Fibrinogen Gamma Chain Mutations Provoke Fibrinogen and Apolipoprotein B Plasma Deficiency and Liver Storage

Abstract

p.R375W (Fibrinogen Aguadilla) is one out of seven identified mutations (Brescia, Aguadilla, Angers, Al du Pont, Pisa, Beograd, and Ankara) causing hepatic storage of the mutant fibrinogen γ. The Aguadilla mutation has been reported in children from the Caribbean, Europe, Japan, Saudi Arabia, Turkey, and China. All reported children presented with a variable degree of histologically proven chronic liver disease and low plasma fibrinogen levels. In addition, one Japanese and one Turkish child had concomitant hypo-APOB-lipoproteinemia of unknown origin. We report here on an additional child from Turkey with hypofibrinogenemia due to the Aguadilla mutation, massive hepatic storage of the mutant protein, and severe hypo-APOB-lipoproteinemia. The liver biopsy of the patient was studied by light microscopy, electron microscopy (EM), and immunohistochemistry. The investigation included the DNA sequencing of the three fibrinogen and APOB–lipoprotein regulatory genes and the analysis of the encoded protein structures. Six additional Fibrinogen Storage Disease (FSD) patients with either the Aguadilla, Ankara, or Brescia mutations were investigated with the same methodology. A molecular analysis revealed the fibrinogen gamma p.R375W mutation (Aguadilla) but no changes in the APOB and MTTP genes. APOB and MTTP genes showed no abnormalities in the other study cases. Light microscopy and EM studies of liver tissue samples from the child led to the demonstration of the simultaneous accumulation of both fibrinogen and APOB in the same inclusions. Interestingly enough, APOB-containing lipid droplets were entrapped within the fibrinogen inclusions in the hepatocytic Endoplasmic Reticulum (ER). Similar histological, immunohistochemical, EM, and molecular genetics findings were found in the other six FSD cases associated with the Aguadilla, as well as with the Ankara and Brescia mutations. The simultaneous retention of fibrinogen and APOB-lipoproteins in FSD can be detected in routinely stained histological sections. The analysis of protein structures unraveled the pathomorphogenesis of this unexpected phenomenon. Fibrinogen gamma chain mutations provoke conformational changes in the region of the globular domain involved in the “end-to-end” interaction, thus impairing the D-dimer formation. Each monomeric fibrinogen gamma chain is left with an abnormal exposure of hydrophobic patches that become available for interactions with APOB and lipids, causing their intracellular retention and impairment of export as a secondary unavoidable phenomenon.

Hepatocellular type II fibrinogen inclusions in a patient with severe COVID-19 and hepatitis

“…6 In rare cases, a few mutations clustered in exons 8-9 of FGG result in the intracellular accumulation of misfolded fibrinogen in hepatocytes, chronic liver disease of various severity, and hypofibrinogenemia. 7 Of note, fibrinogen storage disease without hypofibrinogenemia, which corresponds to the clinical picture presented by our patient, has rarely been associated with acute infections in patients without any hereditary defect of fibrinogen. 8,9 Our patient presented very high plasma fibrinogen levels, making the presence of a known FGG mutation very unlikely, as confirmed by genetic testing.…”

“…5 Thus, insufficient autophagy may contribute to the initiation and progression of WD, in line with the observation that obesity, a state in which autophagy is inhibited due to the excess of nutrients, aggravates WD. 7 Of note, a minimal amount of copper is required for the initiation of the autophagic cascade, as copper binds to and stimulates the kinase activity of the pro-autophagic kinases, Unc-51 like autophagy activating kinases 1 and 2 (ULK1, ULK2). Complete depletion of copper hence blocks autophagy.…”

Structural Characteristics in the γ Chain Variants Associated with Fibrinogen Storage Disease Suggest the Underlying Pathogenic Mechanism

“…Intriguingly, all mutations locate near, but not inside, the interface of homodimerization of two fibrinogen γ molecules. As we showed previously, this interface and other protein regions contain hydrophobic patches that could remain abnormally exposed upon mutation-caused failure in the dimerization of two Aα-Bβ-γ fibrinogen heterotrimers, therefore the mutated γ chain might work as a seed for phenomena of aggregation of hydrophobic peptides and lipid [16]. Previous studies have shown that the mutant γ chains are entirely retained within the hepatocytes [17] and that they are not found in circulation either in Brescia [3], Aguadilla [9], Al du Pont [11], or Thr317Ile (Muncien) [8].…”

“…Based on our structural analysis, we rationalize a common mechanism for Thr371Ile and other variants of fibrinogen γ chain known to give rise to the hepatic storage phenomenon, Brescia Gly284Arg [3,4], Aguadilla Arg375Trp [9], Angers G346_Q350del [10], Al Du Pont Thr314Pro [11], Pisa Asp316Asn and Beograd Gly366Ser [12], and Ankara His340Asp [13]. As we proposed in a previous study, particular mutations cause a failure in the fibrinogen assembly due to conformational changes in the γ-module and consequent abnormal over-exposure of protein hydrophobic patches that could act as seeds for the aggregation of hydrophobic molecules [16]. All mutations cluster near the interface of dimerization (Figure 2).…”

Hereditary Hypofibrinogenemia with Hepatic Storage

“…To date, only 21 index cases characterized at the molecular level, all with a diagnosis of HHHS based on immunohistochemistry and electron microscopy findings, have been described in the English literature. Table 1 lists their main clinical features [ 26 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ].…”