Molecular Diagnosis of Quantitative and Qualitative Fibrinogen Deficiencies: A Portuguese Centre Experience

C. Silva Pinto¹, P. Martinho¹, C. Oliveira², M.F. Rodrigues², R. Salvado³, M. Costa⁴, T. Fidalgo¹, M.L. Ribeiro¹

¹Centro Hospitalar e Universitário de Coimbra, Molecular Hematology Laboratory, Clinical Hematology, Coimbra, Portugal, ²Centro Hospitalar Universitário Lisboa Norte, Hospital Santa Maria, Reference Centre of Congenital Coagulopathies, Lisboa, Portugal, ³Centro Hospitalar Universitário de Coimbra, Reference Centre of Congenital Coagulopathies, Coimbra, Portugal, ⁴Centro Hospitalar Tondela-Viseu, Immunohemotherapy Service, Viseu, Portugal

Abstract Number: PB1187

Meeting: ISTH 2020 Congress

Theme: Hemophilia and Rare Bleeding Disorders » Rare Bleeding Disorders

Background: Congenital fibrinogen deficiencies are rare disorders, classified as quantitative (hypofibrinogenemia/afibrinogenemia, with partial/complete absence of fibrinogen) or qualitative (dysfibrinogenemia and hypodysfibrinogenemia, with normal or reduced fibrinogen levels and/or abnormal functional activity). Clinically, quantitative deficiencies are associated with hemorrhagic events, while dysfibrinogenemias are mainly asymptomatic, or can present with bleeding, thrombosis or both. Fibrinogen is a hexameric protein consisting of three pairs of polypeptide chains (Aa, Bb and g), encoded by FGA, FGB and FGG genes, respectively. Variants in these genes have been reported, both in homo- or heterozygosity.

Aims: Our aim was to identify the molecular diagnosis of patients with fibrinogen anomalies and/or hemorrhagic diathesis of unknown cause by next generation sequencing (NGS).

Methods: Seventeen unrelated patients and 5 relatives were analyzed. The molecular diagnosis was done using a custom panel for NGS (43 genes). Library preparation and sequencing was done using IonS5 (Thermo Fisher Scientific) protocol.

Results: We have identified 13 different variants (pathogenic and potentially pathogenic) in FGA (5), FGB (2) and FGG (6) genes, including five new variants in FGB (p.Glu240Lys) and FGG (p.Tyr27Cys, p.Asp63Val, p.Trp360* and p.Glu422del). The patients were diagnosed as hypofibrinogenemia (n=7), hypodisfibrinogenemia (n=4) and dysfibrinogenemia (n=4). The patients diagnosed with afibrinogenemia (n=2) were homozygous for 2 variants in FGA (complete deletion and p.Arg181*) and had severe hemorrhagic manifestations. Five presented with thrombosis: one was carrier for FGA p.Cys64Tyr, associated with both thrombosis and bleeding. In total, 11 patients were asymptomatic (Table 1 and 2).

Gene	Patients/ Families	Fibrinogen (mg/dL)/ (min-max)	Nucleotide	Amino acid**	Genotype	*In silico*	New Yes/No	Phenotype	Clinical data
FGA	1/1	< 30	Complete deletion		HMZ	P	No	Afibrinogenemia	Spontaneous hemorrhage, intracranial hemorrhage (BS=9) – on prophylaxis
	1/1	< 30	c.541C>T	p.Arg181*	HMZ	P	No		Spontaneous hemorrhage (BS=11) – on prophylaxis
	6/2	(17 – 65)	c.103C>T	p.Arg35Cys	HTZ	P	No (recurrent)	Hypofibrinogenemia	Asymptomatic (BS=0)
	4/3	(<50 - 87)	c.191G>A	p.Cys64Tyr	HTZ	P	No (recurrent)		Bleeding/Asymptomatic/Thrombosis (BS=0;2;6;6)
	2/2	84; 70	c.1543G>A	p.Asp515Asn	HTZ	P	No		Bleeding/Asymptomatic (BS=0; 4)
FGB	1/1	625	c.586C>T	p.Arg196Cys	HTZ	P	No	Dysfibrinogenemia	Thrombosis
	1/1	128	c.718G>A	p.Glu240Lys	HTZ	PP	Yes	Hypodysfibrinogenemia	Bleeding (BS=5)

[Table I. Fibrinogen genetic variants identified in 22 patients with the Thrombosis&Hemostasis (TH)*]

Gene	Patients/ Families	Fibrinogen (mg/dL)/ (min-max)	Nucleotide	Amino acid**	Genotype	*In silico*	New Yes/No	Phenotype	Clinical data
FGG	1/1	256	c.80A>G	p.Tyr27Cys	HTZ	PP	Yes	Dysfibrinogenemia	Thrombosis
	1/1	200	c.140C>T	p.Thr47Ile	HTZ	P	No	Dysfibrinogenemia	Bleeding (BS=5)
	1/1	?	c.188A>T	p.Asp63Val	HTZ	PP	Yes	Hypo-dysfibrinogenemia	Bleeding (BS=5)
	1/1	44	c.901C>T	p.Arg301Cys	HTZ	P	No	Hypo-dysfibrinogenemia	Asymptomatic/Thrombosis/ Spontaneous miscarriage (BS=0)
	1/1	105	c.1080C>A	p.Trp360*	HTZ	P	Yes	Hypo-dysfibrinogenemia	Asymptomatic, 4 years old (BS=0)
	1/1	471	c.1265_1267delAAG	p.Glu422del	HTZ	PP	Yes	Dysfibrinogenemia	Thrombosis
Fibrinogen (mg/dL) – Reference values: 150-400mg/dL; ** Nomenclature HGVS; P – pathogenic; PP – potentially pathogenic; BS – Bleeding Score. Panel TH (43 genes) – Associated with coagulation factors anomalies: F2, F3, F5, F7, F8, F9, F10, F11, F13A1, F13B, LMAN1, MCFD2, FGA, FGB, FGG, GGCX, VKORC1, VWF;* associated with thrombosis: PROC, PROS1, SERPINC1, SERPINE1, SERPINF2, THBD, PLAT, PLG, ANXA5, F12; associated with platelet disorders: GP1BB, GP1BA, GP9, ITGA2B, ITGB3, NBEAL2, NBEA, GP6, TBXA2R, P2RY12, ANKRD26, CYCS, PLA2G4A, TUBB1, PLAU.

[Table II. Fibrinogen genetic variants identified in 22 patients with the Thrombosis&Hemostasis (TH)* (cont.)]

Conclusions: We have identified 5 new variants in the coding region of the fibrinogen cluster, together with high phenotypic variability. A phenotype-genotype correlation was observed in the quantitative deficiencies. In dysfibrinogenemias, it is still a challenge. We add another example of the valuable tool that NGS represents to clinical practice, allowing for a faster and broader diagnosis of fibrinogen hereditary anomalies.

To cite this abstract in AMA style:

Silva Pinto C, Martinho P, Oliveira C, Rodrigues MF, Salvado R, Costa M, Fidalgo T, Ribeiro ML. Molecular Diagnosis of Quantitative and Qualitative Fibrinogen Deficiencies: A Portuguese Centre Experience [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/molecular-diagnosis-of-quantitative-and-qualitative-fibrinogen-deficiencies-a-portuguese-centre-experience/. Accessed December 11, 2023.

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