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Marguerite Neermanarbez - One of the best experts on this subject based on the ideXlab platform.
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heterogeneity of Congenital Afibrinogenemia from epidemiology to clinical consequences and management
Blood Reviews, 2021Co-Authors: Alessandro Casini, Marguerite Neermanarbez, Philippe De MoerlooseAbstract:Fibrinogen is a complex protein playing a major role in coagulation. Congenital Afibrinogenemia, characterized by the complete absence of fibrinogen, is associated with major hemostatic defects. Even though the clinical course is unpredictable and can be completely different among patients, severe bleeding is the prominent symptom. Patients are also at increased risk of thrombosis and sometimes suffer from spontaneous spleen rupture, bone cysts and defective wound healing. Due to the relative rarity of Afibrinogenemia, there are no evidence-based strategies for helping physicians in care of these patients. Fibrinogen supplementation is the keystone to prevent or treat bleeding events. In addition, fibrinogen, a pleiotropic protein with numerous physiological roles in immunity, angiogenesis and tissue repair, is involved in many diseases. Indeed, depletion of fibrinogen in animal models of infections, tumors and neurological diseases has an effect on the clinical course. The consequences for patients with Afibrinogenemia still need to be investigated.
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a homozygous duplication of the fgg exon 8 intron 8 junction causes Congenital Afibrinogenemia lessons learned from the study of a large consanguineous turkish family
Haematologica, 2021Co-Authors: Michel Guipponi, Corinne Di Sanza, Alessandro Casini, Flora Peyvandi, Frederic Masclaux, Frederique Sloanbena, Namik Ozbek, Marzia Menegatti, Baris Malbora, Marguerite NeermanarbezAbstract:Congenital Afibrinogenemia is the most severe Congenital fibrinogen disorder, characterised by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for Congenital Afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short PCR amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from Whole Exome Sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNAs produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons.
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a novel frameshift mutation in the fga gene c 196 delt leading to Congenital Afibrinogenemia
Journal of Pediatric Hematology Oncology, 2020Co-Authors: Sultan Aydin Koker, Marguerite Neermanarbez, Alper Koker, Gokcen Oz Tuncer, Yilmaz Akbas, Tugce Tural Kara, Yasemin CobanAbstract:Background Congenital Afibrinogenemia is characterized by the absence of fibrinogen. Congenital fibrinogen disorders result from several mutations in FGA, FGB, or FGG. Their epidemiology is not well known. Observation The present study reports on 2 children with Congenital Afibrinogenemia. The first child, a male who is now 9 years old, was diagnosed with Afibrinogenemia after spontaneous intracranial bleeding at the age of 3 years. The second child is a 2-year-old female cousin of the first patient, who was diagnosed with Afibrinogenemia after coagulation tests were carried out due to frequent epistaxis and mucocutaneous bleeding. At follow-up, blood samples of the patients and their parents were sent to the Department of Genetic Medicine and Development, University Medical Center, Switzerland, for polymerase chain reaction analysis. In both patients, the novel homozygous frameshift mutation in the FGA exon 3: c.196 delT was detected. The parents of the patients were both heterozygous for the same mutation. Conclusions Congenital Afibrinogenemia is a rare coagulation disease. The molecular epidemiology of Congenital fibrinogen disorders is complex, and the identification of new mutations will help shed light on this complex molecular structure. Therefore, a genetic analysis that includes more centers is needed.
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targeted mutation of zebrafish fga models human Congenital Afibrinogenemia
Blood, 2014Co-Authors: Richard J Fish, Corinne Di Sanza, Marguerite NeermanarbezAbstract:Mutations in the human fibrinogen genes can lead to the absence of circulating fibrinogen and cause Congenital Afibrinogenemia. This rare bleeding disorder is associated with a variable phenotype, which may be influenced by environment and genotype. Here, we present a zebrafish model of Afibrinogenemia. We introduced targeted mutations into the zebrafish fga gene using zinc finger nuclease technology. Animals carrying 3 distinct frameshift mutations in fga were raised and bred to produce homozygous mutants. Using a panel of anti-zebrafish fibrinogen antibodies, fibrinogen was undetectable in plasma preparations from homozygous mutant fish. We observed hemorrhaging in fga mutants and reduced survival compared with control animals. This model will now serve in the search for Afibrinogenemia modifying genes or agents and, to our knowledge, is the first transmissible zebrafish model of a defined human bleeding disorder.
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mutation of the translation initiation codon in fga causes Congenital Afibrinogenemia
Blood Coagulation & Fibrinolysis, 2012Co-Authors: Yordanka Tirefort, Philippe De Moerloose, Olivat Rakoto Alson, Marguerite NeermanarbezAbstract:Congenital Afibrinogenemia is characterized by the complete absence of fibrinogen, the precursor of the major protein constituent of the blood clot, fibrin. Extensive allelic heterogeneity has been found for this disorder and more than 40 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. However, the continuous genetic analysis of additional patients still allows the identification of novel mutations and thus the greater understanding of fibrinogen structure and function. Here we report the identification of a novel missense mutation in FGA exon 1 affecting the translation initiation codon: c.1 A>T (ATG>TTG) M1L, identified in a young boy from Madagascar in compound heterozygosity with a second mutation in FGA exon 4: c.385 C>T (CGA>TGA) R129X. The patient suffered from occasional severe arthralgias (shoulder, knee) most likely caused by intra-articular bleeding with subsequent inflammation.
Philippe De Moerloose - One of the best experts on this subject based on the ideXlab platform.
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heterogeneity of Congenital Afibrinogenemia from epidemiology to clinical consequences and management
Blood Reviews, 2021Co-Authors: Alessandro Casini, Marguerite Neermanarbez, Philippe De MoerlooseAbstract:Fibrinogen is a complex protein playing a major role in coagulation. Congenital Afibrinogenemia, characterized by the complete absence of fibrinogen, is associated with major hemostatic defects. Even though the clinical course is unpredictable and can be completely different among patients, severe bleeding is the prominent symptom. Patients are also at increased risk of thrombosis and sometimes suffer from spontaneous spleen rupture, bone cysts and defective wound healing. Due to the relative rarity of Afibrinogenemia, there are no evidence-based strategies for helping physicians in care of these patients. Fibrinogen supplementation is the keystone to prevent or treat bleeding events. In addition, fibrinogen, a pleiotropic protein with numerous physiological roles in immunity, angiogenesis and tissue repair, is involved in many diseases. Indeed, depletion of fibrinogen in animal models of infections, tumors and neurological diseases has an effect on the clinical course. The consequences for patients with Afibrinogenemia still need to be investigated.
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Congenital Afibrinogenemia identification and characterization of two novel homozygous fibrinogen aα and bβ chain mutations in two tunisian families
Thrombosis Research, 2016Co-Authors: Yessine Amri, Nour El Houda Toumi, Sondess Hadj Fredj, Philippe De MoerlooseAbstract:Abstract Introduction Inherited abnormalities of fibrinogen (FG) are rare coagulation disorders divided into two types: quantitative abnormalities (Afibrinogenemia and hypofibrinogenemia) or qualitative abnormalities (dysfibrinogenemia and hypo-dysfibrinogenemia) of circulating fibrinogen. In particular, Congenital Afibrinogenemia is inherited as an autosomal recessive mode and is usually determined by homozygous or compound heterozygous mutations affecting any of the three fibrinogen genes (FGA, FGB and FGG), resulting in the complete absence or extremely reduced amount of fibrinogen. The aim of the present study was to characterize the fibrinogen abnormalities in two Tunisian families. Methods Coagulation studies were performed on the patients and family members. All the exons and the flanking intron regions of fibrinogen genes were screened by direct sequencing. Results Probands had concomitant bleeding complications with infinitely prolonged standard coagulation assays. Mutational screening of the fibrinogen gene cluster of each proband, disclosed two previously undescribed homozygous point mutations. The first mutation was a major truncation (AαArg252Stop) leads to a severe premature termination codon in the exon 5 of the FGA gene. This mutation defines in vivo the importance of the αC flexible segment in the secretion of a stable fibrinogen molecule. The second afibrinogenemic mutation (BβGly295Ala) occurs in the exon 7 of the FGB gene. This missense mutation would probably lead to significant conformational change not allowing the expression of the fibrinogen protein. Conclusion Current molecular characterization of these two fibrinogen abnormalities confirms the importance of the first portion of αC-region (αC-connector) as well as the Bβ globular domain in the secretion processes.
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mutation of the translation initiation codon in fga causes Congenital Afibrinogenemia
Blood Coagulation & Fibrinolysis, 2012Co-Authors: Yordanka Tirefort, Philippe De Moerloose, Olivat Rakoto Alson, Marguerite NeermanarbezAbstract:Congenital Afibrinogenemia is characterized by the complete absence of fibrinogen, the precursor of the major protein constituent of the blood clot, fibrin. Extensive allelic heterogeneity has been found for this disorder and more than 40 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. However, the continuous genetic analysis of additional patients still allows the identification of novel mutations and thus the greater understanding of fibrinogen structure and function. Here we report the identification of a novel missense mutation in FGA exon 1 affecting the translation initiation codon: c.1 A>T (ATG>TTG) M1L, identified in a young boy from Madagascar in compound heterozygosity with a second mutation in FGA exon 4: c.385 C>T (CGA>TGA) R129X. The patient suffered from occasional severe arthralgias (shoulder, knee) most likely caused by intra-articular bleeding with subsequent inflammation.
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a novel frameshift mutation in fga c 1846 del a leading to Congenital Afibrinogenemia in a consanguineous syrian family
Blood Coagulation & Fibrinolysis, 2011Co-Authors: Emmanuel Levrat, Philippe De Moerloose, Imad Aboukhamis, Jaafar Farho, Sahar Chamaa, G Reber, Alexandre Fort, Marguerite NeermanarbezAbstract:Congenital Afibrinogenemia is a rare autosomal recessive coagulation disorder characterized essentially by bleeding symptoms, but miscarriages and, paradoxically, thromboembolic events can also occur. Most reported mutations leading to Congenital Afibrinogenemia are located in FGA encoding the fibrinogen A α-chain. In this study, we analysed 12 individuals from a consanguineous Syrian family with reduced or absent fibrinogen levels: those with fibrinogen levels around 1 g/l (n = 7) were found to be heterozygous for a novel frameshift mutation in FGA exon 5 (c.1846 del A) and those with undetectable fibrinogen levels (n = 5) were homozygous for the same mutation. This novel frameshift mutation is the most C-terminal causative FGA mutation identified to date in afibrinogenemic patients. The resulting aberrant Aα-chain (p.Thr616HisfsX32) is most likely synthesized, but is less efficiently assembled and/or secreted into the circulation given the phenotype of asymptomatic hypofibrinogenemia in heterozygous individuals and bleeding diathesis in homozygous individuals.
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identification and functional characterization of a novel nonsense mutation in fga accounting for Congenital Afibrinogenemia in six egyptian patients
Blood Coagulation & Fibrinolysis, 2010Co-Authors: Magy Abdel Wahab, Richard J Fish, Philippe De Moerloose, Marguerite NeermanarbezAbstract:Congenital Afibrinogenemia is a rare coagulation disorder attributed to over forty mutations found either in homozygosity or in compound heterozygosity, the majority localized in FGA encoding the fibrinogen Aalpha-chain. Despite the number of genetic analyses performed the study of additional patients still allows the identification of novel mutations and a better understanding of fibrinogen structure and function. Here we report the identification and functional analysis of a novel nonsense mutation in FGA exon 5: c.718C>T (CAG>TAG) p.Q240X (Q221X in the mature chain lacking the signal peptide), accounting for fibrinogen deficiency in six Egyptian patients. Expression of the mutant Aalpha-chain cDNA in combination with wild-type Bbeta-chain and gamma-chain cDNAs demonstrated that although the mutant chain could be detected in the cell media of transfected COS-7 cells it was less secreted in comparison to the wild-type Aalpha-chain. Our patients were all homozygous for p.Q240X(Q221X) yet their clinical spectrum varied considerably in their onset of presentation or severity, with bleeding ranging from moderate mucous membrane bleeds in adolescence to life threatening intracranial hemorrhage in infancy.
Maria Luisa Tenchini - One of the best experts on this subject based on the ideXlab platform.
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the molecular basis of quantitative fibrinogen disorders
Journal of Thrombosis and Haemostasis, 2006Co-Authors: Rosanna Asselta, Stefano Duga, Maria Luisa TenchiniAbstract:Summary. Hereditary fibrinogen disorders include type I deficiencies (Afibrinogenemia and hypofibrinogenemia, i.e. quantitative defects), with low or unmeasurable levels of immunoreactive protein; and type II deficiencies (dysfibrinogenemia and hypodysfibrinogenemia, i.e. qualitative defects), showing normal or altered antigen levels associated with reduced coagulant activity. While dysfibrinogenemias are in most cases autosomal dominant disorders, type I deficiencies are generally inherited as autosomal recessive traits. Patients affected by Congenital Afibrinogenemia or severe hypofibrinogenemia may experience bleeding manifestations varying from mild to severe. This review focuses on the genetic bases of type I fibrinogen deficiencies, which are invariantly represented by mutations within the three fibrinogen genes (FGA, FGB, and FGG) coding for the three polypeptide chains Aα, Bβ, and γ. From the inspection of the mutational spectrum of these disorders, some conclusions can be drawn: (i) genetic defects are scattered throughout the three fibrinogen genes, with only few sites appearing to represent relative mutational hot spots; (ii) several different types of genetic lesions and pathogenic mechanisms have been described in affected individuals (including gross deletions, point mutations causing premature termination codons, missense mutations affecting fibrinogen assembly/secretion, and uniparental isodisomy associated with a large deletion); (iii) the possibility to express recombinant fibrinogen mutants in eukaryotic cells is rapidly shedding light into the molecular mechanisms responsible for physiologic and pathologic properties of the molecule; (iv) though mutation analysis of the fibrinogen cluster does not yield precise information for predicting genotype/phenotype correlations, it still provides a valuable tool for diagnosis confirmation, identification of potential carriers, and prenatal diagnosis.
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Congenital Afibrinogenemia intracellular retention of fibrinogen due to a novel w437g mutation in the fibrinogen bβ chain gene
Biochimica et Biophysica Acta, 2003Co-Authors: Silvia Spena, Rosanna Asselta, Stefano Duga, Massimo Malcovati, Flora Peyvandi, Pier Mannuccio Mannucci, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 Afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in Afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.
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Congenital Afibrinogenemia first identification of splicing mutations in the fibrinogen bβ chain gene causing activation of cryptic splice sites
Blood, 2002Co-Authors: Silvia Spena, Rosanna Asselta, Stefano Duga, Massimo Malcovati, Flora Peyvandi, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in Afibrinogenemia, 17 in the Aα, 6 in the γ, and only 2 in the Bβ fibrinogen–chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bβ-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bβ-chain gene splicing mutations described in Afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bβ-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase–polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bβ chain for fibrinogen assembly and secretion.
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Congenital Afibrinogenemia mutations leading to premature termination codons in fibrinogen aα chain gene are not associated with the decay of the mutant mrnas
Blood, 2001Co-Authors: Rosanna Asselta, Stefano Duga, E Santagostino, Massimo Malcovati, Flora Peyvandi, Pier Mannuccio Mannucci, S Spena, Gavino Piseddu, Roberto Targhetta, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare coagulation disorder with autosomal recessive inheritance, characterized by the complete absence or extremely reduced levels of fibrinogen in patients' plasma and platelets. Eight afibrinogenemic probands, with very low plasma levels of immunoreactive fibrinogen were studied. Sequencing of the fibrinogen gene cluster of each proband disclosed 4 novel point mutations (1914C>G, 1193G>T, 1215delT, and 3075C>T) and 1 already reported (3192C>T). All mutations, localized within the first 4 exons of the A alpha-chain gene, were null mutations predicted to produce severely truncated A alpha-chains because of the presence of premature termination codons. Since premature termination codons are frequently known to affect the metabolism of the corresponding messenger RNAs (mRNAs), the degree of stability of each mutant mRNA was investigated. Cotransfection experiments with plasmids expressing the wild type and each of the mutant A alpha-chains, followed by RNA extraction and semiquantitative reverse-transcriptase-polymerase chain reaction analysis, demonstrated that all the identified null mutations escaped nonsense-mediated mRNA decay. Moreover, ex vivo analysis at the protein level demonstrated that the presence of each mutation was sufficient to abolish fibrinogen secretion.
Rosanna Asselta - One of the best experts on this subject based on the ideXlab platform.
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genetic variants in the fgb and fgg genes mapping in the beta and gamma nodules of the fibrinogen molecule in Congenital quantitative fibrinogen disorders associated with a thrombotic phenotype
International Journal of Molecular Sciences, 2020Co-Authors: Tomas Simurda, Monika Brunclikova, Jana Zolkova, Dusan Loderer, Ingrid Skornova, Jan Hudecek, Sonia Caccia, Rosanna Asselta, Zuzana Kolkova, Zora LasabovaAbstract:Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bβ, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bβ, and γ chain, respectively). Depending on the type and site of mutations, Congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of Congenital Afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.
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the molecular basis of quantitative fibrinogen disorders
Journal of Thrombosis and Haemostasis, 2006Co-Authors: Rosanna Asselta, Stefano Duga, Maria Luisa TenchiniAbstract:Summary. Hereditary fibrinogen disorders include type I deficiencies (Afibrinogenemia and hypofibrinogenemia, i.e. quantitative defects), with low or unmeasurable levels of immunoreactive protein; and type II deficiencies (dysfibrinogenemia and hypodysfibrinogenemia, i.e. qualitative defects), showing normal or altered antigen levels associated with reduced coagulant activity. While dysfibrinogenemias are in most cases autosomal dominant disorders, type I deficiencies are generally inherited as autosomal recessive traits. Patients affected by Congenital Afibrinogenemia or severe hypofibrinogenemia may experience bleeding manifestations varying from mild to severe. This review focuses on the genetic bases of type I fibrinogen deficiencies, which are invariantly represented by mutations within the three fibrinogen genes (FGA, FGB, and FGG) coding for the three polypeptide chains Aα, Bβ, and γ. From the inspection of the mutational spectrum of these disorders, some conclusions can be drawn: (i) genetic defects are scattered throughout the three fibrinogen genes, with only few sites appearing to represent relative mutational hot spots; (ii) several different types of genetic lesions and pathogenic mechanisms have been described in affected individuals (including gross deletions, point mutations causing premature termination codons, missense mutations affecting fibrinogen assembly/secretion, and uniparental isodisomy associated with a large deletion); (iii) the possibility to express recombinant fibrinogen mutants in eukaryotic cells is rapidly shedding light into the molecular mechanisms responsible for physiologic and pathologic properties of the molecule; (iv) though mutation analysis of the fibrinogen cluster does not yield precise information for predicting genotype/phenotype correlations, it still provides a valuable tool for diagnosis confirmation, identification of potential carriers, and prenatal diagnosis.
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Congenital Afibrinogenemia intracellular retention of fibrinogen due to a novel w437g mutation in the fibrinogen bβ chain gene
Biochimica et Biophysica Acta, 2003Co-Authors: Silvia Spena, Rosanna Asselta, Stefano Duga, Massimo Malcovati, Flora Peyvandi, Pier Mannuccio Mannucci, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 Afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in Afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.
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Congenital Afibrinogenemia first identification of splicing mutations in the fibrinogen bβ chain gene causing activation of cryptic splice sites
Blood, 2002Co-Authors: Silvia Spena, Rosanna Asselta, Stefano Duga, Massimo Malcovati, Flora Peyvandi, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in Afibrinogenemia, 17 in the Aα, 6 in the γ, and only 2 in the Bβ fibrinogen–chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bβ-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bβ-chain gene splicing mutations described in Afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bβ-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase–polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bβ chain for fibrinogen assembly and secretion.
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Congenital Afibrinogenemia mutations leading to premature termination codons in fibrinogen aα chain gene are not associated with the decay of the mutant mrnas
Blood, 2001Co-Authors: Rosanna Asselta, Stefano Duga, E Santagostino, Massimo Malcovati, Flora Peyvandi, Pier Mannuccio Mannucci, S Spena, Gavino Piseddu, Roberto Targhetta, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare coagulation disorder with autosomal recessive inheritance, characterized by the complete absence or extremely reduced levels of fibrinogen in patients' plasma and platelets. Eight afibrinogenemic probands, with very low plasma levels of immunoreactive fibrinogen were studied. Sequencing of the fibrinogen gene cluster of each proband disclosed 4 novel point mutations (1914C>G, 1193G>T, 1215delT, and 3075C>T) and 1 already reported (3192C>T). All mutations, localized within the first 4 exons of the A alpha-chain gene, were null mutations predicted to produce severely truncated A alpha-chains because of the presence of premature termination codons. Since premature termination codons are frequently known to affect the metabolism of the corresponding messenger RNAs (mRNAs), the degree of stability of each mutant mRNA was investigated. Cotransfection experiments with plasmids expressing the wild type and each of the mutant A alpha-chains, followed by RNA extraction and semiquantitative reverse-transcriptase-polymerase chain reaction analysis, demonstrated that all the identified null mutations escaped nonsense-mediated mRNA decay. Moreover, ex vivo analysis at the protein level demonstrated that the presence of each mutation was sufficient to abolish fibrinogen secretion.
Stefano Duga - One of the best experts on this subject based on the ideXlab platform.
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the molecular basis of quantitative fibrinogen disorders
Journal of Thrombosis and Haemostasis, 2006Co-Authors: Rosanna Asselta, Stefano Duga, Maria Luisa TenchiniAbstract:Summary. Hereditary fibrinogen disorders include type I deficiencies (Afibrinogenemia and hypofibrinogenemia, i.e. quantitative defects), with low or unmeasurable levels of immunoreactive protein; and type II deficiencies (dysfibrinogenemia and hypodysfibrinogenemia, i.e. qualitative defects), showing normal or altered antigen levels associated with reduced coagulant activity. While dysfibrinogenemias are in most cases autosomal dominant disorders, type I deficiencies are generally inherited as autosomal recessive traits. Patients affected by Congenital Afibrinogenemia or severe hypofibrinogenemia may experience bleeding manifestations varying from mild to severe. This review focuses on the genetic bases of type I fibrinogen deficiencies, which are invariantly represented by mutations within the three fibrinogen genes (FGA, FGB, and FGG) coding for the three polypeptide chains Aα, Bβ, and γ. From the inspection of the mutational spectrum of these disorders, some conclusions can be drawn: (i) genetic defects are scattered throughout the three fibrinogen genes, with only few sites appearing to represent relative mutational hot spots; (ii) several different types of genetic lesions and pathogenic mechanisms have been described in affected individuals (including gross deletions, point mutations causing premature termination codons, missense mutations affecting fibrinogen assembly/secretion, and uniparental isodisomy associated with a large deletion); (iii) the possibility to express recombinant fibrinogen mutants in eukaryotic cells is rapidly shedding light into the molecular mechanisms responsible for physiologic and pathologic properties of the molecule; (iv) though mutation analysis of the fibrinogen cluster does not yield precise information for predicting genotype/phenotype correlations, it still provides a valuable tool for diagnosis confirmation, identification of potential carriers, and prenatal diagnosis.
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Congenital Afibrinogenemia intracellular retention of fibrinogen due to a novel w437g mutation in the fibrinogen bβ chain gene
Biochimica et Biophysica Acta, 2003Co-Authors: Silvia Spena, Rosanna Asselta, Stefano Duga, Massimo Malcovati, Flora Peyvandi, Pier Mannuccio Mannucci, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 Afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in Afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.
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Congenital Afibrinogenemia first identification of splicing mutations in the fibrinogen bβ chain gene causing activation of cryptic splice sites
Blood, 2002Co-Authors: Silvia Spena, Rosanna Asselta, Stefano Duga, Massimo Malcovati, Flora Peyvandi, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in Afibrinogenemia, 17 in the Aα, 6 in the γ, and only 2 in the Bβ fibrinogen–chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bβ-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bβ-chain gene splicing mutations described in Afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bβ-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase–polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bβ chain for fibrinogen assembly and secretion.
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Congenital Afibrinogenemia mutations leading to premature termination codons in fibrinogen aα chain gene are not associated with the decay of the mutant mrnas
Blood, 2001Co-Authors: Rosanna Asselta, Stefano Duga, E Santagostino, Massimo Malcovati, Flora Peyvandi, Pier Mannuccio Mannucci, S Spena, Gavino Piseddu, Roberto Targhetta, Maria Luisa TenchiniAbstract:Congenital Afibrinogenemia is a rare coagulation disorder with autosomal recessive inheritance, characterized by the complete absence or extremely reduced levels of fibrinogen in patients' plasma and platelets. Eight afibrinogenemic probands, with very low plasma levels of immunoreactive fibrinogen were studied. Sequencing of the fibrinogen gene cluster of each proband disclosed 4 novel point mutations (1914C>G, 1193G>T, 1215delT, and 3075C>T) and 1 already reported (3192C>T). All mutations, localized within the first 4 exons of the A alpha-chain gene, were null mutations predicted to produce severely truncated A alpha-chains because of the presence of premature termination codons. Since premature termination codons are frequently known to affect the metabolism of the corresponding messenger RNAs (mRNAs), the degree of stability of each mutant mRNA was investigated. Cotransfection experiments with plasmids expressing the wild type and each of the mutant A alpha-chains, followed by RNA extraction and semiquantitative reverse-transcriptase-polymerase chain reaction analysis, demonstrated that all the identified null mutations escaped nonsense-mediated mRNA decay. Moreover, ex vivo analysis at the protein level demonstrated that the presence of each mutation was sufficient to abolish fibrinogen secretion.
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missense mutations in the human β fibrinogen gene cause Congenital Afibrinogenemia by impairing fibrinogen secretion
Blood, 2000Co-Authors: Stefano Duga, Rosanna Asselta, E Santagostino, Sirous Zeinali, Tatjana Simonic, Massimo MalcovatiAbstract:Congenital Afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of Congenital Afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen Aalpha-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with Afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bbeta-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bbeta fibrinogen gene could cause Congenital Afibrinogenemia by impairing fibrinogen secretion. (Blood. 2000;95:1336-1341)