Identification of a dysfibrinogenemia of γR275C (Fibrinogen Fukushima)

Clinica Chimica Acta 325 (2002) 151 – 156 www.elsevier.com/locate/clinchim

Identification of a dysfibrinogenemia of gR275C (Fibrinogen Fukushima) Yuji Imafuku a,*, Kyoko Tanaka b, Kiyoaki Takahashi b, Kazuei Ogawa c, Minoru Sanpei d, Hidekazu Yamada d, Akira Sato d, Hiroshi Yoshida a a

Department of Clinical Laboratory Medicine, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1247, Japan b Central Clinical Laboratory, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1247, Japan c First Department of Internal Medicine, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1247, Japan d Department of Obstetrics and Gynecology, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1247, Japan Received 16 April 2002; received in revised form 12 August 2002; accepted 13 August 2002

Abstract Background: Various dysfibrinogenemias have been identified worldwide. This paper describes a case of dysfibrinogenemia recently identified in our laboratory. Patient: A 34-year-old pregnant woman without any clinical complaints was admitted to our hospital for delivery. She had an extremely low fibrinogen concentration as determined by the thrombin time method though immunoassay showed a titer within the reference range. Dysfibrinogenemia was suspected and further analyses were performed including on her family. Thrombin time was measured using human and bovine thrombin with and without calcium ion. Reptilase time was also measured. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analyzed for mutation-rich regions in the fibrinogen gene. Results: The subject, her mother and her two infants showed the same pattern of results while her father showed a regular pattern. Thrombin time calculated using both human and bovine thrombin and reptilase time was elongated in the propositus. The extent of the elongation was decreased in the presence of calcium ion. DNA sequencing showed heterogeneous fibrinogen gR275C mutations in the propositus, mother and two children. The father showed no mutation. Conclusions: A case of dysfibrinogenemia gR275C without any clinical symptoms was found by routine coagulation testing and was genetically identified. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Dysfibrinogenemia; Thrombin time; Coagulation test; Immunoblotting; DNA sequencing; gR275C

1. Introduction More than 250 cases of dysfibrinogenemia have been documented worldwide [1,2]. About half were

*

Corresponding author. Tel.: +81-24-548-2111x3553; fax: +8124-548-6016. E-mail address: [email protected] (Y. Imafuku).

reported to be asymptomatic and the rest to involve bleeding or thrombosis. Most were found by chance during routine coagulation tests, often because of remarkably low fibrinogen levels as measured by the thrombin time method, which is routinely measured in many clinical laboratories. We obtained data suggesting dysfibrinogenemia from a routine laboratory examination and confirmed the diagnosis with further tests.

0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 2 9 3 - 0

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2. Case report A 34-year-old pregnant woman was admitted to our hospital for delivery. She had not shown any symptoms of coagulation disorder or thrombosis. Routine biochemical and hematological laboratory examinations including coagulation tests revealed no abnormalities in prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time, etc., but on extremely low fibrinogen concentration (53 mg/dl). The immunoassay for fibrinogen showed a level of 469 mg/dl. Dysfibrinogenemia was suspected and further analyses were performed including on her family.

3. Materials and methods 3.1. Sample collection The purpose of this investigation was explained fully to all subjects and informed consent was obtained. Peripheral blood was taken from the propositus, her father, mother, husband and two infants, and four healthy subjects. Nine samples of blood were collected into plastic tubes containing 3.2% trisodium citrate. Plasma was separated by centrifugation at 1700
g for 15 min at 4 jC.

tion plasma, Dade Behring), APTT (Platelin LS, Organon Teknika, Durham, NC and Behring Coagulation System, Dade Behring, mixing 50 Al of sample plasma, 50 Al of silicate and phospholipid reagent and 50 Al of 25 mmol/l CaCl2, monitoring at 405 nm), bleeding time (Duke’s method) and platelet agglutination (Hematracer 212, MC medical, Tokyo, Japan) were also measured. Thrombin time was measured using human and bovine thrombin. Briefly, 0.1 ml of plasma was preincubated at 37 jC for 1 min and coagulation time was measured by adding either 0.2 ml of human athrombin solution (American Diagnostica, Greenwich, CT) adjusted to 50 IU/ml in imidazole buffer, pH 7.3, with or without 25 mmol/l CaCl2 or bovine thrombin solution (Wako, Tokyo, Japan) adjusted to 20 U/ml in imidazole buffer without CaCl2 and compared with that of the healthy controls. Coagulation time was measured with an analyzer (Thrombotrack, Sanko, Tokyo, Japan) by detecting the movement of a metal ball dropped in plasma. Reptilase time was measured using reptilase reagent (Pentapharm, Basel, Switzerland) according to the manufacturer’s instructions. Reptilase time with calcium ion was measured by a modified method using reptilase reagent reconstructed with 25 mmol/l CaCl2 instead of distilled water. Single measurements were performed for the quantitative analyses mentioned above.

3.2. Coagulation tests 3.3. Immunoblot analysis Plasma fibrinogen was measured by the thrombin time method (Multifibren-U and Behring Coagulation System, Dade Behring, Tokyo, Japan) and nephelometry (Behring Nephelometer II, Dade Behring). Briefly, thrombin time method was performed by mixing of 50 Al of sample plasma and 100 Al of bovine thrombin reagent (50 IU/ml) and monitoring of absorbance at 405 nm. Fibrinogen standard (Dade Behring) was used as standard. Nephelometry was performed by the instrument using N-antiserum to human fibrinogen (Dade Behring) reagent and Nprotein standard PY (Dade Behring) as standard. Serum fibrinogen antigen level was also determined by nephelometry. PT (Thromborel S and Behring Coagulation System, Dade Behring, mixing 50 Al of sample plasma and 100 Al of 50 g/l tissue factor and Ca reagent, monitoring at 405 nm, standard: Calibra-

Immunoblotting was performed to analyze the abnormality in the Aa, Bh and g chains of fibrinogen according to the procedure described by Terasawa et al. [3]. Briefly, plasma diluted 1:30 with Laemmli sample buffer (Bio-Rad, Tokyo, Japan) containing 5% 2-mercaptoethanol (Wako) was heated at 100 jC for 5 min and applied to a 12.5% homogenous polyacrylamide gel (Bio-Rad) using Tris –glycine SDS buffer (pH 8.3, Bio-Rad). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at 40 mA for 60 min. After electric transfer to a nitrocellulose membrane at 60 V for 180 min, blocking was done by adding 10% skimmed milk (Dainippon Pharm, Osaka, Japan). Reduced fibrinogen chains on the membrane were detected using horseradish peroxidase (HRP)conjugated rabbit antihuman fibrinogen antibody

Y. Imafuku et al. / Clinica Chimica Acta 325 (2002) 151–156

(Dako, Tokyo, Japan). An HRP conjugate substrate kit (Bio-Rad) with 4-chloro-1-naphtol and hydrogen peroxide was used to develop the blot. 3.4. DNA sequencing analysis To identify the genetic mutation responsible for the dysfibrinogen, genomic DNA was extracted from the peripheral blood of the propositus and her family. Previously reported dysfibrinogen-related mutationrich regions [1,2], fibrinogen gene Aa exon 2, Bh exon 2 and g exon 8, were amplified by PCR. Oligonucleotide primers were designed as follows: forward primer for Aa exon 2, 5V-GCTCTCCTTAATCTCTGTGA-3V; reverse primer for Aa exon 2, 5V-GAGGTAAATAAACAGTGCTC-3V; forward primer for Bh exon 2, 5V-TCACTATCACCAACCAGCCA-3V; reverse primer for Bh exon 2, 5V-GATGGTAATGTGGGTCAGTG-3V; forward primer for g exon 8, 5VTCTATTGCCTCTTGCCAG-3Vand reverse primer for g exon 8, 5V-CTTACCAGTGCTTGCCTTCTCT-3V. PCR was performed in 100 Al of 10 mmol/l Tris – HCl, pH 9.0, containing 2.5 mmol/l MgCl2, 0.5 Amol/l each of the forward and reverse primers, 200 Amol/l each of dATP, dTTP, dCTP and dGTP (TaKaRa, Tokyo,

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Japan) and 2.5 U of Taq DNA polymerase (Promega, Tokyo, Japan). Thirty cycles of amplification were performed on a DNA thermalcycler (PE-2000, Applied Biosystems, Tokyo, Japan) under the following conditions: denaturation at 93 jC for 1 min, annealing at 55 jC for 1 min and extension at 72 jC for 2 min for 30 cycles. Amplified PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Target PCR products containing each exon (stained band at 418 bp for Aa exon 2, 411 bp for Bh exon 2 and 881 bp for g exon 8) were extracted from agarose gel with a commercial extraction kit using NaI and glass beads (Easytrap, TaKaRa). The extracted DNA was then sequenced directly using a Taq Dye Deoxy Terminator Sequencing Kit (Applied Biosystems) and a DNA sequencer (ABI PRISM 377 Genetic Analyzer, Applied Biosystems). Each primer indicated above was used also as sequencing primer.

4. Results Laboratory data on coagulation for the propositus and her family members are shown in Table 1. A low plasma fibrinogen concentration was obtained in the

Table 1 Coagulation analysis of the family

Plasma fibrinogen (mg/dl) thrombin time method nephelometry Thrombin time (s) bovine thrombin without Ca+ + human thrombin without Ca+ + with Ca+ + Reptilase time (s) without Ca+ + with Ca+ +

Prothrombin time (s, %) APTT (s) BT (min) Platelet agglutination

Propositus

Father

Mother

Child 1 (day 1)

Child 2 (day 1)

Healthy controla

53 469

271 231

< 31 215

< 31 172

< 31 129

150 – 330

15.7

3.6 – 4.7

18.1 9.7

4.6 – 5.6 3.3 – 4.1

42.4 27.9

12.0 – 12.7 11.0 – 11.5

12.2 (98.3%) 29.6 2.5 normal

19.4 (50.6%) 89.8

20.9 (44.5%) 90.0

(60 – 160%) 31 – 45 < 4.0

a Reference values were obtained in our laboratory except for thrombin time and reptilase time which were determined from four healthy subjects.

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propositus by the thrombin time method; however, values higher than the upper reference range (370 mg/ dl in our laboratory) of around 470 mg/dl were obtained by the nephelometric method. The mother and two neonates showed similar patterns, low or undetectable levels by the thrombin time method and levels within the reference range by the immunological (nephelometry) method (Table 1). Serum concentration of fibrinogen antigen was < 15 mg/dl. Thrombin time, using human and bovine thrombin, and reptilase time were elongated in the propositus but not with the healthy controls. The extent of elongation was decreased in the presence of calcium ion. Immunoblotting showed three bands indicating Aa, Bh and g chains but no difference in migration was observed compared with the healthy controls. DNA sequencing revealed a heterogeneous mutation in the gene corresponding to the fibrinogen g chain, a change from g275 Arg to Cys (CGC to TGC), in the propositus, her mother and her two infants (Fig. 1).

Fig. 1. DNA sequence exon 8 of the fibrinogen g gene in the propositus and her father. A heterogeneous point mutation of CGC to TGC was found in the propositus corresponding to a change in the g chain from Arg 275 to Cys (gR275C). The same pattern was obtained in the mother and two infants. Her father showed a normal pattern.

5. Discussion Of the various cases of dysfibrinogenemia reported previously, 12 were of type gR275C (Table 2) [2,4 – 14]. In 8 out of these 12 cases, there were no clinical symptoms. The four other patients had venous thrombosis including Factor V Leiden in one case. This gR275C mutation by itself seems to cause no clinical symptoms as also shown in our case. Our case showed normal PT and APTT, but five out of six cases elongated PT and two out of six showed elongated APTT. Fibrinogen Tochigi I was normal in both PT and APTT as in our patient. Normal PT and APTT are thought to be due to the assay conditions. Namely, higher fibrinogen and Ca concentration and lower ionic strength increase fibrin polymerization [15]. Fibrinogen concentration in assay condition is the same in thrombin time, PT and APTT (all three-fold dilution of plasma). In PT and APTT, added Ca can enhance fibrin polymerization. On the other hand, thrombin time method is free from Ca, so it may explain the discrepancy in PT, APTT and thrombin time. Though ionic strength is larger in PT and APTT than TT because of the addition of CaCl2 in PT and APTT, the presence of Ca seems to accelerate fibrin polymerization. It seems to be so as a result of balance between accelerating action of Ca and inhibitory effects of increased ionic strength as shown by the shortened thrombin time or reptilase time with supplemented Ca compared with those without Ca. Another possibility is that the difference in the amount of thrombin produced in plasma in the reaction of PT and APTT was relatively small compared with the excess of thrombin added for the thrombin time method. About half of the plasma fibrinogen was thought to be normal in heterogeneous gR275C, so a relatively small amount of thrombin may cause the reaction of PT and APTT similar to normal plasma. As only a relatively small volume of plasma was available and purified fibrinogen was not obtained from this patient, we performed immunoblotting to analyze abnormalities in the Aa, Bh and g chains. Previous reports on purified dysfibrinogenemia gR275C showed no abnormality in the pattern of SDS-PAGE [4,5,8], isoelectric focusing [5] and twodimensional electrophoresis [9] though Fibrinogen

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Table 2 Dysfibrinogenemia gR275C reported previously Patient Opportunity of found

Symptom

(1) Tokyo II

39 F

no

27

195

(2) Baltimore IV

56 M

no

32

192

(3) Morioka (4) Tochigi I (5) Osaka II

child 51 M 48 M

no no no

44 < 40

142 normal

(6) Milano V

51 F

no

113

364

(7) Vilajoyosa

27 F

no

76

330

(8) Bologna (9) Matsumoto III

20 F 66 F

DVTb no

39

291

(10) Bellingham

40 M

DVT

38

356

(11) Cedar Rapids

26 F

55

304

(12) Hannover IV (13) Fukushima

34 F

DVT (with Factor V Leiden) hemorrhage no

53

469

a b

routine (pre-ope) routine (pre-ope)

routine routine (pre – ope) routine (pre-ope) routine (pre-ope) unknown unknown examination for DVT examination for DVT unknown routine (pre-delivery)

Fibrinogen (mg/dl)

PT

APTT

Reference

15.8 s (11.3a) 22 s (13 – 17)

37.9 s (21.3) PTT 100 s (88)

[4]

normal

normal

Thrombin time Immunological

[5]

[6] [7] [8] [9]

45% 32 s [10] (70 – 120) (30 – 32) [11] 12.5 s 21.1 s [12] (10 – 12) (24 – 37) 24 s 39 s [13] (13 – 16) (35 – 42) [14] [2] 98.3% 29.6 s (60 – 160) (31 – 45)

Reference value. Deep vein thrombosis.

Tochigi showed an extra high-molecular g band [7]. The cause of these differences is unclear. High concentration of fibrinogen antigen in this case is thought to be due to the final stage of gestation because fibrinogen is known to increase normally in this stage. The elongation of thrombin time in a case of fibrinogen gR275C was reported to be due to impaired polymerization [16]. Recent crystallographic analyses have shown the importance of g275R in fibrin polymerization process. Amino acid residue g275R is placed on the domain D of fibrinogen and forms D:D binding interface with other amino acids and plays a central role in forming hydrogen bonds between two fibrinogen fragment D (asymmetrical binding as g275R on molecule A and g300S on molecule B, and in turn g280Y on molecule A and g275R on molecule B) in fibrin polymerization process, leading to an end-to-end polymerization to form double-stranded protofibrils [17,18]. Dysfibrinogen with g275C instead of g275R may not form the

hydrogen bond enough to form tight D:D interaction, then fibrin formation will progress slowly and thrombin time will be elongated. This theory can be well estimated especially when additional free Cys is attached to g275C by disulfide bond, resulting in the interposition of this additional Cys between D:D interface as reported in dysfibrinogenemia Osaka II [8] though the presence of additional Cys was not disclosed in our case and other gR275C cases. As these cases of dysfibrinogenemias without clinical symptoms were found only by chance during routine examination, the careful observation of clinical data is important.

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