In studying the molecular defects responsible for cross-reacting material- positive hemophilia A, we have identified two patients in whom the nonfunctional factor VIII-like protein has abnormal, slower-moving heavy or light chains on SDS/PAGE. Both patients have severe hemophilia A (<1% of normal factor VIII activity) with a normal plasma level of factor VIII antigen. The molecular defects were identified by denaturing gradient gel electrophoresis screening of PCR-amplified products of the factor VIII-coding DNA sequence followed by nucleotide sequencing of the abnormal PCR products. In patient ARC-21, a methionine-to-threonine substitution at position 1772 in the factor VIII light chain creates a potential new N-glycosylation site at asparagine-1770. In patient ARC-22, an isoleucine-to-threonine substitution at position 566 creates a potential new N-glycosylation site at asparagine- 564 in the A2 domain of the factor VIII heavy chain. The mobility of these chains on SDS/PAGE was normal after N-Glycanase digestion and procoagulant activity was generated-to a maximum of 23% and 45% of control normal plasma. Abnormal N-glycosylation, blocking factor VIII procoagulant activity, represents a newly recognized mechanism for the pathogenesis of severe hemophilia A.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Jun 15 1992|
- denaturing gradient gel electrophoresis
- factor VIII
ASJC Scopus subject areas