Yeling Lu

Paired immunoglobulin-like receptor B regulates platelet activation.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.

The status of carrier and prenatal diagnosis of haemophilia in China

Summary.  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.

Clinical features and molecular basis of 102 Chinese patients with congenital dysfibrinogenemia

INTRODUCTION Congenital dysfibrinogenemia (CD) is a rare qualitative disorder of fibrinogen (Fg) with heterogeneous clinical manifestations. We aimed to analyze clinical phenotype and molecular basis of 102 Chinese CD patients and to evaluate the application of thromboelastography (TEG). MATERIALS AND METHODS Clinical manifestations were recorded and quantified using the consensus ISTH bleeding assessment tool. Kaolin activated TEG and functional Fg TEG were applied in 30 patients. Genetic analysis of Fg genes were performed by direct sequencing. RESULTS 27.5% patients experienced bleeding, 3.9% had thrombosis and 68.6% were asymptomatic. Females were more prone to experience bleeding (P=0.01). Significant difference (P<0.05) in TEG results were found between patients with hot-spot mutations at AαArg35(16) and γArg301(275), but were not identified between patients with and without bleeding. Normal TEG results were found in patients with mutations at AαArg35(16), AαPro37(18) or AαArg38(19). Six novel mutations were identified, including AαGly33(14)del, AαAsp57(38)_Trp60(41)delIVS2+1_+2GTdel, AαPhe742(723)Tyr, γAsn334(308)Thr, γGly335(309)Cys and γTrp395(369)Leu. CONCLUSIONS CD patients have similar clinical manifestations and hot-spot mutations worldwide with no ethnic difference. TEG results could not indicate the bleeding risk in patients, but priority of mutation screening at thrombin cleavage site or polymerization site on Aа chain may be given if TEG results are normal.

Vps33b regulates Vwf‐positive vesicular trafficking in megakaryocytes

Mutations of vacuolar protein sorting‐associated protein 33b (VPS33B) cause arthrogryposis, renal dysfunction, and cholestasis syndrome, and a lack of platelet α‐granules in the affected patients. Conditional Vps33b knockout mice were developed to investigate the function(s) of Vps33b in platelet α‐granule formation. We found that early embryonic deletion of Vps33b was lethal. PF4‐Cre‐driven megakaryocyte‐targeted Vps33b gene deletion greatly diminished Vps33b expression in platelets, but had no effect on platelet α‐granule formation and protein content. Tamoxifen‐induced, haematopoietic stem cell (HSC)‐specific Vps33b deletion completely depleted Vps33b in platelets, caused the absence of α‐granules, and increased the number of vacuoles in platelets and megakaryocytes. VPS33B association with VIPAS39, α‐tubulin, and SEC22B was identified by co‐immunoprecipitation, mass spectra, and immunoblotting in human embryonic kidney 293T (HEK293T) cells. Also, pull‐down experiments revealed that VIPAS39 bound to intact VPS33B; in contrast, α‐tubulin and SEC22B separately interacted with the sec1‐like domains of VPS33B. Vps33b deficiency in megakaryocytes disturbs the redistribution of Vipas39 and Sec22b to proplatelets, and interrupted the co‐localization of Sec22b with Vwf‐positive vesicles. The data presented in this study suggest that Vps33b is involved in α‐granule formation possibly by facilitating the Vwf‐positive vesicular trafficking to α‐granule‐related vacuoles in megakaryocytes. Copyright

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.  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.

Thromboelastography predicts risks of obstetric complication occurrence in (hypo)dysfibrinogenemia patients under non-pregnant state

Congenital (hypo)dysfibrinogenemia patients may have obstetric complications during their pregnancies. This study aimed to evaluate thromboelastography (TEG) as a potential tool for assessing the tendency for obstetric complications in those patients in a non‐pregnant state. A total of 22 female subjects with congenital (hypo)dysfibrinogenemia were recruited. Nine subjects had histories of obstetric complications and the other 13 subjects had at least one uneventful pregnancy without obstetric complications as yet. Detailed clinical investigation and phenotype/genotype detection were carried out, and both kaolin‐activated TEG and functional fibrinogen TEG (FF‐TEG) were applied in all subjects. Significant differences were identified in all TEG parameters except for R and angle between these two groups (P < 0.05) by covariance analysis. Receiver operating characteristic (ROC) analysis of discrimination between these two groups of patients was performed for TEG parameters. Significantly high odds ratio (OR) of obstetric complications occurrence were demonstrated in K ≥ 3.8 min, maximum amplitude (MA) ≤ 54.2 mm, comprehensive index (CI) ≤ −3 (11.67, 95% CI 1.527–89.121, P < 0.05 in all), and MA‐CFF ≤ 12.1 mm (20.00, 95% confidence interval (95% CI) 1.967–203.322, P = 0.002). Moreover, MA‐CFF had better prognostic performance, with a corresponding area under the receiver operating curve of 0.923 (range 0.815–1.031, P = 0.001). This study suggests that (hypo)dysfibrinogenemia patients with values outside of the cut‐off values of TEG assays under non‐pregnant state may have a higher risk of obstetric complications occurring when they are pregnant. No parameters under non‐pregnant state in clinical laboratory have ever been reported to be risk factors for obstetric complication occurrence in (hypo)dysfibrinogenemia patients. This study explored such parameters in TEG assays and found that parameters of TEG assays under non‐pregnant status might predict the occurrence of obstetric complications, which could provide physicians with important information about whether fibrinogen replacement therapy is required, so as to prevent the occurrence of obstetric complications, especially for patients who are asymptomatic in daily life.

Identification of a novel splicing mutation in the fibrinogen Aα chain gene leading to hypofibrinogenaemia in a Chinese pedigree

Fibrinogen is the focal point of the coagulation cascade, which results in the conversion of fibrinogen to fibrin monomer and the eventual covalent crosslinking of the fibrin polymer. Fibrinogen, a 340-kD glycoprotein, is synthesized in the liver as a dimer with each half composed of three different polypeptide chains (Aa, Bb and c). These chains are linked by a network of 29 disulfide bonds to form the circulating protein. Each chain is encoded by a separate gene, FGA, FGB and FGG, which are clustered in a 50-kb region on chromosome 4q32.1. Fibrinogen abnormalities can be classified according to whether there are low or no circulating levels of normal protein (hypofibrinogenaemia or afibrinogenaemia), a mutated species (dysfibrinogenaemia), or a combination (hypodysfibrinogenaemia). Congenital hypofibrinogenaemia is characterized by functional and immunoreactive fibrinogen levels lower than 1.5 g L [1]. Here, we studied a Chinese patient with hypofibrinogenaemia. The proband had low plasma levels of functional and immunoreactive fibrinogen. Sequencing of fibrinogen genes, including exon–intron boundaries and promoter regions, allowed us to identify a heterozygous G>C transition at position +1 of intron 2 of the a-chain gene (FGA IVS2+1G>C). Production of the mutant transcript in transfected HEK 293 cells demonstrated the existence of an aberrant splicing of fibrinogen a-mRNA, leading to a major a-chain truncation associated with hypofibrinogenaemia. The 32-year-old proband had four times of ectopic pregnancy; after ectopic pregnancy, she did not bleed abnormally, did not have anaemia or any other haemostatic challenges. The proband had no acute or chronic liver disorder. No history of bleeding disorders and consanguineous marriages were described in her family (Fig. 1). The results of coagulation tests of all the family members are shown in Table 1. As shown in the table, the proband had prolonged TT and low fibrinogen level (both functional and immunoreactive) whereas all the coagulation tests of other family members were in the normal range. FGA IVS2+1G>C was detected in the patient (Fig. 2). No other mutations were identified in the branch sites and the consensus splicing sequences of other introns. The splice site mutation FGA IVS2+1G>C was not found in any of the other family members. To determine the splicing patterns associated with the FGA IVS2+1G>C mutation, we isolated total RNA from peripheral blood leucocytes of the patient and analysed the transcripts by RT-PCR. Only one band was observed on the agarose gel after electrophoretic separation of RT-PCR products and this band was the same as that of normal control (407 bp) (Fig. 3). The RT-PCR products from the patient were then cloned and sequenced. However, no aberrant transcripts were ever found in 30 individual clones. To verify whether the donor splice-site mutation (FGA IVS2+1G>C) determines the retention of intron 2 in mature mRNA, we made FGA-wt and FGA-mut constructs and transfected them into HEK 293 T cells respectively, total mRNA was extracted after 16 h culture. RT-PCR performed on RNA extracted from FGA-wt-transfected HEK 293 cells allowed us to detect the expected 243-bp long fragment (including a 101-bp exon 2 and a 142-bp Correspondence: Hongli Wang, No.197 Ruijin Second Road, Shanghai 200025, China. Tel.: +86 21 64370045*610609; fax: +86 21 64743206; e-mail: wanghongli602@163.com; wwbzxt@hotmail.com

AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A

BACKGROUND To develop a digitalized intron 22 inversion (Inv22) detection in patients with severe haemophilia A. METHODS The design included two tests: A genotyping test included two multiplex pre-amplification of LD-PCR (PLP) with two combinations of five primers to amplify wild-type and chimeric int22h alleles; a carrier mosaicism test was similar to the genotyping test except only amplification of chimeric int22h alleles by removing one primer from each of two combinations. AccuCopy detection was used to quantify PLP products. RESULTS PLP product patterns in the genotyping test allowed identifying all known Inv22. Quantitative patterns accurately represented the product patterns. The results of 164 samples detected by the genotyping test were consistent with those obtained by LD-PCR detection. Limit of detection (LOD) of the carrier mosaicism test was at least 2% of heterozygous cells with Inv22. Performing the test in two obligate mothers with negative Inv22 from two sporadic pedigrees mosaic rates of blood and hair root of the mother from pedigree 1 were 8.3% and >20%, respectively and negative results were obtained in pedigree 2. CONCLUSIONS AccuCopy quantification combined with PLP (AQ-PLP) method was confirmed to be rapid and reliable for genotyping Inv22 and highly sensitive to carrier mosaicism detection.

Drospirenone enhances GPIb-IX-V-mediated platelet activation.

Epidemiologic studies recently revealed that using drospirenone (DRSP)‐containing contraceptives is associated with an increased risk of thrombosis in women. However, the underlying causality is unclear.