Molecular characterization and subcellular localization of Tyr478del: a pathogenic in‐frame deletion in coagulation factor V

Abstract

Factor V (FV) deficiency is a rare, autosomal recessive disorder with symptoms ranging from mild to a severe bleeding diathesis. This report describes our investigation of a male infant with a severe bleeding disorder and an FV activity level of 2%. His father's FV activity is 50% and his mother's is 68%.All testing was performed with informed consent and institutional review board approval. Genotyping and sequencing of the FV gene was performed as previously described [1], with annotations as per Jenny et al. [2]. The proband is a compound heterozygote with a B-domain frameshift mutation (T2952del) on the paternal allele, leading to a premature termination codon and a 3 bp deletion in exon 10 (nucleotides 1606-1608) on the maternal allele. The latter is in-frame and results in the deletion of tyrosine residue Y478 in the A2 domain. Direct structural information for the A2 domain of the precursor protein is not available. However, Villouteix and Dahlback have used the structure of ceruloplasmin and theoretical models for FVIII to build a theoretical model for the A domains of FV [3]. Based on their model, the location of Y478del in the A2 domain shows that the deletion is the terminal residue of a b-strand within a region joined by a disulfide bridge between Cys472 and Cys498.The proband's FV activity of 2% suggests that Y478del may be contributing to his severe bleeding diathesis, so FV expression studies were undertaken to further characterize the functional consequences of the Y478del mutation. R. J. Kaufman (Howard Hughes Medical Institute, University of Michigan) kindly provided the pMT2/FV expression plasmid, containing the full-length FV cDNA insert. Site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA, USA) was performed to create the pMT2/FV-Y478del mutant plasmid. Transient transfection of COS-1 cells using either the pMT2/FV-unmutated or the pMT2/FV-Y478del plasmids was generally as previously described [4,5].FV coagulant activity was determined in conditioned medium by a one-stage clotting assay; no Y478del secretion could be detected, whereas the unmutated construct demonstrated robust secretion of functional FV. FV antigen levels in conditioned media and in cell lysates determined with a commercial sandwich enzyme-linked immunosorbent assay kit (Cedarlane Laboratories, Hornby, Canada) showed that the Y478del cells had only 50% as much intracellular FV protein as the unmutated cells, and secreted only a very small amount of Y478del protein into the medium. Quantitative reverse transcription polymerase chain reaction was used to measure FV gene expression in the cell lysates on an ABI PRISM 7700 (Applied BioSystems, Foster City, CA, USA), using TaqMan chemistry. The Y478del and the unmutated transfections yielded equivalent levels of FV mRNA expression.Taken together, these results demonstrate a 50% decrease in the level of intracellular FV, and a near total failure of secretion. Immunofluorescent (IF) subcellular localization studies were performed to gain in...

Factor V Deficiency

“…Among rare inherited coagulopathies, FV deficiency is one of the least characterized from the molecular point of view, with only 56 genetic defects hitherto described. 12,[25][26][27][28][29][30][31][32][33][34][35][36][37][38] All mutations are located in F5, and those fully published to date are listed in Fig. 1.…”

“…This approach was extremely useful in defining a common denominator for missense mutations: expressed FV mutant proteins have been in most cases associated with both a secretion impairment and intracellular degradation, probably because of their impaired folding and/or conformational changes leading to retention by the quality control system of the secretory pathway. 25,33,35,36 With respect to splicing defects, FV platelet and peripheral blood mononuclear cell expression was exploited to provide easy access to mature F5 mRNA. When suitable specimens from FV-deficient patients were unavailable to perform studies at the RNA level, expression of appropriate minigene constructs in eukaryotic cells proved to be an effective tool to unravel the consequences of splicing mutations.…”

Molecular characterisation of Tyr530Ser and IVS16–1G>T mutations causing severe factor V deficiency

“…The het-Thrombosis and Haemostasis 104.3/2010 erozygotes are usually asymptomatic, whereas homozygous cases show mild, moderate, or severe bleeding symptoms. According to the online FV mutation database (14), about 100 mutations have been so far reported for FV gene, but only a few of them have been characterised at molecular level (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).…”

Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern

“…5 To date, 42 genetic defects, all located in F5, have been reported as the cause of severe FV deficiency. 1,[6][7][8][9][10][11] Among these, only four variations affecting splicing have so far been identified. 10,[12][13][14] It has been estimated that one-third of hereditary genetic diseases as well as many forms of cancer are caused by mutations resulting in the generation of transcripts bearing a premature termination codon (PTC + transcripts) in their open reading frame.…”