Factor XII Ofunato: Lys346Asn mutation associated with blood coagulation factor XII deficiency causes impaired secretion through a proteasome-mediated degradation.

Background: Congenital blood coagulation factor XII (FXII) deficiency is a rare coagulation disease and an autosomal recessive trait. It is found by chance in many cases. We identified a novel mutation (Lys346Asn) in the FXII gene of a patient with FXII deficiency, designated as Factor XII Ofunato.

Methods: The proband was a 75-year-old Japanese woman with a prolonged activated partial thromboplastin time (52.8s). The FXII activity and antigen were greatly reduced (activity, 5%; antigen, 4.5%). We analyzed FXII gene of this patient using a direct sequencing method and characterized mutant FXII through in vitro expression studies.

Results: Sequence analysis of the FXII gene revealed a G-->C point mutation at nucleotide 9845, resulting in Lys346 (AAG)-->Asn (AAC) replacement in the catalytic domain. Expression studies in Chinese hamster ovary cells demonstrated that mutant FXII (346N-FXII) showed a lower level of accumulation in the cells than wild-type. Secretion of 346N-FXII was greatly reduced in culture medium. We also investigated mRNA expression levels of wild-type and 346N-FXII in transfected cells using quantitative RT-PCR. Both mRNA expressions were equivalent levels. Pulse-chase experiments showed that 346N-FXII was extensively degraded intracellularly compared to wild-type. Using membrane-permeable inhibitors, we observed that degradation occurred in the pre-Golgi compartment and that proteasome apparently plays a central role in this process.

Conclusions: These results show that most 346N-FXII is degraded intracellularly through endoplasmic reticulum-associated degradation as the protein quality control system, resulting in an insufficient secretion phenotype.