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(α Ⅱ b),CD61(β3),and CD42b(GP Ⅰ b)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 α Ⅱ b and β3 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 α Ⅱ b and β3 were only 0.25％and 9.76％,respectively,on the platelet surface of the proband,whereas GP Ⅰ b 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.S1 60-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 α Ⅱ b subunit showed that the β-propeller domain of the p.S 160-S192 deletion lost two β-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 β chain of the extracellular Calf-2 domain.The total α Ⅱ b expression of the proband was absent,and the relative expression of β3 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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