In-vitro expression and functional characterization of four mutations causing factor XI deficiency

Coagulation factor XI (FXI) is the zymogen of a serine protease that, when converted to its active form, contributes to blood coagulation through proteolytic activation of factor IX. The 23-kb long FXI gene (F11) is located on chr4q35.2 and consists of 15 exons. FXI deficiency, characterized by bleeding symptoms mainly associated with injury or surgery, is rare in most populations (incidence 1:106) except in Ashkenazi Jews, in whom the heterozygote frequency may be as high as 10%. While in Jews two prevalent mutations (Glu117stop and Phe283Leu), account for the majority of abnormal alleles, in non-Jewish patients the mutational spectrum is wide. In most cases FXI functional and immunologic levels are proportionally decreased (cross-reactive material negative, CRM- defects), while only one CRM+ mutation has been up-to-now fully published. We here report the molecular characterization of four missense mutations (three novel, Ala91Thr, Val371Ile, and Pro382Thr, and the previously reported Gly460Arg) in F11 identified in the heterozygous state in four Italian patients affected by mild FXI deficiency. All patients were CRM- except the one carrying the Val371Ile mutation, who had normal immunologic FXI levels associated with reduced activity, distinctive of a CRM+ defect. Expression of mutant FXI proteins in COS-1 cells demonstrated that Pro382Thr and Gly460Arg were each sufficient to impair FXI secretion, while FXIs bearing Ala91Thr and Val371Ile were normally secreted. The availability of platelet RNA from the Ala91Thr patient allowed the demonstration of the role of the underlying nucleotide substitution (325G>A, numbering from the ATG start codon of NM_000128) on splicing by RT-PCR experiments. Concerning Val371Ile-FXI, its specific activity was one half that of the wild-type protein, confirming the diagnosis of CRM+ deficiency. Given the proximity of Val371 to FXI activation site, a possible interference on FXI activation was postulated. Preliminary results confirmed a reduced Val371Ile-FXI activation by FXIIa and thrombin.