Mathieu Fiore

Glanzmann thrombasthenia: a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models

Characterized by mucocutaneous bleeding arising from a lack of platelet aggregation to physiologic stimuli, Glanzmann thrombasthenia (GT) is the archetype-inherited disorder of platelets. Transmitted by autosomal recessive inheritance, platelets in GT have quantitative or qualitative deficiencies of the fibrinogen receptor, αIIbβ3, an integrin coded by the ITGA2B and ITGB3 genes. Despite advances in our understanding of the disease, extensive phenotypic variability with respect to severity and intensity of bleeding remains poorly understood. Importantly, genetic defects of ITGB3 also potentially affect other tissues, for β3 has a wide tissue distribution when present as αvβ3 (the vitronectin receptor). We now look at the repertoire of ITGA2B and ITGB3 gene defects, reexamine the relationship between phenotype and genotype, and review integrin structure in the many variant forms. Evidence for modifications in platelet production is assessed, as is the multifactorial etiology of the clinical expression of the disease. Reports of cardiovascular disease and deep vein thrombosis, cancer, brain disease, bone disorders, and pregnancy defects in GT are discussed in the context of the results obtained for mouse models where nonhemostatic defects of β3-deficiency or nonfunction are being increasingly described.

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real‐time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440‐13_c.1440‐1del in intron 14 of ITGA2B causing exon skipping in seven unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits, thereby interfering with integrin maturation and/or function. Our study extends knowledge of GT and the pathophysiology of an integrin.

Glanzmann thrombasthenia-like syndromes associated with Macrothrombocytopenias and mutations in the genes encoding the αIIbβ3 integrin.

Glanzmann thrombasthenia (GT) is the most widely studied inherited disorder of platelets; it is caused by the absence of platelet aggregation due to quantitative and/or qualitative deficiencies of the αIIbβ3 integrin coded by the ITGA2B and ITGB3 genes located at 17q21-23. Although platelet count and platelet volume (and morphology) are normal in classic GT, some reports have inferred a role for αIIbβ3 in megakaryocytopoiesis and some novel but rare point mutations in either of the ITGA2B and ITGB3 genes have been associated with an altered platelet production and selective deficiencies in platelet function. This was brought to light by the discovery of mutations at Arg995 in αIIb and Asp723 in β3 that lead to platelet anisotropy (increased size variation) and thrombocytopenia. Significantly, Arg995 and Asp723 form a salt linkage binding the cytoplasmic tails of αIIbβ3 together keeping the integrin in a bent resting state. Mutations weakening this link (if not abolishing it) increase the activation state of αIIbβ3 and interfere with megakaryocytopoiesis. Other mutations affecting platelet production involve extracellular but membrane proximal domains of β3. Our purpose is to review the mutations in the ITGA2B and ITGB3 genes that lead to anisotropy and to discuss mechanisms by which this can be brought about.

Natural history of platelet antibody formation against αIIbβ3 in a French cohort of Glanzmann thrombasthenia patients.

Summary.  Treatment of the bleeding syndrome in Glanzmann thrombasthenia (GT) is often complicated by naturally occurring isoantibodies directed against the αIIbβ3 integrin that cause the removal of or render ineffective transfused donor platelets. Such antibodies are produced after transfusion or pregnancy when the patient’s immune system comes into contact with normal platelets. Despite many reports of anti‐αIIbβ3 antibodies in GT patients, there is no consensus pertaining to their frequency, their long‐term evolution in the circulation, or their formation in relation to either (i) the extent of the αIIbβ3 deficiency in the patient’s platelets or (ii) the nature of the genetic defect (ITGA2B or ITGB3 genes). Antibody screening was performed on a large series of 24 GT patients in South‐West France dividing the patients into two cohorts: (i) 16 patients with the French gypsy mutation (c.1544 + 1G>A) within ITGA2B that gives platelets totally lacking αIIbβ3 and (ii) 8 patients carrying other defects of ITGA2B or ITGB3 with different expression levels of αIIbβ3. Our results confirm that patients with premature termination mutations resulting in platelets lacking αIIbβ3 are the most susceptible to form isoantibodies, a finding that may be useful in deciding the choice of therapy between platelet transfusion and the use of recombinant factor VIIa (FVIIa).

Founder effect and estimation of the age of the French Gypsy mutation associated with Glanzmann thrombasthenia in Manouche families

The c.1544+1G>A substitution at the 5′ splice donor site of intron 15 of the ITGA2B gene, called the French Gypsy mutation, causes Glanzmann thrombasthenia, an inherited hemorrhagic disorder transmitted as an autosomal recessive trait and characterized by an altered synthesis of the platelet αIIbβ3 integrin. So far, this mutation has only been found in affected individuals originating from French Manouche families, strongly suggesting a founder effect. Our goal was to investigate the origin of the French Gypsy mutation. We estimated the age of the mutation by a likelihood-based method that uses the length of the shared haplotypes among a set of patients. For this, we genotyped 23 individuals of Manouche origin; consisting of 9 Glanzmann thrombasthenia patients homozygous for the French Gypsy mutation, 6 heterozygous carriers and 8 homozygous wild-type individuals. They were genotyped for four single-nucleotide polymorphisms using high-resolution melting curve analysis, and for two CA repeats in the BRCA1 and THRA genes at chromosome 17, using fragment analysis gels. We found that a haplotype of five polymorphic loci covering a 4-cM region was strongly associated with the French Gypsy mutation, suggesting a founder effect. The estimated age of this founder mutation was 300–400 years (range 255–552 years). Thus, all carriers of the French Gypsy mutation c.1544+1G>A at intron 15 descended from a common ancestor 300–400 years ago.

Genetic testing in the diagnostic evaluation of inherited platelet disorders.

Inherited disorders of platelets give rise to rare bleeding syndromes through defects of platelet function and/or platelet production. Platelet function testing by biological and immunologic assays can identify the loss or abnormal functioning of specific receptor systems, signaling pathways, storage organelles, or enzymatic activities essential for adhesion, activation, and aggregation. In vitro culture of megakaryocytes can help identify the origin of familial thrombocytopenias, and electron microscopy can point to ultrastructural defects and giant platelet syndromes. But a full diagnosis can only be complete when the genetic defect has been defined for each patient. Glanzmann thrombasthenia (GT) and the Bernard-Soulier syndrome are the most studied of the membrane disorders and a wide range of mutations identified. We will use GT as an example to show how genetic studies can help understand the cell biology, pathophysiology, and management of a group of rare diseases where many of the genetic causes remain to be elucidated. Knowledge of the mutation in GT and whether it affects either of the ITGA2B or ITGB3 genes will be essential as we enter the period where accurate prenatal diagnosis and gene therapy may become viable options.

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 + progenitors

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.

Clinical utility gene card for: Glanzmann thrombasthenia

Glanzmann thrombasthenia (Glanzmann–Naegeli syndrome, platelet αIIbβ3 deficiency, platelet fibrinogen receptor deficiency). OMIM# of the disease 273800. Analyzed genes or DNA/chromosome segments ITGA2B (αIIb) and ITGB3 (β3) – chromosome 17q21.32. OMIM# of the gene(s) 607759 (ITGA2B) and 173470 (ITGB3). Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for mutations in diagnostic and prenatal settings, and for risk assessment in relatives.

A diagnostic approach that may help to discriminate inherited thrombocytopenia from chronic immune thrombocytopenia in adult patients

Abstract Inherited thrombocytopenia (IT) is a heterogeneous group of rare diseases that are often confused with immune thrombocytopenia (ITP). The objective of this study was to supply clinicobiological elements that allow a distinction to be drawn between IT and chronic ITP. We then compared 23 adult patients with IT and 9 patients with chronic ITP. Our study revealed six discriminating criteria: (i) an age of discovery <34 years: positive predictive value (PPV) = 88.2% [63.6; 98.5], (ii) a family history of thrombocytopenia: PPV = 100.0% [82.4; 100.0], (iii) a personal history of bleeding: PPV = 100% [76.8; 100.0], (iv) a mean platelet volume >11 fL: PPV = 93.3% [68.1; 99.8], (v) an excess of giant platelets on blood smear: 100.0% [76.8; 100.0], and (vi) a percentage >44% of platelets with a surface area >4 µm2 in electron microscopy: PPV = 83.3% [58.6; 96.4]. If at least three of these criteria were combined, it was possible to distinguish IT from chronic ITP with 91.3% [72.0; 98.9] sensitivity and PPV = 100.0% [66.4; 100.0] specificity. The secondary objective of this study was to assess the prevalence of potential IT diagnosis in patients with chronic thrombocytopenia of uncertain origin. Applying our diagnostic approach to a series of 20 cases allowed us to estimate that 40% of them could be suffering from IT. Finally, our diagnostic approach may help to correctly distinguish IT from chronic ITP, particularly in the context of macrothrombocytopenia.

Rapid diagnosis of the French gypsy mutation in Glanzmann thrombasthenia using high-resolution melting analysis

Rapid diagnosis of the French gypsy mutation in Glanzmann thrombasthenia using high-resolution melting analysis -