Resolving Differential Diagnostic Problems in von Willebrand Disease, in Fibrinogen Disorders, in Prekallikrein Deficiency and in Hereditary Hemorrhagic Telangiectasia by Next-Generation Sequencing

Abstract

Diagnosis of rare bleeding disorders is challenging and there are several differential diagnostics issues. Next-generation sequencing (NGS) is a useful tool to overcome these problems. The aim of this study was to demonstrate the usefulness of molecular genetic investigations by summarizing the diagnostic work on cases with certain bleeding disorders. Here we report only those, in whom NGS was indicated due to uncertainty of diagnosis or if genetic confirmation of initial diagnosis was required. Based on clinical and/or laboratory suspicion of von Willebrand disease (vWD, n = 63), hypo-or dysfibrinogenemia (n = 27), hereditary hemorrhagic telangiectasia (HHT, n = 10) and unexplained activated partial thromboplastin time (APTT) prolongation (n = 1), NGS using Illumina platform was performed. Gene panel covered 14 genes (ACVRL1, ENG, MADH4, GDF2, RASA1, F5, F8, FGA, FGB, FGG, KLKB1, ADAMTS13, GP1BA and VWF) selected on the basis of laboratory results. We identified forty-seven mutations, n = 29 (6 novel) in vWD, n = 4 mutations leading to hemophilia A, n = 10 (2 novel) in fibrinogen disorders, n = 2 novel mutations in HHT phenotype and two mutations (1 novel) leading to prekallikrein deficiency. By reporting well-characterized cases using standardized, advanced laboratory methods we add new pieces of data to the continuously developing "bleeding disorders databases", which are excellent supports for clinical patient management.

Keywords: APTT prolongation; differential diagnosis; hemophilia A; hereditary hemorrhagic telangiectasia; next generation sequencing; prekallikrein deficiency; rare bleeding disorders; von Willebrand disease.

Figure 1

Patterns of ristocetin-induced platelet aggregation and multimer analysis of a type 2B vWD patient (Patient 10/1 in Supplementary Table S2). (A) Normal aggregation response to 1.2 mg/mL ristocetin. (B) Response to low dose of ristocetin (0.6 mg/mL). (C) Normal distribution of vWF multimers. vWF multimer analysis was performed by SDS-agarose gel electrophoresis. P = patient plasma, C = control plasma.

Figure 2

Family chart of a type 2N vWD family (Family 21 in Supplementary Table S2). The variants that occur in the family are c.2269_70delCT (p.Leu757Valfs*22) in exon 17; c.2365A>G (p.Thr789Ala) in exon 18 and c.2561G>A (p.Arg854Gln) in exon 20.

Figure 3

Rotational thromboelastometry (ROTEM) analysis in a patient with a novel FGB mutation (patient 13/1 in Supplementary Table S3). FIBTEM, EXTEM, INTEM and APTEM tests were performed detailed in the text. ROTEM curves for (A). FIBTEM, (B). EXTEM, (C). INTEM, (D). APTEM tests.

Figure 4

Homology analysis of novel missense mutations. Affected amino acids are indicated in red squares.