Clinical Heterogeneity Analysis Of Glanzmann's Thrombasthenia And A Pilot Study Of Gene Editing

Chapter? Clinical and genetic characteristics of 97 patients with Glanzmann's thrombasthenia in the Chinese Han populationGlanzmann's thrombasthenia(GT)is a rare bleeding disorder characterized by spontaneous mucocutaneous bleeding and abnormally prolonged bleeding in response to injure and trauma.It is caused by quantitative or qualitative defects in integrin ?IIb?3(encoded by ITGA2 B and ITGB3)on the platelet membrane.The disorder is more common in consanguineous populations.We analyzed 97 patients from 93 families with GT in the Han population in China.This analysis showed a lower consanguinity(18.3%)in Han patients than other ethnic populations in GT-prone countries.Compared with other ethnic populations,there is no significant difference in the distribution of GT types.Han females suffered more severe bleeding and had a poorer prognosis,some Han patients(25.2%)was less severe with age,and the overall mortality rate was higher.We identified a total of 43 different ITGA2 B and ITGB3 variants in 45 patients,of which 25 were novel.These variants included 14 missense,four nonsense,four frameshift and three splicing site variants.Among them,11 were “pathogenic variants”,and 14 were “likely pathogenic variants”.Fifteen patients were homozygotes,twenty-one were compound heterozygotes while seven patients were heterozygous,and two were not identified mutations in ITGA2 B and ITGB3.We identified c.1750C>T(p.R584X),c.2671C>T(p.Q891X),c.2333 A > C(p.Q778P)in ITGA2 B and c.1199G>A(p.C400Y)in ITGB3 in 7,4,7 and 2 unrelated families,respectively.Patients with the same genotype generally manifested the same GT type but presented with different bleeding severities.This suggests that GT clinical phenotype is not solely dependent on genotype.Our study provides an initial yet important clinical and molecular characterization to understand the disease heterogeneity in GT in the Chinese Han population.Chapter ? Molecular analysis of heterogeneity of Glanzmann's thrombastheniaIn order to further explore the molecular mechanisms of the heterogeneity of Glanzmann's thrombasthenia,a Glanzmann's thrombasthenia patient with unique bleeding were selected for further study.GT39 is a 25-years-old male who had only one bleeding episode in his life.He was identified carrying a compound heterozygous variants c.2570G>T(p.C857F)and c.1769G>A(p.R590Q)in ITGA2 B.Experiment in vitro showed that c.2570G>T(p.C857F)and c.1769G>A(p.R590Q)plasmids decreased the expression of ?IIb moderately,but deprived its function completely,indicating that these two variants were pathogenic mutations of GT39.The mechanism may be involved in destroying the C857-C911 disulfide bond and changing the polarity of the amino acid at the 590 site of ?IIb.This case suggest that the heterogeneity of hemorrhage in Glanzmann's thrombasthenia is not only related to the different mutations on ITGA2 B and ITGB3,but also to other factors.Chapter ? Genetic analysis of three pedigrees of Glanzmann's thrombasthenia with nonsense mutationNonsense mutation in ITGA2 B or ITGB3 gene is a common cause of Glanzmann's thrombasthenia.Patients are often type I GT,whose ?IIb?3 is significantly reduced on the surface of platelet.However,the bleeding manifestations of these patients are heterogeneous.In order to further explore the pathogenesis of Glanzmann's thrombasthenia with nonsense mutation and explore the causes for its heterogeneity,we studied three pedigrees of Glanzmann's thrombasthenia with nonsense mutations.We analyzed their DNA,c DNA,total ?IIb protein and surface ?IIb protein expression.Our results showed that there were two pathogenic mechanisms in Glanzmann's thrombasthenia with nonsense mutation.One is nonsense mediated m RNA degradation(NMD)and the other is nonsense-associated altered splicing(NAS).These two ways existed simultaneously.As a result,inhibiting NMD or NAS may be a potential therapeutic approach for Glanzmann's thrombasthenia caused by nonsense mutation.Our research provided a theoretical basis for the precise treatment of Glanzmann's thrombasthenia.A pilot study: gene editing in Glanzmann's thrombastheniaTo explore the gene therapy for Glanzmann's thrombasthenia,we used CRISPR/Cas9 technique to establish a nonsense mutation model of GT in Meg01 cell line.We designed guide RNA,constructed plasmid,transformed the plasmid into DH5?,and then transfected the plasmid into Meg01 cells.DNA sequencing analysis showed that we identified an effective guide RNA to specifically break ITGA2 B gene into double stranded DNA.