Yongqiang Zhao

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.

α1-antitrypsin Pittsburgh in a family with bleeding tendency

A rare but naturally occurring mutation turns alpha1 antitrypsin into a potent antithrombin resulting in a bleeding tendency. This extremely rare phenomenon has been described before, but this additional family provides an interesting insight into the balance of coagulation. We describe a 16-year-old girl and her 41-year-old father who both had a bleeding tendency, dramatic prolongation of all standard clotting assays, undetectable levels of plasma protein C activity, and low or borderline levels of factors X, XI and XII. Plasma and serum electrophoresis revealed a minor peak following the main α1 globulin peak, of which the proportion was increased. Platelet aggregation by thrombin (final concentration 1 U/mL) was absent in both patients, but this inhibition can be overcome by increasing the concentration of thrombin (4 U/mL). The molecular defect responsible for these coagulation abnormalities was identified by genomic sequencing. Both patients are heterozygous for α1-antitrypsin Met 358 to Arg (α1-antitrypsin Pittsburgh). Seven other members of this pedigree had normal coagulation tests and do not carry the same genetic mutation. This unique family with α1-antitrypsin Pittsburgh sheds some light on the study of this extremely rare mutation and its inheritance.

Low-dose tertiary prophylactic therapy reduces total number of bleeds and improves the ability to perform activities of daily living in adults with severe haemophilia A: a single-centre experience from Beijing.

Full-dose prophylaxis treatment for persons with haemophilia is not affordable in China due to its economic constraints, particularly in adults requiring higher clotting factor (CFC) doses. Low-dose tertiary prophylaxis for adults with severe haemophilia A (SHA) in Beijing became feasible and implemented when government insurance covering 85% CFC cost in Beijing began in December 2009. The aim of this study was to evaluate the benefits of low-dose tertiary prophylaxis in SHA adults. Analysis of data on 33 patients on low-dose tertiary prophylaxis (5–10 IU/kg, two to three times per week) at the Haemophilia Treatment Center, Peking Union Medical College Hospital between December 2009 and December 2013. The 33 patients (age 18–60 years, mean 33.4) were on prophylaxis for 20.8 ± 9.9 months (compared with prior on-demand therapy for 20.0 ± 11.7 months). Prophylaxis resulted in significant decrease in annual bleeding rate (ABR, 11.8 ± 7.6 vs. 41.5 ± 20.7, 71.1% reduction, P < 0.0001), and significant improvement in Functional Independence Score in Haemophilia (FISH) measurement reflecting improvement in self-care and mobility. Radiologic (Pettersson) joint score was neither improved nor deteriorated. Ten of the 33 patients originally wheel chair and bed-bound began to walk and function independently in their daily lives. Low-dose tertiary prophylaxis for adults with SHA in China is feasible and beneficial. Although the average ABR remained high, a significant improvement in self-care and mobility measured by FISH was observed. These promising clinical experiences form the basis for further formal studies with more defined therapeutic protocol and outcome measures for affordable prophylaxis regimens in haemophilia adults in China.

A Novel Mutation (G2172→C) in the Factor V Gene in a Chinese Family with Hereditary Activated Protein C Resistance

BACKGROUND Activated protein C resistance (APC-R) was a major risk factor for venous thromboembolism(VTE) in Caucasians, and at least 90% of APC-R were associated with the point mutation of factor V (FV) gene (Arg506-->Gln, FV Leiden). However, this genetic defect was extremely rare in Asian population. OBJECTIVE To identify the genetic defect of FV in a Chinese family with APC-R associated with VTE. METHODS We describe a Chinese family with a history of venous thrombosis. Blood samples were obtained from five family members (including the proband) for screening APC-R by coagulation assay and the genetic defect of FV using direct sequencing. RESULTS Four out of five members had APC-R. We identified a novel mutation (G2172-->C) in exon 13 of the FV gene, which was present in all the individuals with APC-R but was absent in the individual without APC-R. This mutation is predicted to result in the replacement of glutamate by aspartate at position 666, close to one of the APC cleavage sites. CONCLUSIONS We have identified, for the first time, a novel mutation (G2172-->C) of FV that was associated with APC-R in a Chinese family with VTE. We speculate that this mutation interferes with cleavage at Arg679 by APC. The incomplete penetrance of thrombotic phenotype in this family, similar to that conferred by FV Leiden, suggests that it might be a weak risk factor for VTE.

Telomerase gene mutation screening and telomere overhang detection in Chinese patients with acute myeloid leukemia.

Abstract Loss-of-function mutations in telomerase complex genes reduce telomerase activity, and can clinically manifest as bone marrow failure disease, which predisposes to acute myeloid leukemia (AML). Telomerase dysfunction also leads to short telomeric overhang, which is a crucial telomeric structural component, and potentially results in chromosome instability. We screened variants in telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) genes, and investigated the 3’-overhang length in bone marrow samples from 72 Chinese patients with AML (61 de novo, 11 secondary, excluding M3), aged 13–77. Cytogenetics, disease severity and short-term survival were evaluated. Three TERT mutations (n896G>A, n1079C>G and n1451G>C) were identified. Mutation carriers had short overhangs and a poor prognosis. We found that overhang lengths were much shorter in AML compared to normal controls (p < 0.001). Short overhangs were related to a high percentage of karyotype abnormalities and poor prognosis (73.8% in short overhang group vs. 30% in normal group, p=0.001). Multivariant analysis showed that overhang length, age and unfavorable chromosome abnormalities served as independent prognostic markers in AML (Cox regression, p=0.001). These data raise the possibility that short overhang length may predict poor prognosis in patients with AML. These findings would have to be confirmed in large, prospective studies.

The performance of age-adjusted D-dimer cut-off in Chinese outpatients with suspected venous thromboembolism

BACKGROUND D-dimer testing has been widely used in the exclusion of venous thromboembolism (VTE), but its clinical usefulness is limited in older patients because of a lower specificity. OBJECTIVE To evaluate the diagnostic performance of STA-Liatest D-dimer assay and validate the age-adjusted cut-off value in Chinese outpatients with suspected VTE in a prospective non-interventional study. METHODS Symptomatic patients suspected of having deep venous thrombosis or pulmonary embolism were recruited from 2 participating centers. STA-Liatest D-dimer assay, clinical pretest probability assessment and diagnostic imaging test including complete compression ultrasonography or computed tomography pulmonary angiography were performed among all participants. The performance of D-dimer test was assessed with an age-adjusted D-dimer cut-off (age×0.01μg/ml in patients aged>50years) and with conventional cut-off (0.5μg/ml at all ages). RESULTS A total of 594 eligible outpatients were included in this study and VTE was diagnosed in 195 (32.8%) patients. In those patients with a low or moderate pretest probability (n=373), the increase in the proportion of patients with a D-dimer below the age-adjusted cut-off value compared with the conventional cut-off value was 5.9% (95% confidence interval; 3.8%-8.7%). The sensitivity, specificity and negative predictive value of STA-Liatest D-dimer test were 95.0% (83.5% - 98.6%), 84.1%(79.8%-87.6%) and 99.3%(97.5% - 99.8%), respectively, using the age-adapted diagnostic strategy. CONCLUSIONS The application of age-adjusted cut-off of D-dimer test combined with clinical probability greatly increases the proportion of Chinese older outpatients in whom VTE can be safely excluded.

Evaluating and monitoring the efficacy of recombinant activated factor VIIa in patients with haemophilia and inhibitors.

Although the use of bypassing agents has dramatically improved the management of haemophilia in patients with inhibitors, questions remain regarding optimal dosing regimens and methodology for monitoring their clinical effectiveness. In this study, we evaluated the efficacy and safety of two different doses of recombinant activated factor VIIa (rFVIIa) in patients with haemophilia and inhibitors and assessed the feasibility of using thromboelastography (TEG) and thrombin generation assays (TGA) for monitoring the response to rFVIIa. Six patients aged 9–49 years with congenital or acquired haemophilia with inhibitors who experienced a total of nine bleeding episodes were included. Seven episodes were treated with conventional rFVIIa dosing (72.7–109.1 &mgr;g/kg), and two episodes were treated with a single high-dose regimen (254.6–264.0 &mgr;g/kg). Clinical and haemostatic responses were evaluated. Haemostasis was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), factor VII coagulant activity (FVII:C), TEG, and TGA. Six out of seven (85.7%) bleeding episodes responded to conventional rFVIIa dosing, and half (50%) responded to the high-dose regimen. No relationships between PT, aPTT, and FVII:C levels and clinical outcome were observed. However, changes in TEG and TGA parameters tended to correspond to clinical response, although large inter-individual variation in rFVIIa efficacy was noted. A good response was seen with rFVIIa in treating acute bleeding episodes in patients with haemophilia and inhibitors. Because changes in TEG and TGA may correlate with clinical outcomes of rFVIIa, TEG and TGA may be useful for monitoring rFVIIa activity in inhibitor-positive haemophilia.

Multiple cycles of recombinant human thrombopoietin therapy in a patient with chronic refractory idiopathic thrombocytopenic purpura.

We describe a 41-year-old woman with chronic idiopathic thrombocytopenic purpura who received recombinant human thrombopoietin (rhTPO) therapy. rhTPO was administrated subcutaneously at a dosage of 1.0 μg/kg daily for a maximum of 14 days until the platelet count was more than 50 × 109/l. The patient received three cycles (six, 13, and eight doses each) of rhTPO, each initiated when the platelet counts was less than 10 × 109/l. The platelet count increased to above 50 × 109/l on days 5, 11 and 8, and peaked at 456 × 109/l, 130 × 109/l and 82 × 109/l on days 9, 15 and 13 in the three respective cycles, each followed by a gradual decline. The durations of platelet counts at more than 50 × 109/l in the three cycles were 13, 7 and 10 days, respectively. rhTPO was well tolerated with no adverse event observed. Antibodies to rhTPO by enzyme-linked immunosorbent assay were not detected. Our observations suggested that rhTPO could transiently increase the peripheral platelet count in patients with chronic refractory idiopathic thrombocytopenic purpura. The reasons why the peak platelet counts decreased and the duration of response shortened after successive cycles of treatment were unclear.

The Efficacy of Recombinant FVIII Low-Dose Prophylaxis in Chinese Pediatric Patients With Severe Hemophilia A: A Retrospective Analysis From the ReCARE Study

Objective: This study explores the efficacy of recombinant factor VIII (rFVIII) low-dose prophylaxis in Chinese pediatric patients with severe hemophilia A from the Retrospective Study in Chinese Pediatric Hemophilia A Patients With rFVIII Contained Regular Prophylaxis (ReCARE) population. Methods: This is additional analysis of the multicenter, retrospective ReCARE study, in which the annual bleeding rate (ABR), annual joint bleeding rate (AJBR), and safety of >12-week, low dose (10-30 IU/kg/wk) rFVIII prophylaxis divided into primary, secondary, and tertiary groups based on the joint status and joint bleeding history were analyzed. Results: A total of 57 patients (median age: 8.2 [0.4-17.3] years) from the ReCARE study receiving primary (n = 3), secondary (n = 21), and tertiary (n = 33) prophylaxes were included. Low-dose prophylaxis had significant bleeding reduction in all 3 groups compared to the baseline (S = 408.5, P < .001), with median ABR rates of −4.0 (−8.0 to −3.1), −4.0 (−24.0 to 8.0), and −13.9 (−110.6 to 20.6) in the primary, secondary, and tertiary groups, respectively, with a significant difference between the secondary and tertiary groups (P = .008). Median AJBR reduction rates were −2.3 (−6.3 to 8.4) and −14.9 (−61.5 to 19.1) in the secondary and tertiary groups, respectively. But there was no significant difference in AJBRs between the secondary and tertiary groups (P = .061), which was related to damaged joint status. Hence, longer prophylaxis was associated with better prevention of joint bleeding (P = .024). Conclusion: Despite significant ABR/AJBR reduction in all 3 groups, the efficacy of the primary prophylaxis was better than the secondary and tertiary prophylaxes.

A novel mutation in the porphobilinogen deaminase gene in an extended Chinese family with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficiency of porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthetic pathway. Establishing accurate diagnoses of the patient and asymptomatic family members with AIP involves identifying the PBGD enzyme mutations directly. Genetic testing provides a precise diagnosis for the patient and other asymptomatic family members, and thereby proper treatments can be initiated to prevent the disease from progressing. In this study, we report a novel PBGD missense mutation, A G-to-C, at the position 988 resulting in Alanine to Proline (Ala330Pro), in a Chinese family.