Sequence Analysis of Six Candidate Genes in Miniature Schnauzers with Primary Hypertriglyceridemia.

Miniature Schnauzers are predisposed to primary hypertriglyceridemia (HTG). In this study, we performed whole genome sequencing (WGS) of eight Miniature Schnauzers with primary HTG and screened for risk variants in six HTG candidate genes: LPL, APOC2, APOA5, GPIHBP1, LMF1, and APOE. Variants were filtered to identify those present in ≥2 Miniature Schnauzers with primary HTG and uncommon (<10% allele frequency) in a WGS variant database including 613 dogs from 61 other breeds. Three variants passed filtering: an APOE TATA box deletion, an LMF1 intronic SNP, and a GPIHBP1 missense variant. The APOE and GPIHBP1 variants were genotyped in a cohort of 108 Miniature Schnauzers, including 68 with primary HTG and 40 controls. A multivariable regression model, including age and sex, did not identify an effect of APOE (estimate = 0.18, std. error = 0.14; p = 0.20) or GPIHBP1 genotypes (estimate = -0.26, std. error = 0.42; p = 0.54) on triglyceride concentration. In conclusion, we did not identify a monogenic cause for primary HTG in Miniature Schnauzers in the six genes evaluated. However, if HTG in Miniature Schnauzers is a complex disease resulting from the cumulative effects of multiple variants and environment, the identified variants cannot be ruled out as contributing factors.

Evaluation of post-transfusion RBC alloimmunization in dogs using a gel-column crossmatch with and without anti-canine globulin enhancement.

A blood crossmatch is essential to ensure RBC compatibility for previously transfused dogs. There is no gold standard crossmatch method for dogs, although the standards used most commonly by academic institutions and reference laboratories are the tube and gel-column crossmatches. Addition of anti-canine globulin (ACG) has been suggested to increase detection of RBC incompatibilities. Our objective was to determine if there is a correlation between results of a standard and an ACG-enhanced gel-column crossmatch in detecting post-transfusion RBC alloimmunization. Pre- and post-transfusion serum or plasma samples were obtained from 33 dogs for major crossmatches to 1-6 (median: 3) blood donors. Crossmatches were performed with (n = 202) and without (n = 202) ACG, with results scored by 4 observers, 3 of whom were anonymized. Ten of 33 (30%) dogs had major crossmatch incompatibilities post-transfusion. RBC incompatibilities (2-4+ agglutination) were detected only with ACG in 4 dogs, only without ACG in 3 dogs, and with both methods in 3 dogs. There was fair correlation between crossmatch methods for determination of compatibility (ρ = 0.34; p < 0.001) and incompatibility (ρ = 0.35; p < 0.001) scores. Among 4 observers, there was near-perfect agreement in determining compatibility (κ = 0.97; p < 0.001) and substantial agreement in overall scoring of incompatibility (κ = 0.77; p < 0.001). Our results suggest that detection of RBC incompatibilities in dogs can be maximized by performing a gel-column crossmatch both with and without ACG enhancement.

Effect of donor blood storage on gel column crossmatch in dogs.

BACKGROUND: Compared with fresh blood, stored equine donor blood results in spurious tube crossmatch incompatibilities. Interpretation of blood crossmatch results is considered subjective. OBJECTIVES: We aimed to determine if the duration of canine donor blood storage impacts compatibility testing using a standard gel column crossmatch and evaluate interobserver variation in the interpretation of crossmatch results. METHODS: Observational study. Whole blood segments were obtained from 23 canine packed red blood cell (RBC) units for use in crossmatches after storage for 0, 7, 14, 21, 28, and 35 days. Major and minor crossmatches were performed using serum and RBCs, respectively, from two to three healthy "recipient" dogs per unit. All crossmatch results were interpreted by four observers, of whom three were blinded. RESULTS: All major crossmatches (n = 61) were compatible on day 0 and remained compatible through day 35 of storage. All minor crossmatches (n = 69) were compatible at all time points, except for five donor pairs with 1 to 3+ agglutination. Repeat testing of these five donor pairs confirmed crossmatch incompatibilities on days 0 through 35, with no change in the degree of incompatibility over time. There was substantial agreement among four observers in determining compatibility (κ = 0.94) and scoring incompatibility (κ = 0.76). CONCLUSIONS: The current practice of performing canine crossmatches with whole blood segments stored for up to 35 days is acceptable, with no spurious changes in compatibility expected over time. The substantial interobserver agreement suggests that the gel column is suitable for performing canine crossmatches in a laboratory setting with multiple personnel.

An EHPB1L1 Nonsense Mutation Associated with Congenital Dyserythropoietic Anemia and Polymyopathy in Labrador Retriever Littermates.

In this report, we describe a novel genetic basis for congenital dyserythropoietic anemia and polymyopathy in Labrador Retriever littermates characterized by incidental detection of marked microcytosis, inappropriate metarubricytosis, pelvic limb weakness and muscle atrophy. A similar syndrome has been described in English Springer Spaniel littermates with an early onset of anemia, megaesophagus, generalized muscle atrophy and cardiomyopathy. Muscle histopathology in both breeds showed distinctive pathological changes consistent with congenital polymyopathy. Using whole genome sequencing and mapping to the CanFam4 (Canis lupus familiaris reference assembly 4), a nonsense variant in the EHBP1L1 gene was identified in a homozygous form in the Labrador Retriever littermates. The mutation produces a premature stop codon that deletes approximately 90% of the protein. This variant was not present in the English Springer Spaniels. Currently, EHPB1L1 is described as critical to actin cytoskeletal organization and apical-directed transport in polarized epithelial cells, and through connections with Rab8 and a BIN1-dynamin complex generates membrane vesicles in the endocytic recycling compartment. Furthermore, EHBP1L1 knockout mice die early and develop severe anemia. The connection of EHBP1L1 to BIN1 and DMN2 functions is particularly interesting due to BIN1 and DMN2 mutations being causative in forms of centronuclear myopathy. This report, along with an independent study conducted by another group, are the first reports of an association of EHBP1L1 mutations with congenital dyserythropoietic anemia and polymyopathy.

Congenital dyserythropoiesis and polymyopathy without cardiac disease in male Labrador retriever littermates.

BACKGROUND: Two Labrador retriever littermates were identified based on incidentally noted marked microcytosis and inappropriate metarubricytosis. Muscle atrophy was noted and associated with distinctive pathological findings in biopsy samples from 1 dog studied. The disorder represents a rare clinical entity of suspected congenital dyserythropoiesis and polymyopathy. Clinicopathologic changes were similar to a previously reported syndrome of congenital dyserythropoiesis, congenital polymyopathy, and cardiac disease in 3 related English Springer Spaniel (ESS) dogs, but the dogs reported here did not have apparent cardiac disease. INTERVENTIONS: Bone marrow aspiration, electromyography, muscle biopsies, and an echocardiogram were performed on dog 1. Results supported dyserythropoiesis and congenital polymyopathy similar to reports in ESS dogs, but did not identify obvious cardiac disease. CONCLUSION: The clinicopathologic changes of dyserythropoiesis and polymyopathy provide an easily recognizable phenotype for what appears to be a low morbidity syndrome. Early recognition may decrease unnecessary testing or euthanasia.