Δ mutation are more likely to develop hematologic toxicity associated with vincristine than ABCB1 wild-type dogs.
Animals: Thirty-four dogs diagnosed with lymphoma were included in this study.
Methods: Cheek swab samples were obtained from dogs diagnosed with lymphoma that were to be treated with vincristine. DNA was extracted from cheek swabs and the ABCB1 genotype was determined. Hematologic adverse drug reactions were recorded for each dog and graded according to the Veterinary Comparative Oncology Group's criteria for adverse event reporting (Consensus Document). In order to avoid possible bias, ABCB1 genotype results for a particular patient were not disclosed to oncologists until an initial adverse event report had been submitted.
Results: Dogs heterozygous or homozygous for the ABCB1-1Δ mutation were significantly more likely to develop hematologic toxicity, specifically neutropenia ( P = .0005) and thrombocytopenia ( P = .0001), after treatment with vincristine than ABCB1 wild-type dogs.
Conclusions and Clinical Implications: At currently recommended dosages (0.5–0.7 mg/M²), vincristine is likely to cause hematologic toxicity in dogs with the ABCB1-1Δ mutation, resulting in treatment delays and unacceptable morbidity and mortality. Assessing the ABCB1-1Δ genotype before vincristine administration and decreasing the dosage may prevent toxicity and treatment delays resulting from neutropenia or thrombocytopenia.     

ABCB1-1Delta polymorphism can predict hematologic toxicity in dogs treated with vincristine.

BACKGROUND: Dogs that harbor the naturally occurring ABCB1-1Delta polymorphism experience increased susceptibility to avermectin-induced neurological toxicosis as a result of deficient P-glycoprotein function. Whether or not the ABCB1-1Delta polymorphism affects susceptibility to toxicity of other P-glycoprotein substrate drugs has not been studied. HYPOTHESIS: Dogs that possess the ABCB1-1Delta mutation are more likely to develop hematologic toxicity associated with vincristine than ABCB1 wild-type dogs. ANIMALS: Thirty-four dogs diagnosed with lymphoma were included in this study. METHODS: Cheek swab samples were obtained from dogs diagnosed with lymphoma that were to be treated with vincristine. DNA was extracted from cheek swabs and the ABCB1 genotype was determined. Hematologic adverse drug reactions were recorded for each dog and graded according to the Veterinary Comparative Oncology Group's criteria for adverse event reporting (Consensus Document). In order to avoid possible bias, ABCB1 genotype results for a particular patient were not disclosed to oncologists until an initial adverse event report had been submitted. RESULTS: Dogs heterozygous or homozygous for the ABCB1-1Delta mutation were significantly more likely to develop hematologic toxicity, specifically neutropenia (P= .0005) and thrombocytopenia (P= .0001), after treatment with vincristine than ABCB1 wild-type dogs. CONCLUSIONS AND CLINICAL IMPLICATIONS: At currently recommended dosages (0.5-0.7 mg/M(2)), vincristine is likely to cause hematologic toxicity in dogs with the ABCB1-1Delta mutation, resulting in treatment delays and unacceptable morbidity and mortality. Assessing the ABCB1-1Delta genotype before vincristine administration and decreasing the dosage may prevent toxicity and treatment delays resulting from neutropenia or thrombocytopenia.     

THE INSENSITIVITY OF 99mTc PERTECHNETATE FOR DETECTING METASTASES OF A FUNCTIONAL THYROID CARCINOMA IN A DOG

This report describes the use of ^99mtechnetium pertechnetate (^99mTcO₄⁻) and ¹³¹[for imaging of a metastatic thyroid carcinoma in a dog. The ¹³¹] imaging showed metastatic lesions undetected by the ^99mTcO₄⁻ imaging on 2 separate occasions. The possible mechanisms for the discrepancies between ¹³¹I and ^99mTcO₄⁻ imaging of thyroid carcinomas are discussed. The use of ¹³¹I for the imaging of functional thyroid carcinomas in the dog is recommended.