Cobalamin (vitamin B12) deficiency (CD) is a common disorder in the Chinese Shar Pei (Shar Pei) and is suspected to be hereditary. As the severity of clinical symptoms and the age of onset of CD vary widely, identification of a genetic marker co-segregating with CD would facilitate the development of a DNA-based test for early identification of affected dogs, facilitating further research into this condition in Shar Peis. The objective of this study was to identify genetic linkage of microsatellite markers to phenotypic CD in Shar Peis.
Whole blood and serum were collected from a total of 42 unrelated Shar Peis. Owners were asked to fill out a questionnaire regarding the current health status of each dog. Serum cobalamin concentration (reference range 252-908 ng/L) was measured for each dog and DNA was extracted from whole blood. A total of 327 microsatellite markers, 326 belonging to the canine minimal screening set 2 (MSS-2), and a single microsatellite marker not contained in the MSS-2, were amplified by polymerase chain reaction (PCR). PCR products were resolved by automated capillary electrophoresis, and sized relative to an internal standard followed by analysis of genotype data by a commercially available software package. Linkage analysis was conducted with a Fisher's exact test, and statistical significance was set at p < 0.0001 (p < 1×10-4).
Undetectable serum cobalamin concentrations (< 150 ng/L) were observed in 14 of 42 dogs (33.3%). These dogs were considered to be cobalamin deficient. Serum cobalamin concentrations were within the reference range in the remaining 28 dogs. Allele 356 of the microsatellite marker DTR13.6 on chromosome 13 was found significantly more frequently in cobalamin-deficient Shar Peis (19 of 28 dogs or 67.9%) than in Shar Peis with normal serum cobalamin concentrations (10 of 56 dogs or 17.9%; p-value = 8×10-6). Allele 315 of microsatellite REN13N11 on chromosome 13, which is not contained in the MSS-2, was found significantly more frequently in cobalamin-deficient Shar Peis (18 of 28 dogs or 64.3%) than in Shar Peis with normal serum cobalamin concentrations (10 of 56 dogs or 17.9%; p-value = 3×10-5).
Both microsatellite markers on canine chromosome 13, DTR13.6 and REN13N11, showed significant linkage disequilibrium, revealing linkage of alleles 356 and 315, respectively, to serum CD in Shar Peis. This association will facilitate further evaluation of the genomic loci in close proximity to both microsatellite markers on chromosome 13 in Shar Peis, which in turn may lead to further characterization of this condition.