Novel GATA1 Variant Causing a Bleeding Phenotype Associated with Combined Platelet α-/δ-Storage Pool Deficiency and Mild Dyserythropoiesis Modified by a SLC4A1 Variant.

Germline defects in the transcription factor GATA1 are known to cause dyserythropoiesis with(out) anemia and variable abnormalities in platelet count and function. However, damaging variants closely located to the C-terminal zinc finger domain of GATA1 are nearly unknown. In this study, a 36-year-old male index patient and his 4-year-old daughter suffered from moderate mucocutaneous bleeding diathesis since birth. Whole exome sequencing detected a novel hemizygous GATA1 missense variant, c.886A>C p.T296P, located between the C-terminal zinc finger and the nuclear localization sequence with non-random X-chromosome inactivation in the heterozygous daughter. Blood smears from both patients demonstrated large platelet fractions and moderate thrombocytopenia in the index. Flow cytometry and electron microscopy analysis supported a combined α-/δ (AN-subtype)-storage pool deficiency as cause for impaired agonist-induced platelet aggregation (light transmission aggregometry) and granule exocytosis (flow cytometry). The absence of BCAM in the index (Lu(a-b-)) and its low expression in the daughter (Lu(a-b+)) confirmed a less obvious effect of defective GATA1 also on erythrocytes. Borderline anemia, elevated HbF levels, and differential transcription of GATA1-regulated genes indicated mild dyserythropoiesis in both patients. Furthermore, a mild SLC4A1 defect associated with a heterozygous SLC4A1 c.2210C>T p.A737V variant maternally transmitted in the daughter may modify the disease to mild spherocytosis and hemolysis.

Prothrombotic immune thrombocytopenia after COVID-19 vaccination.

We report 5 cases of prothrombotic immune thrombocytopenia after exposure to the ChAdOx1 vaccine (AZD1222, Vaxzevria) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients presented 5 to 11 days after first vaccination. The spectrum of clinical manifestations included cerebral venous sinus thrombosis, splanchnic vein thrombosis, arterial cerebral thromboembolism, and thrombotic microangiopathy. All patients had thrombocytopenia and markedly elevated D-dimer. Autoantibodies against platelet factor 4 (PF4) were detected in all patients, although they had never been exposed to heparin. Immunoglobulin from patient sera bound to healthy donor platelets in an AZD1222-dependent manner, suppressed by heparin. Aggregation of healthy donor platelets by patient sera was demonstrated in the presence of buffer or AZD1222 and was also suppressed by heparin. Anticoagulation alone or in combination with eculizumab or intravenous immunoglobulin (IVIG) resolved the pathology in 3 patients. Two patients had thromboembolic events despite anticoagulation at a time when platelets were increasing after IVIG. In summary, an unexpected autoimmune prothrombotic disorder is described after vaccination with AZD1222. It is characterized by thrombocytopenia and anti-PF4 antibodies binding to platelets in AZD1222-dependent manner. Initial clinical experience suggests a risk of unusual and severe thromboembolic events.

PF4-Dependent Immunoassays in Patients with Vaccine-Induced Immune Thrombotic Thrombocytopenia: Results of an Interlaboratory Comparison.

BACKGROUND: Coronavirus disease 2019 vaccine ChAdOx1 nCov-19 may rarely lead to vaccine-induced thrombotic thrombocytopenia (VITT). Antibody-mediated, platelet factor 4 (PF4)-dependent platelet activation appears to resemble a key mechanism in VITT, partially comparable to heparin-induced thrombocytopenia. The use of PF4/heparin immunoassays has been proposed as part of a diagnostic approach, but their sensitivity has not been established. METHODS: Sera from 12 well-defined VITT patients were first studied by two different laboratories in functional assays. Sera where then used for an interlaboratory comparison, in which five different PF4/heparin immunoassays were used by four laboratories. RESULTS: Results for functional testing were highly concordant. VITT antibodies were also reliably detected by PF4/heparin enzyme-linked immunosorbent assays (ELISAs) (92-100%). In contrast, only 25% of VITT antibodies were reactive in a particle gel immunoassay (PaGIA), and 8% in a lateral flow assay (LFA). An automated chemiluminescence immunoassay (CLIA) was negative for all sera tested (0%). CONCLUSION: It seems feasible to establish functional antibody testing for the confirmation of VITT. For the initial screening of suspected VITT cases, PaGIA, LFA, and CLIA are useless when applied as single tests. Only ELISA-based PF4/heparin immunoassays are sensitive enough to be incorporated in the diagnostic workup. However, a combination of a positive ELISA and a negative CLIA may be useful to identify VITT antibodies in the absence of confirmatory functional assays.

Prevention of Pregnancy Complications in Antiphospholipid Syndrome.

Despite a lot of research on antiphospholipid antibodies (aPL), standardization of test systems, and better definition of its clinical symptoms, the pathomechanism of this acquired autoimmune disease is not yet fully explained. Progress in treatment increased the live birth rate in 70 to 80% of women suffering from obstetric antiphospholipid syndrome (OAPS). However, still 20 to 30% will develop adverse pregnancy outcome. Lack of awareness of this disorder as the cause for pregnancy complications is very harmful to mothers and to their newborns. Complications can be avoided or minimized by proper treatment. The aim of this article is to increase the awareness of gynecologists and medical personal for OAPS.

Acquired haemophilia caused by non-haemophilic factor VIII gene variants.

The aetiology of anti-factor VIII (FVIII) autoantibody formation in acquired haemophilia remains unknown. We hypothesised that encounter of antigenically different, allogeneic FVIII may challenge inhibitor formation after presentation on MHC class II. Eighteen consecutive cases with acquired haemophilia were enrolled (nine females, nine males). A control group comprised 50 male and 50 female healthy blood donors. The coding region of the FVIII gene and the HLA-DRB1 genotype were studied. The presentation of foreign FVIII variants on the patient's MHC class II alleles was predicted using SYFPEITHI algorithm. A rare FVIII variant (E2004K) was found in one patient with acquired haemophilia after massive transfusion; the 2004 K allele was predicted to be presented on the patient's HLA-DRB1*0101. Moreover, distribution of a polymorphism (D1241E) was significantly skewed comparing patients and controls. Three of three patients with transfusion-associated disease carried 1241D in homozygous or hemizygous form and were predicted to present 1241E (foreign), but not 1241D (self), on their HLA-DRB1*0301. Therefore, encounter of 1241E may result in the presentation of a new T cell epitope in these patients. The same conditions were not found in any patient with acquired haemophilia of other causes. The expected frequency in the general Caucasoid population undergoing transfusion is 3% to 4%. In conclusion, encounter of variant allogeneic FVIII presented on a suitable MHC background could be a risk factor for inhibitor formation.

Antigen-recognition sites of micromanipulated T cells in patients with acquired aplastic anemia.

OBJECTIVE: Acquired aplastic anemia (AA) is a rare disorder characterized by pancytopenia and hypocellular bone marrow. Though experimental and clinical data suggest that AA represents a T cell-mediated disease, neither the immune response nor the nature of inciting antigen(s) have been characterized so far. The identification of a restricted T cell repertoire by PCR techniques in total lymphocyte populations supports an antigen-driven T cell response. In order to investigate the clonal composition, we analyzed the gene rearrangements of the T cell receptor (TCR) variable beta chain (Vbeta) at the single-cell level. PATIENTS AND METHODS: CD3(+) T lymphocytes were micromanipulated from peripheral blood and bone marrow samples of 8 AA patients and healthy controls. Subsequently amplified VDJ gene segments of the TCRVbeta chain were analyzed for functional rearrangements. More than 500 functionally rearranged TCR loci were studied for Vbeta/Jbeta gene segment usage and molecular composition of the complementary-determining region 3 (CDR3). RESULTS: In comparison to healthy controls, the Vbeta sequences confirmed a highly restricted T cell repertoire in AA patients at the single-cell level. Both in bone marrow and peripheral blood a predominance of Vbeta13 and Jbeta2S7 was observed. Furthermore, individual clonal T-cell expansion was identified in the majority of patients. However, deduced CDR3 amino acid sequences revealed a high variability without common motifs among the 8 patients. CONCLUSION: Individual clonal T-cell expansion with high diversity of the antigen-binding sites among the analyzed patients argues for the predominance of private inciting epitopes in AA.

Evolution of FLT3-ITD and D835 activating point mutations in relapsing acute myeloid leukemia and response to salvage therapy.

Internal tandem duplications (ITDs) of the juxtamembrane region of the FLT3 tyrosine kinase receptor are the most frequent genetic alterations in acute myeloid leukemia (AML). The presence of this mutation has been recognized as an independent poor prognostic factor. In this study, we compared the FLT3 mutational status between diagnosis and subsequent relapses in 31 patients with AML. At diagnosis, seven patients were identified to contain FLT3-ITD mutations and one patient to harbor the D835 mutation. Five patients remained FLT3-ITD positive throughout the disease course (+/+). Three patients lost the FLT3 gene mutation at first (one FLT3-ITD, one D835 mutation), or second relapse (one FLT3-ITD) (+/-). One additional patient lost a small FLT3-ITD positive clone at relapse and at the same time gained an apparently unrelated FLT3-ITD positive clone. One patient without FLT3 mutation at diagnosis relapsed with an FLT3-ITD mutation (-/+). A shorter median duration of first remission (6 months versus 11.5 months) and a higher relapse rate after salvage therapy (e.g. allogeneic peripheral blood stem cell transplantation) resulted in a lower leukemia-free survival in the FLT3 mutated group (11% versus 31%). The loss of a clone with a mutation in the FLT3 gene at relapse did not improve the prognosis.

Evidence for allelic evolution of C/EBPalpha mutations in acute myeloid leukaemia.

Transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is mutated in 6-10% of patients with acute myeloid leukaemia (AML). Recently, we reported the emergence of an N-terminal C/EBPalpha mutation after chemotherapy in a patient with secondary AML. The clone carrying the mutation became the dominant clone at relapse. This observation prompted us to compare the C/EBPalpha mutational status of 26 de novo non-core binding factor AML patients at diagnosis and at relapse after induction and consolidation chemotherapy. Four mutations in the C/EBPalpha gene were identified in two out of 26 patients. In both these cases, a biallelic mutation was present at diagnosis and at relapse: an amino-terminal frameshift mutation and a mutation of the fork/leucine finger 1 region. In patient 1, the amino-terminal frameshift mutation was duplicated and found on both alleles at relapse. In patient 2, the amino-terminal frameshift mutation and a mutation in the fork region were found either alone or combined on the same allele, suggesting a subclone formation. None of the patients without a C/EBPalpha mutation at diagnosis showed a mutation at relapse. This is the first report of an evolution of the C/EBPalpha gene between diagnosis and relapse in AML.

The deletion polymorphism in the angiotensin-converting enzyme gene is a moderate risk factor for venous thromboembolism.

ACE displays potent vasoconstrictive effects, attenuation of fibrinolysis, and platelet activation and aggregation, thus possibly promoting venous thromboembolism (VTE). The ACE gene contains an insertion (I) or deletion (D) polymorphism accounting for 50% of the variation in serum ACE concentration. To evaluate the role of the I/D polymorphism in VTE, its prevalence was determined in 931 patients with VTE and 432 blood donors. The prevalence of the DD genotype was 27.6% in patients and 21.3% in controls (OR 1.4; p < 0.02). In multivariate analysis there was a trend of the DD genotype to be an independent risk factor (OR 1.4; p = 0.08). No differences in DD genotype prevalence according to exogenous risk factors were found. Coinheritance of FV G1691A, PT G20210A mutation, and PS deficiency with the DD genotype increased the relative risk of VTE. Thus, the ACE DD genotype is a moderate risk factor of hereditary thrombophilia. Exogenous risk factors did not alter the manifestation of VTE among carriers of the DD genotype, whereas coinheritance of the DD genotype with the aforementioned defects increased the risk for VTE considerably.