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Technical Briefs |
16Arg
His Mutation
a author for correspondence: fax 64-3-364-0545, e-mail hridgway{at}chmeds.ac.nz
We have now identified mutations in 17 families with
dysfibrinogenemias. Over half of these families have the
A
16Arg
His mutation. This mutation is the most commonly reported
cause of dysfibrinogenemia and, like other dysfibrinogenemias, is
readily detected because of the associated prolonged thrombin and
reptilase times (1)(2)(3). The mutation alters the
thrombin cleavage site such that release of fibrinopeptide A is
delayed. However, fibrinopeptide release assays are difficult and do
not directly confirm the molecular basis of the impaired fibrinopeptide
release. We have therefore designed a rapid and technically simple
PCR-based method for detection of the A16ArgHis mutation. This
allows reliable identification of a common dysfibrinogenemia that, in
its heterozygous form, is usually asymptomatic and does not pose any
substantial threat to the health of the patient. Application of this
method will allow clinical laboratories to determine the molecular
defect in many of the cases that they detect during coagulation
studies.
We examined nine families with the A16ArgHis mutation. These had
been referred for further investigation when routine coagulation
studies were consistent with dysfibrinogenemia. All procedures were
carried out in accordance with the guidelines of our local ethics
committee. Blood samples were collected into Na+
citrate Vacutainer Tubes (Becton Dickinson), and coagulation studies
were performed by routine clinical tests for thrombin and reptilase
times. There was considerable variation both within and between
families in thrombin times [range 36–70 s (reference range 20 ±
2)] and reptilase times [range 45–72 s (reference range 20 ±
2)]. In each case fibrinopeptide release assays (4)
demonstrated reduced fibrinopeptide A concentrations with an additional
earlier eluting peak. Either amino acid analysis of the abnormal
fibrinopeptide or DNA sequence analysis then confirmed the mutation.
Genomic DNA was isolated from whole blood (5). The
oligonucleotides Fn1111 (ATT GCT GTT GCT CTC TTT TG) and Fn1309
(AAT CTC CTG CTT CCC CCG CT) were used to amplify a 199-bp region
spanning exon 2 of the A gene by PCR (6). Each 100-µL
amplification reaction contained 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH
8.3, 1.5 mmol/L MgCl2, 200 µmol/L of each dNTP, 1
µmol/L of each primer, 1 µg of DNA template, and 2 units of
Taq DNA polymerase (Boehringer Mannheim). Amplification was
for 30 cycles with denaturation for 30 s at 94 °C, annealing
for 30 s at 60 °C, and extension for 1 min at 72 °C with a
final extension at 72 °C for 7 min. The PCR products were digested
for 4 h at 37 °C with 5 units of NlaIII according to
the manufacturer's instructions (New England Biolabs). Typically 7
µL of PCR product was diluted to 10 µL by the addition of 0.5 µL
of 10 units/µL NlaIII, 1 µL of NEBuffer 4 (New England
Biolabs), 1 µL of 1 mg/mL bovine serum albumin, and 0.5 µL of
sterile distilled water. Digestion was assayed by gel electrophoresis
in 2% agarose, 50 mmol/L Tris base, 45 mmol/L boric acid, 0.5 mmol/L
EDTA for 30–40 min at 100 V. Products were visualized by staining in
20 µg/mL ethidium bromide for 5 min followed by transillumination at
302 nm.
The A16ArgHis mutation changes the sequence CGTG to CATG creating
an NlaIII cleavage site near the middle of the PCR product
(Fig. 1
, lower panel). Cleavage at this site generated 104-bp and 95-bp
products that were not resolved on the agarose gel, but were clearly
separated from the uncut product. DNA from apparently healthy
individuals remained uncut. The upper panel of Fig. 1
shows the
restriction pattern produced from apparently healthy individuals (lanes
2 and 5) and the pattern produced by individuals heterozygous for the
A16Arg His (CGTCAT) mutation (lanes 3, 4, and 6).
Additionally, the assay should be able to detect homozygotes because no
uncut product should remain; however, appropriate controls were not
available.
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The mutation A16ArgHis affects the thrombin cleavage site at the
N-terminal of the A chain. Normal cleavage at this site exposes the
Gly-Pro-Arg (A) site, which interacts with a preformed, complementary
site located in the C-terminal of the 
chain, thereby initiating
polymerization (7). The net effect of replacing the
arginine at position 16 of the A chain is only to delay the
thrombin-catalyzed exposure of the A polymerization site. Therefore, it
is not surprising that this mutation is usually asymptomatic. Despite
this, two reported cases have been associated with mild bleeding
tendencies (8)(9). In these cases, the
bleeding tendency generally can be attributed to additional
abnormalities in other coagulation proteins. In fibrinogen Milano VI,
the patient showed defective platelet aggregation (8),
whereas in fibrinogen Birmingham, abnormalities in von Willebrand
factor were seen (9). The only reported case of this
mutation in its homozygous form, fibrinogen Giessen I, is associated
with more severe symptoms and displays a severe bleeding tendency and
miscarriage (10).
Dysfibrinogenemias with the A16Arg His mutation are usually
detected by prolonged thrombin-clotting times. Once detected, the
mutation can be characterized by reversed-phase monitoring of
fibrinopeptide release (4). In patients with the
A16ArgHis mutation, the A peptide peak is reduced by half, and
there is an additional earlier-eluting peak that represents the
histidine-containing A peptide. Subsequent protein sequencing of the
abnormal peptide is required to confirm this mutation. Although the
method does provide a definitive result, the apparatus and technical
expertise required are well beyond the scope of most clinical
laboratories. With the method described here, the detection of this
mutation, which in our experience accounts for 50% of all cases of
dysfibrinogenemia, is straightforward, requiring only a simple PCR and
restriction digest. The absolute identification of this mutation will
enable the clinician to reassure the patient that their
dysfibrinogenemia is unlikely to cause any bleeding disorder.
Acknowledgments
This work was supported by the Health Research Council of New Zealand.
Footnotes
Molec. Pathol. Lab., Christchurch Hosp., Canterbury Health Ltd., Christchurch, New Zealand
References
20ValAsp). J Clin Invest 1995;96:2854-2858.
C domains of fibrin in clot formation. Biochemistry 1994;33:6986-6997.
[Medline]
[Order article via Infotrieve]
16 ArgHis) with bleeding tendency. Eur J Haematol 1990;45:26-30.
[ISI][Medline]
[Order article via Infotrieve]
16ArgHis) containing heterodimeric molecules. Blood 1988;71:613-618.
16ArgHis substitution. Am J Hematol 1987;25:479-482.
[ISI][Medline]
[Order article via Infotrieve]
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X174/HaeIII
molecular mass marker; lanes 2 and 5, DNA from
apparently healthy individuals; lanes 3, 4, and
6, DNA from heterozygous individuals. Lower
panel, DNA sequence showing the normal and the mutated sequence of
A