RESUMEN

Objective: The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored. Methods: The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (ß3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and ß3 in platelets were analyzed by Western blot. Results: Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and ß3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5'SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the ß-propeller domain of the p.S160-S192 deletion lost two ß-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a ß chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of ß3 was 11.36% of the normal level. Conclusion: The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.

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RESUMEN

Glanzmann Thrombasthenia (GT) is an inherited platelet disorder caused by defects in platelet integrin αIIbß3 (GPIIb/IIIa), which is a platelet receptor essential for the binding of fibrinogen. This can lead to severe bleeding, especially after trauma or perioperatively, and to microcytic anemia because of chronic blood loss. We report on a 40-year-old female patient with extensive bleeding complications and platelet antibody formation who presented in Homburg and Freiburg for extensive platelet function analyses and molecular genetic analyses. According to platelet aggregometry, the patient had previously been diagnosed with Glanzmann Thrombasthenia (GT). In addition, an MRI scan had been performed due to an unsteady gait and had revealed bilateral para-ophthalmic aneurysms of both internal carotid arteries (ICAs). Assuming a 5% rupture risk per 5 years for each aneurysm, the patient was offered and accepted endovascular treatment. Next-generation sequencing (NGS) panel analysis identified a previously undescribed homozygous one-base-pair deletion in ITGA2B, which leads to a loss of function of the αIIb-subunit of the receptor. This case illustrates the difficulties that can arise regarding the treatment of patients with rare platelet bleeding disorders, and supports the importance of continuous medical care by a specialized hemophilia center for these patients.

RESUMEN

We recently reported mutation analysis of the largest cohort of Glanzmann thrombasthenia (GT) patients so far examined. Sanger sequencing of coding regions, splice sites, upstream and downstream regions of the ITGA2B and ITGB3 genes identified 78 causal genetic variants (55 novel); 4 large deletions or duplications were also detected. We have now analysed the expression of non-causal gene polymorphisms in the sequenced regions of both genes in selected members of this cohort. We identified 10 mostly silent variants in ITGA2B and 37 in ITGB3; all were present in control donor databases. Three non-synonymous single nucleotide polymorphisms present were human platelet alloantigen (HPA) variants. A series of haplogroups, often including HPA-3b in ITGA2B, repeated with little variation across unrelated families of wide geographical origins and with different GT-causing mutations whether in ITGA2B or ITGB3. In contrast, a deleterious heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B shared a common ITGA2B haplogroup composed of at least five gene polymorphisms and re-occurred in seven European families with no known family relationships. Our results highlight the value of gene polymorphism analysis in GT and are consistent with the bulk of disease-causing mutations in GT being of recent origin.

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