Xuefeng Wang

A rapid multifluorescent polymerase chain reaction for genetic counselling in Chinese haemophilia A families.

Summary.u2002 Linkage analysis is a widely used strategy for genetic counselling in haemophilia A (HA) families. We attempted to develop more informative markers closely linked to factor VIII (FVIII) gene and establish a rapid multifluorescent polymerase chain reaction (PCR) method with these markers. Five extragenic (DXS15, DXS9901, G6PD, DXS1073 and DXS1108) and one intragenic (F8Civs13) markers were examined in 118 healthy individuals and 12 HA families which had been diagnosed before. Five extragenic markers were within an interval of about 1.5u2003Mb to FVIII gene and located on each side of the gene. The expected heterozygote rate (HR) of DXS15, DXS9901, G6PD, DXS1073, DXS1108 and F8Civs13 were 74.97%, 79.77%, 56.06%, 59.92%, 39.97% and 47.61%, while the observed HR were 88.24%, 82.35%, 21.57%, 62.75%, 35.29% and 52.94%. When six polymorphic markers were combined together, all the studied females were informative in at least one of these markers and 29.41% of them were detected informative in three markers with the highest frequency. The diagnostic rates of DXS15, DXS9901, G6PD, DXS1073, DXS1108 and F8Civs13 in 12 haemophilia families were 75.00%, 91.67%, 41.67%, 75.00%, 33.33% and 66.67% respectively. All the genetic diagnosis was consistent with the result we analysed before and no recombination was observed. Family 1 was given as an example in this study and was found to be informative in three polymorphic markers DXS15, DXS9901 and DXS1073. The patients sister was detected the same allele as the proband, but her male fetus did not inherit the affected allele from her, which was consistent with the result of sequencing. It was demonstrated that the multifluorescent PCR method established in this study was convenient and efficient and can be applied to carrier detection and prenatal diagnosis in HA families.

Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees

Summary.u2002 To investigate the molecular defects in two Chinese pedigrees with inherited factor V (FV) deficiency.

MR Molecular Imaging of Thrombus: Development and Application of a Gd-based Novel Contrast Agent Targeting to P-selectin:

Molecular imaging of thrombus formation at initial stage requires a robust thrombus-specific contrast agent with high sensitivity. In this study, we report a novel P-selectin-targeted paramagnetic molecular imaging agent and the agent’s potential to sensitively detect occult microthrombi on the intimal surface of endothelium. Platelet clots and blood clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that was improved with increasing gadolinium level. In vivo contrast enhancement under part of circulation conditions was assessed in dogs. The micro-thrombi around the femoral vein of dog demonstrated higher signal intensities than the control clots and the adjacent muscle. Histology was performed on regions likely to contain thrombus as indicated by MRI. These results suggest that molecular imaging of P-selectin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of P-selectin and may allow for early, direct identification of microthrombi, leading to early diagnosis.

Molecular characterization of two novel mutations causing factor X deficiency in a Chinese pedigree

Summary.u2002 Factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait. In this study, we investigated the molecular basis of FX deficiency in a Chinese pedigree. The proposita showed a markedly prolonged activated partial thromboplastin time and a mild prolongation of prothrombin time. The levels of FX antigen and FX activity were 58.6% and 2.5%, respectively. Molecular analysis revealed that the proposita was compound heterozygous for two novel mutations: IVS1u2003+u20031Gu2003> A and G1185A (Arg347His). The aberrant transcripts from the IVS1u2003+u20031Gu2003>u2003A mutant allele were not detected by analyzing the splicing pattern of ectopic transcripts in leukocytes of the patient with nested polymerase chain reaction after reverse transcription. We thus hypothesize that the mRNA molecules originating from the IVS1u2003+u20031Gu2003>u2003A mutation were rapidly destroyed in vivo. Site‐directed mutagenesis of FX cDNA was used to introduce FXG1185A mutation, and wild‐type as well as mutant FX proteins were expressed by transient transfection in HEK 293 cells. Normal FX antigen levels both in the conditioned media of cells expressing the mutant and in cell lysates were detected by an enzyme‐linked immunoadsorbent assay. Evaluation of wild‐type and mutant coagulant activity demonstrated that the FX molecules carrying the Arg347His mutation have dramatically decreased activity.

Prothrombin Shanghai: hypoprothrombinaemia caused by substitution of Gla29 by Gly

Summary.u2002 Prothrombin deficiency is a rare bleeding disorder inherited as an autosomal recessive trait. In this study, we reported a Chinese family with hereditary prothrombin deficiency. The proposita had a prolonged activated partial thromboplastin time (APTT, 71.6u2003s) and prothrombin time (PT, 28.0u2003s). The coagulation factors activities were normal except that prothrombin coagulation activity was markedly reduced, and the prothrombin antigen level was moderately decreased. Nucleotide sequencing of amplified DNA revealed a novel mutation, Glu (GAG) to Gly (GGG) at residue 29, which normally undergoes γ‐carboxylation within the Gla domain of prothrombin. The proposita was identified as homozygous, while her father, mother and maternal grandmother were heterozygous for the mutation. Gla29 has been demonstrated as one of the key residue for Ca2+‐binding, membrane interaction and biological activity of prothrombin.

Molecular mechanism for hereditary protein C deficiency in two Chinese families with thrombosis.

Protein C (PC), the key component of the PC anticoagulant system, plays an important role in the regulation of the hemostatic system. After activation by the thrombin-thrombomodulin complex, activated protein C (APC) inhibits blood coagulation in the presence of protein S, phospholipids, and calcium ions through the proteolytic inactivation of factor (F)Va and FVIIIa. Inherited PC deficiency is caused by mutations in the PC gene and a wide variety of genetic mutations can lead to PC deficiency and PC molecular abnormality [1–3]. Phenotypically, congenital PC deficiency is classified into type I (quantitative) and type II (qualitative) [2]. In this study, we describe the genetic defects and the molecular mechanisms underlying PC deficiency in two Chinese families. Proband 1 from family 1 was a 32-year-old female diagnosed with deep vein thrombosis (DVT) of the left leg at the age of 24 years. In 2003, at 30 years, she developed mesenteric venous thrombosis and 8 months later suffered from recurrent DVT of the left leg. The mother of proband 1 had a history of mesenteric venous thrombosis and DVT of the left leg and subsequently died of disseminated thrombosis. Proband 2 from family 2 was a 19-year-old male diagnosed with DVT of both legs. No thromboembolic episodes were noted in this family. A routine hemostatic investigation showed that two family members’ platelet count, activated partial thromboplastin time, prothrombin time, thromboplastin time, fibrinogen, and FVIII, FIX, FX, von Willebrand factor, protein S and antithrombinwere within normal ranges. Neither member exhibited APC resistance. The PC:Ag and PC:A levels for members of the two families are shown in Fig. 1A,B. Proband 1 (II-7, Fig. 1A) showed severe PC deficiency (PC:Ag 0.0 mg L, PC:A 1.2%) and three othermembers (I-1, I-4, III3) asymptomatic from this family had approximately 50% of normal values for both PC antigen and activity, suggestive of type I PC deficiency. Proband 2 (II-1, Fig. 1B) had an approximately 50% reduction of both PC:Ag and PC:A, also suggestive of type I PC deficiency. DNA sequencing demonstrated that proband 1 had compound heterozygous missense mutation in exon 5 (C to G transition at nucleotide 3135, which caused Cys64Trp) and in exon 7 (T to G transition at nucleotide 6128, which caused Phe139Val [4–6]). We have designated this novel variant Cys64Trp as PC Shanghai. Further analysis showed that in the family, I-1 was heterozygous for Cys64Trp, whereas I-4 was heterozygous for Phe139Val. III-3, proband’s daughter, had compound heterozygous mutations (Cys64Trp inherited from proband 1 and Lys150 or Lys151 deletion mutation (DLys150 orDLys151 [4,7]) in exon 7 inherited from II-8). Individuals I-5, II-11, and II-12 were also heterozygous for DLys150 or DLys151 (Fig. 1A). In family 2, individuals I-2, I-3, and proband 2 were identified as heterozygous for the DLys150 or DLys151 mutation. Three polymorphic sites within the promoter region of the PC gene at positions -1654, -1641, and -1476 were screened in family 2. Proband 2 was found to be homozygous at all three sites (CC/GG/TT), whereas individuals I-1, I-2, and I-3 were found to be heterozygous at all three sites (TC/GA/AT). Heterozygosity for PC Shanghai (Cys64Trp) would account for a reduction of approximately 50% in proband 1 and individual I-1’s PC antigen/activity. Heterozygosity for Phe139Val would account for the remaining reduction in PC antigen/activity in the proband, as well as reduced PC antigen/ activity in her father and daughter. Transient expression experiments with COS-7 cells showed that intracellular levels of mutant PC-Trp64 protein were 29.6% of that of wild-type recombinant protein and no PCTrp64 protein was secreted into the conditioned medium. Immunohistochemical and protein degradation inhibitor experiments showed that mutant PC-Trp64 was locatedmainly in pre-Golgi compartment and degraded intracellularly through the proteosome pathway, whilst wild-type PC appeared to reside mainly in the rough ER and was secreted rapidly after undergoing modification at the Golgi, as reported previously [8]. These results suggest that mutation at Cys64Trp led to impaired secretion of the mutant and that the mutant did not accumulate but was partially degraded intracellularly through the proteosome pathway. Correspondence: H-L. Wang, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, No. 197 Ruijin II Road, Shanghai 200025, China. Tel.: + 86 21 64370045 ext. 610602; fax: + 86 21 64743206; e-mail: wanghongli602@163.com

Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation.

Aims: To identify the mutation and study the molecular mechanism of inherited protein C (PC) deficiency in a Chinese pedigree. Methods: The plasma levels of PC activity (PC:A) and antigen (PC:Ag) were measured by chromogenic assay and ELISA, respectively. The PROC gene was amplified and sequenced for mutational screening. Wild type and Pro275Ser mutant PC cDNA expression plasmids were constructed and transfected into HEK 293T cells and COS 7 cells, respectively. The expression and transcription of PC were investigated by ELISA, Western blot and real time RT-PCR. Immunofluorescence staining was utilised to analyse the intracellular distribution of PC, and pulse-chase experiments were used to detect the intracellular stability of the mutant PC. Results: The probands plasma PC:A and PC:Ag were 5% and 13.9%, respectively. A missense mutation (p.Pro275Ser) was identified in exon 9 of PROC gene. In vitro expression study showed that Pro275Ser variant was present at 22.6% and 78.9% of wild type levels in culture supernatants and cell lysates, respectively. No significant differences in the molecular weights, mRNA levels or intracellular stability were observed between the mutant and wild type PC. Immunofluorescence staining revealed that the mutant protein was mainly located in the endoplasmic reticulum. Conclusions: A homozygous Pro275Ser mutation was identified in a Chinese pedigree of PC deficiency. Impaired secretion of the mutant PC might be the molecular mechanism of PC deficiency caused by Pro275Ser mutation.

Spectrum of F9 mutations in Chinese haemophilia B patients: identification of 20 novel mutations

AIMSnHaemophilia B (HB) is an X-linked recessive haemorrhagic disorder caused by F9 mutations. In this study, we performed molecular analysis of 107 Chinese HB patients and analysed the F9 mutation spectrum of Chinese HB patients.nnnMETHODSn107 Chinese HB patients were enrolled in this study. Direct sequencing of the whole F9 or mutation scanning of exon 1 to exon 7 by high resolution melting (HRM) curve analysis combined with direct sequencing of exon 8 was used to identify the mutations in these patients.nnnRESULTSn78 different F9 mutations were identified in the 107 HB patients. The mutations were composed of 50 missense mutations, 14 nonsense mutations, nine small deletions, three splice site mutations and two small insertions. Forty-three of 78 mutations were located in factor IX (FIX) catalytic domain. Among the 78 mutations, 20 have not been previously reported.nnnCONCLUSIONSnThe F9 mutations were heterogenous and the missense mutations were the most prevalent gene defects in Chinese HB patients.Aims: Haemophilia B (HB) is an X-linked recessive haemorrhagic disorder caused by F9 mutations. In this study, we performed molecular analysis of 107 Chinese HB patients and analysed the F9 mutation spectrum of Chinese HB patients. Methods: 107 Chinese HB patients were enrolled in this study. Direct sequencing of the whole F9 or mutation scanning of exon 1 to exon 7 by high resolution melting (HRM) curve analysis combined with direct sequencing of exon 8 was used to identify the mutations in these patients. Results: 78 different F9 mutations were identified in the 107 HB patients. The mutations were composed of 50 missense mutations, 14 nonsense mutations, nine small deletions, three splice site mutations and two small insertions. Forty-three of 78 mutations were located in factor IX (FIX) catalytic domain. Among the 78 mutations, 20 have not been previously reported. Conclusions: The F9 mutations were heterogenous and the missense mutations were the most prevalent gene defects in Chinese HB patients.

Clinical observation on safety and efficacy of a plasma- and albumin-free recombinant factor VIII for on-demand treatment of Chinese patients with haemophilia A.

Summary.u2002 Recombinant FVIII (rFVIII) has become the best choice for treating bleeding of haemophilia A patients. A plasma‐ and albumin‐free recombinant FVIII (rAHF‐PFM, ADVATE®), as the third generation rFVIII, virtually eliminates the risk of blood‐borne disease transmission by excluding all human blood derived additives throughout cell culture, purification and formulation. In this multicentre prospective clinical study we evaluated the efficacy, safety and immunogenicity of ADVATE® in Chinese patients with haemophilia A. Fifty‐eight patients enrolled and received ADVATE® treatment. Of the patients enrolled, eight (13.79%) had severe haemophilia, 45 (77.59%) had moderate haemophilia and five (8.62%) had mild haemophilia. Fifty‐four patients completed 6u2003months of observation. A total of 781 bleeds occurred in these 58 subjects, all evaluable per‐protocol. A total of 984 infusions were administered with a mean of 17.0u2003±u200311.1 infusions per patient. On average, each patient received a mean of 15030.2u2003± 7972.7u2003IU ADVATE® (median 13u2003625u2003IU, range 9500–19u2003750u2003IU) during 6u2003months. The majority of bleeding episodes (95.9%) were successfully treated with one or two infusions of ADVATE®. Overall, response to the first ADVATE® treatment was rated as either ‘excellent’ (82.8%) or ‘improved’ (17.2%) in all subjects. All patients tolerated ADVATE® infusions well. One patient (1/58, 1.7%) developed an inhibitor of 4u2003Betheseda units at day 180 visit. The results of this clinical observational study support that ADVATE® is efficacious, safe and well tolerated in the treatment of Chinese patients with haemophilia A.

Type I coagulation factor V deficiency caused by compound heterozygous mutation of F5 gene.

A 16‐year‐old Chinese female with prolonged bleeding after surgery has been studied. Routine clotting tests revealed a prolonged activated partial thromboplastin time (APTT; 126.6u2003s) and prothrombin time (PT; 42.8u2003s). The coagulation factors activities were normal except for factor V, which was only 0.3% of normal. DNA analysis of the FV gene revealed five nucleotide substitutions in exons, including two silent mutations (G327A and A5112G), one polymorphism (G1628A), a G1348T missense mutation and 4887∼8delG. These abnormalities were associated with her FV deficiency, perhaps by causing a Gly392Cys substitution in FV amino acid sequence or by introducing a premature stop codon at amino acid position 1390. This is the third case in which FV deficiency is caused by compound heterozygous mutation of F5 gene, and is the first report from a Chinese family.