Ding Ql

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

Summary.  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.  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: IVS1 + 1G > A and G1185A (Arg347His). The aberrant transcripts from the IVS1 + 1G > A 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 IVS1 + 1G > A 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.  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, 71.6 s) and prothrombin time (PT, 28.0 s). 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.

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

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.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.

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; 126.6 s) and prothrombin time (PT; 42.8 s). 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.

Prevalence of the factor 8 gene intron 1 inversion in Chinese haemophiliacs and its application to carrier detection and prenatal diagnosis in haemophilia A families.

1 Zhang L, Li H, Zhao H et al. Retrospective analysis of 1312 patients with hemophilia and related disorders in a single Chinese institute. Hemophilia 2003; 9: 696–702. 2 White GC 2nd, McMillan CW, Kingdon HS et al. Use of recombinant antihaemophilic factor in the treatment of two patients with classic hemophilia. N Engl J Med 1989; 320: 166–70. 3 Shi J, Zhao Y, Wu J, Sun J, Wang L, Yang R. Safety and efficacy of a sucrose-formulated recombinant factor VIII product for the treatment of previously treated patients with hemophilia A in China. Hemophilia 2007; 13: 351–6. 4 Bray GL, Gomperts ED, Courter S et al. A multicenter study of recombinant factor VIII (recombinate): safety, efficacy, and inhibitor risk in previously untreated patients with hemophilia A. The Recombinate Study Group. Blood 1994; 83: 2428–35. 5 Scharrer I, Ehrlich HJ. Lack of evidence for increased inhibitor incidence in patients switched from plasma-derived to recombinant factor VIII. Haemophilia 2001; 7: 346–8.

Recombination in a Chinese haemophilia A family

Sir,Haemophilia A (HA) is the most common X-linked, recessively inherited bleeding disorder which results from the deficiency of procoagulant factor VIII (FVIII). This disease affects approximately...

[Identification of genetic defects in a Chinese pedigree with factor XIII deficiency: case report and literature review].

OBJECTIVE To perform phenotypic diagnosis, genetic diagnosis and prenatal diagnosis of inherited coagulation factor XIII (FXIII)deficiency in a Chinese family also provide a review of inherited coagulation F XIII deficiency. METHODS The activity levels of F XIII (F XIII:C) of proband and family members were measured by clot solubility test and REA-chrom F XIII kit. The antigen levels of F XIII(FXIII:Ag) were measured by enzyme-linked immunosorbent assay. Thrombelastography (TEG) test was used to make a comprehensive evaluation of coagulation status in the proband. All 15 exons and exon-intron boundaries of the F13A1 gene were amplified by PCR, and DNA sequencing was performed then. The mutation identified in the proband was screened in the family members. Furthermore, the related literatures were reviewed to provide a profile of clinical manifestation, gene mutations, the relationship between the mutations and phenotype, and treatments of inherited coagulation F XIII deficient cases. RESULTS The clot solubility test was positive in the proband. The FXIII:Ag level of the proband was less than 1% and the FXIII:C level was below the lower limit of detection (<3%). Two compound heterozygous missense mutations (p.Arg662* and p.Trp665*) were identified in the proband. Family study showed that the two mutations were both inherited from the parents. The fetus also carried two compound heterozygous mutations, the same as the proband, and was diagnosed with severe F XIII deficiency. As reported in the literatures, most mutations were missense mutations and nonsense mutations, and no hot spot was found. The clinical pattern of F XIII deficiency varied among patients, with potentially fatal consequences from severe bleeding complications. CONCLUSION Better understanding of F XIII biochemical properties and function and developing of FXIII laboratory assays and genetic detection could prevent missed diagnosis, and patients moght benefit from better care.目的 对1个遗传性凝血因子XIII(FXIII)缺陷症家系进行表型诊断、基因诊断和产前诊断，并进行文献回顾。 方法 用尿素溶解法以及REA-chrom FXIII试剂盒检测患者及其家系成员血浆FXIII活性(FXIII:C)，用双抗体夹心法检测FXIII抗原(FXIII:Ag)，采用血栓弹力图(TEG)对先证者凝血功能进行评估。PCR扩增F13A1基因的15个外显子及其侧翼序列，PCR产物纯化后直接测序，并对家系成员F13A1基因相应的突变序列进行检测。 结果 先证者FXIII尿素溶解试验阳性，FXIII:Ag<1%，FXIII:C低于检测下限(<3%)。基因检测发现先证者F13A1基因14号外显子存在双杂合突变(p.Arg662＊和p.Trp665*)，先证者母亲及父亲均存在相应位点的单杂合突变，胎儿携带与先证者相同的双杂合突变。 结论 加强对FXIII结构与功能的了解，开展相关实验室诊断和基因诊断，可使患者得到及时的诊断和治疗。