Molecular defects in the factor X gene caused by novel heterozygous mutations IVS5+1G>A and Asp409del

Summary Factor X (FX) deficiency is a rare autosomal‐recessive bleeding disorder caused by diverse mutations in the F10 gene. To investigate the molecular basis of severe FX deficiency in a mildly hemorrhagic patient, variants of the F10 gene were detected by sequencing. A missense mutation was analysed by in vitro expression and modelling analysis, and a splice mutation using ectopic transcript analysis. The levels of activity of FX (FX:C) were A and Asp409del) were identified in the F10 gene. Ectopic transcript expression combined with informative marker (heterozygous Asp409del) analysis of the splice mutation (IVS5+1G>A) revealed and confirmed that the transcript from the mutated allele was absent, likely caused by the nonsense‐mediated mRNA decay pathway. In vitro expression analysis showed that the Asp409del mutant led to a loss of enzymatic activity rather than impaired expression. Molecular modelling analysis confirmed that the Asp409del mutant dramatically altered the conformation of the 185–189 loop and impaired binding of the loop to sodium ions (Na+), diminishing the enzymatic activity of FXa. This is the first report to clarify the molecular mechanisms of two naturally occurring F10 gene variants that cause severe FX deficiency.