Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis.

The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5' TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3' TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.