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A novel fibrinogen gamma chain mutation (c.1096C>G; p.His340Asp), fibrinogen Ankara, causing hypofibrinogenaemia and hepatic storage

2017; Elsevier BV; Volume: 49; Issue: 5 Linguagem: Inglês

10.1016/j.pathol.2017.03.007

1465-3931

Francesco Callea, Isabella Giovannoni, Sinan Sarı, Aysel Ünlüsoy Aksu, Guldal Esendagly, Buket Dalgıç, Renata Boldrini, Gülen Akyol, Paola Francalanci, Emanuele Bellacchio,

Blood properties and coagulation

Hereditary hypofibrinogenaemia with hepatic storage of fibrinogen was identified as an endoplasmic reticulum storage disease (ERSD) in 1987.1Callea F. De Vos R. Pinackat J. et al.Hereditary hypofibrinogenemia with hepatic storage of fibrinogen: A new endoplasmic reticulum storage disease.in: Lowe G.D.O. Douglas J.T. Forbes C.D. Henschen A. Fibrinogen 2: Biochemistry, Physiology, and Clinical Relevance. Elsevier, Amsterdam1987: 75-78Google Scholar, 2Brennan S.O. Wyatt J. Medicina D. Callea F. George P.M. Fibrinogen brescia: hepatic endoplasmic reticulum storage and hypofibrinogenemia because of a gamma284 Gly-->Arg mutation.Am J Pathol. 2000; 157: 189-196Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Initially the entity was accepted with skepticism by fibrinogen experts not only because of its unusual presentation, i.e., hypofibrinogenaemia in the absence of overt dysfibrinogenaemia or coagulation or thrombosis manifestations, but also because of its unexpected pathogenesis, i.e., plasma deficiency secondary to a defective export and consequential liver storage.3Callea F. Brisigotti M. Fabbretti G. Bonino F. Desmet V.J. Hepatic endoplasmic reticulum storage diseases.Liver. 1992; 12: 357-362Crossref PubMed Scopus (82) Google Scholar The discovery of this disease entity has revealed the aetiology of a disease that otherwise would remain cryptogenic, in analogy to alpha-1-antitrypsin. Indeed all reported fibrinogen storage disease (FSD) cases have been detected by morphological examination of liver tissue specimens obtained for unexplained liver disease. So far FSD has been reported with six fibrinogen mutant variants, fibrinogen Brescia (p.Gly284Arg), Aguadilla (p.Arg375 Trp), Angers (del372-376), Al DuPont (p.Thr314Pro),4Casini A. Sokollik C. Lukowski S.W. et al.Hypofibrinogenemia and liver disease: a new case of Aguadilla fibrinogen and review of the literature.Haemophilia. 2015; 21: 820-827Crossref PubMed Scopus (22) Google Scholar Pisa (p.Asp316Asn) and Beograd (p.Gly366Ser).5Asselta R. Robusto M. Braidotti P. et al.Hepatic fibrinogen storage disease: identification of two novel mutations (p.Asp316Asn, fibrinogen Pisa and p.Gly366Ser, fibrinogen Beograd) impacting on the fibrinogen gamma-module.J Thromb Haemost. 2015; 13: 1459-1467Crossref PubMed Scopus (30) Google Scholar All cases have displayed similar morphological features, i.e., cytoplasmic inclusions in the vast majority (or all) hepatocytes, which reacted positively for fibrinogen and displayed a characteristic fingerprint-like appearance under the electron microscope (EM).2Brennan S.O. Wyatt J. Medicina D. Callea F. George P.M. Fibrinogen brescia: hepatic endoplasmic reticulum storage and hypofibrinogenemia because of a gamma284 Gly-->Arg mutation.Am J Pathol. 2000; 157: 189-196Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Here we report on a novel mutation of Fibrinogen gamma chain (FGG) (p.His340Asp, fibrinogen Ankara) displaying peculiar histopathological, immunohistochemical features and disruptive effects on the D domain of the protein. The proband was a 5.5-year-old Turkish girl, first born from non-consanguineous parents, presenting with persistent hypertransaminasaemia and very low plasma fibrinogen measured by immunological methods. Low plasma fibrinogen levels were present also in her father, whilst mother and sister had normal values. Prothrombin (PT), partial thromboplastin time (PTT), international normalised ratio (INR) and thrombin clotting time were within the normal range (Table 1). Serology for hepatitis (HAV, HBV, HCV, HEV), Epstein–Barr virus (EBV) and cytomegalovirus (CMV) was negative. Full blood count, erythrocyte sedimentation rate (ESR), C reactive protein, ceruloplasmin and alpha-1-antitrypsin (AAT) were normal. On physical examination, the child was well appearing with height and weight for age above 97%, BMI 17.2 kg/m2 for age and gender was 87.5% (SD±1.15). Due to persistent liver function test abnormalities, after 2 years, a needle liver biopsy was obtained. Peripheral blood samples from the patient and other members of the family were collected for molecular genetic analysis with an informed consent.Table 1Patient and family main laboratory resultsResults (reference interval)PatientSisterMotherFatherAge5.5 y10 m24 y31 yAST (<40 IU/L)50–60231521ALT ( G mutation in the proband (Supplementary Fig. 2, Appendix A). The mutation in exon 8 replaced histidine 340 with aspartic acid (p.His340Asp). Familial analysis of the FGG gene revealed that the patient's father also had this novel mutation. No mutations were detected in mother and sister (Supplementary Fig. 2, Appenix A). The Ankara mutation is located in the globular domain of the fibrinogen gamma chain nearby the region of 'end-to-end' interaction that is crucial for the formation of D:D dimers (Fig. 2). By giving rise to a π-π interaction with the proximal His 343, His 340 has a role in defining the conformation of the D:D interface. The replacement of the conserved His 340 with an aspartic acid residue modifies these interactions and has two main implications: changes in the conformation of gamma chains at the level of the D:D interface and in the interactions of the same chains with knob 'A'. These effects are expected to impair D:D dimer formation. The newly discovered fibrinogen mutation, fibrinogen Ankara, fulfils all criteria for ERSD, similar to AAT deficiency.3Callea F. Brisigotti M. Fabbretti G. Bonino F. Desmet V.J. Hepatic endoplasmic reticulum storage diseases.Liver. 1992; 12: 357-362Crossref PubMed Scopus (82) Google Scholar Fibrinogen Ankara indeed displays all retention patterns first identified in AAT deficiency (Pi MZ or ZZ), designated as type Ia, Ib, Ic, II and III (Supplementary Fig. 3, Appendix A).6Callea F. Immunohistochdemical stuy on alpha-1-antitrypsin. KU Leuven, Leuven1983: 1-113Google Scholar, 7Callea F. Fevery J. De Groote J. Desmet V.J. Detection of Pi Z phenotype individuals by alpha-1-antitrypsin (AAT) immunohistochemistry in paraffin-embedded liver tissue specimens.J Hepatol. 1986; 2: 389-401Abstract Full Text PDF PubMed Scopus (27) Google Scholar Like in AATD, type Ic, type II and type III immunoreactive inclusions can be easily missed due to their small size, or focal occurrence or sampling error. Fibrinogen and AAT share an intrinsic tendency to aggregate because the sites for the D:D non-covalent interactions, which stabilise the end-to-end arrangement of adjacent monomers, are exposed in the non-secreted, non-thrombin activated fibrinogen molecule. In ZAAT, polymerisation and fibril formation initiate the insertion of the reactive site loop of one mutant molecule into the five-strand beta sheet of a neighbouring molecule. A similar mechanism has been demonstrated with fibrinogen Brescia in which the perturbation of the β structure may allow intermolecular strand insertion leading to fibrinogen fibril formation and blockage of its secretion.2Brennan S.O. Wyatt J. Medicina D. Callea F. George P.M. Fibrinogen brescia: hepatic endoplasmic reticulum storage and hypofibrinogenemia because of a gamma284 Gly-->Arg mutation.Am J Pathol. 2000; 157: 189-196Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Regarding the potential toxicity of accumulated misfolded proteins, either AAT or fibrinogen, most authors have proven that both disorders can cause chronic liver disease,8Callea F. Fabbretti G. Bonetti M. Brisigotti M. Desmet V.J. Alpha-1-antitrypsin deficiency.in: Schmid R. Bianchi L. Gerok W. Maier K.P. Extrahepatic Manifestations in Liver Diseases. Springer, Amsterdam1993: 315-330Google Scholar whilst others believe that additional environmental or genetic factors are required for the development of liver disease.4Casini A. Sokollik C. Lukowski S.W. et al.Hypofibrinogenemia and liver disease: a new case of Aguadilla fibrinogen and review of the literature.Haemophilia. 2015; 21: 820-827Crossref PubMed Scopus (22) Google Scholar The latter view has been suggested by heterozygous individuals who can be asymptomatic or never display signs of liver disease. However for ERSD the genotype/phenotype correlation should not follow the sequence: mutation > disease, but rather mutation > hepatocytic storage. The storage process per se represents the elementary lesion as well as the histological sign of cell damage.6Callea F. Immunohistochdemical stuy on alpha-1-antitrypsin. KU Leuven, Leuven1983: 1-113Google Scholar Either in AAT or FSD Ankara heterozygous patients the low amount of retained protein can be due to efficient degradation of the misfolded protein, either via endoplasmic reticulum-associated degradation (ERAD) or autophagy.9Kruse K.B. Dear A. Kaltenbrun E.R. et al.Mutant fibrinogen cleared from the endoplasmic reticulum via endoplasmic reticulum-associated protein degradation and autophagy: an explanation for liver disease.Am J Pathol. 2006; 168: 1299-1308Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10Perlmutter D.H. Alpha-1-antitrypsin deficiency: importance of proteasomal and autophagic degradative pathways in disposal of liver disease-associated protein aggregates.Annu Rev Med. 2011; 62: 333-345Crossref PubMed Scopus (91) Google Scholar Alternatively, the low amounts of retained mutant protein could be due to the lack of the 'Recruitment-Secretory Block' (R-SB) phenomenon.11Callea F. Fevery J. Massi G. Lievens C. de Groote J. Desmet V.J. Alpha-1-antitrypsin (AAT) and its stimulation in the liver of PiMZ phenotype individuals. A "recruitment-secretory block" ("R-SB") phenomenon.Liver. 1984; 4: 325-337Crossref PubMed Scopus (26) Google Scholar Thus, the immunohistological patterns of the novel mutation appear to represent the histological hallmark for the diagnosis and a reference for detection of further FSD cases. Like all other mutations, Ankara mutation localises in the D domain of the gamma chain causing conformational changes at the 'end-to-end' sites employed in D:D dimer formation. Although the latter process takes place after fibrinogen secretion and its cleavage by thrombin, the D-region, which is normally exposed in the ER, when incorrectly folded would cause abnormal interactions and aggregation of fibrinogen within this same cellular compartment. The mutation-caused aggregation of fibrinogen in the ER is congruent with the immunohistochemical results observed with Brescia mutation in which retained gamma chain were detected by using monoclonal anti-gamma and anti-D domain antibodies12Medicina D. Fabbretti G. Brennan S.O. George P.M. Kudryk B. Callea F. Genetic and immunological characterization of fibrinogen inclusion bodies in patients with hepatic fibrinogen storage and liver disease.Ann NY Acad Sci. 2001; 936: 522-525Crossref PubMed Scopus (26) Google Scholar as well as with the absence of abnormal gamma monomers in the plasma from the same patient.3Callea F. Brisigotti M. Fabbretti G. Bonino F. Desmet V.J. Hepatic endoplasmic reticulum storage diseases.Liver. 1992; 12: 357-362Crossref PubMed Scopus (82) Google Scholar The normal thrombin clotting time in our Ankara patient would also suggest that no mutant fibrinogen could reach the circulation. In the present case, the analysis of the crystal structure of fibrinogen has provided insights on the molecular mechanism of disturbance of the His340Asp mutation. His 340 is engaged in multiple interactions with surrounding residues determining the fold in a portion of the C-terminal region of the fibrinogen gamma chain presenting intrinsic tendency to non-covalent self-association, as known from its participation in the D:D dimer formation (Fig. 2). These interactions are likely to be severely altered upon the replacement of His 340 with an aspartic acid residue. As the outcome, the consequential conformational changes should dramatically enhance the D domain aggregation capability. This is supported by the observations that fibrinogen accumulates in the ER and that the translocation of the mutated protein along the secretory pathway is prevented. In conclusion, FSD should be suspected when hypofibrinogenaemia is associated with an unexplained chronic liver disease. Liver biopsy represents the mainstay for the diagnosis. Confirmatory molecular analysis is strongly suggested, especially in difficult histological cases like fibrinogen Ankara which do not present the usual massive hepatic storage. Without bearing in mind the peculiar immunostaining patterns described with fibrinogen Ankara, FSD patients would be missed and categorised as having a cryptogenic liver disease. Due to its nature of rare disease we believe that is important that pathologists continue to record case reports. The authors are grateful to Federica Salutari, Riccardo Mariani and Marta Stefanelli for expert technical assistance. The authors state that there are no conflicts of interest to disclose.