Oligonucleotides were computer designed to amplify by the polymerase chain reaction (PCR) the coding region, splice junctions, 112 bp of the 5' flanking region and 279 bp surrounding the polyadenylation site of the
factor IX gene for analysis by denaturing gradient gel electrophoresis (DGGE). Forty-four unselected
haemophilia B patients were studied of whom 24 had severe
haemophilia and 20 had a mild to moderate form of the disease. Potential mutations were identified in 40 (91%) of the 44 cases. A defect could not be detected in three severe and one mild haemophiliac by DGGE analysis and direct sequencing of all the PCR fragments from these patients revealed no
nucleotide alteration supporting the DGGE results. A total of 37 point mutations, two complete gene deletions and a duplication of 26 bp were found. The 37 point mutations included 35 single
nucleotide substitutions, a deletion and an insertion of one
nucleotide. The 35 single
nucleotide substitutions included 26 missense mutations, seven
nonsense mutations, a G (-6) to A transition in the promoter region and a G (30154) to A transition within the donor splice site of the last intron. Fifteen of these
nucleotide substitutions involved CpG dinucleotides. Fifteen point mutations were found at
codons where
nucleotide substitutions had not been detected before. An insertion of a single
nucleotide T at position 6370 and deletion of a G at
nucleotide 30845 resulted in frameshift mutations creating
stop codons at
amino acid positions -2 and 250, respectively. A duplication of 26 bp (17747-17772) in exon V was found in a severe
haemophilia patient resulting in a
termination codon in exon VI. The detection of the mutation by the combined use of PCR, DGGE and direct sequencing was important for carrier diagnosis of 20 families with no prior history of
haemophilia B.