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Analysis of the molecular defects causing haemophilia B in six patients

University of British Columbia

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 the 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 Leamington resulted in the conversion of glycine^ to alanine. Activity and antigen levels of FDC Leamington are both below 1%. Glycines is situated in the second EGF-like domain of factor IX. From sequence homology and models based on human EGF and the first EGF-like domains of FIX and FX, glycines is conserved and likely occupies the third position in a type II |3 turn. Alanine at this position is thought to disrupt the turn and possibly affect disulfide bonding between cysteinem and cysteine 124. In FIX Creston, arginineiso is mutated to proline due to a G to C alteration at nt. position 20,519. Although normal levels of FIX were present, only -2% activity was exhibited. The mutation disrupted the arginineiso-valineisi cleavage site required by FXIa and FVIIa to activate FIX. In FIX Edmonton 3, a G to A transition at nt. position 30,150 converted alanine233 to threonine and resulted in a mutant enzyme with 15% activity. Computer models of the catalytic domain (based on crystal structures of the pancreatic serine proteases) show alanine233 to be removed from the active site and substrate binding pocket. The threonine mutation may therefore disrupt FVIIIa binding rather than interfere with catalysis. Alternatively, according to computer models the leucine379 to phenylalanine mutation (A to C at nt. position 31,258) is situated near the active site region in FIX Brantford. Phenylalanine, having a larger and more constrained side chain, likely disrupts the surrounding tightly packed residues thus affecting substrate binding and/or catalysis. FIX Brantford exhibited 5% activty.

Thesis/Dissertation

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2008-12-18

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http://hdl.handle.net/2429/3112

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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