Missense mutations in the human β fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion

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 A-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 Bβ-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 Bβ fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion.

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Genetic Analysis of Afibrinogenemia and Hypofibrinogenemia: Novel Mutations in the FGB Gene in the Turkish Population

Acta Haematologica ◽

10.1159/000505174 ◽

2020 ◽

Vol 143 (6) ◽

pp. 529-532

Author(s):

Didem Torun Özkan ◽

Nazan Sarper ◽

Nejat Akar

Keyword(s):

Amino Acid ◽

Autosomal Recessive ◽

Adenine Nucleotide ◽

Gene Mutations ◽

Autosomal Recessive Disorder ◽

Turkish Population ◽

Chain Gene ◽

Congenital Afibrinogenemia ◽

Low Levels ◽

First Time

Introduction: 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. Hypofibrinogenemia is characterized by fibrinogen levels <1.5 g/L. Objective: In this study, we analyzed fibrinogen beta chain gene mutations in Turkish afibrinogenemia and hypofibrinogenemia patients. Methods: We evaluated 20 afibrinogenemia and hypofibrinogenemia patients and 80 healthy controls. We have sequenced all exons of the FGB gene using the DNA isolated from the peripheral blood samples of patients and controls. Results and Conclusion: We found a nonsense mutation in exon 4 at nucleotide 630 that encoded serine amino acid, and in the same exon a missense mutation of T to C at nucleotide 647, resulting in a transition from leucine to proline (p.L198P) in a child with hypofibrinogenemia. These mutations have been shown for the first time in the same patient of Turkish descent. Furthermore, there was a novel heterozygous guanine-to-adenine nucleotide change in exon 3. This caused the change of arginine amino acid to threonine amino acid at position 136 (p.A136T) in a protein, which has not been described in the literature before.

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Congenital afibrinogenemia: identification and expression of a missense mutation in FGB impairing fibrinogen secretion

Blood ◽

10.1182/blood-2003-06-2141 ◽

2003 ◽

Vol 102 (13) ◽

pp. 4413-4415 ◽

Cited By ~ 22

Author(s):

Dung Vu ◽

Paula H. B. Bolton-Maggs ◽

Jeremy R. Parr ◽

Michael A. Morris ◽

Philippe de Moerloose ◽

...

Keyword(s):

Missense Mutation ◽

Gene Mutations ◽

Great Majority ◽

Autosomal Recessive Disorder ◽

Homozygous Deletion ◽

Chain Gene ◽

Missense Mutations ◽

Exon 2 ◽

Congenital Afibrinogenemia ◽

The Media

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease: a homozygous deletion of approximately 11 kb of the fibrinogen α-chain gene (FGA). Subsequent studies revealed that the great majority of afibrinogenemia mutations are localized in FGA, but mutations were also found in FGG and FGB. Apart from 3 missense mutations identified in the C-terminal portion of FGB, all fibrinogen gene mutations responsible for afibrinogenemia are null. In this study, a young boy with afibrinogenemia was found to be a compound heterozygote for 2 mutations in FGB: an N-terminal nonsense mutation W47X (exon 2) and a missense mutation (G444S, exon 8). Coexpression of the FGB G444S mutant cDNA in combination with wild-type FGA and FGG cDNAs demonstrated that fibrinogen molecules containing the mutant β chain are able to assemble but are not secreted into the media, confirming the pathogenic nature of the identified mutation. (Blood. 2003;102:4413-4415)

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Prenatal diagnosis for congenital afibrinogenemia caused by a novel nonsense mutation in the FGB gene in a Palestinian family

Blood ◽

10.1182/blood-2002-10-3116 ◽

2003 ◽

Vol 101 (9) ◽

pp. 3492-3494 ◽

Cited By ~ 29

Author(s):

Marguerite Neerman-Arbez ◽

Dung Vu ◽

Bassam Abu-Libdeh ◽

Isabelle Bouchardy ◽

Michael A. Morris

Keyword(s):

Prenatal Diagnosis ◽

Nonsense Mutation ◽

Autosomal Recessive ◽

Direct Sequencing ◽

Autosomal Recessive Disorder ◽

Causative Mutation ◽

Chain Gene ◽

Congenital Afibrinogenemia ◽

The Media ◽

Homozygous Deletions

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease, homozygous deletions of approximately 11 kb of the fibrinogen alpha chain gene (FGA). Subsequent analyses revealed that most afibrinogenemia alleles are truncating mutations of FGA, although mutations in all 3 fibrinogen genes, FGG, FGA andFGB have been identified. In this study, we performed the first prenatal diagnosis for afibrinogenemia. The causative mutation in a Palestinian family was a novel nonsense mutation in theFGB gene, Trp467Stop (W467X). Expression of the Trp467Stop mutant FGB cDNA in combination with wild-typeFGA and FGG cDNAs showed that fibrinogen molecules containing the mutant beta chain are not secreted into the media. The fetus was found to be heterozygous for the Trp467Stop mutation by direct sequencing and by linkage analysis, a result that was confirmed in the newborn by intermediate fibrinogen levels.

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Abstract TP269: Distinct Molecular Mechanisms of Htra1 Mutants in Manifesting Heterozygotes With Carasil

Stroke ◽

10.1161/str.48.suppl_1.tp269 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Hiroaki Nozaki ◽

Taisuke Kato ◽

Megumi Nihonmatsu ◽

Yohei Saito ◽

Ikuko Mizuta ◽

...

Keyword(s):

Autosomal Recessive ◽

Molecular Mechanisms ◽

Small Vessel Disease ◽

Gel Filtration ◽

Gene Mutations ◽

Missense Mutations ◽

Inherited Disease ◽

Wild Type ◽

Vessel Disease ◽

Activation Cascade

Introduction: Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an autosomal recessive inherited cerebral small vessel disease (CSVD), involves severe leukoaraiosis, multiple lacunar infarcts, early-onset alopecia, and spondylosis deformans. High-temperature requirement serine peptidase A1 (HTRA1) gene mutations cause CARASIL by decreasing HTRA1 protease activity. Although CARASIL is a recessive inherited disease, heterozygous mutations in the HTRA1 gene were recently identified in 11 families with CSVD. Because CSVD is frequently observed in elderly individuals, it is unclear which mutants truly contribute to CSVD pathogenesis. Here, we found heterozygous mutations in the HTRA1 gene in individuals with CSVD and investigated the differences in biochemical characteristics between these mutant HTRA1s and mutant HTRA1s observed in homozygotes. Methods: We recruited 113 unrelated index patients with clinically diagnosed CSVD. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. Results: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in CARASIL (A252T and V297M) did not inhibit wild- type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer- associated HTRA1 activation. Conclusions: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.

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A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽

10.1182/blood.v96.7.2501 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2501-2505 ◽

Cited By ~ 22

Author(s):

Maurizio Margaglione ◽

Rosa Santacroce ◽

Donatella Colaizzo ◽

Davide Seripa ◽

Gennaro Vecchione ◽

...

Keyword(s):

Amino Acid ◽

Rna Splicing ◽

Messenger Rna ◽

Stop Codon ◽

Premature Stop Codon ◽

Autosomal Recessive Disorder ◽

Chain Gene ◽

Hemorrhagic Diathesis ◽

Reading Frame ◽

Congenital Afibrinogenemia

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

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Homozygous truncation of the fibrinogen Aα chain within the coiled coil causes congenital afibrinogenemia

Blood ◽

10.1182/blood.v96.2.773.014k07_773_775 ◽

2000 ◽

Vol 96 (2) ◽

pp. 773-775 ◽

Cited By ~ 2

Author(s):

Andrew P. Fellowes ◽

Stephen O. Brennan ◽

Randi Holme ◽

Helge Stormorken ◽

Frank R. Brosstad ◽

...

Keyword(s):

Transcription Initiation ◽

Coiled Coil ◽

Initiation Site ◽

Chain Gene ◽

Severe Bleeding ◽

Nucleotide Change ◽

Total Absence ◽

Affected Member ◽

Congenital Afibrinogenemia ◽

A Chain

The molecular basis of a novel congenital afibrinogenemia has been determined. The proposita, the only affected member in a consanguineous Norwegian family, suffers from a moderate to severe bleeding disorder due to the total absence of any detectable fibrinogen. Dot blots of solubilized platelets revealed a small amount of γ chain but no A or Bβ chains, whereas no chains were detected in plasma dot blots. DNA sequencing of the A chain gene revealed a homozygous C→T transversion 557 nucleotides from the transcription initiation site. This nucleotide change predicts the nonsense mutation A 149 Arg (CGA)→stop (TGA). Early truncation of the A chain appears to result in defective assembly or secretion of fibrinogen, probably due to the removal of the C-terminal disulfide ring residues that are critically required for the formation of a stable 3-chained half molecule.

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A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Blood ◽

10.1182/blood-2004-12-4935 ◽

2005 ◽

Vol 105 (12) ◽

pp. 4700-4706 ◽

Cited By ~ 62

Author(s):

Ilia Voskoboinik ◽

Marie-Claude Thia ◽

Joseph A. Trapani

Keyword(s):

Functional Analysis ◽

Hemophagocytic Lymphohistiocytosis ◽

Autosomal Recessive ◽

Missense Mutations ◽

Wild Type ◽

Lytic Function ◽

Functional Defect ◽

Target Cell Membrane ◽

And Function ◽

Prf1 Gene

Abstract Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms. (Blood. 2005;105:4700-4706)

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MAP1LC3B overexpression protects against Hermansky-Pudlak syndrome type-1-induced defective autophagy in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00213.2015 ◽

2016 ◽

Vol 310 (6) ◽

pp. L519-L531 ◽

Cited By ~ 13

Author(s):

Saket Ahuja ◽

Lars Knudsen ◽

Shashi Chillappagari ◽

Ingrid Henneke ◽

Clemens Ruppert ◽

...

Keyword(s):

Autosomal Recessive ◽

Alveolar Epithelial Cell ◽

Critical Role ◽

Autosomal Recessive Disorder ◽

Lamellar Bodies ◽

Wild Type ◽

Alveolar Epithelial ◽

Hermansky Pudlak Syndrome

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, and some patients with HPS develop pulmonary fibrosis, known as HPS-associated interstitial pneumonia (HPSIP). We have previously reported that HPSIP is associated with severe surfactant accumulation, lysosomal stress, and alveolar epithelial cell type II (AECII) apoptosis. Here, we hypothesized that defective autophagy might result in excessive lysosomal stress in HPSIP. Key autophagy proteins, including LC3B lipidation and p62, were increased in HPS1/2 mice lungs. Electron microscopy demonstrated a preferable binding of LC3B to the interior of lamellar bodies in the AECII of HPS1/2 mice, whereas in wild-type mice it was present on the limiting membrane in addition to the interior of the lamellar bodies. Similar observations were noted in human HPS1 lung sections. In vitro knockdown of HPS1 revealed increased LC3B lipidation and p62 accumulation, associated with an increase in proapoptotic caspases. Overexpression of LC3B decreased the HPS1 knockdown-induced p62 accumulation, whereas rapamycin treatment did not show the same effect. We conclude that loss of HPS1 protein results in impaired autophagy that is restored by exogenous LC3B and that defective autophagy might therefore play a critical role in the development and progression of HPSIP.

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The haemochromatosis protein HFE induces an apparent iron-deficient phenotype in H1299 cells that is not corrected by co-expression of beta2-microglobulin

Biochemical Journal ◽

10.1042/bj20021607 ◽

2003 ◽

Vol 370 (3) ◽

pp. 891-899 ◽

Cited By ~ 21

Author(s):

Jian WANG ◽

Guohua CHEN ◽

Kostas PANTOPOULOS

Keyword(s):

Cell Surface ◽

Autosomal Recessive ◽

Regulatory Protein ◽

Autosomal Recessive Disorder ◽

Inducible Promoter ◽

Wild Type ◽

Type Molecule ◽

Iron Deficient ◽

Hfe Mutations

HFE, an atypical MHC class I type molecule, has a critical, yet still elusive function in the regulation of systemic iron metabolism. HFE mutations are linked to hereditary haemochromatosis type 1, a common autosomal recessive disorder of iron overload. Most patients are homozygous for a C282Y point mutation that abrogates the interaction of HFE with β2-microglobulin (β2M) and, thus, impairs its proper processing and expression on the cell surface. An H63D substitution is also associated with disease. To investigate the function of HFE we have generated clones of human H1299 lung cancer cells that express wild-type, C282Y or H63D HFE under the control of a tetracycline-inducible promoter. Consistent with earlier observations in other cell lines, the expression of wild-type or H63D, but not C282Y, HFE induces an apparent iron-deficient phenotype, manifested in the activation of iron-regulatory protein and concomitant increase in transferrin receptor levels and decrease in ferritin content. This phenotype persists in cells expressing wild-type HFE after transfection with a β2M cDNA. Whereas endogenous β2M is sufficient for the presentation of at least a fraction of chimeric HFE on the cell surface, this effect is stimulated by approx. 2.8-fold in β2M transfectants. The co-expression of exogenous β2M does not significantly affect the half-life of HFE. These results suggest that the apparent iron-deficient phenotype elicited by HFE is not linked to β2M insufficiency.

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A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽

10.1182/blood.v96.7.2501.h8002501_2501_2505 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2501-2505

Author(s):

Maurizio Margaglione ◽

Rosa Santacroce ◽

Donatella Colaizzo ◽

Davide Seripa ◽

Gennaro Vecchione ◽

...

Keyword(s):

Amino Acid ◽

Rna Splicing ◽

Messenger Rna ◽

Stop Codon ◽

Premature Stop Codon ◽

Autosomal Recessive Disorder ◽

Chain Gene ◽

Hemorrhagic Diathesis ◽

Reading Frame ◽

Congenital Afibrinogenemia

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

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