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284 Gly
Arg Mutation
From the Molecular Pathology Laboratory,*
Christchurch
Hospital, Christchurch, New Zealand; and the 1st Servizio di Anatomia
Patologica,
Spedali Civili di Brescia,
Brescia, Italy
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Abstract |
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CGG (Arg) at codon 284 of the 
-chain gene.
However, examination of purified fibrinogen chains by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis,
reverse-phase high-performance liquid chromatography,
ion-exchange high-performance liquid chromatography, and
isoelectric focusing, failed to show any evidence of the mutant
Br chain in plasma fibrinogen. This finding was
substantiated by electrospray ionization mass spectrometry,
which showed only a normal (and Bß) chain mass, but a
large increase in the portion of their disialo isoforms. We speculate
that misfolding of the variant protein causes hepatic retention and the
subsequent hypofibrinogenemia, and that the functional defect
(dysfibrinogenemia) results from hypersialylation of otherwise normal
Bß and chains consequent to the liver cirrhosis. These
conclusions were supported by studies on six other family members with
hypofibrinogenemia, and essentially normal clotting
times, who were heterozygous for the 284 GlyArg
mutation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Bß, and ) with nominal
molecular weights of 66, 55, and 48 kd, respectively.1,2
The symmetrical molecule consists of a central E domain joined to two
peripheral D domains by triple-helix spacers. Single biantennary
oligosaccharide side chains, of sequence,
-NAcGlc2-Man-(Man-NAcGlc-Gal-
NAcNeu)2, are attached to the Bß and chains
at Asn 364 and 52, respectively.3
After activation by thrombin, the fibrin monomer spontaneously
polymerizes through D:E interactions to form a half-staggered
bimolecular array that is stabilized by noncovalent end-to-end D:D
interactions between adjacent monomer units.1,2
Covalent
cross-links are subsequently inserted to buttress this interaction and
form an insoluble clot. The C-terminal of the chain is intimately
involved in each of these interactions; however, the finely balanced
process of clot formation can be disrupted by both genetic and acquired
perturbations of molecular structure. The acquired dysfibrinogenemias
result from alterations in the pattern of posttranslational
modification and are associated with chronic liver
disease.4
Their impact on function or thrombin clotting
time (TCT) is through elevation of the level of sialylation of the
biantennary oligosaccharide.5
Specific genetic mutations in fibrinogen have long been associated with bleeding and thrombosis and more recently with extracellular amyloid deposits that can result in fatal renal disease.6,7 Putative undefined mutations have also been suspected of causing hepatic endoplasmic reticulum (ER) storage disease and consequent hypofibrinogenemia. In 1987 we described a family from Brescia, Italy, who suffered from hereditary hypofibrinogenemia with intrahepatic fibrinogen storage.8 A similar clinical and pathological picture had been reported earlier in two German families.9,10 Subsequent investigations of other families with cryptogenic chronic hepatitis/cirrhosis established that fibrinogen can be stored in the liver as four different distinguishable morphological types.11
Z-antitrypsin deficiency is perhaps the most well-recognized and
characterized ER storage disease, and blockage of secretion is because
of a single 342 GluLys mutation in the 55-kd
inhibitor.12
Accumulation and subsequent liver disease
results from polymerization of the Z protein within the ER. This
polymerization and fibril formation is initiated by insertion of the
mobile reactive site loop of one molecule into a five-stranded (A) ß
sheet of a neighbor.13
By analogy it seemed that the different morphological fibrinogen
inclusions might result from a series of mutations in one or another of
the three fibrinogen genes. Here we investigate the molecular basis of
the type I inclusions in fibrinogen Brescia and identify a novel 284
GlyArg mutation in the five-strand ß-sheet structure of the -D
domain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liver specimens were fixed in Holland’s fluid and embedded
in paraffin. Serial 4-µm sections were stained with hematoxylin and
eosin (H&E), periodic acid-Schiff (PAS) with and without previous
amylase digestion, Masson’s trichrome, and with a conventional
immunoperoxidase technique using commercial polyclonal antibodies
against fibrinogen, -1-antitrypsin, -1-antichymotrypsin,
albumin, complement C3/C4, haptoglobin, -2-macroglobulin, and IgG
(DAKO, Copenhagen, Denmark). Both standard and immunoelectron
microscopy were carried out on dewaxed material as reported
previously.14
Coagulation Studies
Standard clinical coagulation tests, including thrombin and reptilase clotting times and antigenic fibrinogen levels, were performed on citrated plasma. Functional fibrinogen levels were determined by the Clauss method and gravimetric concentrations by fibrinopeptide quantitation.15
Fibrinogen Purification and Protein Analysis
Fibrinogen was purified by precipitation with 22% saturated ammonium sulfate. The pellet was washed three times with 25% saturated ammonium sulfate and redissolved at 5 mg/ml in distilled water.15
Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% reducing and 4% nonreducing) was essentially as described by Laemmli.16 For isoelectric focusing fibrinogen was first reduced in 0.1 mol/L dithiothreitol, 8 mol/L urea, 5 mmol/L Tris/HCl, pH 8.0, and the chains focused over a pH range of 4 to 8 in 7% acrylamide gels in 8 mol/L urea, 0.5% Triton X-100.17
Ion-exchange high-performance liquid chromatography (HPLC) was performed on a 10-cm CX-300 column (Brownlee, Santa Clara, CA) in 8 mol/L urea, 10 mmol/L phosphate buffer, pH 6.5, and 0.1% mercaptoethanol. Gradient elution was with this same buffer made 0.2 mol/L with respect to NaCl. The flow rate was 0.75 ml/min and the absorbance was monitored at 280 nm. Fifty µl of fibrinogen (5 mg/ml) in 8 mol/L urea, 10 mmol/L phosphate buffer, pH 6.5, and 10% mercaptoethanol was acidified with 0.25 µl H3PO4 immediately before injection.18
For reverse-phase separations, fibrinogen (5 mg/ml) was dissociated by reduction (4 hours at 37°C) in 8 mol/L urea, 0.1 mol/L Tris/HCl, pH 8.0, 15 mmol/L dithiothreitol, and individual chains were resolved by HPLC on a Phenomenex (Torrance, CA) C-4 column (25 x 0.45 cm). The column was monitored at 215 nm and developed with a 0.05% trifluoroacetic acid/acetonitrile gradient system at a flow rate of 0.75 ml/min.19
Electrospray ionization mass spectrometry (ESI MS) was preformed on a
VG Platform II quadrupole analyzer (Micromass, Manchester, UK) as
previously outlined.19,20
Peak crests from reverse-phase
purified chains were collected and 20 µl of each was directly
injected into the ion source at a flow rate of 5 µl/minute. The probe
was charged at + 3500 volts and the source maintained at 60°C. The
mass range 700 to 1500 m/z was scanned every 2 seconds and a cone
voltage ramp of 30 to 60 volts was applied over this range. Up to 120
scans were averaged in acquiring the raw data. Calibration was made
over this same m/z range using the charge series generated by human
globin. Data were acquired and processed using Mass-Lynx software and
transformed on to a true molecular mass scale using maximum entropy
(Max-Ent) software.19,20
DNA Amplification and Sequence Analysis
Genomic DNA was extracted from whole blood21
and the
coding regions and the intron/exon boundaries of all three fibrinogen
chain genes were amplified by polymerase chain reaction22
and sequenced. Exon 8 of the gene was amplified for 30 cycles using
the oligonucleotides g5515 (5' TAT CTA TTG CCT CTT GCC A) and g6126 (5'
ACT TGG TAT TAT CCA CTT CC) with cycling parameters of denaturation at
94°C for 20 seconds, annealing at 56°C for 20 seconds, and
extension at 72°C for 20 seconds. The polymerase chain reaction
products were purified before sequencing using a High Pure kit
(Boehringer Mannheim, Mannheim, Germany). Cycle sequencing of
exon 8 was performed with the internal primer g5607 (5' CCT ACG AAG
AGG GAA CTT C), using 33P-radiolabeled
terminators and Thermosequenase (Amersham, Arlington Heights,
IL) according to the manufacturer’s instructions.
Screening for the G284R mutation used a mutagenic polymerase chain
reaction/BbrPI digestion procedure. The upstream mutagenic
primer 5725BbrPI (5' G TAC CGC CTA ACA TAT GCC TAC TTC
GCA CGT; mismatches underlined) and
5837 (5' CTG CAT GCC ATT ATG GGA TG) were used to amplify a 114-bp
fragment using the above conditions. On digestion with BbrPI
(Roche Diagnostics, Indianapolis, IN) the product of the normal allele
is specifically hydrolyzed to 85- and 29-bp fragments, whereas the
variant product remains uncut.
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Results |
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In 1983 the proposita, at age 64, underwent an abdominal operation consisting of a cholecystectomy for gall-bladder stones and a duodenal polypectomy. A wedge liver biopsy was performed because of the unexpected finding of liver cirrhosis. The postoperative course was uneventful; in particular there were no problems with hemostasis or wound healing. At the time of the operation there was a mild transaminase elevation, a Clauss fibrinogen of 0.2 mg/ml (normal, 1.0 to 3.0), an immunoreactive fibrinogen of 1.0 mg/ml, a prolonged TCT of 47 seconds, and HBsAg was negative. She had esophageal bleeding because of varices in 1977. Plasma fibrinogen levels have remained consistently low throughout the last 15 years (0.2 to 0.6 mg/ml) and presently the patient shows signs of decompensated cirrhosis (aspartate aminotransferase 59; alanine aminotransferase 65); her fibrinogen is currently 0.5 mg/ml, TCT 37 seconds, and she is hepatitis C virus antibody-negative.
Morphological Studies
Histological examination of the liver biopsy revealed features of
early cirrhosis with thin connective tissue septa linking portal tracts
and surrounding parenchymal nodules. Portal tracts contained
inflammatory cells with evidence of piecemeal necrosis. The vast
majority of hepatocytes contained cytoplasmic inclusions (arrow in
Figure 1
) appearing either as round or
polygonal globules with an irregular outline, often surrounded by a
clear halo, at times centered by small lipid droplets, or as acicular
structures (asterisk). The latter filled the entire cytoplasm and
imparted a fiberglass appearance to the cell. The inclusions were
weakly stained on H&E preparations (Figure 1b)
and faintly positive on
PAS staining (Figure 1c)
. Under electron microscopy the globular
inclusions corresponded to dilated rough endoplasmic reticulum (RER)
cisternae filled with densely packed tubular structures arranged in
curved bundles resulting in a fingerprint-like appearance (Figure 1, f and g)
. The acicular fiberglass inclusions corresponded to
longitudinally cut cisternae of the RER containing tubular structures
arranged in a parallel fashion. The electron microscopy observations
confirmed the presence of lipid droplets within the large
intracisternal inclusions. Neither the smooth endoplasmic reticulum,
the Golgi, or the secretory vesicles, showed signs of dilation or
endoluminal precipitates and the remaining organelles (mitochondria,
lysosomes, and peroxisomes) were normal in size and number. The cytosol
contained free ribosomes and a variable number of glycogen particles.
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and
electron microscopy level (not shown). No immunoreaction for fibrinogen
was observed outside the RER and the tubular intracisternal material
was negative on immunostaining for all other proteins tested. Family Studies
Plasma and DNA were obtained from fifteen other family members and
the coagulation results and genotypes are summarized in Figure 2
. All but one direct descendant of the
proposita (II-2) had normal plasma fibrinogen concentrations and normal
TCTs; none of these individuals had inherited the 284 mutation (see
below). The exception (III-6) had both hypofibrinogenemia and liver
cirrhosis, however he died in 1985 and archival material was not
available for genetic analysis. The sister (II-1) and all but one of
her descendants had hypofibrinogenemia together with a very slightly
extended TCT and these were later found to be heterozygous for the
fibrinogen Brescia mutation. To exclude the possibility of
coincidental segregation of the 284 mutation with the
hypofibrinogenemia we also examined 20 randomly selected Italians using
mutagenic primer amplification followed by a BbrPI
digestion; none of the 40 alleles examined had the Brescia mutation.
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Examination of purified plasma fibrinogen showed the expected 340-
and 305-kd protein bands on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and after reduction, a normal pattern of A, Bß,
and chains was observed. Reverse-phase HPLC separations also
showed a normal elution profile of A, Bß, and chains.
Further examination of the HPLC-purified chains from the proposita by
ESI MS indicated a single normal A peak of mass 66,228 d (control,
66,230 d) and as expected the Bß and chains were heterogeneous,
each displaying both mono- and disialo-isoforms. Again the masses of
these chains did not differ significantly from the controls; 54,510 d
for the disialo Bß chain versus a control of 54,504 d, and
48,404 d for the monosialo chain versus two independent
control values of 48,404 d and 48,408 d (Figure 3)
. What was surprising however was the
preponderance of the disialo isoforms of the Brescia Bß (not shown)
and chains. The Brescia Bß chains were 77% fully sialylated (not
shown) and the chains 46% (Figure 3)
compared to normal control
values of 45% and 24%, respectively.
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CGG
(Arg) mutation at codon 284 of the chain gene (Figure 4)
.
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chain. Although the analysis would not have been expected to resolve
the two forms, in a 50/50 heterozygous mixture of mutant
Br and normal A
chains the average chain mass would have been expected to increase by
99/2 d. Indeed we have previously measured the predicted 60/2 d change
associated with heterozygosity for a 280 TyrCys mutation at
precisely 31 d.23
The implication from this is that
the mutant Brescia chain is not expressed to a significant extent
in plasma fibrinogen.
This possibility was confirmed by a more detailed analysis of plasma
fibrinogen chains by ion exchange chromatography and isoelectric
focusing in the presence of 8 mol/L urea. Because the GlyArg
mutation introduces a charge increase of +1, these procedures would
also be expected to detect Br chains if
present. Positive controls were included in these experiments and
heterozygotes for a Bß 14 ArgCys mutation showed two clear 50/50
peaks on CX-300 chromatography and a 279 AlaAsp heterozygote was
clearly identified by a -1 anodal shift of the components on
isoelectric focusing (Figure 5)
. However,
in the Brescia case only a single normal peak was observed on
CX-300 chromatography (not shown) and a normal distribution of
isoforms on isoelectric focusing. Specifically there was no new
cathodal band that would be expected if the Br
chain, with its new Arg, was actually present in circulating fibrinogen
molecules (Figure 5)
.
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CGG
mutation using mutagenic primer amplification followed by a
BbrPI digestion and six carriers were identified. Two
additional affected family members (II-1 and III-1) and two normals
(IV-2 and III-3) were further analyzed by ESI MS. The average monosialo
chain mass of the two affected individuals was 48,389 d and that of
the normals was 48, 391 d and the amount of disialo isoform
averaged 29% and 23% for the affected and normals, respectively. The
level of Bß chain sialylation averaged 46% and 35%, respectively.
These results suggest that there was no significant plasma expression
of the Br chain in these heterozygotes and no
significant increase in sialylation of the A
chain. This was substantiated by isoelectric focusing of purified
fibrinogen from the heterozygotes III-1 and III-2. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain mutation.
The 284 GlyArg substitution was inexorably linked to the
hypofibrinogenemia because the seven proven carriers had an average
plasma fibrinogen of 0.7 mg/ml and the nine normal family members had
average concentrations of 2.4 mg/ml (Figure 2)
. Although the hepatic
storage seems to predispose to the development of cryptogenic chronic
liver disease, the link between the mutation and storage is not as
strong as that with hypofibrinogenemia. There was however, no other
discernible cause of the chronic liver disease in the proposita, and it
seemed to segregate with the fibrinogen mutation because her only
direct descendant with hypofibrinogenemia (III-6) died from
decompensated cirrhosis. Whereas none of the other surviving carriers
of the 284 mutation has yet developed any symptoms, this is similar
to Z-antitrypsin deficiency where only a minority of individuals
develop significant liver injury.12,24
In individuals with the MZ-antitrypsin phenotype the Z allele
contributes 15% of the plasma antitrypsin, however no
Br chains were present in circulatory
fibrinogen from Brescia heterozygotes. Because of the dimeric nature of
fibrinogen, random incorporation of the Br
chain into nascent dimers in the ER would mean that 75% of molecules
would contain at least one variant chain and potentially contribute to
inclusions. With this scenario we would expect the mutation to induce a
more severe hypofibrinogenemia than would result from the simple
nontranslation of a gene; this was indeed the case because carriers had
one-third the fibrinogen concentration of their nonaffected relatives.
The pathology associated with the 284 GlyArg mutation seems to be
of liver cirrhosis rather than of bleeding or thrombosis. For the
latter, aberrant molecules would first have to be present in plasma and
the evidence from ion-exchange HPLC, isoelectric focusing, and ESI MS
is that they are not. How then do we explain the functional defect
implied by the prolonged TCT (37 s) if the proposita has no significant
plasma expression of the variant? The polymerization defect can best be
explained by the hypersialylation of the Bß and chains because
Martinez et al4
have shown that the acquired
dysfibrinogenemia associated with chronic liver disease is because of
excess sialic acid on these chains. They showed that whereas normal
fibrinogen contained six residues of sialic acid, their five subjects
with liver disease had between 1.4 and 3.4 residues more per molecule
and this extended their thrombin times by between 12 and 22 seconds.
Here ESI MS shows Bß and chains of normal mass but with an
increased proportion of disialo isoforms. Indeed if we assume a maximum
of eight sialic acids per (A2
Bß2 )2 molecule, the
data shows 5.4 residues/mole for normal controls and 6.5 for the
patient (Table 1)
. This might be expected
to increase the TCT by approximately 12 seconds, which is reasonably
consistent with the observed increase of 15 seconds.
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284 is absolutely conserved, not only in all known
-chain sequences, but also in all Bß and extended E
chains.25
This conservation between homologous chains
suggests a structural rather than functional importance for the site,
because the C-terminals of the Bß and E chains do not have the
critical role in polymerization that the C-terminal of the chain
does, but all three chains have similar three-dimensional
structures.26
Although located in the functionally important D domain, X-ray
structures25
indicate that residue 284 is situated away
from regions directly involved in D:E or D:D polymerization sites and
therefore the mutation would not be expected to have a direct effect on
function. The Gly residue itself lies at the end of a ß
strand27
that starts at 280 Tyr and extends away from
the D:D interface to Ala 286. Tyr 280 is however intimately involved in
D:D polymerization and forms the closest contact across this
surface.23,25
The strand containing Gly 284 is the most
distal of five that make up an extended ß-sheet structure (Figure 6)
.25,27
It seems that this
strand might be quite mobile because in the double D structure of
Spraggon et al26
residues 280 to 284 appear as a random
coil. This sheet structure is reminiscent of the A sheet in antitrypsin
that undergoes a transition from a metastable five-stranded structure
to stable six-stranded structure on cleavage within its reactive center
loop.28
The Z mutation at position 342 is at the hinge
point of this loop-sheet insertion and the GluLys substitution
facilitates intermolecular loop to sheet insertion leading to
antitrypsin Z-fibril formation and the blockage of hepatic
secretion.13
The two other rare antitrypsin mutations that
result in ER storage, 52 Phe deleted and 53 SerPhe, also seem to
perturb the movement of strands in the A sheet by destabilizing its
supporting structure.29
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284 GlyArg mutation may perturb the
ß-sheet array and allow intramolecular strand insertions leading to
fibrinogen fibril formation and the blockage of its secretion.
Certainly the electron microscopy images of fibrinogen Brescia
inclusions show a degree of order similar to Z-antitrypsin fibrils. This is the first fibrinogen mutation identified that results in hypofibrinogenemia and it seems that defective secretion may account for the hepatic storage and plasma deficiency. Although this new form of ER storage disease maybe an uncommon cause of cirrhosis, fibrinogen inclusions have been detected in eight out of 700 biopsies on patients with hepatitis.11 This suggests that it is an unrecognized entity. Fibrinogen storage should be excluded in appropriate cases and, when immunoperoxidase stains are positive, genetic analysis undertaken. The recognition of four distinct morphological forms also suggests that several different mutations may be involved.
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Acknowledgements |
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Footnotes |
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Supported by the Canterbury Medical Research Society.
Accepted for publication April 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain gene in a kindred with renal amyloidosis. Blood 1996, 87:4197-4203 1 antitrypsin deficiency. Hepatology 1996, 24:1504-1516[Medline]
20 ValAsp). J Clin Invest 1995, 96:2854-2858
330AspVal impairs fibrin polymerization. Blood 1986, 67:1751-1756 chains. Thromb Haemost 1998, 80:263-265[Medline]
280 TyrCys): a new variant with defective polymerization. Br J Haematol 1998, 101:24-31[Medline]
EC domain from human fibrinogen-420. Proc Natl Acad Sci USA 1998, 95:9099-9104 chain of human fibrinogen. Structure 1997, 5:125-138[Medline]
1 antitrypsin Siiyama further evidence for intracellular loop sheet polymerization. J Biol Chem 1993, 268:15333-15335This article has been cited by other articles:
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