Investigation On The Molecular Pathogenesis Of Inherited FⅫ Deficiency And Two Other Platelet Associate Diseases

Five Chinese pedigrees with congenital factor Ⅻ(FⅫ) deficiency were enrolled in the present study, and studies on the clinical manifestations, family survey, biochemical examinations and gene diagnosis of these pedigrees were performed. Three mutations(c.776G>A, c.799C>G, c.1561G>A) of the F12 gene were identified in the five pedigrees. To figure out the pathogenic mechanisms of the three mutations, the structure and function of three FⅫ mutants were studied in this study. In addition, in the present study we also explored the underlying mechanism of the regulatory effects of the GPIbβ intracellular domain on GPIbα shedding, as well as the gene diagnosis of a patient with refractory ITP with the application of whole exome sequencing.FⅫ is the initiation factor of endogenous coagulation system. Congenital FⅫ deficiency is a rare, autosomal recessive coagulation disease caused by mutations in the F12 gene. The clinical manifestations of FⅫ deficiency are asymptomatic, and these patients were always dignosed due to a prolonged activated partial thromboplastin time(APTT) during normal cogulation tests. The incidence rate of FⅫ deficiency is not clear. In the present study, we have investigated the underlying mechanism of the FⅫ deficiency phenotype in these pedigrees. All five patients had prolonged APTT, as well as markedly decreased FⅫ activity and antigen level. Gene analysis identified three mutations [c.776G>A(p. G259E), c.799G>C(p. R267G) and c.1561G>A(p. E521K)] in the F12 gene of the five patients, two of whom were with heterozygous mutations(R267RG and E521 EK, respectively). c.776G>A(p. G259E) and c.799G>C(p. R267G) are two novel missense mutations causing FⅫ deficiency. In vitro studies in transiently transfected HEK 293 T cells demonstrated that these mutations significantly lowered the FⅫ levels in the culture media, suggesting that these mutations were the causing reasons for the FⅫ deficiency in these patients. Results from real-time PCR showed that these mutations did not impact on the transcription level of FⅫ proteins; further protein degradation inhibition experimentswith various inhibitors suggested that the three mutants were degraded intracellularly through the proteasome pathway in the pre-Golgi compartment,resulting in the significant decrease of FⅫ proteins.Moreover,R267G mutation exhibited dominant negative effects leading to FⅫ deficiency,as evident by the decreased wild type FⅫ level when co-transfected with mutant FⅫ,consistent with the phenotypes in the heterozygous carriers,but such dominant negative effects were not due to the dimerization of FⅫ.The GPIb-IX-V complex is receptor protein involved the first step of platelet activation under high shear stress, and plays important roles in haemostasis and thrombosis. GPIbα is the most important subunit in the GPIb-IX-V complex. Its ectodomain shedding plays a negative regulatory role in the process of thrombosis, and is also pivotal in the platelet storage and clearance. However, the underlying regulatory mechanism of GPIbα shedding is not clear. Our previous studies showed that the membrane-proximal region of the GPIbβ cytoplasmic domain could inhibit GPIbα shedding by interacting with an unknown protein. Therefore, to elucidate the unknown protein, in the present study, we have expressed and purified wild type or key mutant(R151E/R152 E or R149E/L150E) GPIbβ cytoplamsic domain in E. coli, and pull-down experiment in combination with MS were used to identify the proteins in the platelet lysates that could interact with wild type but not mutant GPIbβ. Our results showed that several proteins, including moesin and thrombospondin, could ben pulled down only by the wild type GPIbβ, but the specific mechanism of the interactions between these proteins with GPIbβ requires further investigation.Immune thrombocytopenic purpura(ITP) is an autoimmune disease in which antiplatelet antibodies accelerate the destruction of platelets. In addition, platelet production can be impaired. Clinical manifestations include mucocutaneous or visceral hemorrhage. In this study, we found a case of refractory ITP, who is ineffective with the treatment of glucocorticoid. In order to investigate the pathogenic mechanism of the drug resistance, we performed whole exome sequencing of the patient’s both peripheral blood DNA and bone marrow DNA, find only exist in the bone marrow DNA in peripheral blood in DNA negative variation. With bioinformatics analysis, a set of candidate pathogenic genes was selected. Further verification by Sanger sequencing, we hypothesized that the S240 SF mutation in Ct BP2, which might result in the defect of megakaryocyte maturation, might be the causing reason of the thrombocytopenia of the patient.