TY - JOUR
AU - George, Peter, M
AB - We have now identified mutations in 17 families with dysfibrinogenemias. Over half of these families have the Aα16Arg→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 Aα16Arg→His 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 Aα16Arg→His 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 Fn1111α (ATT GCT GTT GCT CTC TTT TG) and Fn1309α (AAT CTC CTG CTT CCC CCG CT) were used to amplify a 199-bp region spanning exon 2 of the Aα 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 Aα16Arg→His 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 Aα16Arg →His (CGT→CAT) 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. The mutation Aα16Arg→His affects the thrombin cleavage site at the N-terminal of the Aα 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 γ chain, thereby initiating polymerization (7). The net effect of replacing the arginine at position 16 of the Aα 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 Aα16Arg → 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 Aα16Arg→His 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. Molec. Pathol. Lab., Christchurch Hosp., Canterbury Health Ltd., Christchurch, New Zealand Figure 1. Open in new tabDownload slide Aα chain exon 2. Upper panel, restriction digest of amplified Aα chain exon 2 from affected and unaffected family members run on a 2% agarose gel for 30–40 min at 100 V. Lane 1, φX174/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 Aα chain exon 2. The mutation produces an NlaIII recognition site (underlined), and the arrow denotes where the enzyme cleaves. Numbers designate the amino acid position within the Aα chain. Figure 1. Open in new tabDownload slide Aα chain exon 2. Upper panel, restriction digest of amplified Aα chain exon 2 from affected and unaffected family members run on a 2% agarose gel for 30–40 min at 100 V. Lane 1, φX174/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 Aα chain exon 2. The mutation produces an NlaIII recognition site (underlined), and the arrow denotes where the enzyme cleaves. Numbers designate the amino acid position within the Aα chain. This work was supported by the Health Research Council of New Zealand. 1 Bithell TC. Hereditary dysfibrinogenemia. Clin Chem 1985 ; 31 : 509 -516. Crossref Search ADS PubMed 2 Henschen AH. Human fibrinogen—structural variants and functional sites. Thromb Haemost 1993 ; 70 : 42 -47. Crossref Search ADS PubMed 3 Matsuda M, Yoshidi N, Terukina S, Kensuke Y, Maekawa H. Molecular abnormalities of fibrinogen—the present status of structure elucidation. Matsuda M Iwanaga S Takada A Henschen A eds. Fibrinogen 4: current basic and clinical aspects 1990 : 139 -151 Elsevier Science Publishers Amsterdam. . 4 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (Aα20Val→Asp). J Clin Invest 1995 ; 96 : 2854 -2858. Crossref Search ADS PubMed 5 Ciulla TA, Sklar RM, Hauser SL. A simple method for DNA purification from peripheral blood. Anal Biochem 1988 ; 174 : 485 -488. Crossref Search ADS PubMed 6 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988 ; 239 : 487 -491. Crossref Search ADS PubMed 7 Gorkun OV, Veklich YI, Medved LV, Henschen AH, Weisel JW. Role of the αC domains of fibrin in clot formation. Biochemistry 1994 ; 33 : 6986 -6997. Crossref Search ADS PubMed 8 Bögli C, Cofrancesco E, Cortellaro M, Della Volpe A, Hofer A, Furlan M, Zanussi C. Fibrinogen Milano VI: a heterozygous dysfibrinogenaemia (Aα16 Arg→His) with bleeding tendency. Eur J Haematol 1990 ; 45 : 26 -30. PubMed 9 Siebenlist KR, Prchal JT, Mosesson MW. Fibrinogen Birmingham: a heterozygous dysfibrinogenemia (Aα16Arg→His) containing heterodimeric molecules. Blood 1988 ; 71 : 613 -618. Crossref Search ADS PubMed 10 Alving BM, Henschen AH. Fibrinogen Giessen I: a congenital homozygously expressed dysfibrinogenemia with Aα16Arg→His substitution. Am J Hematol 1987 ; 25 : 479 -482. Crossref Search ADS PubMed © 1997 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Rapid Detection of the Fibrinogen Aα16Arg→His Mutation
JF - Clinical Chemistry
DO - 10.1093/clinchem/43.11.2184
DA - 1997-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/rapid-detection-of-the-fibrinogen-a-16arg-his-mutation-2qCrL0dvY6
SP - 2184
EP - 2186
VL - 43
IS - 11
DP - DeepDyve
ER -