|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1336-1341
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGYAU#0
Missense mutations in the human  fibrinogen gene cause
congenital afibrinogenemia by impairing fibrinogen
secretion
Stefano Duga,
Rosanna Asselta,
Elena Santagostino,
Sirous Zeinali,
Tatjana Simonic,
Massimo Malcovati,
Pier Mannuccio Mannucci, and
Maria Luisa Tenchini
From the Department of Biology and Genetics for Medical Sciences,
the Institute of Veterinary Physiology and Biochemistry, the
Angelo Bianchi Bonomi Hemophilia and Thrombosis
Center, and the Fondazione Luigi Villa and the Department of Internal
Medicine, University of Milan; the IRCCS Maggiore Hospital, Milan,
Italy; and the Department of Biotechnology, Pasteur Institute of
Tehran, Iran.
 |
Abstract |
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
B -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
B fibrinogen gene could cause congenital afibrinogenemia by
impairing fibrinogen secretion.
(Blood. 2000;95:1336-1341)
© 2000 by The American Society of Hematology.
| |
Introduction |
Fibrinogen is a 340-kd glycoprotein synthesized
primarily by hepatocytes and secreted as a hexamer composed of 3 pairs
of polypeptide chains (A , B, and  ) (for review,
see1). It has a trinodular structure with a central nodule
(E-domain) that contains the N-terminus of each chain and 2 lateral
globular domains (D-domains) that contain the C-terminus of B- and
-chains. The E-domain is linked to the 2 D-domains by a
coiled-coil triple helix structure.2 The 3 chains are encoded by different genes, clustered in a region of
approximately 50 kb on chromosome 4q28.3 Fibrinogen
participates in hemostasis by forming the insoluble fibrin clot and
mediating platelet aggregation.4 Moreover, fibrinogen is a
class II protein involved in the acute phase response to injury and
stress (for review, see5).
Congenital fibrinogen disorders include afibrinogenemia,
hypofibrinogenemia, and dysfibrinogenemia, characterized by the
complete absence or extremely low levels of plasma fibrinogen, by
reduced amounts of plasma fibrinogen, or by the presence of
dysfunctional fibrinogen molecules, respectively. Congenital
afibrinogenemia (MIM 202 400) is a rare autosomal recessive disorder
with a high rate of consanguinity within affected families, and it is
characterized by bleeding manifestations that often start at birth with
uncontrolled umbilical cord hemorrhages. Bleeding from mucosal
membranes, hematomas, hemarthroses, and hemorrhages after trauma and
surgery are relatively frequent.6-8 However, the
disease usually can be controlled by fibrinogen replacement therapy.
Although several missense mutations in the 3 fibrinogen genes have been
identified as the cause of dysfibrinogenemia,
hypofibrinogenemia, or both,9 congenital
afibrinogenemia, originally described in 1920,10 has been
associated only with a recently reported homozygous 11-kb deletion of
the A-chain gene.11 However, the existence of different
genetic alterations in inherited afibrinogenemia was suggested by a
previous study on 2 patients with afibrinogenemia who had no gross
alterations within the fibrinogen cluster as revealed by Southern blot
analysis.12
In this study 2 patients with afibrinogenemia, an Italian and an
Iranian, each the offspring of a marriage between first cousins, were
studied. Both had plasma fibrinogen levels that could not be measured
by functional assay, but extremely low levels (between 0.05% and 0.5%
of the average normal value) were detected by enzyme immunoassay. Two
different homozygous missense mutations (Leu353Arg and Gly400Asp),
neither of which has yet been described, were identified in exons 7 and
8 of the fibrinogen B-chain gene. In vitro expression of the mutant
proteins demonstrated that each mutation abolished fibrinogen
secretion, thus causing the extremely reduced fibrinogen levels
observed in the patients.
| |
Methods |
Materials
Full-length A, B and cDNA, cloned in the
expression vector pRSV-Neo, were kind gifts of Dr C. M. Redman
(Lindsley F. Kimball Research Institute, New York Blood Center, New
York, NY). Rabbit polyclonal antibodies to human fibrinogen were from
DAKO (Copenhagen, Denmark). Oligonucleotides were purchased from Life
Technologies (Inchinnan, Paisley, UK).
Coagulation tests
Fibrinogen was measured in plasma by a functional assay based on
fibrin polymerization time using a commercial kit (Laboratoire Stago,
Asnieres, France), and it was measured in plasma and washed platelets
by enzyme immunoassay.13 The sensitivity of the functional assay was 5 mg/dL and that of the immunoassay was 0.02 mg/dL.
DNA extraction
All examined subjects signed informed consent. Genomic DNA was
extracted from blood samples using the Nucleon BACC1 kit (Amersham Pharmacia Biotech, Uppsala, Sweden).
Linkage analysis
The polymorphic locus used for linkage analysis was the
tetranucleotide repeat FGA-i3, located in intron 3 of the fibrinogen A-chain gene.14 Polymerase chain reaction (PCR) was
performed on genomic DNA under standard conditions, and amplified
products were separated using an automated 370A DNA sequencer (PE
Biosystems, Foster City, CA). Genotyping was performed using the
Genescan 3.1 software (PE Biosystems) and the LINKAGE
package was used for linkage analysis.15 The
average maximum expected LOD score was calculated using the
SIMULATE software.16
Sequence analysis
DNA sequencing was performed on both strands, either directly on
purified-PCR products or on plasmids using the Taq dye-deoxy terminator method and an automated 370A DNA sequencer (PE Biosystems). All primers used for sequencing were designed on the basis of known
sequences of the fibrinogen genes and intergenic regions (Genbank
accession numbers M64 982, M64 983, M10 014, and U36 478). Factura
and Sequence Navigator software packages (PE Biosystems) were used for
mutation detection.
Allele-specific oligonucleotide hybridization
20 ng PCR products, corresponding to regions in exon 7 or 8 of the
fibrinogen B-chain gene, were blotted onto nylon membranes (Amersham
Pharmacia Biotech) and hybridized to 32P-labeled
allele-specific oligonucleotides (ASO) according to standard
procedures.17 Wild-type and mutant probes are listed in
Table 1.
Site-directed mutagenesis
Leu353Arg and Gly400Asp mutations were introduced in the
pRSV-Neo-B plasmid by the Quick-change site-directed mutagenesis kit
(Stratagene, La Jolla, CA), according to the manufacturer's instructions, using the oligonucleotides L353R-F, L353R-R, and G400D-F,
and the oligonucleotide G400D-R, respectively (Table 1).
Cell cultures, transfections, and metabolic labeling
The human hepatoma cell line HepG2 was cultured in Dulbecco's
modified Eagle's medium (DMEM) and Ham's F12 media (1:1, vol/vol), supplemented with 10% fetal calf serum. The African green monkey kidney cell line COS-1 was cultured in DMEM containing 10% fetal calf
serum. Antibiotics (100 IU/mL penicillin and 100 µg/mL
streptomycin) and glutamine (1%) were added to the media. Cells were
grown at 37°C in a humidified atmosphere of 5% CO2 and
95% air. Using CELL FECTIN (Life Technologies), semiconfluent COS-1
cells were transfected with equimolar amounts (5 µg
each) pRSV-Neo-A, pRSV-Neo-, and pRSV-Neo-B plasmids, the latter either wild-type or mutant.
Thirty-six hours after transfection, the cells were labeled by
incubating each 100-mm dish for 3 hours with 5 mL methionine- and
cysteine-free DMEM (ICN Biomedicals, High Wycombe, Berks, UK)
supplemented with 150 µCi
L-[35S] methionine and 60 µCi
L-[35S] cysteine (Amersham Pharmacia
Biotech), with 10% dialyzed fetal calf serum, 0.1 mg/mL heparin, and
1% glutamine. In pulse-chase experiments, cells were pulse labeled for
3 hours as described above and then chase incubated for various periods
of time (30, 90, 180 minutes) with fresh medium containing 10 mmol/L
methionine and 10 mmol/L cysteine.
Immunoprecipitation and sodium dodecyl
sulphate- polyacrylamide gel electrophoresis
After metabolic labeling, culture media were collected and a
protease inhibitor mixture was added (Complete; Roche, Basel, Switzerland). Five milliliters medium from each plate was centrifuged to remove cell debris and concentrated using a Centricon Plus-20 column
(Millipore, Bedford, MA). Cells were washed 3 times with prechilled
phosphate-buffered saline (PBS) and lysed for 1 hour on ice with lysis
buffer containing 1 × PBS, 1% Triton X-100, and the protease
inhibitor mixture. Cell lysates were centrifuged to remove cell debris.
Rabbit antihuman fibrinogen antibodies, preadsorbed with protein
G-Sepharose (Sigma Chemical, St. Louis, MO) at room temperature for 3 hours, were added to cell lysates and media and incubated for 4 hours
on ice. Pellets were collected by centrifugation for 3 minutes and
washed 3 times with lysis buffer. The immunoprecipitated proteins were
released from protein G-Sepharose by boiling for 5 minutes in sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) loading
buffer. Samples were analyzed by SDS-PAGE according to
Laemmli18 and were detected by autoradiography or by a
STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
| |
Results |
Patient data
The proband of the Italian family was a 17-year old boy whose
parents were first cousins (Figure 1A). The
diagnosis of afibrinogenemia was made at birth because the patient had
life-threatening bleeding from the umbilical cord, which rendered
necessary transfusion with whole blood and fibrinogen concentrates.
After that he had relatively mild symptoms, such as epistaxis and
posttraumatic muscle hematomas. His parents, sister, and other family
members available for study were asymptomatic. In the proband, plasma fibrinogen levels could not be measured by functional assay (less than
5 mg/dL; normal range, 160-400 mg/dL), and they were found to be very
low by enzyme immunoassay (0.13 mg/dL; normal range, 160-400 mg/dL).
Platelet levels of immunoreactive fibrinogen were also very low
(0.35 × 109 platelets; normal range, 60-190 µg/109 platelets). His parents and sister had
approximately half the normal levels of plasma fibrinogen and normal
intraplatelet levels.
View larger version (23K):
[in this window]
[in a new window]
| Fig 1.
Family pedigrees of the 2 probands with congenital
afibrinogenemia analyzed in this study.
(A) Pedigree of the Italian and (B) of the Iranian family. Plasma
functional fibrinogen levels (mg/dL), immunoreactive fibrinogen levels
(mg/dL), intraplatelet levels (µg/109 platelets) and
haplotypes of the FGA-i3 tetranucleotide repeat marker (alleles
numbered from the largest to the smallest) are indicated in this order
below each symbol. Arrows indicate the probands. nd, not done.
|
|
The proband of the Iranian family (Figure 1B) was a
24-year-old man, also born of a consanguineous marriage. He bled at
birth from the umbilical cord and later during circumcision, and he was
treated with whole blood and fresh-frozen plasma on both occasions. Subsequently, he suffered repeatedly from muscle hematomas and hemarthroses that occurred spontaneously or after minor trauma. His
parents, 2 brothers, and 2 sisters were asymptomatic, and there was no
history of bleeding in the other family members not investigated. In
the proband, fibrinogen could not be measured by functional assay, and
very low values (1.6 mg/dL) were detected by the more sensitive
immunoassay. His father's plasma fibrinogen level was half the normal
level, and his mother had a low borderline level of functional plasma
fibrinogen and a normal level of immunoreactive fibrinogen. Platelet
samples could not be obtained.
Mutational analysis
The absence of gross deletions in the fibrinogen cluster has been
verified by Southern blot analysis and by long PCR amplification of
each fibrinogen gene (data not shown). To verify whether the fibrinogen
cluster was still a candidate for mutation screening, linkage analysis
using the tetranucleotide repeat FGA-i3 marker located within the
A-chain gene was performed in the Italian family. Evaluation of
plasma levels of functional fibrinogen allowed us to classify
phenotypically the 9 available members of this family as homozygous
normal (more than 160 mg/dL) or heterozygous (less than 160 mg/dL)
(Figure 1A). Linkage analysis resulted in an LOD score of 2.02 at
 = 0. This LOD score, close to the average maximum expected LOD
score of 2.37 calculated for this pedigree, suggested the existence of
linkage to the fibrinogen cluster. Because of the small number of
available members, the Iranian pedigree could not be considered for
this analysis. The proband received the same FGA-i3 allele from both
parents, as expected for the affected offspring of a consanguineous
marriage (Figure 1B).
On the basis of these results in each proband, the entire coding region
was sequenced, including exon-intron boundaries and approximately 300 bp of the promoter region of each fibrinogen gene. In the Italian
proband, a single homozygous T-G transversion was found in
exon 7 of the fibrinogen B-chain gene at position 7156 (numbering
according to Genbank accession number M64983) (Figure
2A). This resulted in a missense mutation
leading to a Leu-Arg substitution at position 353 (L353R). The
proband's parents were heterozygous for the mutation (Figure 2A). All
available family members were tested for the mutation by dot blot
hybridization with ASO probes. Besides the proband's parents, the
mutation was present in the heterozygous state in the proband's
grandmother and in phenotypically heterozygous relatives (Figure 2B).
Two hundred aploid genomes from unrelated persons in a northern Italian control population were also analyzed, and the mutation was absent. In
the Iranian proband, a single G-A homozygous transition at position 7915 was identified in exon 8 of the fibrinogen B-chain gene, replacing glycine with aspartate at position 400 (G400D) (Figure
2C). This mutation was also present in the heterozygous state in the
proband's parents (Figure 2C), but not in 200 unrelated Iranian aploid
control genomes. Both amino acid substitutions are nonconservative and
reside in the C-terminal globular portion of the B-chain.
View larger version (18K):
[in this window]
[in a new window]
| Fig 2.
L353R and G400D mutations.
(A) Electropherograms showing the mutation identified in the Italian
proband with afibrinogenemia. The T-G transversion leading to the L353R
mutation, indicated by an arrow, was present in the heterozygous state
in both parents. (B) ASO hybridization showing cosegregation of the
L353R mutation and clinical phenotype in the Italian kindred. Family
members were phenotypically classified as homozygous normal or
heterozygote on the basis of plasma fibrinogen levels. Individuals
within the pedigree are positioned above the corresponding lanes.
Wild-type (FGB-L353) and mutant (FGB-R353) probes are listed in Table
1. (C) Electropherograms showing the mutation identified in the Iranian
proband with afibrinogenemia. The G-A transition leading to the G400D
mutation, indicated by an arrow, was present in the heterozygous state
in both parents. Family members are labeled as in Figure 1. FGB,
fibrinogen B-chain gene; K, G or T nucleotide; R, A or G
nucleotide.
|
|
In vitro protein expression
To determine whether the L353R and G400D mutations affect fibrinogen
assembly or secretion, mutant proteins were transiently expressed in
COS-1 cells that do not express fibrinogen. The 2 point mutations were
independently introduced by site-directed mutagenesis in a B-chain
expression plasmid (pRSV-Neo-B)19 and checked by
sequencing. Each mutated plasmid was transiently cotransfected in COS-1
cells, together with plasmids pRSV-Neo-A and pRSV-Neo- expressing
the wild-type A- and -chains, respectively.20 Transfections with equal amounts of wild-type and each of the mutated
B-chain cDNA were also performed, to mimic the heterozygous state.
Transfected cells were incubated with L-[35S]
methionine and L-[35S] cysteine. Fibrinogen
expression was analyzed by immunoprecipitation using polyclonal
antihuman fibrinogen antibodies and protein G-Sepharose and then by
SDS-PAGE.
After SDS-PAGE under nonreducing conditions, a specific fibrinogen band
was observed in cell lysates and culture media of COS-1 cells
transfected with plasmids containing wild-type fibrinogen cDNA (Figures
3A, 3B). The same band was also observed in
untransfected control fibrinogen-expressing HepG2 cells (Figures 3A,
3B). By contrast, in cells transfected with plasmids expressing L353R or G400D B-chains, hexameric fibrinogen was detectable only in cell
lysates (Figure 3A) but was undetectable in the corresponding culture
media (Figure 3B). Cotransfection of wild-type and each of the mutated
B-chain cDNA restored fibrinogen secretion to the medium (Figure
3B). Mock-transfected (pUC18) COS-1 cells showed only background bands
in cell lysate and medium (Figures 3A, 3B). Immunoprecipitated proteins
from the same culture media as in Figure 3B were also separated on
SDS-PAGE under reducing conditions. The expected pattern of A, B
and -chains was observed in fibrinogen-containing media (Figure 3C).
View larger version (41K):
[in this window]
[in a new window]
| Fig 3.
Expression of wild-type and mutant fibrinogen chains in
COS-1 cells.
Intracellular and secreted fibrinogen from COS-1 cells transfected with
wild-type A and -chains together with wild-type and/or L353R or
G400D B, chains have been analyzed. Metabolic labeling,
immunoprecipitation, and SDS-PAGE were carried out as described in
"Materials and Methods." (A) Immunoprecipitable proteins in cell
lysates separated on 4% SDS-PAGE under nonreducing conditions. The
arrowhead indicates the 340-kd fibrinogen complex. (B)
Immunoprecipitable proteins in the culture media separated on 4%
SDS-PAGE under nonreducing conditions. The arrowhead indicates the
340-kd fibrinogen complex. (C) Immunoprecipitable proteins in the
culture media separated on 10% SDS-PAGE under reducing conditions. The
positions of A, B, and chains are indicated. The position of
molecular weight markers (MW) in kd is denoted at the right-hand side
of the figure.
|
|
To evaluate whether the observed absence of mutant fibrinogens in
culture media resulted from an impairment of fibrinogen secretion or
from increased intracellular or extracellular degradation, a
pulse-chase experiment was performed. Transfected COS-1 cells were
labeled for 3 hours with L-[35S] methionine
and L-[35S] cysteine and were chase
incubated with an excess of the corresponding unlabeled amino acids for
various periods up to 3 hours. As shown in Figure
4A, in lysates from cells transfected with
the 3 wild-type cDNA, immunoprecipitated radioactive fibrinogen
markedly declined at 90 minutes and disappeared at 180 minutes. By
contrast, in lysates from cells transfected with plasmids expressing
L353R or G400D B-chains, hexameric fibrinogen persisted
intracellularly up to 3 hours (Figure 4A). Secreted fibrinogen was
present, at the analyzed chase-periods, in culture media from COS-1
cells cotransfected with A, B, and wild-type cDNA, whereas it
was absent in culture media from cells cotransfected with A and wild-type chains and either B mutant chain expressing plasmids (Figure 4B).
View larger version (43K):
[in this window]
[in a new window]
| Fig 4.
Intracellular retention of L353R and G400D mutant
fibrinogens.
COS-1 cells were incubated with L-[35S]
methionine and L-[35S] cysteine for 3 hours
and then chase incubated with 10 mmol/L L-methionine and
L-cysteine for various periods of time up to 3 hours. At
the specified chase periods (0, 30, 90, and 180 minutes), radioactive
fibrinogen was immunoprecipitated from cell lysates and from the
corresponding culture media, as described in "Materials and
Methods." Intracellular (A) and secreted (B) immunoprecipitable
proteins were separated by 4% SDS-PAGE under nonreducing conditions.
The arrowheads indicate the 340-kd fibrinogen complex. Intracellular
and secreted fibrinogen at the end of the pulse period from control
HepG2 cells was also analyzed. In both panels, pUC18 lane contains
immunoprecipitable proteins at the end of the pulse period from COS-1
cells transfected with the unrelated pUC18 plasmid.
|
|
| |
Discussion |
Inherited afibrinogenemia is a rare recessive bleeding disease
characterized by fibrinogen deficiency in plasma and platelets. Even
though, strictly speaking, the term should refer to the complete absence of fibrinogen, minute amounts of fibrinogen can sometimes be detected through sensitive immunoassays. Asymptomatic heterozygotes usually have approximately half the normal levels of functional and
immunoreactive fibrinogen. The 2 probands studied here, both the
offspring of consanguineous marriages, had unmeasurable, clottable fibrinogen and very low plasma levels of immunoreactive fibrinogen (0.13 mg/dL and 1.59 mg/dL). Genotypically heterozygous relatives had
approximately half the normal fibrinogen levels and a good concordance
between functional and immunologic values. The only notable exception
was the mother of the Iranian proband, who had low borderline levels of
functional plasma fibrinogen (192 mg/dL) with 50% higher
immunoreactive fibrinogen (303 mg/dL). Some kindred afibrinogenemic
putative carriers with normal fibrinogen levels have been
described7; however, there is no clear explanation for the
discrepancies between functional and immunologic test results except
those that can be attributed to methodologic variability. In the
Italian family, intraplatelet fibrinogen was also measured, and levels
were normal in the 3 heterozygous members (III-7, III-8, and IV-2)
studied. The intraplatelet fibrinogen level of the proband was very low
(approximately 0.3% of the average normal level) but was less reduced
than plasma fibrinogen levels (0.05%). This phenomenon may be
explained by the ability of platelets actively to take up fibrinogen
from plasma.21
Inherited fibrinogen deficiencies may have different causes, such as
decreased protein synthesis, increased intracellular or circulatory
degradation, defective assembly or secretion, or a combination of these
defects. The only previously identified genetic basis of congenital
hypofibrinogenemia and afibrinogenemia is a homozygous truncation of
the A-chain gene. The deletion of 25% of the A-chain identified
in fibrinogen Marburg was associated with mild hypofibrinogenemia (60 mg/dL, immunoassay),22 whereas in fibrinogen Otago, the
more severe truncation of 56% of the A-chain was associated with
severe hypofibrinogenemia (6 mg/dL, functional assay).23
The truncation of 26% of the A-chain in fibrinogen Milano III was
associated with normal circulating levels (260 mg/dL, functional
assay).24 All these cases are also characterized by the
presence of dysfunctional fibrinogen molecules and should therefore be
defined as dysfibrinogenemia or hypo-disfibrinogenemia. The severest A-chain truncation is the 11-kb deletion recently identified by Neerman-Arbez et al,11 involving the whole
gene except exon 1. In this case, fibrinogen was undetectable either by
functional assay or immunoassay.
In this article, we report the identification of new pathogenetic
defects underlying congenital afibrinogenemia. Two different missense
mutations (L353R and G400D) were identified in the C-terminal globular
portion of fibrinogen B-chain (Figure
5A). These nonconservative substitutions
involve strictly conserved residues in a region highly conserved among
vertebrates (Figure 5B). Inspection of the fibrinogen crystal
structure2 showed that Leu353 and Gly400 were part of the
same region of the protein, in the hydrophobic core of the B-chain
C-terminal region. Gly400 was solvent inaccessible, in contact with
Phe262, Tyr269, Phe375, Tyr404, and Asn413. Similarly, Leu353 was
buried beneath the protein surface, extensively interacting with the
side chains of Tyr326, Val342, His370, Trp402, Ala410, and Trp437. The
L353R and G400D mutations introduced bulky, charged side chains to
these hydrophobic regions. Clearly, such drastic amino acid
replacements would be expected to perturb the packing of the side
chains in the B-chain core and would likely affect the stability of
the protein, which might be unable to fold correctly, with the ultimate
result of impaired assembly and release of an active fibrinogen
molecule.
View larger version (35K):
[in this window]
[in a new window]
| Fig 5.
Position of the L353R and G400D mutations in the
structure of the fibrinogen B chain.
(A) C-alpha trace of the AB trimer, outlining the position of
Gly400 and Leu353, produced using the coordinates under the Protein
Data Bank entry 1FZA. FGA, FGB, and FGG indicate the A, B, and
fibrinogen chains, respectively. N- and C-terminal residues of each
chain are indicated. Light gray, black, and gray correspond to A,
B, and chains, respectively. (B) Multiple alignment of human,
rat, bovine, xenopus, chicken, and lamprey fibrinogen B chain in the
region containing the 2 identified mutations. Identical amino acids are
boxed. Positions of L353R and G400D mutations are indicated by arrows.
Amino acid sequences were obtained from Swiss-Prot database (accession
numbers: P02675 [human], P14480 [rat], P02676 [bovine], AAA85283
[xenopus], Q02020 [chicken], and FIBB_PETMA_2 [lamprey]) and
numbered; the signal peptide is omitted. Secondary structures shown
below the alignments (with cylinders representing -helices and the
arrow representing the -strand) refer to the human protein,
drawn from Spraggon et al2 data.
|
|
It has been demonstrated, by deletion experiments, that the C-terminal
half of the B-chain, which is part of the D-domain, is not necessary
for the assembly and secretion of fibrinogen.25 Nevertheless the assembly of a misfolded protein could result in severe
impairment of protein secretion. Numerous genetic diseases are known in
which a single amino acid change can lead to failure of protein
export,26 as evidenced in cystic fibrosis,27
juvenile pulmonary emphysema,28 osteogenesis
imperfecta,29 juvenile diabetes insipidus,30
and hypercholesterolemia.31
To assess whether L353R and G400D mutations could be responsible for
cases of congenital afibrinogenemia, the mutant B-chains were
expressed, together with the other 2 wild-type chains, in COS-1 cells.
This cell line does not express fibrinogen and represents a widely used
in vitro system for studying fibrinogen assembly and secretion by
transient or stable transfections.20 Transfection experiments suggested that either mutation abolished fibrinogen secretion because mutant fibrinogen molecules were synthesized and assembled intracellularly but were undetectable in culture media.
The observation that mutant fibrinogen molecules are assembled intracellularly might explain the minute amounts of protein measured in
a patient's plasma using a sensitive immunoassay. This finding is
consistent with the concept that missense mutations do not necessarily
block protein production completely. The hypothesis that the main
defect caused by these 2 mutations was at the secretion level was
confirmed by the results of pulse-chase experiments showing the lack of
cellular depletion of L353R and G400D mutant fibrinogens.
In conclusion, 2 different homozygous missense mutations were
identified in the same region of the B-chain in 2 patients with
afibrinogenemia. Transfection experiments in COS-1 cells with plasmids
expressing the mutant proteins demonstrated, for the first time, that
these mutations can abolish fibrinogen secretion without affecting the
protein synthesis. These results demonstrated that congenital
afibrinogenemia can be caused by point mutations that impair the normal
fibrinogen secretion pathway.
| |
Acknowledgments |
We thank Dr C. Redman for kindly providing A-, B-, and
-chain-expressing pRSV-Neo plasmids. We thank Dr A. Mattevi for helpful discussion on the structural effect of mutations on the protein
folding and Drs A. Clivio, C. Pinter, and B. Pedrotti for helpful
suggestions on expression and immunoprecipitation experiments. We also
thank the members of both families for their participation in this study.
| |
Footnotes |
Submitted August 9, 1999; accepted October 11, 1999.
Supported by the Ministero dell'Università e della Ricerca
Scientifica e Tecnologica (MURST 60%) and by IRCCS Maggiore Hospital, Milan, Italy.
Reprints: Maria Luisa Tenchini, Dipartimento di Biologia e
Genetica per le Scienze Mediche, via Viotti, 3/5-20133 Milano, Italy;
e-mail: marialuisa.tenchini{at}unimi.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Doolittle RF.
Fibrinogen and fibrin.
Ann Rev Biochem.
1984;53:195[Medline]
[Order article via Infotrieve].
2.
Spraggon G, Everse SJ, Doolittle RF.
Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin.
Nature.
1997;389:655[Medline]
[Order article via Infotrieve].
3.
Marino MW, Fuller GM, Elder FF.
Chromosomal localization of human and rat A alpha, B beta, and gamma fibrinogen genes by in situ hybridization.
Cytogenet Cell Gene.
1986;422:36.
4.
Mosesson MW.
Fibrinogen structure and fibrin clot assembly.
Semin Thromb Hemost.
1998;24:169[Medline]
[Order article via Infotrieve].
5.
Kushner I.
The acute phase response: an overview.
Methods Enzymol.
1988;163:373[Medline]
[Order article via Infotrieve].
6.
Mammen EF.
Fibrinogen abnormalities.
Semin Thromb Hemost.
1983;9:1[Medline]
[Order article via Infotrieve].
7.
Al-Mondhiry H, Ehmann WC.
Congenital afibrinogenemia.
Am J Hematol.
1994;46:343[Medline]
[Order article via Infotrieve].
8.
Lak M, Keihani M, Elahi P, Peyvandi F, Mannucci PM.
Bleeding and thrombosis in 55 patients with inherited afibrinogenemia.
Br J Haematol.
1997;107:204.
9.
Martinez J.
Congenital dysfibrinogenemia.
Curr Opin Hematol.
1997;4:357[Medline]
[Order article via Infotrieve].
10.
Rabe F, Salomon E.
Über-faserstoffmangel im Blute bei einem Falle von Hâmophilie.
Arch Int Med.
1920;95:2.
11.
Neerman-Arbez M, Honsberger A, Antonarakis SE, Morris MA.
Deletion of the fibrinogen alpha-chain gene (FGA) causes congenital afibrogenemia.
J Clin Invest.
1999;103:215[Medline]
[Order article via Infotrieve].
12.
Uzan G, Courtois G, Besmond C, et al.
Analysis of fibrinogen genes in patients with congenital afibrinogenemia.
Biochem Biophys Res Commun.
1984;120:376[Medline]
[Order article via Infotrieve].
13.
Cattaneo M, Bettega D, Lombardi R, Lecchi A, Mannucci PM.
Sustained correction of the bleeding time in an afibrinogenaemic patient after infusion of fresh frozen plasma.
Br J Haematol.
1992;82:388[Medline]
[Order article via Infotrieve].
14.
Mills KA, Even D, Murray JC.
Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA).
Hum Mol Genet.
1992;1:779[Free Full Text].
15.
Lathrop GM, Lalouel JM.
Easy calculations of LOD scores and genetic risks on small computers.
Am J Hum Genet.
1984;36:460[Medline]
[Order article via Infotrieve].
16.
Weeks DE, Ott J, Lathrop GM.
Detection of genetic interference: simulation studies and mouse data.
Genetics.
1994;136:1217[Abstract].
17.
Sambrook J, Fritsch EF, Maniatis T.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
18.
Laemmli UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature.
1970;227:680[Medline]
[Order article via Infotrieve].
19.
Roy SN, Mukhopadhyay G, Redman CM.
Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen.
J Biol Chem.
1990;265:6389[Abstract/Free Full Text].
20.
Roy SN, Procyk R, Kudryk BJ, Redman CM.
Assembly and secretion of recombinant human fibrinogen.
J Biol Chem.
1991;266:4758[Abstract/Free Full Text].
21.
Handagama P, Scarborough RM, Shuman MA, Bainton DF.
Endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa).
Blood.
1993;82:135[Abstract/Free Full Text].
22.
Koopman J, Haverkate F, Grimbergen J, Egbring R, Lord ST.
Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA >stop TAA).
Blood.
1992;80:1972[Abstract/Free Full Text].
23.
Ridgway HJ, Brennan SO, Faed JM, George PM.
Fibrinogen Otago: a major alpha chain truncation associated with severe hypofibrinogenaemia and recurrent miscarriage.
Br J Haematol.
1997;98:632[Medline]
[Order article via Infotrieve].
24.
Furlan M, Steinmann C, Jungo M, et al.
A frameshift mutation in Exon V of the A alpha-chain gene leading to truncated A alpha-chains in the homozygous dysfibrinogen Milano III.
J Biol Chem.
1994;269:33,129[Abstract/Free Full Text].
25.
Zhang Z, Redman CM.
Identification of B beta chain domains involved in human fibrinogen assembly.
J Biol Chem.
1992;267:21,727[Abstract/Free Full Text].
26.
Kim PS, Hossain SA, Park YN, Lee I, Yoo SE, Arvan P.
A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: a model of human endoplasmic reticulum storage diseases.
Proc Natl Acad Sci U S A.
1998;95:9909[Abstract/Free Full Text].
27.
Ward CL, Omura S, Kopito RR.
Degradation of CFTR by the ubiquitin-proteasome pathway.
Cell.
1995;83:121[Medline]
[Order article via Infotrieve].
28.
Sifers RN, Finegold MJ, Woo SL.
Molecular biology and genetics of alpha 1-antitrypsin deficiency.
Semin Liver Dis.
1992;12:301[Medline]
[Order article via Infotrieve].
29.
Prockop DJ, Kivirikko KI.
Collagens: molecular biology, diseases, and potentials for therapy.
Annu Rev Biochem.
1995;64:403[Medline]
[Order article via Infotrieve].
30.
Ito M, Jameson JL, Ito M.
Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus: cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum.
J Clin Invest.
1997;99:1897[Medline]
[Order article via Infotrieve].
31.
Pathak RK, Merkle RK, Cummings RD, Goldstein JL, Brown MS, Anderson RG.
Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex.
J Cell Biol.
1988;106:1831[Abstract/Free Full Text].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. Vu, C. Di Sanza, and M. Neerman-Arbez
Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants
Haematologica,
February 1, 2008;
93(2):
224 - 231.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Vu, C. Di Sanza, D. Caille, P. de Moerloose, H. Scheib, P. Meda, and M. Neerman-Arbez
Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia
Hum. Mol. Genet.,
November 1, 2005;
14(21):
3271 - 3280.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D Vu, P de Moerloose, A Batorova, J Lazur, L Palumbo, and M Neerman-Arbez
Hypofibrinogenaemia caused by a novel FGG missense mutation (W253C) in the {gamma} chain globular domain impairing fibrinogen secretion
J. Med. Genet.,
September 1, 2005;
42(9):
e57 - e57.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. V. Kravtsov, P. E. Monahan, and D. Gailani
A classification system for cross-reactive material-negative factor XI deficiency
Blood,
June 15, 2005;
105(12):
4671 - 4673.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Neerman-Arbez, M. Germanos-Haddad, K. Tzanidakis, D. Vu, S. Deutsch, A. David, M. A. Morris, and P. de Moerloose
Expression and analysis of a split premature termination codon in FGG responsible for congenital afibrinogenemia: escape from RNA surveillance mechanisms in transfected cells
Blood,
December 1, 2004;
104(12):
3618 - 3623.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. M. Mannucci, S. Duga, and F. Peyvandi
Recessively inherited coagulation disorders
Blood,
September 1, 2004;
104(5):
1243 - 1252.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. V. Kravtsov, W. Wu, J. C. M. Meijers, M.-F. Sun, M. A. Blinder, T. P. Dang, H. Wang, and D. Gailani
Dominant factor XI deficiency caused by mutations in the factor XI catalytic domain
Blood,
July 1, 2004;
104(1):
128 - 134.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. W. Mosesson
Afibrinogenemia in a compound heterozygote: making sense out of missense
Blood,
December 15, 2003;
102(13):
4247 - 4248.
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Vu, P. H. B. Bolton-Maggs, J. R. Parr, M. A. Morris, P. de Moerloose, and M. Neerman-Arbez
Congenital afibrinogenemia: identification and expression of a missense mutation in FGB impairing fibrinogen secretion
Blood,
December 15, 2003;
102(13):
4413 - 4415.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Neerman-Arbez, D. Vu, B. Abu-Libdeh, I. Bouchardy, and M. A. Morris
Prenatal diagnosis for congenital afibrinogenemia caused by a novel nonsense mutation in the FGB gene in a Palestinian family
Blood,
May 1, 2003;
101(9):
3492 - 3494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Kanno, H. Suzuki, K. Fujiu, Y. Yoshino, A. Ohishi, and M. Gotoh
Hemopneumothorax associated with marfan syndrome and congenital afibrinogenemia
Ann. Thorac. Surg.,
April 1, 2003;
75(4):
1304 - 1306.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Duga, M. C. Montefusco, R. Asselta, M. Malcovati, F. Peyvandi, E. Santagostino, P. M. Mannucci, and M. L. Tenchini
Arg2074Cys missense mutation in the C2 domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein
Blood,
January 1, 2003;
101(1):
173 - 177.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Okumura, F. Terasawa, H. Tanaka, M. Hirota, H. Ota, K. Kitano, K. Kiyosawa, and S. T. Lord
Analysis of fibrinogen gamma -chain truncations shows the C-terminus, particularly gamma Ile387, is essential for assembly and secretion of this multichain protein
Blood,
May 15, 2002;
99(10):
3654 - 3660.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Iwaki, M. J. Sandoval-Cooper, M. Paiva, T. Kobayashi, V. A. Ploplis, and F. J. Castellino
Fibrinogen Stabilizes Placental-Maternal Attachment During Embryonic Development in the Mouse
Am. J. Pathol.,
March 1, 2002;
160(3):
1021 - 1034.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Asselta, S. Duga, S. Spena, E. Santagostino, F. Peyvandi, G. Piseddu, R. Targhetta, M. Malcovati, P. M. Mannucci, and M. L. Tenchini
Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen Aalpha -chain gene are not associated with the decay of the mutant mRNAs
Blood,
December 15, 2001;
98(13):
3685 - 3692.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Bodo, A. Katsumi, E. A. Tuley, J. C. J. Eikenboom, Z. Dong, and J. E. Sadler
Type 1 von Willebrand disease mutation Cys1149Arg causes intracellular retention and degradation of heterodimers: a possible general mechanism for dominant mutations of oligomeric proteins
Blood,
November 15, 2001;
98(10):
2973 - 2979.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Asselta, S. Duga, T. Simonic, M. Malcovati, E. Santagostino, P. L. F. Giangrande, P. M. Mannucci, and M. L. Tenchini
Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation
Blood,
October 1, 2000;
96(7):
2496 - 2500.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Margaglione, R. Santacroce, D. Colaizzo, D. Seripa, G. Vecchione, M. R. Lupone, D. De Lucia, P. Fortina, E. Grandone, C. Perricone, et al.
A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing
Blood,
October 1, 2000;
96(7):
2501 - 2505.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. A. Ploplis, J. Wilberding, L. McLennan, Z. Liang, I. Cornelissen, M. E. DeFord, E. D. Rosen, and F. J. Castellino
A Total Fibrinogen Deficiency Is Compatible with the Development of Pulmonary Fibrosis in Mice
Am. J. Pathol.,
September 1, 2000;
157(3):
703 - 708.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Neerman-Arbez, P. de Moerloose, C. Bridel, A. Honsberger, A. Schonborner, C. Rossier, K. Peerlinck, S. Claeyssens, D. Di Michele, R. d'Oiron, et al.
Mutations in the fibrinogen Aalpha gene account for the majority of cases of congenital afibrinogenemia
Blood,
July 1, 2000;
96(1):
149 - 152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction