Clinical And Laboratory Studies On Three Rare Bleeding Disorders

1.Study on the Clinical Features and Gene Diagnosis of phytosterolemia Causing Macrothrombocytopenia and StomatocytosisSitosterolemia?phytosterolemia?is a rare inherited sterol storage disorder,characterized by significantly elevated plasma levels of plant sterols.The clinical features of sitosterolemia are xanthomas,premature atherosclerosis,arthritis,and,occasionally,liver function impair and hematologic abnormalities.Giant platelet disorders?GPD?are a heterogeneous group of disease with large platelets and variable hemorrhagic phenomena.With the development of DNA sequencing technology,a group of GPD characterized by large platelets has been found to be caused by the abnormal metabolism of lipid known as Sitosterolemia.Recently,we identified 20 patients from 13 unrelated families who presented with large platelets,abnormal erythrocytes and splenomegaly,and were diagnosis as Sitosterolemia by measuring the plasma levels of phytosterols and sequencing the ABCG5 or ABCG8 genes.Investigation of clinical characteristics of Sitosterolemia would be essential to recognize this disorder for doctors.Objective: To make an exact diagnosis and investigate the clinical features of Sitosterolemia.Methods: Patients were collected to perform the routine detection.Erythrocyte and platelet morphology was examined under light microscope;Platelet glycoproteins?GP??Ib/IX and IIb/IIIa?were analyzed using flow cytometry;Platelet aggregation tests were performed by using aggregometer;Plasma phytosterols were measured by high pressure/performance liquid chromatography?HPLC?method;All of ABCG5 and ABCG8 exons and intron-exon boundaries were amplified by PCR and directly sequenced to identify mutations,and to exclude the probability of SNP through SNP data base in NCBI.Results:60% of the patients were female,and 40% male.The mean age of diagnosis was 47?23-67?years old.The reason of 85% patients for medical consultation was thrombocytopenia.All of the patients had a history of splenectomy for splenomegaly,and the mean age of operation was 26.72 years old.90% of the patients presented with xanthomas,25% with premature atherosclerosis,20% with arthritis,and 45% with impaired liver function.The peripheral blood films showed some unique features: large platelets and various abnormal erythrocyte shapes,especially stomatocyte.The patients' erythrocytes showed an increased osmotic fragility with hemolysis beginning at an average concentrations of 0.572 % of saline solution?normal range 0.44-0.48%?.Serum levels of the sitosterol and stigmasterol of the patients were remarkably elevated up to 455.54±227.00 mg/L and 298.50±122.67 mg/L,which were about 15 and 30 times higher than those of normal subjects,respectively.14 gene mutations were found in the patiens and 2/3 mutation were located on ABCG5 gene.The p.?R446Ter?mution was found in the 3 families.Conclusions:1.In various clinical manifestations of our patients,the most prominent features were macrothrombocytopenia,stomatocytic anemia and splenomegaly;xanthomas are most often seen in eyelips and developed with age.2.Sitosterolemia was certainly not as rare as originally thought and the phenomena of macrothrombocytopenia/hemolysis /plenomegaly might represent a special type of Sitosterolemia.2.Clinical characters in a family of Gray platelet syndrome caused by NBEAL2 gene mutationObjective: To investigate clinical and pathological features in a family of gray platelet syndrome.Methods: A gray platelet syndrome family was studied,including clinical presentation,platelet count,platelet aggregation tests,fluorescence intensity of glycoprotein in platelet membrance,optical and transmission electron microscopy,exome sequencing analyses and DNA sanger analysis.Results: The proband presented with severe menometrorrhagia,macrothrombocytopenia,absence of ?-granules,lower responses to agonists,normal glycoprotein of platelet,and gene analysis showed a novel C27713 A mutation in Nbeal2 gene leading to a premature protein in 1726 amino acid residents.The carriers had only large platlets.Conclusion:1.A novel NBEAL2?g.NG₀31914.1?g.27713C>A mutation in NBEAL2 was responsible for the gray platelet syndrome.Our data extend the clinical manifestations and the spectrum of mutations responsible for gray platelet syndrome.2.The production of platelets were slightly affected in the carriers of NBEAL2?g.NG₀31914.1?g.27713C>A mutation.3.Clinical characteristics of two female patients with ?1-antitrypsin PittsburghObjective: To identify clinical and laboratory abnormalities of two patients with ?1-antitrypsin??1-AT?Pittsburgh in a family and review the literatures.Methods: Both plasma clotting time and factor activities were performed using clotting or substrate methods.Platelet aggregation was evaluated using an optical aggregometer.The serum protein electrophoresis was performed on Sebia HYDRASYS by using Agarose gel.The exons of ?1-AT were amplified by using polymerase chain reaction?PCR?and then sequenced and blasted with NCBI gene bank.Results: The proband suffered from everal ruptures of corpus luteum and bleeding after operation,while her daughter had no bleeding history.Both of them showed prolonged coagulation tests which could not be corrected by mixing with the normal plasma.They also showed low levels of plasma coagulation factors,undetected protein C and S activity and abnormal bands of ?1-globulin.The results of gene sequencing demonstrated that they are heterozygous for Met358 to Arg mutation of ?1-antitrypsin gene.Conclusions:1 Comparing with the data of previously described cases,our results confirm the obvious abnormality of coagulation tests and the discrepancy of bleeding tendency of ?1-antitrypsin Pittsburgh patients,and suggest that the rupture of corpus luteum would be a specific characteristic in women of child-bearing age.2 The scanning of serum electrophoresis displayed a very thin band of the 1-globulin where the A1 AT variant had been identified and an abnormal trace following the albumin band that showed to be proalbumin.