A Frameshift Mutation in the Human Fibrinogen Aα-chain Gene (Aα [499] Ala Frameshift Stop) Leading to Dysfibrinogen San Giovanni Rotondo

Maurizio Margaglione; Gennaro Vecchione; Rosa Santacroce; Francesca D’Angelo; Bruno Casetta; Maria Luisa Papa; Elvira Grandone; Giovanni Di Minno

doi:10.1055/s-0037-1616752

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2001; 86(06): 1483-1488
DOI: 10.1055/s-0037-1616752

Review Article

Schattauer GmbH

A Frameshift Mutation in the Human Fibrinogen Aα-chain Gene (Aα [499] Ala Frameshift Stop) Leading to Dysfibrinogen San Giovanni Rotondo

Maurizio Margaglione

¹Unità di Aterosclerosi e Trombosi, I.R.C.C.S. ”Casa Sollievo della Sofferenza“, S. Giovanni Rotondo

²Genetica Medica, Università di Foggia

,

Gennaro Vecchione

¹Unità di Aterosclerosi e Trombosi, I.R.C.C.S. ”Casa Sollievo della Sofferenza“, S. Giovanni Rotondo

,

Rosa Santacroce

¹Unità di Aterosclerosi e Trombosi, I.R.C.C.S. ”Casa Sollievo della Sofferenza“, S. Giovanni Rotondo

,

Francesca D’Angelo

¹Unità di Aterosclerosi e Trombosi, I.R.C.C.S. ”Casa Sollievo della Sofferenza“, S. Giovanni Rotondo

,

Bruno Casetta

³Applied Biosystems, Monza

,

Maria Luisa Papa

⁴Centro di Emofilia e Trombosi, Ospedale ”San Giovanni Bosco”, Napoli

,

Elvira Grandone

¹Unità di Aterosclerosi e Trombosi, I.R.C.C.S. ”Casa Sollievo della Sofferenza“, S. Giovanni Rotondo

,

Giovanni Di Minno

⁵Istituto di Medicina Interna e Geriatria, Università di Palermo, Italy

› Institutsangaben

Abstract

Summary

We have investigated a 53-yr-old asymptomatic white man with decreased functional, but not immunologic, fibrinogen plasma levels together with prolonged thrombin and reptilase times, detected through routine coagulation studies prior to a surgical procedure. A new heterozygous single nucleotide deletion (C) at position Ala⁴⁹⁹ within the Aα-chain gene was identified, which predicted changes of the corresponding aminoacids encoded by the subsequent portion of the exon V and the appearance of a premature stop codon at position 518 (A [499]Ala frameshift stop). The new dysfunctional fibrinogen, San Giovanni Rotondo variant, was confirmed in vivo by SDS-PAGE analysis of HPLC-purified fibrinogen chains. Mass spectrum examination of the abnormal HPLC-purified peak gave an estimated mass (56,088 Da) similar to that predicted by DNA analysis of the mutated Aα-chain gene (56,088 Da) and, after tryptic digestion, the truncated Aα-chain was shown only in the propositus, who also carried normal Aα-chain. In addition, mass spectrum analysis of the tryptic digest of the abnormal chain confirmed the presence of a new and unpaired cysteine at the last position that was predicted to form a disulfide bridge with human serum albumin. Immuno-blot analysis confirmed that fibrinogen San Giovanni Rotondo variant, but not normal fibrinogen, contained substantial amounts of albumin. Present findings confirm that truncated Aα-chain lacking part of the terminal domain may be incorporated into mature fibrinogen molecules and normally secreted in the bloodstream.

Keywords

Dysfibrinogenemia - fibrinogen genes - mass spectrometry - truncated protein

Volltext

Referenzen

References
1 Martinez J. Congenital dysfibrinogenemia. Curr Opin Hematol 1997; 4: 357-65.
2 Imperato C, Dettori AG. Ipofibrinogenemia congenita con fibrinoastenia. Helvetica Pediatrica Acta 1958; 13: 380-99.
3 Henschen A, McDonagh J. Fibrinogen, fibrin and factor XIII. In: Zwaal RFA, Hemker HC. eds. Blood Coagulation. Amsterdam: Elsevier; 1986: 171-241.
4 Hoeprich PD, Doolittle RF. Dimeric half-molecules of human fibrinogen are joined through disulphide bonds in an antiparallel orientation. Biochemistry 1983; 22: 2049-55.
5 Doolittle RF, Watt KWK, Cottrell BA, Strong DD, Riley M. The amino acid sequence of the α-chain of human fibrinogen. Nature 1979; 280: 464-8.
6 Chung DW, Harris JE, Davie EW. Nucleotide sequences of the three genes coding for human fibrinogen. In: Liu CY, Chien S. eds. Fibrinogen, Thrombosis, Coagulation and Fibrinolysis. New York: Plenum; 1990: 39-48.
7 Marino MW, Fuller GM, Elder FFB. Chromosomal localisation of human and rat alpha, beta and gamma fibrinogen genes by in situ hybridation. Cytogenet Cell Genet 1986; 42: 36-41.
8 Buetow KH, Shiang R, Nakamura Y, Brechot C, Evans R, Fukuda M, Schull M, Hulsebos T, Wallner B, Murray JC. A multipoint genetic map and new RFLPs for human chromosome 4. Cytogenet Cell Genet 1989; 51: 973.
9 Kant JA, Fornace AJ, Saxe D, McBride OW, Crabtree GR. Evolution and organization of the fibrinogen locus on chromosome 4: Gene duplication accompanied by transposition and inversion. Proc Natl Acad Sci USA 1985; 82: 2344-8.
10 Kant JA, Lord ST, Crabtree GR. A partial mRNA sequence for human α, β and γ fibrinogen chains: Evolutionary and functional implications. Proc Natl Acad Sci USA 1983; 80: 3953-7.
11 Yu S, Sher B, Kudrik B, Redman C. Intracellular assembly of human fibrinogen. J Biol Chem 1984; 259: 10574-81.
12 Ptashne M. How eukaryotic transcriptional activators work. Nature 1988; 335: 683-9.
13 Margaglione M, Santacroce R, Colaizzo D. et al. A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing. Blood 2000; 96: 2501-5.
14 Vecchione G, Casetta B, Santacroce R, Margaglione M. A comprehensive on-line digestion-LC-MS/CID-MS approach for the characterization of human fibrinogen. RCM. 2001 (in press).
15 Cook NS, Ubben D. Fibrinogen as a major risk factor in cardiovascular disease. TIPS 1990; 11: 444-51.
16 Di Minno G, Mancini M. Measuring plasma fibrinogen to predict stroke and myocardial infarction. Arteriosclerosis 1990; 10: 1-7.
17 Koopman J, Haverkate F, Grimbergen J, Egbring R, Lord ST. Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids Aα 461-610 (Lys 461 AAA-> stop TAA). Blood 1992; 80: 1972-9.
18 Furlan M, Steinmann C, Jungo M. et al. A frameshift mutation in exon V of the Aα-chain gene leading to truncated A -chains in the homozygous dysfibrinogen Milano III. J Biol Chem 1994; 269: 33129-34.
19 Ridgway HJ, Brennan SO, Gibbons S, George PM. Fibrinogen Lincoln: a new truncated α chain variant with delayed clotting. Br J Haematol 1996; 93: 177-84.
20 Ridgway HJ, Brennan SO, Faed JM, George PM. Fibrinogen Otago: a major α chain truncation associated with severe hypofibrinogenemia and recurrent miscarriage. Br J Haematol 1997; 98: 632-9.
21 Neerman-Arbez M, de Moerloose P, Bridel C. et al. Mutation in the fibrinogen Aα gene account for the majority of cases of congenital afibrinogenemia. Blood 1990; 96: 149-52.
22 Fellowes AP, Brennan SO, Holme R, Stormorken H, Brosstad FR, George PM. Homozygous truncation of the fibrinogen Aα chain within the coiled coil causes congenital afibrinogenemia. Blood 2000; 96: 773-5.
23 Collen A, Maas A, Kooistra T. et al. Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aα-chain, alters endothelial capillary tube formation. Blood 2001; 97: 973-80.
24 Veklich YI, Gorkun OV, Medved LV, Nieuwenhuizen W, Weisel JW. Carboxyl-terminal portions of thechains of fibrinogen and fibrin: localization by electron microscopy and the effects of isolated αC fragments on polymerization. J Biol Chem 1993; 268: 13577-85.
25 Thiagarajan P, Rippon AJ, Farrell DH. Alternative adhesion sites in human fibrinogen for vascular endothelial cells. Biochemistry 1996; 35: 4169-75.
26 Uemichi T, Liepnieks JJ, Yamada T, Gertz MA, Bang N, Benson MD. A frameshift mutation in the fibrinogen A-alpha chain gene in a kindred with renal amyloidosis. Blood 1996; 87: 4197-203.
27 Asl LH, Liepnieks JJ, Uemichi T, Rebibou J-M, Justrabo E, Droz D, Mousson C, Chalopin J-M, Benson MD, Delpech M, Grateau G. Renal amyloidosis with a frameshift mutation in the fibrinogen A(alpha)-chain gene producing a novel amyloid protein. Blood 1997; 90: 4799-805.