Mutations in LMAN1 and MCFD2 May Account for All Cases of Combined Deficiency of Factor V and Factor VIII.

Factor V (FV) and factor VIII (FVIII) are two large plasma glycoproteins that function as essential cofactors for the proteolytic activation of prothrombin and factor X, respectively. Efficient biosynthesis of FV and FVIII requires LMAN1 and MCFD2, two proteins localized to the early secretory pathway of the cell. LMAN1 is a 53-kD homo-hexameric transmembrane protein with homology to leguminous mannose-binding lectins. MCFD2 is an EF-hand domain protein that co-localizes with LMAN1 to the ER-Golgi intermediate compartment (ERGIC). MCFD2 interacts with LMAN1 to form a stable, calcium-dependent protein complex that functions as a cargo receptor, ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. Mutations in LMAN1 or MCFD2 cause combined deficiency of factors V and VIII, an autosomal recessive disorder associated with plasma levels of FV and FVIII in the range of 5% to 30% of normal. However, three families were found to have no LMAN1 or MCFD2 mutations, with 2 of these families showing genetic evidence against linkage to either gene, raising the possibility of additional locus heterogeneity and the involvement of a third F5F8D gene. We now report the analysis of 10 previously reported and 9 new F5F8D families. We identified 3 MCFD2 mutations accounting for 6 F5F8D families, and 8 LMAN1 mutations accounting for 8 additional families, including the first-reported single amino acid substitution, replacement of cysteine at amino acid position 475 with arginine (C475R). Cysteine 475 was previously reported to be important in forming an intermolecular disulfide bond required for LMAN1 oligomerization. However, C475R LMAN1 was undetected by Western blot analysis in lymphoblasts derived from a patient hemizygous for this mutation, with only a trace of protein detectable by immunoprecipitation. Thus, the C475R mutation appears to result in an unstable LMAN1 protein that is rapidly degraded. Failure of proteasome inhibitors to increase the intracellular accumulation of this protein suggests an alternative degradation pathway. Finally, two LMAN1 alleles for which no mutations were identified were nonetheless shown to result in no detectable LMAN1 mRNA, indicating a cis-defect in transcription or mRNA stability. Taken together with our previous reports, we have now identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 70 of 75 families. Two of the remaining 5 families are consistent with linkage to the LMAN1 or MCFD2 loci, suggesting mutations in the regulatory region of the genes that were missed by direct sequencing. Reanalysis of the remaining 3 families suggests an initial misdiagnosis, with one reclassified as isolated, mild FV deficiency, and two others as von Willebrand disease. These results suggest that mutations in LMAN1 and MCFD2 account for all cases of F5F8D, with no evidence for a 3rd F5F8D gene.