Genetic Diagnosis And Molecular Pathogenesis Research Of Congenital Factor ? Deficiency And Study On The Procoagulant Property Of Microparticles Generated In Vitro

Prevalent in people of Jewish ancestry approaching 10%,congenital Factor XI(F?)deficiency is a rare bleeding disorder,yet statistics of morbidity rate in other races is not clear currently.Its bleeding manifestations range from absence of symptoms to injury/surgery-related bleedingwhereas F?activity(F?:C)fails to predict clinical bleeding severity,driving related treatment into controversies.Up to 220 mutations has been found till now according to the F?mutation database with E117*and F283L the most commonly found in Jewish.Interestingly,considerable genetic heterogeneity occurs in various ethnic groups.Our group aimed to study the spectrum of F11mutations in China by studying 57 unrelated Chinese patients and their family members diagnosed with F?deficiency.Following the direct sequencing of fifteen exons of F11,mutations wereidentified in respective patients.AccuCopy technique was applied to detect copy number variations(CNVs)in patients whose phenotypes failed to match genotypes.Particularly,mRNA transcripts werecharacterized by RT-PCR to carry out the possible pathological mechanism resulted from splicing mutations.57 successive pedigrees were enrolled in this study.A majority of propositi were detected at a laboratory screening prior to surgeries or routine check-up excluding any secondary deficiency.Among them,only three were diagnosed as partial deficiency(F?:C>20 IU/dL),while the other 54 were severe deficiency.Bleeding histories were only mentioned in 12 severe patients with the commonest incidences of easy bruising and bleedings from surgeries and minor wounds 5/57(8.8%),epistaxis 3/57(5.3%),tooth extraction 2/57(3.5%),menorrhagia 3/40(7.5%of women)and postpartum hemorrhage 1/40(2.5%of women).No direct associations were found between bleeding manifestations of patients and lab diagnosis.A total number of 37 mutations were identified in 57 patients.Of all the participants,17 were homozygotes,32were compound heterozygous individuals and 8 were heterozygotes.These 37 different sequence variants included 19 missense mutations,9 nonsense mutations,6 splice site mutations and 3 small deletions.CNV results turned to be normal in our tested patients.W228*(16 cases,14.0%)turned out to be the most frequent mutation,followed by G400V together with Q263*(14 cases,12.3%respectively)and c.1136-4delGTTG(11 cases,9.6%).Furthermore,13 novel mutationsincluding 4 missense mutations(C182S,R308H,W407L,W501L),a nonsense mutation(Q384*),5 splice site mutations(c.55+2T>C,c.326-1G>A,c.485+1G>A,c.596-8T>A,c.1480+3A>T)and 3 small deletions(c.327delT,c.1325delT,c.1448delT)were also identified.The pathogenesis of two splicing mutations(c.596-8T>A,c.1136-4delGTTG)were explicited through mRNA analysis.Defects in F11result in FXI deficiency with worldwide distribution and it remains a heterogeneous disease at the molecular level.Our study,for the first time,carried out the genetic diagnosis of F?deficiency in a large group,analyzed the relations between clinical phenotypes and genotypes and summarized the hotspot mutations in China.In silicoand in vitro analysis were also applied to elucidate the possible mechanisms of novel mutations.Microparticles,especially tissue factor positive microparticles(TF~+MP)have triggered rising attention recently in the arena of coagulation and thrombosis.Characterized by the diameter of 0.1 to 1?m,they are small membrane vesicles originated from activated or apoptotic cells that can be increased in various diseases and gained the procoagulant property.Hemophilia A(HA)is a sex-chromosomal recessive bleeding disorder resulted from the deficiency of coagulation factor?(F?).Replacement treatment is the first option currently,yet the development of the inhibitor it caused has also become the problem on the top of the agenda.The aim of the study was to explore the possible procoagulant property ofTF~+MP via stimulation of human acute monocytic leukemia cell line(THP-1 cells)by lipopolysaccharide(LPS)through various concentrations in vitro,setting foundations for the possible role of MPs in the treatment of HA,especially in HA with inhibitors.THP-1 cells were cultured in a steady state and treated with various concentrations of LPS,and MPs were separated by gradient centrifugation.Thrombin generation test was applied to measure the respective ability of TF and phosphatidylserine(PS)to generate the thrombin by adding MPs into MP-depleted plasma from normal donors.ZYMUPHEN MP-TF kit was performed to detect TF activity of MPs.0.45?m as well as0.65?m filters were selected to determine the procoagulant activity of distinct diameters(<0.45?m and<0.65?m).The results showed both thrombin generation assay and ZYMUPHEN kit by method of chromogenic substrate demonstrated a procoagulant activity of MPs generated in vitro.Thrombin generation test further showed a dose-dependent procoagulant activity increase with the addition of LPS.MPs that>0.65?m behaved stronger procoagulant activities.We therefore could carefully draw a conclusion that MPs generated by THP-1 cells through stimuli treatment by LPS in vitro demonstrated certain procoagulant property,which set a solid basis on the further study of the application of MPs in the treatment of HA.