Identification of a 6 base pair insertion in West Highland White Terriers with erythrocyte pyruvate kinase deficiency

Objective-To define the disease-causing mutation in West Highland White Ter riers (WHWT) with erythrocyte pyruvate kinase (R-PK) deficiency and to desi gn a genetic test capable of recognizing affected (homozygous) and carrier (heterozygous) dogs. Animals-3 anemic WHWT littermates and 1 unaffected littermate; 16 dogs from the same kennel, including 4 unrelated, phenotypically normal dogs (contro l dogs), and 12 for which PK activity was not known; 2 PK-deficient Basenji s; 2 PK-deficient Beagles; 4 unaffected English Springer Spaniels; and 1 mi xed-breed dog. Procedures-cDNA was cloned and sequenced, and cDNA sequences were compared with the published sequence for canine R-PK cDNA to identify the putative d isease-causing mutation. Genomic DNA spanning the affected region was clone d and sequenced to verify the mutation. Subsequently, polymerase chain reac tion primers were designed to amplify the section of the gene containing th e mutation from DNA in blood or buccal swab samples. C;el electrophoresis a llowed assignment of genotypes on the basis of allele separation. Results-4 single base polymorphisms attributable to sequencing errors in th e published sequence were identified, along with a 6 base pair (bp) inserti on in exon 10 that was recognized as a putative disease-causing mutation. A n identical insertion was found in genomic DNA. Amplification of genomic DN A yielded a 117 bp product for genotypically normal dogs and a 123 bp produ ct for WHWT homozygous for PK deficiency. Carriers had 1 copy of each allel e and variable heteroduplex structures. Conclusions and Clinical Relevance-A 6 bp insertion in the C domain of R-PK was identified in WHWT with PK deficiency. Affected and carrier dogs could be distinguished with a genetic test.