Mutational analysis and coagulation factor VIII sequence in a colt with hemophilia A

Abstract

Hemophilia A is an X-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed based on deficiency in FVIII activity, causative gene mutations have not been identified. The objectives of this project were to determine the sequence for the FVIII gene in the horse, identify the causative mutation in a colt with hemophilia A, and determine if the mutation was spontaneous or due to maternal inheritance. Genomic sequence analysis was compared from amplified PCR products from a colt diagnosed with hemophilia A, the colt’s mare, and a normal horse. PCR reactions were also performed on hepatic cDNA and a western blot on hepatic lysate from the affected colt and normal horse. Based on the results, the mutation was isolated to the non-coding region of FVIII (specifically intron 1). Genomic sequencing of intron 1 suggested maternal inheritance. Transcription of the intron across the 5’ splice site suggested activation of an aberrant splice site in intron 1. It is yet to be determined if this defect is responsible for hemophilia A in other horses.