title: Identification of a 6 base pair insertion in West Highland White Terriers with erythrocyte pyruvate kinase deficiency.
abstract: OBJECTIVE
  To define the disease-causing mutation in West Highland White Terriers (WHWT) with erythrocyte pyruvate kinase (R-PK) deficiency and to design a genetic test capable of recognizing affected (homozygous) and carrier (heterozygous) dogs.
  
  
  METHODS
  3 anemic WHWT littermates and 1 unaffected littermate; 16 dogs from the same kennel, including 4 unrelated, phenotypically normal dogs (control dogs), and 12 for which PK activity was not known; 2 PK-deficient Basenjis; 2 PK-deficient Beagles; 4 unaffected English Springer Spaniels; and 1 mixed-breed dog.
  
  
  METHODS
  cDNA was cloned and sequenced, and cDNA sequences were compared with the published sequence for canine R-PK cDNA to identify the putative disease-causing mutation. Genomic DNA spanning the affected region was cloned and sequenced to verify the mutation. Subsequently, polymerase chain reaction primers were designed to amplify the section of the gene containing the mutation from DNA in blood or buccal swab samples. Gel electrophoresis allowed assignment of genotypes on the basis of allele separation.
  
  
  RESULTS
  4 single base polymorphisms attributable to sequencing errors in the published sequence were identified, along with a 6 base pair (bp) insertion in exon 10 that was recognized as a putative disease-causing mutation. An identical insertion was found in genomic DNA. Amplification of genomic DNA yielded a 117 bp product for genotypically normal dogs and a 123 bp product for WHWT homozygous for PK deficiency. Carriers had 1 copy of each allele and variable heteroduplex structures.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6010, USA.
authors.forename: B J
authors.initials: BJ
authors.lastname: Skelly
authors.forename: M
authors.initials: M
authors.lastname: Wallace
authors.forename: Y R
authors.initials: YR
authors.lastname: Rajpurohit
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: U
authors.initials: U
authors.lastname: Giger
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000097:blood / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004283:Dog Diseases / Q000235:genetics*; D004285:Dogs; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004912:Erythrocytes / Q000201:enzymology*; D006580:Genetic Carrier Screening; D005820:Genetic Testing / Q000662:veterinary; D006720:Homozygote; D009154:Mutation; D011770:Pyruvate Kinase / Q000097:blood / Q000172:deficiency* / Q000235:genetics; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA; D011770:Pyruvate Kinase