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Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive disorder characterized by mild-to-moderate bleeding and reduction in FV and FVIII levels in plasma. F5F8D is caused by mutations in one of two different genes, LMAN1 and MCFD2, which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to the Golgi apparatus. Here, we report the identification of a novel mutation Asp89Asn in the MCFD2 gene in a Tunisian patient. In the encoded protein, this mutation causes substitution of a negatively charged aspartate, involved in several structurally important interactions, to an uncharged asparagine. To elucidate the structural effect of this mutation, we performed circular dichroism (CD) analysis of secondary structure and stability. In addition, CD analysis was performed on two missense mutations found in previously reported F5F8D patients. Our results show that all analysed mutant variants give rise to destabilized proteins and highlight the importance of a structurally intact and functional MCFD2 for the efficient secretion of coagulation factors V and VIII.

IntroductionHemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder.AimIn this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum.MethodsWe screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure.ResultsWe identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles.ConclusionThe mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715

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Inherited factor VII (FVII) deficiency is a rare disorder characterized by a bleeding phenotype varying from mild to severe. To date, more than 200 mutations have been described along the F7 gene encoding for FVII. The aim of this study was the identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia. Mutation detection was performed by sequencing the whole F7 gene coding region, exon-intron boundaries and about 400 bp of the promoter region. We identified 5 mutations in five unrelated families; the novel p.F328Y mutation and the reported mutations: p.R304Q, p.M298I, IVS1aG > A and p.G-39G. For the remaining 5 patients we didn’t identified any mutations using PCR/Sequencing protocol. In conclusion, this study represents the first comprehensive molecular series of FVII deficiency affected patients in Tunisia from the North. We will try in the future to continue the molecular study for Tunisian patients from Center and South provinces in order to have a complete idea about the FVII deficiency mutational profile in our country.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1288044089753085