DETECTION OF A MOLECULAR DEFECT IN 40 OF 44 PATIENTS WITH HEMOPHILIA-B BY PCR AND DENATURING GRADIENT GEL-ELECTROPHORESIS

Oligonucleotides were computer designed to amplify by the polymerase c hain 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 electropho resis (DGGE). Forty-four unselected haemophilia B patients were studie d 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 th e 44 cases. A defect could not be detected in three severe and one mil d haemophiliac by DGGE analysis and direct sequencing of all the PCR f ragments from these patients revealed no nucleotide alteration support ing the DGGE results. A total of 37 point mutations, two complete gene deletions and a duplication of 26 bp were found. The 37 point mutatio ns included 35 single nucleotide substitutions, a deletion and an inse rtion of one nucleotide. The 35 single nucleotide substitutions includ ed 2 6 missense mutations, seven nonsense mutations, a G (-6) to A tra nsition 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 del etion of a G at nucleotide 30845 resulted in frameshift mutations crea ting stop codons at amino acid positions -2 and 250, respectively. A d uplication of 26 bp (17747-17772) in exon V was found in a severe haem ophilia patient resulting in a termination codon in exon VI. The detec tion of the mutation by the combined use of PCR, DGGE and direct seque ncing was important for carrier diagnosis of 20 families with no prior history of haemophilia B.