Molecular Mechanism Of Three Cases Of Congenital Factor X? Deficiency

Objective:Our goal was to analyze the clinical and laboratory characteristics of three patients with congenital factor X? deficiency,identify genetic mutations,and explore the molecular mechanism of coagulation factor X? deficiency.Methods:1.Factor X? activity was measured by a Fluorescent Factor X? assay Kit on a Varioskan? Flash Multimode Reader.2.The protein levels of FX? A2B2,FX?-A,and FX?-B in plasma were detected by ELISA and Western blot.3.All exons and intron-exon boundaries of FX?-A were amplified by PCR and directly sequenced to detect mutations.4.Construct FX?-A mutant plasmids by site-directed mutagenesis.5.The wild-type and mutant FX?-A plasmids were transiently transfected into COS7 cells.The m RNA expression and protein levels of FX?-A were detected separately by RT-PCR or Western blot in supernatants and cell lysates after transfections.6.Confocal immunofluorescence microscopy was used for intracellular localization of FX?-A,and the co-localization coefficient and the Pearson's correlation coefficient were calculated in the ER and Golgi.Results:1.Plasma FX? activities were significantly decreased in three patients FX? deficiency compare to healthy controls.2.The FX?A2B2 antigen levels in three patients were lower than that of healthy control as measured by ELISA.Patients' FX?-B bands were the same as the normal plasma,but their FX?-A bands were almost undetectable.3.Five mutations were detected in three patients by PCR.Patient 1:c.G688 C and c.A2273 G respectively caused W188 C and H374 R.Patient 2: c.A1798 C,c.del723-6 AGAA resulted in T559 P,R202 Fs 206 ter,respectively.Patient 3: c.T1944 C leads to H717 R.4.Four missense mutations(W188C,H374 R,T559P and H717R)were constructed by site-directed mutagenesis and large primer method respectively.These construts were verifiyed by DNA sequencing.5.The m RNA levels of FX?-A of four constructs were normal in transfected COS7 cells.Western blot results showed that the FX?-A protein was expressed in both the wild-type and the mutants in cell lysates,but decreased modestly.However,in the concentrated supernatant,only the wild-type FX?-A protein but not FX?-A munat was expressed.6.Confocal immunofluorescence microscopy showed the expression of FX?-A WT and mutants were normal,and most of which stayed in the ER.The co-localization coefficient in the ER were calculated and showed no difference between WT and variants.Howerever,in the Golgi the co-localization coefficient were decreased in variants compared to WT.Conclusion:1.Three bleeding patients caused by absence of FX?-A protein in plasma were confirmed as type I FX?-A deficiency.Genetic analysis identified W188 C,H374R,T559 P,H717R and R202 Fs 206 ter mutations in FX?-A.2.Confocal analysis suggest that the mutations of W188 C,H374R,T559 P,and H717 R result in a defective transport of FX?-A into the Golgi,and as a result,the secretion of FX?-A was blocked,leading to decreased levels of FX?-A in plasma.