Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and

Essentials Fibrinogen Disorders are characterized by variable expressivity.Patients with fibrinogen disorders can present with bleeding, thrombosis, or both.As previously reported, genotype‐phenotype correlations are difficult to establish.Molecular modeling may help to further understand the effects of mutations on the mature fibrinogen protein. IntroductionFibrinogen is a complex molecule comprised of two sets of Aα, Bβ, and γ chains. Fibrinogen deficiencies can lead to the development of bleeding or thromboembolic events. The objective of this study was to perform DNA sequence analysis of patients with clinical fibrinogen abnormalities, and to perform genotype‐phenotype correlations.Materials and Methods DNA from 31 patients was sequenced to evaluate disease‐causing mutations in the three fibrinogen genes: FGA,FGB, and FGG. Clinical data were extracted from medical records or from consultation with referring hematologists. Fibrinogen antigen and functional (Clauss method) assays, as well as reptilase time (RT) and thrombin time (TT) were obtained for each patient. Molecular modeling was used to simulate the functional impact of specific missense variants on the overall protein structure.ResultsSeventeen mutations, including six novel mutations, were identified in the three fibrinogen genes. There was little correlation between genotype and phenotype. Molecular modeling predicted a substantial conformational change for a novel variant, FGG p.Ala289Asp, leading to a more rigid molecule in a region critical for polymerization and alignment of the fibrin monomers. This mutation is associated with both bleeding and clotting in the two affected individuals.ConclusionsRobust genotype‐phenotype correlations are difficult to establish for fibrinogen disorders. Molecular modeling might represent a valuable tool for understanding the function of certain missense fibrinogen mutations but those should be followed by functional studies. It is likely that genetic and environmental modifiers account for the incomplete penetrance and variable expressivity that characterize fibrinogen disorders.

Combined factor V and factor VIII deficiency (F5F8D) was first recognized in two young brothers by Oeri et al.[1] as a rare, recessive coagulation disorder. Individuals affected with this disease present with a moderate bleeding tendency and plasma levels of factor V and VIII in the range of 5-30% of normal. Extensive genetic analysis of F5F8D patients linked the disorder to causative mutations in the two genes LMAN1 and MCFD [2,3]. Subsequent studies suggest that LMAN1, a mannose-specific membrane lectin, operates as a cargo receptor for the transport of glycoproteins from the endoplasmic reticulum (ER) to Golgi intermediate compartment (ERGIC) indicating that F5F8D could result from a defect in the ER-to-Golgi transport machinery of both coagulation factors. However, in approximately 30% of individuals with this dual deficiency no LMAN1 mutations could be detected, suggesting that another gene may also be involved in this disease [4]. Recently, Zhang et al. [5] reported that mutations in a novel gene named MCFD2 (multiple coagulation factor deficiency gene 2) are also responsible for F5F8D. MCFD2 is an EF-hand domain protein localized to the ERGIC through a direct, calciumdependent interaction with LMAN1. LMAN1 and MCFD2 form a protein complex that directly interacts with FVIII [6].Desmopressin (DDAVP) is a synthetic derivate of the antidiuretic hormone (AVP, l-arginine vasopressin) that has a prolonged and increased antidiuretic effect compared with AVP. On account of these properties, DDAVP is registered for the treatment of diabetes insipidus and enuresis nocturna. In addition, DDAVP increases the concentrations of factor VIII and von Willebrand factor in plasma [7]. Hence, DDAVP is prescribed in patients with mild haemophilia A and von Willebrand disease type 1 prior to minor surgical procedures and for non-life threatening bleedings.In this report, we performed a DDAVP test in a 11-year-old Argentinean (pre-pubertal) girl prior to her beginning menarche. The girl, at 8 years of age, had presented with moderate epistaxis and increased bruising in the legs. At 7 years of age she suffered profuse bleeding following dental trauma. Fortunately, it stopped with local compression alone. Both parents, who are not consanguineous, and the two older brothers are asymptomatic, but the paternal grandmother and a maternal uncle were complained with easy bruising and epistaxis. Laboratory tests, in the girl, revealed a prolongation of both the prothrombin time and the activated partial thromboplastin time in comparison to normal (17.8 ± 0.9 vs. 12.1 ± 0.2 seg. and 56.3 ± 2.1 vs. 29.8 ± 1.1 seg., respectively) with normal fibrinogen and thrombin time. The levels of factor V and factor VIII detected by one-stage procedure in the plasma of the proband were 23.2 ± 3.1% and 18.6 ± 6.1%, respectively (mean of two determinations assayed in three occasions >3 months apart). Furthermore, the presence of weak and strong inhibitors against factor V and factor VIII were analysed by incubation of plasma patient with normal plasma ...

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