Q. Ding

Normal ranges and genetic variants of antithrombin, protein C and protein S in the general Chinese population. Results of the Chinese Hemostasis Investigation on Natural Anticoagulants Study I Group

Background Inherited deficiency of antithrombin, protein C and protein S, three important, naturally occurring coagulation inhibitors, might play a major role in the occurrence of venous thromboembolism in Chinese. The establishment of age- and gender-related normal ranges of these inhibitors is crucial for an accurate diagnosis of these deficiencies. Design and Methods We designed a prospective cross-sectional study recruiting healthy adults from four university–affiliated hospitals in China. Antithrombin, protein C and protein S were studied by measuring their activity. Gene analysis was performed when natural anticoagulant deficiency was suspected. Polymorphisms of the factor V gene were searched for among subjects who were positive for activated protein C resistance. Results In 3493 healthy Chinese adults (1734 men, 1759 women; age 17–83 years), we found higher age-adjusted activities for protein C and protein S in men than in women but no sex difference for antithrombin. In women, mean protein C and protein S activities increased with age. In men, mean protein C levels increased with age up to the age of 49 but decreased after 50 years old; mean protein S levels decreased after 50 years of age. Antithrombin levels remained stable over time in women but decreased significantly after 50 years of age in men. Reference values according to age and sex allowed the identification of 15 genetic variants (protein C: 10, antithrombin: 3, protein S: 2) in subjects with protein activity below the 1st percentile. Conclusions This is the largest survey ever conducted in the healthy general Chinese population. These normal ranges provide the essential basis for the diagnosis and treatment of thrombosis in Chinese.

The prevalence of factor VIII inhibitors and genetic aspects of inhibitor development in Chinese patients with haemophilia A

Summary.u2002 The prevalence of inhibitors in Chinese haemophiliacs has not yet been reported. The aim of this study was to identify the prevalence of factor VIII (FVIII) inhibitors among haemophiliacs who are treated only with plasma‐derived FVIII (pdFVIII), cryoprecipitate or fresh frozen plasma (FFP), and tried to explore the relationship between the generation of inhibitors and particular FVIII deficiency genotypes. Clinical information and blood samples of 1435 patients with haemophilia A (HA) were collected by six haemophilia centres in China. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, long‐range PCR and direct sequencing were performed for genotyping. The overall prevalence of inhibitors in Chinese HA patients was 3.9% and the prevalence of severe haemophiliacs was 4.3%; 18 of the 56 patients with inhibitors had high titres. A total of 38 different mutations were identified in the 55 patients with inhibitors, including 15 intron 22 and 3 intron 1 inversions, seven large deletions, 14 small deletion/insertions, seven nonsense mutations, one splice site mutations and eight missense mutations. Of 38 mutations, 28 were novel. Patients with large deletions and nonsense mutations were prone to have high titre inhibitors, with incidence rates of 57.1% (4/7) and 42.9% (3/7), respectively. In conclusion, the prevalence of inhibitors in Chinese HA patients is much lower than that reported for other ethnic groups and the large deletion and nonsense mutations are high risk factors for high titre inhibitor development.

Characterization of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique

Large deletions in the F8 gene are responsible for approximately 3% of severe hemophilia A (HA) cases. However, only a few breakpoints in large deletions have been characterized.

Female hemophilia A heterozygous for a de novo frameshift and a novel missense mutation of factor VIII

Summary.u2002 Background:u2002Hemophilia A (HA) is an X‐chromosome‐linked recessive disorder. Aim:u2002We report the case of a female HA patient with a moderate decrease of factor (F) VIII activity and antigen (FVIII:C 3.4%, FVIII:Ag 4.2%) and severe bleeding symptoms. Methods:u2002The patients father had mild FVIII deficiency (FVIII:C 6.9%, FVIII:Ag 7.4%), and her mother had normal FVIII activity. The von Willebrand disease antigen and von Willebrand factor ristocetin cofactor activity were normal in all family members. The genomic DNA was extracted from the peripheral blood lymphocytes of the patient and her family members. Long‐distance polymerase chain reaction (PCR) was employed to screen for the intron 22 inversion of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. Results:u2002Two heterozygous mutations were identified in the patient: one a substitution of nucleotide 5981T by C that leads to a missense mutation Leu1975Pro, and the other an insertion of an ‘A’ between nucleotides 3637 and 3638 (3637_3638insA) that shifts the reading frame and predicts a premature stop codon downward. The mutation Leu1975Pro was identified in the fathers F8; however, 3637_3638insA was a de novo mutation that occurred in the patients maternal‐derived F8. Real‐time PCR was applied to analyze the level of ectopically F8 gene transcripts in the peripheral lymphocytes of family members. The ectopic transcripts of F8 of the patient were less abundant than the normal control (patient:normal control ratio 0.67), whereas her parents showed no significant difference from the normal control. Conclusion:u2002The FVIII deficiency of the HA patient resulted from a de novo occurrence of a frameshift 3637_3638insA in her maternal‐derived F8 and a novel missense mutation Leu1975Pro inherited from her father.

The status of carrier and prenatal diagnosis of haemophilia in China

Summary.u2002 Haemophilia A (HA) and haemophilia B (HB) are the most common X‐linked inherited bleeding disorders. It is important to detect the carrier women in families with HA/HB and subsequent antenatal diagnosis of confirmed carriers. This study consists of 102 HA families which include 68 mothers for prenatal diagnosis and 107 female relatives for carrier diagnosis, and 29 HB families which include 16 mothers and 31 female relatives respectively. The rapid fluorescent PCR with two groups of different combined polymorphism markers was applied for linkage analysis in HA and HB families respectively. The Amelogenin gene was added to help the detection of gender diagnosis. Gene sequencing was also used to detect the mutations directly. There were 37 causative F8C mutations (23 novel) and 24 causative F9C mutations (eight novel) found in this cohort of patients. Few of the women could not be diagnosed due to homologous recombination and/or inability to locate the mutation. Complicated cases have been found in some families. With regard to carrier and prenatal diagnosis, it was considered that genetic diagnosis by linkage analysis and direct sequencing was successful. Some special families might require combination of the linkage analysis and gene sequence for a successful diagnosis. New intragenic SNP and STR sites special to Chinese population need to be discovered.

Characterisation and validation of a novel panel of the six short tandem repeats for genetic counselling in Chinese haemophilia A pedigrees.

Summary.u2002 Haemophilia A (HA) is the most common hereditary bleeding disorder caused by F8 gene mutation. Linkage analysis is an auxiliary strategy to direct mutation analysis for genetic counselling of HA. Here we characterize and validate a novel panel of six short tandem repeat (STR) loci for genetic counselling in Chinese HA pedigrees. The panel was analysed in 116 unrelated healthy female patients and 108 male patients, and verified in 169 unrelated pedigrees with HA. The six STR loci in the panel spanned a distance of 0.3u2003Mb from each side of the F8 gene. Three of them, F8Up226, F8Up146 and F8Down48, were first described here. Markers F8Up226, F8Up146, F8Int13, F8Int25, F8Down48 and DXS1073 exhibited the number of alleles 16, 9, 8, 6, 9 and 10, and heterozygosity rates of 74.8%, 44.8%, 60.9%, 42.6%, 61.7% and 62.0% respectively. Haplotype frequencies analysis suggested that the genotypes of haplotype provided a highly informative content (56.5%). The panel was informative in 167 of 169 unrelated haemophilic pedigrees with the combined diagnostic rate of 98.8%. In eight pedigrees could not be diagnosed by mutation detection linkage studies using the panel were informative in all the pedigrees and a reliable diagnosis was made in seven pedigrees. The novel panel of the six STR loci represents a high degree of informativeness and a low fraction of recombination. Linkage analysis using this panel provides an alternative strategy when direct mutation detection is not feasible for genetic counselling in Chinese HA families.

Molecular defects in the factor X gene caused by novel heterozygous mutations IVS5+1G>A and Asp409del

Factor X (FX) deficiency is a rare autosomal‐recessive bleeding disorder caused by diverse mutations in the F10 gene. To investigate the molecular basis of severe FX deficiency in a mildly hemorrhagic patient, variants of the F10 gene were detected by sequencing. A missense mutation was analysed by in vitro expression and modelling analysis, and a splice mutation using ectopic transcript analysis. The levels of activity of FX (FX:C) were <1% in both intrinsic and extrinsic pathway assays and 1.71% in chromogenic assay, the level of FX antigen (FX:Ag) was 53.36% in the proband. Two novel heterozygous mutations (IVS5+1G>A and Asp409del) were identified in the F10 gene. Ectopic transcript expression combined with informative marker (heterozygous Asp409del) analysis of the splice mutation (IVS5+1G>A) revealed and confirmed that the transcript from the mutated allele was absent, likely caused by the nonsense‐mediated mRNA decay pathway. In vitro expression analysis showed that the Asp409del mutant led to a loss of enzymatic activity rather than impaired expression. Molecular modelling analysis confirmed that the Asp409del mutant dramatically altered the conformation of the 185–189 loop and impaired binding of the loop to sodium ions (Na+), diminishing the enzymatic activity of FXa. This is the first report to clarify the molecular mechanisms of two naturally occurring F10 gene variants that cause severe FX deficiency.

Molecular basis of type I antithrombin deficiency in two women with recurrent venous thromboembolism in the first trimester of pregnancy.

Inherited antithrombin (AT) deficiency carries a 50% risk of venous thromboembolism (VTE) during pregnancy. Here, we investigated the molecular basis of type I AT deficiency in two women with recurrent VTE in the first trimester of pregnancy. Phenotype analysis showed both probands had almost 50% of normal AT levels. Two novel heterozygous AT mutations were identified: g.7920C>T resulting in a Trp225Cys mutation in case 1 and g.13863C>A causing an Ala404Asp mutation in case 2. Transient expression of either wild-type (WT) or mutant AT expression vectors in HEK293T and CHO cells showed impaired secretion of both AT mutant proteins. Immunofluorescence analysis revealed that the staining of AT-Trp225Cys in both endoplasmic reticulum (ER) and Golgi apparatus was similar to that of AT-WT, and the staining of AT-Ala404Asp was mainly present in ER but was weaker than that of AT-WT. These results revealed that the type I AT deficiency in two patients was caused by impaired secretion of the AT-Trp225Cys and AT-Ala404Asp mutant proteins, respectively. The two mutations are associated with a high risk of thrombotic onset and women with these AT mutations are prone to VTE in early pregnancy.

Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigree

Summary.u2002 Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572‐1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband’s F7 gene. To investigate the splicing patterns associated with F7 c.572‐1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position ‐1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT‐PCR analysis. The aberrant transcripts from the F7 c.572‐1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT‐PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572‐1G>A mutant allele is responsible for the factor VII deficiency in this pedigree.

A novel Pro126His β propeller mutation in integrin αIIb causes Glanzmann thrombasthenia by impairing progression of pro-αIIbβ3 from endoplasmic reticulum to Golgi

BACKGROUNDnGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3 (glycoprotein IIb/IIIa).nnnPATIENTSnMucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family.nnnOBJECTIVESnTo determine the molecular basis of GT and characterize the mutation by in vitro expression studies.nnnRESULTSnAnalysis of the patients platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of beta3, exposed on her platelet surface, but a complete absence of alphaIIbbeta3. Sequence analysis revealed a novel C470A transversion in exon 4 of the alphaIIb gene predicting a Pro126His alteration in the blade 2 of the alphaIIb beta propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alphaIIb and wild-type beta3 failed to express alphaIIbbeta3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature alphaIIb. Immunoprecipitation with antibody against beta3 demonstrated pro-alphaIIb in the cells expressing the mutant alphaIIbbeta3, indicating pro-alphaIIbbeta3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-alphaIIbbeta3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker.nnnCONCLUSIONSnA novel Pro126His mutation in alphaIIb compromised transport of the pro-alphaIIbbeta3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired alphaIIbbeta3 transport is responsible for the thrombasthenia in this patient.