Gene Mutations Detection And Molecular Genetic Analysis Of Glanzmann's Thrombasthenia

Introduction:Glanzmann's thrombasthenia(GT)is a rare inherited bleeding disease,incidence being approximately one in one million worldwide.GT is characterized by the impaired platelet aggregation to multiple physiologic agonists such as collagen,adenosine diphosphate(ADP),arachidonic acid(AA),but normal reaction to ristocetin.Platelet counts and sizes are within normal range in most patients.Patients presented with bleeding of varing severity,sometimes life-threatening.Bleeding severity varies from person to person,Bleeding symptoms of a single patient could even alter with age.The ITGA2B and ITGB3 gene,located at chromosome 17q21.31-32 closely,encode for ? ?b and ? 3 unit respectively.Pathogenic variants of a ?b or ? 3 could cause the disease.The database of gene mutations is continuously updated on the Internet(http://www.hgmd.org,accessed 2020.4.6);it lists a total of 238 variants of the ITGA2B gene and 175 variants of the ITGB3 gene.Objective:To analyze clinical and laboratory characteristics of GT patients,compare bleeding severity between different groups(based on gender,age and type of GT disease).And to investigate the gene mutations and founder effect of two variants in Chinese GT patients.Methods:In total,102 patients were recruited in our study.Information on baseline characteristics,laboratory tests,bleeding sites and severity were recorded.The severity of bleeding was evaluated by the WHO bleeding scale.The bleeding scores were compared between groups divided according to gender,age and type of GT.Next generation sequencing(NGS)was performed using a custom-made panel for the bleeding and platelet disorders.Prediction of variants were made using bioinformatics tools.The founder effect was assessed in patients with two recurrent mutations using haplotype analysis.Results:Most patients presented with moderate-to-severe bleeding symptoms according to World Health Organization(WHO)bleeding scale,and the median age at diagnosis is 5 years(IQR,2-9 years).The mean bleeding score in female patients is significantly higher than that of male patients(3.21 vs 2.59,p<0.001).There is no significant difference in bleeding scores between each types.Fifty-six different mutations were detected(ITGA2B:n=35;ITGB3:n=21),including 29 missense,11 nonsense,7 splice site and 9 frameshift mutations.Among 56 detected mutations,there were 18 novel pathogenic mutations,12 in ITGA2B(c.2444₂445del,c.2915dupC,c.2758₂759delCA,c.1229C>G,c.2896dupC,c.432G>A,c.454G>A,c.989A>T,c.338₃56del,c.1622T>A,c.1210+1G>A,c.2452₂454del)and 6 in ITGB3(c.361+1G>A,c.1690G>C,c.756delC,c.1288C>T,c.1985G>A,c.1684T>G).Haplotype analysis shows ITGA2B c.2333A>C locates in a strong linkage disequilibrium zone.Conclusion:The clinical manifestations of patients with GT were heterogeneous,and bleeding symptoms varied from person to person.There is no significant difference in the severity of bleeding between different types of GT patients.In total,56 different variants were detected in ITGA2B and ITGB3,18 of which were newly identified.Theses novel findings expand the GT mutation spectrum.The haplotype analysis of several patients with ITGA2B c.2333A>C indicated that founder effect might be involved.