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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Biology and Genetics for Medical
Sciences, University of Milan, Italy; Angelo Bianchi Bonomi Hemophilia
and Thrombosis Center and Fondazione Luigi Villa, Department of
Internal Medicine, University of Milan and IRCCS Maggiore Hospital,
Milan, Italy; Servizio di Coagulazione, Servizio di Prevenzione,
Diagnosi e Terapia delle Malattie Emorragiche e Trombotiche, Azienda
USL n.1, Sassari, Italy; Centro Emofilia e Malattie
Emorragiche, University of Cagliari, Cagliari, Italy.
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 All eukaryotes possess the ability to detect and
selectively degrade messenger RNAs (mRNAs) harboring premature
termination codons. It has been suggested that this mechanism, known as
nonsense-mediated mRNA decay, participates in the control of gene
expression by regulating the stability of selected physiological
transcripts and that it modulates the phenotypic severity of a number
of genetic diseases.1-3 In this respect, the relatively
small size of the Fibrinogen is a complex glycoprotein that consists of 2 sets of 3 different polypeptide chains (A Mutations in the 3 fibrinogen genes responsible for this disease
have been recently described. Apart from an 11-kilobases (kb)
deletion in the A In this study, 5 point mutations (4 novel and 1 previously reported)
were found in 8 afibrinogenemic patients. All mutations were located
within the first 4 exons of the A Materials
Blood collection and coagulation tests
Fibrinogen was measured in plasma by a functional assay based on fibrin polymerization time with the use of a commercial kit (Laboratoire Stago, Asnières, France) and by an enzyme-linked immunosorbent assay.29 The normal range for both tests was 160 to 400 mg/dL; the sensitivities of the functional assay and of the immunoassay were 5 mg/dL and 0.02 mg/dL, respectively. The immunoassay was performed for each proband in at least triplicate on the same plasma samples. DNA sequence analysis Genomic DNA was extracted from whole blood by means of the Nucleon BACC1 Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Plasmid DNA was isolated by QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). DNA sequencing was performed on both strands, either directly on purified polymerase chain reaction (PCR) products or on plasmids, by means of the BigDye Terminator Cycle Sequencing Kit and an automated Abi-310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Primers used for sequencing were designed on the basis of the known sequences of the 3 fibrinogen genes and intergenic regions (GenBank accession numbers: M64982, M64983, M10014, U36478, and AF229198). Details on primers and PCR conditions will be supplied on request. Factura and Sequence Navigator softwares (Applied Biosystems) were used for mutation detection.Haplotype analysis Haplotype analysis was performed with the use of the following A -chain gene polymorphic markers:  128 C>G and 58 G>A polymorphisms (both single-base substitutions),30
FGA-i3 microsatellite (a TCTT repeat),31 and
TaqI polymorphism (a 28-base pair [bp] duplication).32 Analyses were carried out as previously
described.12,30
Construction of expression vectors and mutagenesis A region of human fibrinogen A-chain gene, spanning from exon
1 to intron 5, was inserted as a 5.4-kb PCR-amplified fragment into the
mammalian expression vector pTARGET (Promega, Milan, Italy). This fragment was amplified from genomic DNA with the use of
the primer couple FGA-Ex1-F (5'-TAGGAGCCAGCCCCACCCTAGA-3') and
FGA-In5-R (5'-GTCATGGCTCTGTACTGTTAGGCA-3'). PCR was carried out in a
50-µL reaction mixture containing 100 ng genomic DNA (from a healthy
control individual), by means of the Expand Long 20kb-plus Kit (Roche),
in a PTC-100 thermal cycler (MJ-Research, Watertown, MA). The sample
was subjected to 10 cycles of denaturation at 94°C for 10 seconds,
and elongation at 68°C for 6 minutes, followed by 20 cycles of
denaturation at 94°C for 10 seconds, and elongation at 68°C for 6 minutes plus a time increment of 10 seconds for each cycle. A final
extension step of 10 minutes at 68°C was added after the last cycle.
This PCR product was A-tailed by a modification of the method described
by Marchuck et al33 (with the use of deoxyadenosine
5'-triphosphate instead of deoxythymidine 5'-triphosphate) and
was cloned with the use of the pTARGET Mammalian Expression
T-Vector System Kit (Promega). The pT-A-wild-type (wt) recombinant
plasmid was checked by sequencing. The 5 identified mutations were
independently introduced in pT-A-wt by the QuickChange Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA), according to the
manufacturer's instructions. Each mutant plasmid (pT-A-mut, with
"mut" indicating either Gly13stop, frameshift
[FS]-51stop, Ser100stop, Arg110stop, or Arg149stop
mutation) was checked by sequencing.
"Fluorescent hot-stop" PCR Quantitation of allele ratio was carried out by a modification of the "hot-stop" PCR technique34 (with the use of a fluorochrome-labeled primer instead of a radiolabeled one). Accuracy of the quantitation by the "fluorescent hot-stop" PCR was verified by performing PCR amplifications on DNA mixtures, containing known molar ratios of pT-A-wt and pT-A-Arg149stop plasmids (1:1, 1:4, 1:7,
1:9, 9:1, 7:1, and 4:1). The total amount of template was 0.001 ng in
each experiment. Both the wild-type and the mutant alleles were
PCR-amplified with the use of the primer couple
5'-TGTTAGAGCTCAGTTGGTTGATATGTAA-3' (sense mutagenic primer introducing
an allele-specific MaeIII restriction site) and
5'-CAGAGGTGTGGTGATGTAATG-3' (antisense primer) under standard PCR
conditions. The 309-bp-amplified product was subjected to one further
amplification cycle after the addition of 20 pmol antisense primer,
5'-labeled with 6-Fam. The labeled fragments from each PCR assay were
digested with MaeIII, according to the manufacturer's
instructions. The mutant uncut (309 bp) and the wild-type digested (280 bp) products were separated on an Abi-310 Genetic Analyzer, and peak
areas were measured by means of GeneScan Analysis software 3.1 (Applied Biosystems).
mRNA analysis The African green monkey kidney cell line COS-1 was cultured in Dulbecco modified Eagle medium containing 10% fetal calf serum, antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin), and glutamine (1%). Cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Semiconfluent COS-1 cells were transfected with equimolar amounts (2 pmol each) of pT-A-wt and each
of the pT-A-mut plasmids, by means of Cellfectin (Life
Technologies). Equimolarity of cotransfected plasmids was verified by
fluorescent hot-stop PCR as described above. At 5 hours after
transfection, media were replaced with fresh ones, and cells were
incubated for 60 additional hours. Cells were then washed twice with
phosphate-buffered saline, and total RNA was extracted by means of the
RNAWIZ Kit (Ambion, Austin, TX). First-strand cDNA synthesis, starting
from 200 ng total RNA, was performed with the use of random nonamers and Enhanced Avian RT-PCR Kit (Sigma, St Louis, MO). We used 5 µL of
20 µL as template to PCR-amplify DNA fragments containing the
corresponding mutation. A mutagenic PCR strategy enabling allele-specific restriction enzyme digestion was used to distinguish between the wild-type and mutant transcripts (Table
1). All mutagenic PCRs were performed by
means of the fluorescent hot-stop technique. The labeled fragments,
after digestion with the appropriate restriction endonuclease
(according to the manufacturer's recommendations), were detected and
measured as described above.
Protein analysis Semiconfluent COS-1 cells were transfected with equimolar amounts (1.5 pmol) of pRSV-Neo-B , pRSV-Neo- , and pT-A
plasmids, the latter being either wild type or mutant, by means of
Cellfectin. At 5 hours after transfection, media were replaced
with fresh ones. At 16 hours later, the cells were fed fresh media
without serum and cultured for an additional 48 hours. Conditioned
media were collected and a protease inhibitor mixture was added
(Complete; Roche). Then, 10 mL medium from each plate was centrifuged
to remove cell debris and was concentrated with the use of a Centricon Plus-20 column (Millipore, Bedford, MA). Untransfected human hepatoma HepG2 cells, cultured as previously described,12 were used
as control. The concentrated samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), essentially as
described by Laemmli.35 Polyacrylamide gels were
electroblotted onto polyvinylidene difluoride (PVDF) membranes
(Hybond-P; Amersham Pharmacia Biotech) for 1 hour at 200 mA by means of
a Biometra Fast Blot (Biometra, Göttingen, Germany). PVDF
membranes were soaked in a blocking buffer containing 5% skim milk in
TBST (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween20) at 4°C for
16 hours and incubated with rabbit antihuman fibrinogen antibodies (1:1000 dilution) in TBST buffer at 4°C for 2 hours. The membranes were washed 3 times for 5 minutes with TBST buffer and incubated for 1 hour at room temperature with horseradish peroxidase-conjugated antirabbit immunoglobulin (1:1500 dilution) (Envision; Dako). After 3 washings with TBST buffer, immunoreactive bands were visualized with an
enhanced chemioluminescence kit (ECLplus; Amersham Pharmacia Biotech).
Patient data Eight afibrinogenemic patients, 6 Italian, 1 Iranian, and 1 from Barbados, were studied. The Iranian patient was the only patient belonging to a family with overt consanguinity. All the patients had unmeasurable functional plasma fibrinogen levels, but very low fibrinogen levels were detected by immunoassay (ranging from 0.015 to 4.25 mg/dL) (Table 2). The patients, whose main clinical features are summarized in Table 2, did not suffer from concomitant coagulation disorders. All probands' parents were asymptomatic and had approximately half the normal fibrinogen levels, with good concordance between functional and immunologic values (data not shown).
Sequence analysis Sequencing of the entire coding region as well as of the splicing junctions and of about 500 bp of the promoter region of each fibrinogen gene, performed in all probands, disclosed 5 mutations, all located within the first 4 exons of the fibrinogen A-chain gene. Three
mutations were in the homozygous state: 1914C>G (in proband I3);
3075C>T (in probands P4 and B1); and 3192C>T (in probands C9, S2, and
S3). The remaining 2 mutations were in the heterozygous state: 1193G>T
(in proband F5) and 1215delT (in proband R3). In patients F5 and R3, we
could not detect, in the sequenced regions, the second mutation that is
expected to contribute to the observed afibrinogenemic phenotype.
Southern blot analysis and long PCR amplification of each fibrinogen
gene excluded the presence of gross deletions in the fibrinogen cluster
(data not shown).
Figure 1 shows the position of the
identified mutations in the A
Haplotype analysis In order to check for the existence of a common ancestor, haplotype analysis for markers located in the A-chain gene was performed in probands showing the occurrence of the same mutation (Arg110stop in patients P4 and B1; Arg149stop in patients C9, S2, and
S3). All analyzed probands were homozygous for each marker (Table
3). Probands C9, S2, and S3 from
Sardinia, a relatively isolated Italian island, shared the same
haplotype. In contrast, patients P4 and B1, originating from Palermo
and Barbados, respectively, showed different haplotypes.
mRNA analysis by fluorescent hot-stop PCR In order to evaluate mutant mRNA stability, a 5.4-kb fragment of fibrinogen A-chain gene, spanning from exon 1 to intron 5, was
inserted into the pTARGET expression vector giving rise to
the pT-A-wt plasmid containing the normal A-chain. This construct was used to produce, by site-directed mutagenesis, 5 recombinant vectors each containing one of the identified mutations. Equal amounts
of plasmids expressing wild type and each mutant fibrinogen A mRNA
were transiently cotransfected in COS-1 cells (not expressing fibrinogen). Expression levels of mutant and wild-type transcripts were
compared in each experiment, using a semiquantitative
reverse-transcriptase (RT)-PCR analysis. RT-PCR assays were performed
with primers chosen in adjacent exons, in order to avoid the
amplification of contaminating genomic DNA. Moreover, to distinguish
between mRNAs transcribed from mutant and wild-type alleles, a single
mismatch in one primer of each couple was introduced, thereby creating
an allele-specific restriction endonuclease site (Table 1). The only
exception was represented by the 1215delT mutation, whose presence
creates a restriction site for BstXI enzyme. The
heteroduplex formation that under these conditions usually skews the
results of restriction digestion of PCR products was circumvented by
performing the RT-PCR assays by means of the hot-stop PCR
technique.34 This recently described assay involves the
addition, at the final PCR cycle, of a radiolabeled PCR primer that
excludes heteroduplexes from quantitation, since heteroduplexes do not
contain the labeled primer. We used a slight modification of this
technique, consisting of the use of a fluorochrome-labeled primer
instead of a radiolabeled one and enabling allele quantitation by means
of an automated DNA sequencer (fluorescent hot-stop PCR). This
modification was first tested by 7 independent PCR assays on varying
ratios of pT-A-wt and pT-A-Arg149stop plasmids (the latter
containing the 5.4-kb fragment of A-chain gene carrying the
Arg149stop mutation). Quantitation of 6-Fam-labeled
MaeIII-digested PCR products (see "Materials and
methods") showed that the actual allele ratios were equal to the
expected ones (Figure 2), demonstrating
the accuracy and linearity of the fluorescent hot-stop PCR. This method was therefore used to quantitate the relative amount of wild-type and
mutant transcripts in RNA from cells transfected with equal amounts of
wild-type and mutant plasmids. A mutant-wild-type ratio of
approximately 1 was estimated for each mutation (Figure
3). As control, a similar experiment was
performed on an aliquot of the plasmid DNA mixtures used to carry out
the cotransfection experiments and resulted in a mutant-wild-type
ratio of approximately 1, as expected (data not shown).
Protein studies To demonstrate the causal role of the identified mutations in affecting fibrinogen secretion, each expression vector pT-A-mut was
independently cotransfected with equimolar amounts of pRSV-Neo-B and
pRSV-Neo- plasmids (expressing the wild-type fibrinogen B- and
-chains) in COS-1 cells. As positive controls, COS-1 cells transfected with plasmids expressing the 3 wild-type chains and untransfected fibrinogen-expressing HepG2 cells were used. The presence
of fibrinogen in conditioned media was checked by SDS-PAGE under
nonreducing conditions, followed by Western blot analysis with the use
of polyclonal antihuman fibrinogen antibodies. Secreted fibrinogen was
detected only in HepG2-conditioned medium and in the supernatant of
COS-1 cells expressing the 3 wild-type chains (Figure 3, lanes 1 and
8). In contrast, in culture media of cells expressing each mutant
fibrinogen and in conditioned medium of mock transfected (pUC18) COS-1
cells used as negative control, the secreted molecule was undetectable
(Figure 4, lanes 2-7).
In this study, we analyzed 8 afibrinogenemic patients, whose
bleeding manifestations ranged from the complete absence of symptoms to
severe intracranial hemorrhages. Sequence analysis performed on the 3 fibrinogen genes disclosed 4 nonsense mutations and 1 single-base
deletion, all located in the A The Gly13stop and FS51stop mutations were found, in the heterozygous state, in patients F5 and R3, respectively. In both cases, one parent (the mother of F5 and the father of R3), with half normal functional and immunoreactive plasma fibrinogen levels, was heterozygous for the same mutation present in the corresponding affected offspring. These data demonstrate that Gly13stop and FS51stop mutations are not sufficient, in the heterozygous state, to cause congenital afibrinogenemia. Both the remaining parents (the father of F5 and the mother of R3) were found to be homozygous normal at the corresponding nucleotide position. As they were phenotypically heterozygous, the existence of a second, yet undiscovered, mutation is postulated. The identity of these mutations could not be determined after mutational screening of the 3 fibrinogen genes. Possibly they are located in the intronic regions of the fibrinogen cluster not covered by sequencing. Since premature termination codons are frequently known to affect the
metabolism of the corresponding mRNAs, stability of mutant mRNAs was
evaluated by coexpressing each mutant mRNA in COS-1 cells, together
with the wild-type transcript, and by performing a hot-stop PCR for
linear quantitation of allele ratio. This assay was modified by using a
fluorochrome-labeled primer (fluorescent hot-stop PCR) and an automated
DNA sequencer to separate and quantitate alleles, a simpler method than
that originally proposed by Uejima et al.34 Allele
quantitations demonstrated that none of the identified mutations
induced a selective degradation of the corresponding mRNA. These
results increase the number of cases in which the nonsense-mediated
mRNA decay surveillance system is bypassed.37-40 The
mechanisms by which transcripts carrying premature termination codons
escape nonsense-mediated mRNA decay have not been completely elucidated. It has been demonstrated that premature termination codons
located within a distance of 50 to 55 nucleotides from the 3'-most
exon-intron junction do not trigger mRNA degradation by
nonsense-mediated mRNA decay.1-5 This condition is
fulfilled only by the Arg149stop mutation, which is due to a C>T
transition at position 3192, which is 9 nucleotides upstream of the
donor splice site of intron 4 (Figure 1). This exon-intron junction represents the 3'-most one for the large majority of A Owing to the lack of mutant mRNA degradation, the effect of severely
truncated A In summary, besides confirming that the majority of cases of congenital
afibrinogenemia are due to A
Neerman-Arbez et al26 report the identification of a frameshift mutation (g1185delT) which is identical to 1215delT.
We thank Dr C. M. Redman for kindly providing B
Submitted April 5, 2001; accepted August 1, 2001.
Supported by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST 60%), by Progetto Giovani (1999 and 2000), and by IRCCS Maggiore Hospital, Milan, Italy.
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.
Reprints: Maria Luisa Tenchini, Department of Biology and Genetics for Medical Sciences, via Viotti, 3/5-20133 Milan, Italy; e-mail: marialuisa.tenchini{at}unimi.it.
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-chain gene are not associated
with the decay of the mutant mRNAs
Top
Abstract
Introduction
T.
Blood.
2001;97:1879-1881