Diagnoses And Treatment Of Inherited And Autoimmune FX? Deficiency And Analysis Of The Molecular Mechanism

Objective: To establish a complete FX?D detection and treatment procedure and to analyze the molecular mechanism.Methods: The clinical bleeding manifestations and related family informations of 5 patients were recorded.FX?:Act was tested by the measurement of ammonia released during the transglutaminase reaction.FX?-A:Ag and FX?-B:Ag were tested by ELISA method.The clot solubility assay and mixing test was taken to preliminarily distinguish the inherited FX?D and AHX?.For inherited FX?D patients,all the exons including exon-intron boundaries of F13A1 and F13 B were amplified and directly sequenced.And CNVplex? method for F13A1 and F13 B was established to detect the CNVs.Breakpoints of the F13A1 large deletion were identified by the quantitative primer walking combined long-range PCR?LR-PCR?strategies,and the rearrangement mechanism was analyzed by bioinformatics analysis.For AHX? patients,Bethesda method was used to detect anti-FX? neutralizing antibody,and ELISA method was taken to confirm the autoantibodies against FX?-A.Results:For the 5 patients,positive results were showed in the qualitative method in urea.All the FX?:Act <5%,FX?-A:Ag <3%,and FX?-B:Ag was all in normal range.Two patients of them had bleeding manifestations since childhood.Proband 1 had experienced several severe bleeding,such as umbilical cord hemorrhage,ovarian corpus luteum rupture,and intracranial hemorrhage.Proband 2 had experienced muscle hematoma,hemarthrosis,splenic rupture,and rare spinal cord hematoma,and showed obvious performance of delayed would healing.The two both were born to non-consanguineous parents with no family history.Mixing test got negative results in the 2 patients.Proband 1 was found to be a compound heterozygote for a novel small deletion?c.1147delA?and a previously described missense mutation of Arg383 Ser.Proband 2 was compound heterozygous for a novel large deletion?g.[77815₁12815del;112837₁16628del]?and a previously reported Arg716 Gly mutation in F13A1.Bioinformatics analysis of the large deletion breakpoints predicted that two FoSTeS/MMBIR events with two homologies of TCT and C might be responsible for the complex rearrangement.The other three were elderly patients without bleeding history and related family history.The main bleeding manifestation of them was muscle hematoma.Two of the patients separately had complications of urticaria and herpes zoster,and the other one had no immune disease.Mixing test got positive results in the three patients,and Bethesda method revealed the neutralizing antibody separately at 1.83 BU,1.67 BU,and 1.75 BU.Autoantibodies against FX?-A were confirmed by immunological assay in the three patients.One of the three patients rejected treatment.His hematoma relapsed for three times,and he finally died from intracranial hemorrhage.The other two patients received FX? transfusion and immunotherapy,and no more severe bleeding events happened since then.Conclusions: The comprehensive diagnosis and treatment procedure for inherited FX?D and AHX? was established.2 patients were diagnosed with severe type I inherited FX?D,and the other 3 patients were diagnosed with AHX? caused by low titer autoantibodies against FX?-A.The prophylactic replacement therapy is effective for inherited FX?D patients to avoid severe bleeding events.AHX? could result to severe fatal bleeding,so FX? transfusion and immunotherapy was necessary.