Pharmacokinetic Studies of Factor VIII in Chinese Boys with Severe Hemophilia A: A Single-Center Study / 中华医学杂志(英文版)

<p><b>Background</b>Although much attention has been paid to the pharmacokinetics (PKs) of different factor VIII (FVIII) concentrates in persons with hemophilia A (HA), limited information is available in young boys with severe HA. In this study, we aimed to assess the PK parameters of FVIII products in boys with severe HA in China.</p><p><b>Methods</b>A total of 36 boys (plasma-derived [pd]-FVIII, n = 15; recombinant [r] FVIII, n = 21) were enrolled between January 2015 and May 2016 in Beijing Children's Hospital. PK characteristics of FVIII products were studied according to a reduced 4-sampling time point design (1 h, 9 h, 24 h, and 48 h postinfusion).</p><p><b>Results</b>The mean FVIII half-life (t) was 10.99 ± 3.45 h (range 5.52-20.02 h), the mean in vivo recovery (IVR) was 2.01 ± 0.42 IU/dl per IU/kg (range 1.24-3.02 IU/dl per IU/kg) and mean clearance (CL) of FVIII is 4.34 ± 1.58 ml·kg·h (range 2.29-7.90 ml·kg·h). We also analyzed the influence of several parameters that potentially modulate FVIII PK. The age was closely associated with FVIII half-life (R = 0.32, P < 0.01). The tof FVIII increased by 0.59 h per year. Besides age, von Willebrand factor antigen (VWF:Ag) also was associated with FVIII half-life (R = 0.52, P < 0.01). Patients with blood Group O had a shorter FVIII half-life than patients with non-O blood group (9.40 ± 0.68 h vs. 12.3 ± 0.79 h, t = 2.70, P = 0.01). The FVIII IVR correlated with age (R = 0.21, P < 0.01) and VWF:Ag level (R = 0.28, P < 0.01). CL rates were faster in young patients and in those with low-VWF:Ag levels. CL rates of FVIII are higher in blood Group O versus non-blood Group O persons (5.02 ± 0.38 vs. 4.00 ± 0.32 ml·kg·h, t = 2.53, P = 0.02).</p><p><b>Conclusions</b>Chinese boys with severe HA have similar PK values to other ethnic groups and large differences in FVIII PK between individual patients. Age, blood group, and VWF:Ag levels are important determining factors for FVIII CL.</p>

Correlation of Thrombosis and Prothrombotic State with Coagulation Factor V Gene Polymorphism and APCR. HHcy / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of patients with thrombosis or prothrombotic status with hyperhomocysteinemia (HHcy), activated protein C-resistance(APCR) and gene polymorphism of coagulation factor V.</p><p><b>METHODS</b>Three hundred healthy voluteers were selected as controls, 223 cases of thrombosis (80 cases of cerebral infarction of CT, the MI of 82 cases of myocardial infarction, venous thrombosis of VTE 61 cases), 270 cases of patients with prothrombotic state (76 cases of pregnancy disease of PIH, 62 cases of chronic obstructive pulmonary disease (COPD), 60 cases of diabetes(DM) and 72 cases of cancer) were enrolled in this study. The plasma APCR and hyperhomocysteinemia were detected by APTT coagulation method and cycling enzyme method respectively, and restriction fragment length polymorphism(RFLP) were was used to detect the gene polymorphism of FV G1691-A, G1091-C and A1090-G in the patient and control groups.</p><p><b>RESULTS</b>APCR positive rate was 62.29% and 7.33%, and the positive hyperhomocysteinemia accounted for 68.42% and 10.00% respectively in the group of the patients with venous thrombosis and the normal control group. 3 cases of heterozygous FV gene mutations were found in the APCR-positive patients with venous thrombosis.</p><p><b>CONCLUSION</b>HHcy possitive rate of patients with venous thrombosis is signiticantly higher than that in control, the HHcy is one of the important causes resulting in thrombosis, the patients with venous thrombosis have proved to be with APCR, and the possitive APCR may be related with the coagulation factor V gene polymorphism.</p>

The binding mechanisms of F VIII Trp1707Ser mutation-associated inhibitor / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the binding mechanisms of FVIII Trp1707Ser mutation-associated inhibitor.</p><p><b>METHODS</b>The APPT, PT, TT, Fg and FVIII:C were detected to make phenotypic diagnosis of haemophilia A. Inhibitors titer were measured by Bethesda method. Long distance-PCR (LD-PCR) and sequence-specific PCR were adopted for screening the intron 22 and intron 1 inversions respectively. FVIII coding and boundary sequences were analyzed by direct DNA sequencing. Inhibitor was reacted with different segments of FVIII, including heavy chain and its components A1 and A2, light chain and its components A3, C1 and C2. Corrected test was used to measure the remaining F VIII:C (% ) by adding pooled normal plasmas. After labeling purified inhibitors with biotin, western blot was performed to further confirm the binding reactions between inhibitors and segments.</p><p><b>RESULTS</b>The haemophilia A patient had mild deficiency of FVIII:C (1.1%) and had high FVIII inhibitor titer of 18.4 BU. A mutation c.97223C>G in exon 14 of F8 gene resulted to p.Trp1707Ser was identified by DNA sequencing. Corrected test showed that the remaining F VIII:C was increased when inhibitors reacted with heavy chain and light chain, especially with heavy chain. The remaining FVIII:C was also increased in the A2 and C2 domain reactions. No significant differences were seen in the A1, A3 and C1 domain reactions. Antigen-antibody reaction bands were confirmed by western blots when degenerated B-domain deleted recombinant FVIII, A2 and C2 were used as antigens.</p><p><b>CONCLUSION</b>The binding sites of FVIIITrp1707Ser mutation inhibitor were the A2 domain of heavy chain and C2 domain of light chain. The binding reaction with heavy chain was more intense.</p>

Functional study of abnormal fibrinogen caused by Arg275His mutation in fibrinogen γ chain / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees.</p><p><b>METHODS</b>Routine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity.</p><p><b>RESULTS</b>The coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876T>C (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time.</p><p><b>CONCLUSION</b>We first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.</p>

Genotype and function analyses of four inherited dysfibrinogenemia pedigree caused by Arg16 amino acid substitution in fibrinogen Aα chain / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype, genotype and function in four Chinese pedigrees with inherited dysfibrinogenemia.</p><p><b>METHODS</b>Routing tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), the activities of antithrombin (AT), protein C (PC) and protein S (PS) were detected in four pedigrees. The activity and antigen of plasma fibrinogen were analyzed by Clauss and immunoturbidimetry methods, respectively. The molecular weight of fibrinogen of four probands was assessed by Western blot. The function of abnormal fibrinogen was evaluated by fibrinogen clottability, fibrinogen dynamic polymerization and fibrinolysis velocity, respectively. The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>Four probands had prolonged TT and RT, reduced plasma fibrinogen activity levels and normal antigen levels. The assays of Western blot showed no abnormal molecular weight of fibrinogen. Function tests revealed reduced fibrinogen clottability, delayed and decreased fibrinogen dynamic polymerization and reduced fibrinolysis velocity. Aα chain Arg16His and Arg16Cys mutations were identified in the four probands, respectively.</p><p><b>CONCLUSION</b>The four probands with dysfibrinogenemia were caused by the mutations of Aα chain Arg16His or Arg16Cys. Mutation of the fibrinogen induced dysfunction of plasma fibrinogen.</p>

Phenotype and genotype analysis of two Chinese pedigrees with type 3 von Willebrand diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype of two Chinese pedigrees with von Willebrand diseases, and to investigate the molecular pathogenesis.</p><p><b>METHODS</b>Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor-ristocetin cofactor (vWF:Rco), von Willebrand factor antigen (vWF:Ag), von Willebrand factor activity (vWF:A), von Willebrand factor collagen binding assay (vWF:CB) and multimer analysis were used for phenotype diagnosis. DNA was extracted. All of the 52 exons and exon-intron bounda ries of the VWF gene were amplified with polymerase chain reaction(PCR) and analyzed by direct sequencing.</p><p><b>RESULTS</b>APTT and BT were prolonged. Plasma RIPA, vWF:Rco, vWF:Ag, vWF:A and vWF:CB was significantly decreased. No VWF multimer can be found by plasma VWF multimer analysis. Homozygous insertional mutation g.82888_82889insCATG in exon 17 was found in proband A. Compound heterozygous mutations g.94865 G to A (Trp856stop) in exon 20 and g.110698_110699delinsG in exon 28 were found in proband B.</p><p><b>CONCLUSION</b>Homozygous insertional mutation g.82888_82889insCATG and compound heterozygous mutations g.94865G to A(Trp856X) and g.110698_110699delinsG probably have respectively induced type 3 von Willebrand diseases in the two probands.</p>

The activity levels and prevalence of deficiency of protein C, protein S and antithrombin in Chinese Han population / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the distribution and influence factors of protein C (PC), protein S (PS) and antithrombin (AT) activities and to determine the prevalence of their deficiencies in the Chinese Han healthy population.</p><p><b>METHODS</b>Healthy volunteers including blood donors and individuals for routine check-up were recruited from 4 Chinese medical centers. The plasma levels of PC, PS and AT activities were measured. The plasma levels of activities were measured by chromogenic substrate assay (AT and PC) and clotting assay (PS).</p><p><b>RESULTS</b>A total of 3493 healthy Chinese adults had been recruited in this study. Males had higher PS and PC activities than females, especially for PS (P < 0.01). PC activities increased with age in both sexes but decreased in men after 50 years old. There was no significant change with age were of PS in 50 years old, while there was a decline in males and a rise in females above 50 years old. AT tended to increase with age in women but decreased with age in men after 50 years old. Based on the age and gender, the general prevalence of PC, PS and AT deficiencies in the general Chinese Han population were 1.15%, 1.49% and 2.29%, respectively.</p><p><b>CONCLUSION</b>PC, PS and AT activities have correlation with age and gender in Chinese Han population. Reference range should be laid down and deficiencies should be identified</p>

The molecular mechanism of haemophilia B caused by the Arg327Ile novel mutation in FIX gene in vitro expression / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg327Ile (R327I) in FIX gene.</p><p><b>METHODS</b>The R327I, R327Ala(A), R327Lys(K), R327Asn(N) and a replacement mutant (FIXβFVII), in which FIX β strand 324-329 was replaced by that of FVII 298-303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type (WT) FIX expression plasmid. The HEK293 cell was transiently transfected, then the activity of FIX (FIX:C) was assayed by one stage method in the conditioned medium, while the FIX:Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells.</p><p><b>RESULTS</b>FIX:C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced (CRMR). The viable cell fluorescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX:C of the R327A, R327K, R327N and FIXβFVII mutants was reduced compared to that of WT, the reduction of FIX:C of FIXβFVII was the most significantly amount among all the mutants in medium. FIX:Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium.</p><p><b>CONCLUSION</b>The abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as the β strand domain of R327 located play important roles of the specific function of FIX.</p>

Analysis of phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia.</p><p><b>METHODS</b>Laboratory tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), and the activities of antithrombin (AT:C), protein C (PC:C) and protein S(PS:C) were detected in three pedigrees. The activity and antigen of plasma fibrinogen (Fg) were analyzed by Clauss and immunoturbidimetry methods, respectively. The Fg of three probands was assessed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of all the exons and exon-intron boundaries of the three Fg genes FGA, GFB and FGG were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>Three probands had normal APTT, PT, PC:C, PS:C and AT:C, but prolonged TT and RT. The activity levels of the 3 probands's plasma Fg were reduced, but antigen levels were normal. Western blot and SDS-PAGE showed no abnormal molecular weight of Fg. The 3 heterozygous mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys were identified in the 3 probands, respectively.</p><p><b>CONCLUSION</b>The three probands with dysfibrinogenemia were caused by the mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys, respectively. Both Aα Pro18Leu and Aα Arg16Cys were first reported in Chinese population.</p>

Molecular analysis of a patient with hemophilia A caused by FVIII His99Arg mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).</p><p><b>METHODS</b>FVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.</p><p><b>RESULTS</b>The proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.</p><p><b>CONCLUSION</b>Both the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.</p>

Molecular mechanisms of recurrent venous thrombosis in two pedigrees with type I antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency.</p><p><b>METHODS</b>The routine coagulation screening tests were detected, thrombin generation tests was performed to evaluate the hypercoagulation. Anticardiolipin antibody (ACA) and lupus anticoagulant (LA) were detected with enzyme-linked immunosorbent assay (ELISA) and diluted viper venom time assay (DVVT), respectively. The activities of protein C, protein S and AT (PC:A, PS:A, AT:A) were tested with chromogenic substrate assay or clotting method. The antigen of AT (AT:Ag) was performed with immunoturbidimetry methods. Western blot was used to analyze the molecular weight (MW) and the plasma levels of AT:Ag. All 7 exons and the flanking sequences were amplified by PCR. The mutation of AT gene and thrombophilia associated gene polymorphisms were analyzed by direct DNA sequencing. The expression plasmid of Ala404Asp mutant was constructed with site-directed mutagenesis method based on the wild-type (WT) AT cDNA contained in pcDNA 3.1 vector, and transiently expression of AT WT and the Ala404Asp mutant was performed using HEK293T cells. Cultured supernatant and cell lysates were collected and measured for AT:Ag by ELISA and Western blot.</p><p><b>RESULTS</b>The results of routine coagulation tests in two probands were normal, thrombin generation tests indicated that proband 1 presented hypercoagulable state with 2.8 and 1.5 times higher of the endogenous thrombin potential (ETP) and peak height compared with that of normal, respectively. The levels of PC:A, PS:A, ACA and LA were normal. AT:A in proband 1 and proband 2 were 45% and 32%, and AT:Ag were almost half of the normal (121 mg/L and 158 mg/L), respectively. The results of Western blot showed that both probands' plasma levels of AT:Ag were lower than the normal pooled plasma and MW was normal. Two heterozygous mutations of g.3291C→T(Thr98Ile), g.13863C > A(Ala404Asp) were identified in the probands, respectively. No proband had venous thrombosis associated gene polymorphisms. Expression in vitro showed that AT:Ag in culture media and lysates of Ala404Asp are 4.8% and 60.6% of that of WT, respectively.</p><p><b>CONCLUSION</b>Thr98Ile and Ala404Asp mutation of AT gene significantly correlate with recurrent venous thrombosis in the two probands, respectively. Ala404Asp has not been described before. The mutant Ala404Asp protein can not be expressed due to impaired secretion and increased intracellular degradation, resulting in type I AT deficiency.</p>

Phenotype and genotype analysis of three Chinese pedigrees with von Willebrand disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism.</p><p><b>METHODS</b>Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor (vWF): ristocetin cofactor (RCof) (vWF:RCof), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB), vWF and Factor VIII (FVIII) binding assay (vWF:FVIII:B) and multimer analysis were used for phenotype diagnosis. Genomic DNA was extracted from the peripheral blood (PB). All the 52 exons and flanking sequences of the probands' vWF gene were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>APTT were prolonged in all three probands, while BT were normal excepting for proband 3. Plasma RIPA, vWF:RCo, vWF:Ag, vWF:A and vWF:CB were decreased in different extents. In multimer analysis, proband 3 lost the large and intermediate molecular weight multimers, while proband 1 and 2 were normal. Gene analysis in the three probands revealed three heterozygous missense mutations of 144067 G→A (R2287Q) in exon 39, 110374G→A (R1374H) and 110770C→T (S1506L) in exon 28 and heterozygous polymorphism 110667G→A (D1472H) in exon 28, respectively.</p><p><b>CONCLUSION</b>The three heterozygous mutations (R2287Q, R1374H and S1506L) and an heterozygous polymorphism (D1472H) are genetic defects of the hereditary vWD of the three pedigrees respectively. R2287Q is a novel mutation reported for the first time in the literature.</p>

Two new mutations of AT gene in type I inherited antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the clinical phenotype and gene mutation in two kindreds with type I inherited antithrombin (AT) deficiency.</p><p><b>METHODS</b>The coagulation and anticoagulation testing and thrombophilia screening were used for phenotypic diagnosis and immunonephelometry and chromogenic assay for plasma level of AT antigen (AT:Ag) and AT activity (AT:A), respectively. All of the seven exons and intron-exon boundaries and untranslation regions of AT gene were amplified by PCR, and the PCR products analysis was by direct sequencing. The corresponding gene sites of the two family members and healthy individuals were detected according to the gene mutation sites.</p><p><b>RESULTS</b>The plasma levels of AT:Ag of proband 1 and proband 2 were 126 mg/L and 117 mg/L, and AT:A was 49% and 48%, respectively. Heterozygotic deletion of 3239-3240delCT in proband 1 and nonsense mutation 3206A-->T (K70Stop) in proband 2 were rchaacterized in exon 2 of AT gene. And some of their family members were also detected with the heterozygotic gene mutation.</p><p><b>CONCLUSION</b>Type I inherited antithrombin deficiency of the two probands were caused by AT gene mutation 3239-3240delCT and 3206A-->T (K70Stop).</p>

Analysis of phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V (FV) deficiency.</p><p><b>METHODS</b>The tests of activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) were used for phenotype diagnosis. All the exons and exon-intron boundaries of F5 gene were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>The APTT and PT in each of the four probands were obviously prolonged, and both activity and antigen of FV in the four probands were extremely lower compared with that of normal mixed plasma. Sequencing of F5 gene in proband 1 identified a heterozygous mutation, G16088C (Asp68His), and four polymorphisms, T35788C (Met385Thr), A47295G (His1299Arg), A58668G (Met1736Val) and A74083G (Asp2194Gly), which were located in the same chromosome; proband 2 was homozygous for two mutations, C46253T (Arg952Cys) and C46724T(Gln1109stop); the F5 gene of proband 3 showed a homozygous missense mutation, C67793G(Pro2006Ala); and proband 4 was homozygous for one missense mutation, C74022T (Arg2174Cys).</p><p><b>CONCLUSION</b>Five mutations (Asp68His, Arg952Cys, Gln1109stop, Pro2006Ala and Arg2174Cys) and four polymorphisms (Met385Thr, His1299Arg, Met1736Val and Asp2194Gly) may lead to type I inherited FV deficiency for these four probands, respectively. Gln1109stop, Pro2006Ala and Arg2174Cys haven't been identified before.</p>

Intron 1 and 22 inversions in factor VIII gene in patients with haemophilia A / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze intron 1 and 22 inversions in factor VIII (FVIII) gene in hemophilia A (HA) patients and and their families and to investigate the correlation between intron inversion and FVIII antibody.</p><p><b>METHODS</b>All patients were detected FVIII: C and FVIII antibody. In addition, 81 unrelated HA patients were directly detected by multiplex PCR and long-distance PCR for intron 1 and 22 inversions in FVIII gene. Pedigree investigation for some patients were conducted.</p><p><b>RESULTS</b>In 81 unrelated HA patients, 3 severe cases were found intron 1 inversion which accounted for 4.6% of total 65 severe cases. Of the 3 cases, one was FVIII antibody positive. Two female family members of a intron 1 inversion patient were identified as one carrier and one non-carrier. Twenty five of 65 (38.5%) severe cases were found intron 22 inversion. Of the 25 cases 1 was FVIII antibody positive. Nine female members in 5 HA families which had patients with intron 22 inversion were identified as 7 carries and 2 non-carriers.</p><p><b>CONCLUSION</b>Besides intron 22 inversion, intron 1 inversion was another important molecular defect in resulting in severe HA. Intron inversion analysis can also be used for deviation rectification of experiment grouping in HA patients. Intron 1 and 22 inversions may be one of the higher risk factors for resulting in FVIII antibodies.</p>

Detection of factor IX gene mutation in patients with hemophilia B by DNA sequencing / 中国实验血液学杂志

In order to investigate the patterns of FIX gene mutation in 3 unrelated hemophilia B (HB) patients, the activated partial thromboplastin time (APTT) and FIX activity (FIX: C) tests were adopted for phenotype diagnosis. All of the eight exons and their flank of FIX gene were amplified by polymerase chain reaction (PCR), the nucleic acid sequences were detected by dideoxymediated chain-termination method. The results indicated that as compared with normal control, the APTT value significantly increased, FIX: C value obviously decreased, PT value was normal. Sequencing results showed that all of 3 HB patients had the changes of gene sequences, among 3 patients the G22119A point mutation of exon 6 existed in case No.1, the G7932C point mutation of exon 2 was detected in case No.2 and the T32685C point mutation of exon 8 was found in case No.3. In conclusion, the relevant changes of gene sequences in all of 3 HB patients were detected, which provides some evidences for molecular mechanism of gene deficiency in HB patients.

Molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation.</p><p><b>METHODS</b>All exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy.</p><p><b>RESULTS</b>The alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi.</p><p><b>CONCLUSIONS</b>The L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.</p>

Analysis of clinical features and genotype in three Chinese pedigrees with Glanzmann thrombasthenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees.</p><p><b>METHODS</b>Platelet counts (BPC), blood film, bleeding time, platelet aggregation and flow cytometry were used for phenotype diagnosis of all the patients. All the exons of alpha II b and beta 3 genes were amplified by polymerase chain reaction (PCR) and direct sequencing was performed for mutational screening. One hundred and three healthy blood donors were as normal controls.</p><p><b>RESULTS</b>Three probands showed normal BPC, defective platelets aggregation, prolonged bleeding time and significantly reduced platelet aggregation to ADP, epinephrine, and collagen, while relatively normal aggregation to ristocetin. Flow cytometry showed platelet surface expressed alpha II b beta 3 was strongly reduced in proband 1 and proband 3 and mildly reduced in the amount of surface expressed alpha II b beta 3 (63%) in proband 2. Sequencing results showed that proband 1 had a G10A homozygous mutation in alpha II b, and a G1412T homozygous mutation in beta3. Compound heterozygous mutations in beta3, G1199A and 1525delC were identified in proband 2. No mutations in alpha II b beta 3 gene were identified in proband 3.</p><p><b>CONCLUSIONS</b>Compound homozygous mutations, GI0A in alpha II b and G1412T in beta3, lead to GT in proband 1. Compound heterozygous mutations in beta3, G1199A and 1525delC, lead to GT in proband 2. The mutations of G10A, G1412T and 1525delC were reported for the first time in GT patients.</p>

The application of thrombin generation tests to warfarin anticoagulation monitoring / 中华血液学杂志

<p><b>OBJECTIVES</b>To explore the thrombin generation capacity in patients on warfarin therapy with different prothrombin time international normalized ratio (PT-INR), the capacity in relation to bleeding, and the application of thrombin generation tests to warfarin therapy monitoring.</p><p><b>METHODS</b>Seventy eight blood samples were taken from patients on warfarin therapy for more than 3 months owing to valve replacement or atrial fibrillation. The patients' case history and PT-INR were collected and thrombin generation tests were performed in all samples.</p><p><b>RESULTS</b>Patients were ranked into three groups according to different PT-INR. There were 23 patients in group I with PT-INR from 1.51 to 2.00, 39 patients in group II with PT-INR from 2.01 to 3.00, and 16 patients in group III with PT-INR from 3.01 to 4.26. There were significant differences between each two of the three groups in lag time, peak, and ttpeak (time to peak) (P <0.01). There was a significant difference between group I and group II in endogenous thrombin potential (ETP) (P = 0.0001), but not between group II and group III (P= 0.06). Five patients developed bleeding and their ETP was less than 15% of normal control.</p><p><b>CONCLUSION</b>In patients on warfarin therapy, when the PT-INR was more than 3.0, increasing the dose of warfarin doesn' t decrease the thrombin generation, but increase bleeding risk. PT-INR combined with ETP may better reflect patient's coagulation status, therefore be of more significance in preventing bleeding.</p>

Gene diagnosis of 3 haemophilia B families / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore factor IX gene mutations and molecular mechanism of haemophilia B in 3 unrelated families.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT) and FIX activity (FIX: C) assay were used for phenotypic diagnosis. The STR loci gene polymorphisms for genetic linkage analysis in the patients and their family members were assayed. All of the 8 exons and the exon-intron boundaries of FIX gene were amplified by polymerase chain reaction (PCR) and direct sequencing.</p><p><b>RESULTS AND CONCLUSION</b>Mutations were found in the FIX gene of the propositi. Proband 1 had a G22119A mutation in exon 6, proband 2 a G7392C mutation in exon 2 and proband 3 a T32685C mutation in exon 8.</p>