Detection of mutations in ectopic factor VIII transcripts from nine haemophilia A patients and the correlation with phenotype.

Haemophilia A is a common X-linked recessive disorder of bleeding caused by deleterious mutations in the gene for clotting factor VIII. The large size of the factor VIII gene, the high frequency of de novo mutations and its tissue-specific expression complicate the detection of mutations. We have used a combination of reverse transcription/polymerase chain reaction (RT-PCR) of ectopic factor VIII transcripts and PCR of genomic DNA to amplify the entire essential sequence of the factor VIII gene. Chemical mismatch cleavage analysis and direct sequencing have then be employed in order to facilitate a comprehensive search for mutations. In this report, we describe the characterisation of nine potentially pathogenic mutations, six of which are novel. The mutations include six single base substitutions (five missense, viz. D56E, V162M, G701D, A1834T and R1869I, and one nonsense, viz. R-5X), a single base deletion (5697delC), a gross deletion of exon 16 and one mRNA abnormality characteristic of the common intron-22-embedded F8A-mediated DNA inversion. In each case, a correlation of the genotype with the observed phenotype is presented. In order to evaluate the pathogenicity of the five missense mutations, we have analysed them for evolutionary sequence conservation and for their involvement with sequence motifs catalogued in the PROSITE database of protein sites and patterns. Analysis of the sequences in the immediate vicinity of the mutations has revealed sequence features that may have had a possible role in mutagenesis.