| With the tremendous advances including the human genome sequence mapcompletion and molecular biology in hemostasis and thrombosis research, almost all genesof the blood coagulation factors and platelet membrane glycoproteins and other ingredientshave been cloned, with its structure and function relationship being greatly clarified. Manyhereditary bleeding disorder gene abnormalities have been found, genetic diagnosis hasalso become the main direction of the future development of hereditary hemorrhagicdisease research. Congenital afibrinogenemia is a rare autosomal recessive disease,50%occurring in the consanguineous marriage pedigrees, the incidence rate is about onemillionth. Umbilical cord bleeding is often the first symptom, followed by subcutaneoushemorrhage, epistaxis, hematuria, women can present excessive menstrual blood, andintracranial bleeding is its primary cause of death. Bernard-Soulier syndrome (BSS), a rarecongenital platelet disease is autosomal recessive, mainly due to the defects of plateletmembrane GPIb, GPIX and GPV. Prolonged bleeding time, reduced platelet count andlarge platelet size are its characteristics. This study is divided into two parts:Part1: Congenital afibrinogenemia caused by a novel insertionmutation in the FGB geneTo investigate the genetic defect and its mechanism in a congenital afibrinogenemiapatient with kin marriage of his parents, phenotypic diagnosis, gene analysis, functionaltesting and in vitro cell experiments were performed to explore the molecularpathogenesis.The plasma fibrinogen concentration of the patient was determined using both theClauss method and immuno-nephelometric assay. Genomic DNA was isolated fromperipheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCRfollowed by direct sequencing. Thrombin fibrin aggregation curve were detected in theplasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed,and then transfected into COS-7cells. The wild-type and mutant proteins from the culturemedia and cell lysates were tested by Western blot method and ELISA method. The resultsshowed that APTT﹥200s, PT﹥100s, TT﹥100s in the proband. Plasma fibrinogen couldnot be detected using the Clauss method and immuno-nephelometry, the proband’father,mother, younger brother and son showed plasma slightly lower fibrinogen activity than thenormal control. Gene analysis revealed that a novel homozygous GTTT insertion betweennucleotides2833and2834in FGB exon2in the proband. The proband’s father, mother,brother and son were heterozygous. The polymerization curves of the patient did not showa lag phase or final turbidity, compared with the normal controls. Western blot analysisshowed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen inpatient’s plasma under non-reducing conditions. It also could not detect the truncated Bβchain under reducing conditions.Abnormal fibrinogen molecule (molecule weight﹥340KDa) were found intransfected mutant COS-7cells by Western blot, which indicated that the mutation causethe abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absentin culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen werecomparable with those of wild-type fibrinogen in cell lysates by ELISA analysis. However,the levels of the mutant fibrinogen were significantly lower than those of wild typefibrinogen in culture media. Congenital afibrinogenemia was caused by this frameshiftmutation in exon2of FGB. This mutation is a novel mutation which has not been reportedbefore. The mutation causes the fibrinogen abnormal molecular assembly and secretion,but did not affect the intracellular synthesis.Part2: Studying molecular biology on glycoprotein Ibα genedefect in Bernard-Soulier syndromeMolecular biology methods was used to confirm the GPIbα genetic c.1444del A as acause of BSS, and to investigate the significance of the mutation in the pathogenesis ofBSS. The blood samples of patient and his parents were collected to measure routine bloodbleeding time, ADP, collagen, ristocetin and epinephrine-induced platelet aggregation. They were also underwent bone marrow aspirate examination. PCR amplification of thefull-length coding of the three genes GPIbα, GPIbβ and GPIX, PCR products were directlysubjected to DNA sequencing analysis. Flow cytometry tested the platelet GPIb and GPIXpositive percentage of patient and control. Western blot was used to detect patient andcontrol platelet lysates. GPIbα mutant plasmid was constructed. The plasmids containingthe GPIbα, GPIbβ with GPIX wild-type or GPIbα mutant full-length coding sequence weretransfected into Chinese hamster ovary (CHO) cells. Transfected CHO GPIb-GPIXcytoplasmic expressions were analyzed using Western blot. Patient exhibited heavybleeding since childhood, usually required frequent platelet transfusions for hemostasis.The proband’s parents were first cousin, and his sister died of serious bleeding at the age offour. Examinations showed thrombocytopenia and giant platelets in peripheral blood smear.Bleeding time was more than20minutes. Platelet aggregation were able to be induced byADP, collagen, and epinephrine-induced, but not by ristocetin. Coagulation test wasnormal and bone marrow megakaryocytes were in the normal range. Tested by GPIb,GPIX and GPV gene analysis, the proband had homozygous frameshift mutation of GPIbαgene (c.1444del A), which produced early termination of amino acid sequence in510(p.Thr452ProfsX58) location. The father and mother of the proband were heterozygous.The percentage and fluorescence intensity of GPIbα was significantly decreased onpatient’s platelet membrane by flowcytometry. Expression of GPIb-GPIX complex inmutant CHO cells were absent in the cytoplasma. Our results indicated the mutation of theGPIbα gene(c.1444del A) was the cause of the BSS patient, which affected the synthesisand assembly of the GPIbα-GPIX complex. |
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