A Thr(359)Met mutation in factor VII of a patient with a hereditary deficiency causes defective secretion of the molecule

We elucidated the genetic basis responsible for factor VII deficiency in an Italian woman with a severe bleeding diathesis, In the allele inherited from the patient's father, we identified a G to A mutation at nucleotide 6070 at the 5' splice site of intron 4 and a G to A substitution at nucleotide 10976 resulting in the Arg(353)Gln polymorphism. The maternal allele demonstrated a C to T substitution at nucleotide 10994 resulting in Thr(359)Met, The mutation at nucleotide 6070 alters an invariant GT dinucleotide and disrupts normal mRNA processing, To investigate the mechanism by which Thr(359)Met reduces factor VII levels, we expressed wild type factor VII cDNA (FVIIwt) and a mutant factor VII cDNA containing the base substitution resulting in Met(359) (FVII359M) in Chinese hamster ovary cells (CHO), In cells transfected with the mutant factor VII cDNA, FVII359M accumulated intracellularly, and no factor VII was detected in the media after 3 hours of chase. The carbohydrate side chains associated with FVII359M were sensitive to Endo H digestion, which indicates that the protein is retained in the endoplasmic reticulum, Analysis of cell lysates also showed that FVII359M was associated with the 78 kD protein corresponding to GRP78/BiP, We conclude that a Thr(359)Met mutation in factor VII results in a severe secretion defect that probably results from abnormal folding of the molecule. (C) 1996 by The American Society of Hematology.