[Correlation Analysis of Fâ §Gene Mutation and the Production of Fâ § Inhibitor with Severe Hemophilia A Patients in a Single Medical Center].

OBJECTIVE: To investigate the relationship between the type of FⅧgene mutation and the development of FⅧ inhibitors in patients with severe haemophilia A (HA). METHODS: The medical records of 172 patients with severe hemophilia A from January 2009 to September 2020 were reviewed. The types of FⅧgene mutations and the production of factor Ⅷ inhibitors were collected and divided into high-risk mutation group ( intron 1 inversions, large deletions, nonsense mutations), low-risk mutation group (missense mutations, small deletions and insertions, splice site mutations) and intron 22 inversions group. The correlation of FⅧgenotype and the production of FⅧ inhibitors in patients with HA were analyzed. RESULTS: Among 172 patients with severe HA, 21 cases(12.21%) developed FⅧ inhibitors. The cumulative incidence of FⅧ inhibitor development was 32%(10/31) in high risk group (75% patients with large deletions, 43% patients with intron 1 inversions, 20% patients with nonsense mutations) and 5%(2/43) in low risk group(6% patients with missense mutations, 5% patients with small deletions or insertions and 0% patient with a splice site mutation) and 9%(9/98) in intron 22 inversions group. Compared with the risk of FⅧ inhibitor development in intron 22 inversions group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝4.7, 95% CI： 1.7-13.0), the risk of FⅧ inhibitor development in low risk group was equal (OR＝0.5, 95% CI： 0.1-2.3). Compared with the risk of inhibitor development in low risk group, the risk of FⅧ inhibitor development in high risk group was higher (OR＝9.8, 95% CI： 2.0-48.7). CONCLUSION: Gene mutations of patients with severe HA in high-risk group which include intron 1 inversions, large deletions, nonsense mutations are a risk factor for FⅧ inhibitor production.

Generation an induced pluripotent stem cell line SXMUi001-A derived from a hemophilia B patient carries variant F9 c.223C＞T(p.R75X).

Hemophilia B (HB) is an X chromosome-linked recessive disorder caused by a quantitative or qualitative defect of coagulation zymogen factor IX. In this study, urine cells were collected from a patient with HB who carries variant F9 c.223C > T (p.R75X), and reprogrammed into induced pluripotent stem cells (iPSCs) using the reprogramming factors, OCT4, SOX2, m-MYC, and KLF4. The HB-iPSC line (SXMUi001-A) has characteristics similar to human embryonic stem cell, namely, pluripotency and the potential to differentiate into three germ layers. This cell line can be used as a disease model for exploring the molecular mechanism and readthrough treatment of HB.

Molecular mechanism of a novel Ser362Asn mutation causing inherited FX deficiency in a Chinese family.

Factor X (FX) deficiency is an inherited autosomal recessive bleeding disorder. Here, we analyzed a proband with FX deficiency in a Chinese family. Genetic analysis revealed that the proband and his affected sister was homozygous for c.1085G>A mutation, corresponding to a Ser362Asn substitution. In vitro expression experiments showed that the FX Ser362Asn mutation led to a significant reduction in activity levels in the culture medium. This Ser to Asn substitution may change the shape of the active site. Moreover, simulations of molecular dynamics indicated that the binding energy of the FX Ser362Asn to the substrate is higher than that of wild type and the side-chain conformation of the catalytic residue His276 (His42) is changed. This impairs the conformational switch of the protein from zymogen to proteinase, thus causing the functional defect of FX protein. Our findings suggest that the Ser362Asn substitution is a pathogenic mutation that causes inherited FX deficiency.

Spectrum of Molecular Defects in 216 Chinese Families With Hemophilia A: Identification of Noninversion Mutation Hot Spots and 42 Novel Mutations.

Hemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the factor VIII gene ( F8). Our aim is to identify the causative mutations in a large HA cohort from China. We studied 216 unrelated HA families. Molecular analyses of F8 were performed using a combination of molecular techniques, including polymerase chain reaction, direct sequencing, and multiplex ligation-dependent probe amplification. The deleterious consequences of the unreported missense mutations were evaluated using various bioinformatics approaches. Causative mutations in F8 were identified in 209 families, intron 22 inversion (Inv22) was identified in 89 severe families, and intron 1 inversion (Inv1) was positive in 5 severe families; 95 mutations were detected among 115 noninversion families, of which 42 were novel, including 29 null variations and 13 missense mutations for which causality was demonstrated via bioinformatics. Among the 53 previously reported mutations, more nonsense (5 of 9) and missense (10 of 26) mutation sites were found to occur at Arginine (Arg) sites and multiple small deletions/insertions (5 of 10) located within the poly-A runs of the B domain. The majority of these sequence variants frequently recurred in the database. The odds ratios for the likelihood of developing inhibitors significantly increased in the presence of nonsense mutation. Our F8 defect spectrum was heterogeneous. Small deletions/insertions in the poly-A runs of the B domain and nonsense and missense mutations at Arg sites were identified as mutation hot spots. Nonsense mutation increased the risk of developing inhibitors.

Hemophilia care in China: review of care for 417 hemophilia patients from 11 treatment centers in Shanxi Province.

AIMS: We analyzed the clinical features of 417 patients with hemophilia from 11 Hemophilia Treatment Centers in Shanxi Province (SP) in China. METHODS: We used data collected in the national registry of hemophilia A and hemophilia B in SP from January 2010 to December 2013. RESULTS: Ratio of hemophilia A:hemophilia B patients was 5:1, of which 48% (200/417) were severe, 31% (129/417) moderate and 21% (88/417) mild. Episodes of joint bleeding occurred in 73% (305/417) of patients. Only 4% (15/417) of patients received tertiary prophylaxis. Three percent of patients (2/72) were hepatitis B virus-Ab positive, and 7% (5/72) of patients were hepatitis C virus-Ab positive. The incidence of inhibitors was 6% (11/182). CONCLUSION: The ability to manage hemophilia in SP remains suboptimal. However, due to limited data, the evaluation and extrapolation of large hemophilia populations in SP are restricted, therefore, further studies with a large cohort are needed.

[Correlation analysis of thrombin-activatable fibrinolysis inhibitor single nucleotide polymorphism with venous thromboembolism].

This study was aimed to explore the change of single nucleotide polymorphism (SNP) of thrombin-activatable fibrinolysis inhibitor (TAFI) and its correlation of 2 sites (505a/g, 1040c/t) in its gene-coding region with venous thromboembolism (VTE). The genotype distribution of TAFI in 80 patients with VTE and 80 normal controls was detected by allele-specific PCR. The results showed that the distribution of each genotype of 505a/g polymorphism was not significantly different between the VTE and control groups (P > 0.05). However, t allele frequency of 1040c/t in VTE group decreased significantly as compared with the control group (40% vs 53.75%, P < 0.05), mainly due to the decrease of the proportion of tt homozygous in VTE group. It is concluded that obvious relationship is found between the polymorphism of 1040c/t in TAFI gene and VTE patients. t allele genotype may paly a protective role in VTE. The polymorphism of TAFI 505a/g may be not associated with VTE.

[1059 g/c gene polymorphism of C-reactive protein in patients with deep vein thrombosis].

This study was aimed to investigate the distribution of 1059 G/C gene polymorphism of C-reactive protein(CRP) in deep vein thrombus (DVT) and its clinical significance. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to screen 1059 G/C polymorphism in exon 2 of C-reactive protein gene in 61 cases of DVT and 60 healthy controls. The frequency of mutation was calculated. The results showed that there was no statistical difference of 1059 G/C genotype and mutation frequency of allele between deep vein thrombosis group and control group (p > 0.05). It is concluded that the 1059 G/C gene polymorphism of CRP displays certain difference in races and areas, and whether 1059 G/C gene polymorphism of CRP is a dangerous factor for deep vein thrombosis, which needs to be deeply explored.

[Expression and gene polymorphisms of B cell activating factor in patients with idiopathic thrombocytopenic purpura].

OBJECTIVE: To analyze the polymorphisms of B cell activating factor (BAFF) gene and the plasma levels of BAFF in patients with idiopathic thrombocytopenic purpura (ITP), and to investigate their roles in the pathogenesis of ITP. METHODS: Alleles specific polymerase chain reaction (AS-PCR) and agarose gel electrophoresis were used to identify polymorphisms -871C/T of BAFF promotor in 133 ITP patients and 117 healthy controls. The plasma levels of BAFF were assayed by ELISA. RESULTS: In ITP group, the frequency of C/C, C/T and T/T was 33.1%, 42.1% and 24.8%, respectively, the corresponding frequency in control group was 55.6%, 33.3% and 11.1%, respectively. The allele frequency of T in ITP and control groups was 45.9% and 27.4%, respectively. There was a significant difference in the BAFF -871C/T genotypic frequency between the ITP and control groups (P < 0.05). BAFF antigen in untreated ITP, treated patients and controls was 875.86 pg/ml, 502.59 pg/ml and 736.88 pg/ml, respectively, being also a significant difference among the three groups (P < 0.05). BAFF antigen in homozygous T/T was higher than that in homozygous C/C and heterozygous C/T, but the difference was not statistically significant (P > 0.05). CONCLUSIONS: Over expression of BAFF may be a risk factor for ITP patients. There is a correlation of the BAFF promotor polymorphism -871C/T with ITP, but the polymorphism does not affect the expression of BAFF.

[Significance of detection of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura].

OBJECTIVE: To detect the frequencies of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura (ITP) and non-immune thrombocytopenia, and to evaluate their roles in the diagnosis of ITP and their clinical significance. METHODS: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody and platelet-specific antibody in 58 ITP patients, 33 non-ITP patients and 31 healthy controls were tested by Enzyme-linked Immunospot Assay (ELISPOT) and modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) respectively. RESULTS: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody in ITP patients \[(6.6 ± 4.2)/10(5) PBMNC\] were significantly increased (P < 0.05) than that of the controls \[(1.3 ± 0.5)/10(5) PBMNC\] and non-immune thrombocytopenic purpura patients \[(2.2 ± 2.0)/10(5) PBMNC\]. However there was no apparent difference between the latter two groups (P > 0.05). ELISPOT had a sensitivity of 70.69%, a specificity of 90.91% for the diagnosis of ITP, the sensitivity being higher than that of modified MAIPA's (43.10%) (χ(2) = 7.03, P < 0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.886. CONCLUSION: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody may reflect the pathogenesis of ITP. ELISPOT assay have high sensitivity and specificity than modified MAIPA for the diagnosis of ITP and the guidance for clinical therapy.

[The clinical significance of immune-related marker detection in idiopathic thrombocytopenic purpura].

OBJECTIVE: To assess the clinical significance of detecting the immune markers in idiopathic thrombocytopenic purpura (ITP). METHODS: The frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombopoietin in 64 ITP patients and 31 healthy controls were measured with enzyme-linked immunospot assay (ELISPOT), modified monoclonal antibody immobilization of platelet antigens assay (MAIPA), flow cytometry and sandwich enzyme-linked immunosorbent assay respectively. RESULTS: Compared with the controls [1.3 ± 0.5/10(5) peripheral blood mononuclear cell (PBMC), (0.33 ± 0.06, 0.41 ± 0.03), (22.08 ± 4.54)% and (8.19 ± 2.46)%], the frequencies of circulating B cells secreting platelet-specific antibody (7.6 ± 4.6/10(5) PBMC in acute ITP group, 5.3 ± 3.0/10(5) PBMC in chronic ITP group), platelet-specific antibody (including the anti-GPIIb/IIIa antibody, anti-GPIb/IX antibody) (0.51 ± 0.11, 0.48 ± 0.06 in acute ITP group; 0.49 ± 0.10, 0.46 ± 0.09 in chronic ITP group), the percentage of CD(8)(+) T Lymphocyte (27.09 ± 9.86)%, the percentage of reticulated platelet in ITP patients [the megakaryocyte cytosis group (24.85 ± 19.18)%, the normal megakaryocyte group (23.89 ± 18.90)%] were significantly increased (all P < 0.05). The frequencies of circulating B cells secreting platelet-specific antibody in acute ITP patients were notably increased (P < 0.05) compared to the chronic ITP patients. In T lymphocyte subsets, the percentage of CD(3)(+) T lymphocyte and CD(4)(+) T lymphocyte and the ratio of CD(4)(+)/CD(8)(+) in the patients with ITP [(60.88 ± 14.59)%, (28.41 ± 10.55)%, 1.18 ± 0.59] were notably decreased than those in the healthy controls [(69.89 ± 6.43)%, (35.38 ± 5.05)%, 1.64 ± 0.29, P < 0.05]. There was no apparent difference of the level of thrombopoietin between ITP patients with megakaryocyte cytosis (72.09 ± 41.64) and health controls (75.37 ± 26.32, P > 0.05), however, the level of thrombopoietin of ITP patients with normal megakaryocyte apparently increased (118.60 ± 70.72, P < 0.05). CONCLUSION: Detecting the frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombopoietin in the patients with ITP may improve the diagnosis and guide clinical therapy.

[Significance of B cell activating factor of TNF family promoter polymorphisms in patients with immune thrombocytopenic purpura].

The study was aimed to examine the B cell activating factor promoter polymorphism of the TNF family (BAFF)-871 C/T in patients with immune thrombocytopenic purpura (ITP) and to explore its correlation with ITP and the relationship between the blood platelet count of newly diagnosed patients with ITP and genotypes of BAFF-871 C/T polymorphisms. Alleles specific polymerase chain reaction (ASP-PCR) and agarose gel electrophoresis were used to identify polymorphisms -871 C/T of BAFF promotor in 133 ITP patients and 117 healthy controls, and determine the genotype of subjects. Meantime, the frequency of genotype and alleles were analyzed. The results indicated that out of 133 patients with ITP, 33.1% patients exhibited C/C, 42.1% patients were heterozygous C/T, and 24.8% patients were homozygous T/T. The corresponding frequencies in 117 healthy controls were 55.6% C/C, 33.3% C/T and 11.1% T/T. The allele frequency of T in ITP patients and healthy controls were 45.9% and 27.4% respectively. There was significant difference in the BAFF-871 C/T genotypic frequency between the ITP patients and healthy controls (p < 0.05). The allele frequency of T in ITP patients was higher than that in healthy controls. There was no significant difference in the blood platelet counts between the various genotype (p > 0.05). It is concluded that the polymorphism -871 C/T of BAFF promoter is correlated with the pathogenesis of ITP. However, there is no significant difference in blood platelet counts between the various genotype, so the polymorphism -871 C/T of BAFF promoter can not be referred as the analysis index for evaluating the severity of ITP.

[Correlation of inflammatory marker and coagulation factors with deep vein thrombosis].

This study was purposed to investigate the correlation of deep vein thrombosis (DVT) with C-reactive protein (CRP), fibrinogen (Fg), coagulation factor VIII (FVIII:C), coagulation factor IX (FIX:C) and to explore the effect of inflammation and coagulation as well as their interaction in DVT and its mechanism. 59 patients with DVT undergoing selective venous ultrasonography and 26 healthy individuals as controls were enrolled in this study. The plasma level of CRP was detected by immunoturbidimetry, FVIII:C, FIX:C levels were determined by a one-stage assay and fibrinogen level was measured by full-automatic biochemical apparatus. The results showed that the mean levels of plasma CRP, Fg, FVIII:C and FIX:C were significantly higher in deep vein thrombosis group than that in controls [CRP (2.67 +/- 0.91) vs (0.14 +/- 0.08) mg/dl; Fg (4.73 +/- 1.36) vs (2.79 +/- 0.66)g/L; FVIII:C (126.71 +/- 28.10) vs (81.35 +/- 20.77)%; FIX:C (81.01 +/- 23.60) vs (70.71 +/- 11.3)%] (p < 0.01), and the level of plasma CRP was strongly correlated with Fg, FVIII:C and FIX:C (r(s) = 0.432, 0.571 and 0.544, p < 0.01). It is concluded that the DVT and inflammation are closely related, increased level of plasma CRP may be a predictor of DVT. Increased plasma levels of Fg, FVIII:C and FIX:C all are important risk factors to DVT. Interaction between inflammation and coagulation promote the incidence of DVT, which may be one of DVT pathogenesis.

[Gene mutation analysis of one case with von willebrand disease type 2A].

Objective of this study was to identify gene mutation involved in a patient with type 2A von Willebrand disease (vWD). The bleeding time, vWF:Ag, FVIII:C, RIPA and multimeric assay were used for phenotypic diagnosis. All of the 52 exons and the exon-intron boundaries of vWF gene were amplified by polymerase chain reaction (PCR) and direct sequencing was carried out. The results indicated that the levels of vWF:Ag, FVIII:C and RIPA decreased in this patient, the vWF multimer with high and intermediate molecular weight was absent in plasma. The sequencing of genomic DNA revealed a C4738G (L1580V) missense mutation in the vWF gene from the patient. In conclusion, the C4738G (L1580V) missense mutation effecting the form of vWF multimer was responsible to molecular mechanism in this patient with vWD.

[Correlation analysis of tissue factor promotor polymorphism -1208I/D and venous thromboembolism].

The study was aimed to investigate the correlation of tissue factor promotor polymorphism -1208I/D with the venous thromboembolism in patients. Tissue factor promotor polymorphism -1208 was detected by polymerase chain reaction (PCR) and DNA sequencing in 96 cases of DVT, 14 cases of PE and 59 nonthrombosis normal individuals. The results showed that the allele containing a 18-bp nucleotides insertion at -1208. 67.8% of normal individuals exhibited D/D, 25.4% were heterozygous I/D, and 6.8% were homozygous for I/I. DVT group and PE group exhibited a similar distributions (62.5%D/D, 29.8% I/D, 8.3% I/I and 57.1% D/D, 35.7% I/D, 7.1% I/I). The allele frequencies of D and the allele frequencies of I in the normal control, DVT and PE groups were 80.5%, 77.1%, 75.0% and 19.5%, 22.9%, 25.0% respectively. There was no significant difference in the TF-12081/D genotype frequency between the groups of patients and normal individuals. In conclusion, there is no correlation of the tissue factor promotor polymorphism -1208I/D in the patients with venous thromboembolism. The gene of promoter -1208I/D may not be a major susceptible gene of VTE in Chinese Han. Further investigations would be necessary to define accurately tissue factor gene polymorphisms.

[Detection of factor IX gene mutation in patients with hemophilia B by DNA sequencing].

In order to investigate the patterns of FIX gene mutation in 3 unrelated hemophilia B (HB) patients, the activated partial thromboplastin time (APTT) and FIX activity (FIX: C) tests were adopted for phenotype diagnosis. All of the eight exons and their flank of FIX gene were amplified by polymerase chain reaction (PCR), the nucleic acid sequences were detected by dideoxymediated chain-termination method. The results indicated that as compared with normal control, the APTT value significantly increased, FIX: C value obviously decreased, PT value was normal. Sequencing results showed that all of 3 HB patients had the changes of gene sequences, among 3 patients the G22119A point mutation of exon 6 existed in case No.1, the G7932C point mutation of exon 2 was detected in case No.2 and the T32685C point mutation of exon 8 was found in case No.3. In conclusion, the relevant changes of gene sequences in all of 3 HB patients were detected, which provides some evidences for molecular mechanism of gene deficiency in HB patients.