Next-generation sequencing reveals novel variants and large deletion in FANCA gene in Polish family with Fanconi anemia.

BACKGROUND: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. However, establishing its molecular diagnosis remains challenging. Chromosomal breakage analysis is the gold standard diagnostic test for this disease. Nevertheless, molecular analysis is always required for the identification of pathogenic alterations in the FA genes. RESULTS: We report here on a family with FA diagnosis in two siblings. Mitomycin C (MMC) test revealed high level of chromosome breaks and radial figures. In both children, array-Comparative Genomic Hybridization (aCGH) showed maternally inherited 16q24.3 deletion, including FANCA gene, and next generation sequencing (NGS) disclosed paternally inherited novel variants in the FANCA gene-Asn1113Tyr and Ser890Asn. A third sibling was shown to be a carrier of FANCA deletion only. CONCLUSIONS: Although genetic testing in FA patients often requires a multi-method approach including chromosome breakage test, aCGH, and NGS, every effort should be made to make it available for whole FA families. This is not only to confirm the clinical diagnosis of FA in affected individuals, but also to enable identification of carriers of FA gene(s) alterations, as it has implications for diagnostic and genetic counselling process.

Next-Generation Sequencing of Connective Tissue Genes in Patients with Classical Ehlers-Danlos Syndrome.

BACKGROUND: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been described. MATERIAL AND METHODS: The study group included 59 patients of Polish origin, diagnosed with cEDS. The analysis was performed on genomic DNA (gDNA) with NGS technology, using an Illumina sequencer. Thirty-five genes related to connective tissue were investigated. The pathogenicity of the detected variants was assessed by VarSome. RESULTS: The NGS of 35 genes revealed variants within the COL5A1, COL5A2, COL1A1, and COL1A2 genes for 30 of the 59 patients investigated. Our panel detected no sequence variations for the remaining 29 patients. DISCUSSION: Next-generation sequencing, with an appropriate multigene panel, showed great potential to assist in the diagnosis of EDS and other connective tissue disorders. Our data also show that not all causative genes giving rise to cEDS have been elucidated yet.

Selected Parameters of Angiogenesis and the JAK2, CALR, and MPL Mutations in Patients With Essential Thrombocythemia.

The aim of the study was to evaluate selected angiogenic factors in patients with essential thrombocythemia (ET) depending on JAK2V617F, calreticulin gene (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations. Sixty ET patients and 20 healthy volunteers were enrolled in the study. The following tests were performed: vascular endothelial growth factor- A (VEGF-A), soluble vascular endothelial growth factor receptor-1 (sVEGFR-1),soluble vascular endothelial growth factor receptor-2 (sVEGFR-2), platelet-derived growth factor( PDGF-BB), and stromal-derived factor-1α (SDF-1α). We observed an increased PDGF-BB level in patients with ET compared to the controls. Patients with CALR mutation had significantly higher concentration of PDGF-BB and lower concentration of SDF-1α than patients with JAK2V617F mutation. High concentration of PDGF-BB and low concentration of SDF-1α in patients with CALR(+) ET may indicate a contribution of these chemokines in disturbed Ca2+ metabolism in platelets.

Extrinsic blood coagulation pathway and risk factors for thrombotic events in patients with essential thrombocythemia.

INTRODUCTION    The clinical course of essential thrombocythemia (ET) is varied, and some patients do not exhibit any clinical signs of the disease at the time of diagnosis. The most frequent complications that occur during the course of ET are hemostasis abnormalities manifesting as hemorrhagic or thrombotic events. The mechanism of thrombotic events in patients with ET is complex and not fully understood. OBJECTIVES    The aim of the study was to evaluate the concentration and activity of tissue factor (TF) and tissue factor pathway inhibitor (TFPI), depending on the most important risk factors of thrombotic complications (age >60 years, history of thrombotic episodes, presence or absence of the JAK2 V617F mutation, and increased leukocyte count). PATIENTS AND METHODS    The study group included 113 patients with diagnosed ET, and the control group, 30 healthy volunteers matched for age and sex. The concentration and activity of TF and TFPI were measured using enzyme-linked immunosorbent assays. RESULTS    Patients with ET had a significantly higher activity and concentration of TF and increased activity of TFPI, as compared with controls. The analysis of the studied parameters in relation to risk factors revealed that patients with ET with a history of thrombotic events had a significantly higher concentration of TF, and patients with the JAK2 V617F mutation had a lower TFPI activity, as compared with patients without the mutation. CONCLUSIONS    Our study showed that in patients with ET who have a history of thrombosis or the JAK2 V617F mutation, the enhanced risk of thrombosis may result from an increased TF concentration or decreased TFPI activity.

Activation of the tissue factor-dependent extrinsic pathway and its relation to JAK2 V617F mutation status in patients with essential thrombocythemia.

Thrombotic complications may occur in 7.6-29.4% of patients with essential thrombocythemia. According to the cellular theory, tissue factor (TF) activating extrinsic blood coagulation pathway is essential for the activation of blood clotting. The aim of the study was to evaluate the activation of the TF-dependent extrinsic pathway in patients with essential thrombocythemia, depending on the presence or absence of the Janus kinase 2 (JAK2) V617F mutation. The study included 74 newly diagnosed patients (F/M: 47/27; mean age 61 years) with essential thrombocythemia (Tefferi and Vardiman, Leukemia 2008; 22(1):14-22). Patients were diagnosed in the Department of Clinical Hematology and Hematological Malignancies University Hospital No. 2, Bydgoszcz, Poland. The control group consisted of 30 healthy volunteers (F/M: 17/13; mean age 49 years). The concentration and activity of TF and TF pathway inhibitor (TFPI) were measured using ELISA method. In patients with essential thrombocythemia, we observed a higher concentration of TF [median (Me) = 686.90 vs 164.28 pg/ml] and over 10-fold higher activity of TF (Me = 46.05 vs 4.01 pmol/l) when compared with the control group. We also reported significantly higher activity of TFPI compared with the control group (Me = 1.93 vs 1.78 U/ml). Moreover, a concentration of TFPI was significantly lower in patients with essential thrombocythemia with JAK2 V617F mutation as compared with patients without the mutation (Me = 1.90 vs 2.16 U/ml; P = 0.039639). Increased TF activity and concentration is responsible for higher procoagulant potential in patients with essential thrombocythemia. Reduced activity of TFPI in patients with essential thrombocythemia with JAK2 V617F mutation indicates an increased prothrombotic risk in this group of patients.