Collagen remodelling and plasma ascorbic acid levels in patients suspected of inherited bleeding disorders harbouring germline variants in collagen-related genes.

INTRODUCTION: Variants in collagen-related genes COL1A1, COL3A1, COL5A1 and COL5A2 are associated with Ehlers-Danlos syndrome (EDS), a heterogeneous group of connective tissue disorders strongly associated with increased bleeding. Of patients with incompletely explained bleeding diathesis, a relatively high proportion were shown to harbour at least one heterozygous variant of unknown significance (VUS) in one of these genes, the vast majority without meeting the clinical criteria for EDS. AIM: To investigate the functional consequences of the identified variants by assessing the formation and degradation of types I, III and V collagen, in addition to plasma levels of ascorbic acid (AA). METHODS: A total of 31 patients harbouring at least one heterozygous VUS in COL1A1, COL3A1, COL5A1 or COL5A2 and 20 healthy controls were assessed using monoclonal antibodies targeting neo-epitopes specific for collagen formation and degradation. Plasma AA levels were measured in patients using high-performance liquid chromatography. RESULTS: Serum levels of C5 M (degradation of type V collagen) were decreased in patients compared with healthy controls (p = .033). No significant differences were found in biomarkers for remodelling of types I and III collagen. A significant negative correlation between bleeding (ISTH-BAT score) and plasma AA levels was shown (r = -.42; r2  = .17; p = .020). Suboptimal or marginally deficient AA status was found in 8/31 patients (26%). CONCLUSION: Functional investigations of collagen remodelling were not able to identify any clear associations between the identified variants and increased bleeding. The negative correlation between plasma AA levels and ISTH-BAT score motivates further investigations.

A rare case of IgE kappa monoclonal gammopathy of undetermined significance identified in a Swedish female.

Monoclonal gammopathies involving immunoglobulin E (IgE) is a very rare phenomenon, with less than 70 cases being previously described in the literature. The IgE monoclonal gammopathies include malignant plasma cell disorders such as IgE multiple myeloma (MM), as well as the associated premalignant condition IgE monoclonal gammopathy of undetermined significance (MGUS). We report a case of a 41-year-old woman presenting with an IgE kappa monoclonal protein following routine laboratory testing. Serum protein electrophoresis (SPEP) initially showed a monoclonal protein in the beta-2 fraction, at an estimated concentration of <4 g/L. Subsequent serum immunofixation electrophoresis (SIFE) including antisera to Ig heavy chains delta and epsilon confirmed the presence of an IgE kappa monoclonal protein. Analysis of serum free light chains (FLCs) showed increased levels of kappa FLC, resulting in an abnormally elevated kappa/lambda FLC ratio. No Bence-Jones proteinuria was present. Bone marrow aspiration showed 6% plasma cells, and no sign of myeloma-associated end-organ damage was evident. Consequently, the patient was diagnosed with IgE kappa MGUS. In the present report, the clinical characteristics of the patient are compared to previous descriptions of IgE monoclonal gammopathy. The report further emphasizes the importance of considering the presence of monoclonal IgD or IgE when SIFE shows a clear band positive for a light chain but is negative for Ig heavy chains gamma, alpha and mu.

Genetic screening of children with suspected inherited bleeding disorders.

INTRODUCTION: Genetic screening using high-throughput DNA sequencing has become a tool in diagnosing patients with suspected inherited bleeding disorders (IBD). However, its usefulness and diagnostic efficacy in children is unclear. AIM: To evaluate the diagnostic efficacy of genetic screening for IBD in children and downstream further testing. METHODS: After informed consent, children (<18 years) with suspected IBD underwent genetic screening with 94 selected genes. RESULTS: A total of 68 heterozygous class 3-5 variants were detected in 30 children, 2.3 variants per patient. Directed specific functional testing was performed after genetic screening in a subset of patients. Adhering to the ACMG guidelines, the results of functional testing together with family history and previous publications classified three variants as likely disease causing (class 4) and two variants as disease causing (class 5), all in children with thrombocytopenia. The overall diagnostic rate was 16.7% (5/30). Children with thrombocytopenia had a significantly higher rate of significant genetic findings, 5/9 (55.6%) vs. 0/21 (0%; P = .0009). CONCLUSION: We conclude that performing genetic screening in children is an effective tool especially for children with inherited thrombocytopenia and has the possibility to diagnose platelet disorders adequately early in life. Children with bleeding diathesis, normal coagulation work-up and without thrombocytopenia are unlikely to be diagnosed by genetic screening. Ethical issues such as incidental findings, variants associated with cancer and the interpretation of the genetic results into clinical practice remain problematic.

Germline heterozygous variants in genes associated with familial hemophagocytic lymphohistiocytosis as a cause of increased bleeding.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3-5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised platelet function. Therefore, we aimed to investigate platelet degranulation in patients heterozygous for variants in UNC13D, STX11 and STXBP2. During the work-up of patients referred to the Coagulation Unit, Skåne University Hospital, Malmö, Sweden and the Department of Hematology, Rigshospitalet, Copenhagen, Denmark due to bleeding tendencies, 12 patients harboring heterozygous variants in UNC13D, STX11 or STXBP2 were identified using targeted whole exome sequencing. Transmission electron microscopy (TEM) was used to assess the secretion of platelet dense granules following thrombin stimulation. Platelet degranulation, activation and aggregation were further assessed by flow cytometry (FC) and light transmission aggregometry (LTA) with lumi-aggregometry. In total, eight out of twelve (67%) patients showed impaired degranulation by at least one of the assays (TEM, FC and LTA). In the 12 patients, eight different heterozygous variants were identified. One variant was strongly associated with impaired degranulation, while four of the variants were associated with impaired granule secretion to a slightly lesser extent. One additional variant was found in six out of the twelve patients, and was associated with varying degrees of degranulation impairment. Accordingly, six out of the eight (75%) identified variants were associated with impaired platelet degranulation. Our results suggest that heterozygous variants in UNC13D, STX11 and STXBP2 are sufficient to cause platelet secretion defects resulting in increased bleeding.

Severe Congenital Thrombocytopenia Characterized by Decreased Platelet Sialylation and Moderate Complement Activation Caused by Novel Compound Heterozygous Variants in GNE.

Background: Hereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack. Methods: Sialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays. Results: The proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis. Conclusion: We report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.

A rare heterozygous variant in FGB (Fibrinogen Merivale) causing hypofibrinogenemia in a Swedish family.

: Fibrinogen is essential for normal hemostasis. Congenital fibrinogen disorders (afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia and hypodysfibrinogenemia), caused by pathogenic variants in the genes FGA, FGB and FGG, have the potential of causing bleeding diathesis and/or thrombotic events of variable severity. We describe a case of familial hypofibrinogenemia in a Swedish family. The proband is a 27-year-old woman, with a history of significant bleeding diathesis. She was diagnosed with moderate hypofibrinogenemia (0.8 g/l), and genetic screening identified a rare heterozygous missense variant in FGB (c.854G>A, p.Arg285His) (Fibrinogen Merivale) previously described in a New Zealand European family with symptomatic hypofibrinogenemia. The father, sister and brother of the proband also harbored the FGB variant, segregating with hypofibrinogenemia (0.9-1.2 g/l). The proband showed a more severe bleeding phenotype compared with her other hypofibrinogenemic family members; this was attributed to a concomitant platelet dysfunction, also present in her normofibrinogenemic mother.