Analysis of the molecular defects causing haemophilia B in six patients

Beatrice M. Tam
1992
The factor IX genes from six haemophilia B patients were analyzed in order to determine the molecular defect responsible for causing the disease in each of the cases. Blood samples were obtained from the six patients and genomic DNA was extracted from the white blood cells. Regions of the factor IX gene were amplified from the genomic DNA via the polymerase chain reaction (PCR) for use in either single stranded conformational polymorphism (SSCP) analysis or for subcloning. The exons, including
more » ... he intron/exon splice junctions, were selectively amplified. DNA representing 150 base pairs 3' of the first exon (containing the putative promoter region) was amplified together with exon 1. SSCP analysis was performed on the amplified exons to screen rapidly for the presence of base pair mutations as compared to wild type FIX sequence. The exact nature and locations of the mutations were then determined by DNA sequencing of the subcloned exons. The remaining exons of each FIX gene were also subcloned and sequenced and no other sequence discrepancies were found. A single base pair alteration was found in each FIX gene and was therefore assumed to cause the defect in factor DC and thus cause haemophilia B. Factor IX antigen levels in the patients' plasmas were determined using sandwich ELISA assays with polyclonal anti-factor DC antibodies. The coagulant activity of the mutant factor DC polypeptides were determined using the standard APTT (activated partial thromboplastin time) assays. A C to A change at nt. position 17,700 was found in FIX Edmonton 1. The predicted amino acid sequence at residue 95 changed from cysteine to a stop codon. Likewise, in FDC Edmonton 2, a C to T change at nt. position 31,133 introduced a stop codon in place of arginine at residue 338. Both haemophilias are severe with less than 1% activity and antigen. Disruption of protein structure probably caused these two truncated polypeptides to be degraded within the hepatocytes. A G to C mutation at nt. position 17,756 in FDC Le [...]
doi:10.14288/1.0086598 fatcat:jy5grmbz3rcvnisbz2uphrwrrq