Cancer Associated Cells in Tumor Microenvironment of Esophageal Carcinoma： A Review / 中国实验方剂学杂志

Esophageal carcinoma is one of the malignant tumors with a high mortality rate， accounting for nearly 570，000 cancer deaths worldwide annually， and this number is increasing year by year. In recent years， despite the continuous improvement of treatment programs for esophageal carcinoma， the overall five-year survival rate of esophageal carcinoma is still less than 20% due to the development of drug resistance and the tolerance of patients during the treatment process. Tumor microenvironment （TME） is composed of various cells and related components with tumor cells as the core and featured by hypoxia， acidosis， chronic inflammation and immunosuppression， which plays an important role in the progression of tumors. Studies have found that tumor-associated fibroblasts， tumor-associated macrophages， myeloid-derived suppressor cells and regulatory T cells in TME can promote the proliferation， migration， invasion， and lymph node metastasis of cancer cells by secreting cytokines and activating pro-inflammatory pathways， and promote cancer progression by inducing the drug resistance of cancer cells and evading immunosuppression. Because cancer-associated cells in TME are genetically more stable than cancer cells， have fewer mutations and have lower chance of drug resistance， targeting cancer-associated cells in TME by regulating TME is a new research direction of cancer therapy. Traditional Chinese medicine has the advantages of multi-component and multi-target. It can participate in the regulation of TME through multiple ways， reduce the number of cancer-associated cells in TME， inhibit crosstalk between TME and cancer cells， and restore immune cell function. It is an important source for the regulation of TME and the research and development of drugs targeting cancer-associated cells in TME. In this paper， the role of cancer-associated cells in the TME of esophageal cancer and the current application of traditional Chinese medicine targeting cancer-associated cells in TME are reviewed， so as to provide reference for the research and development of TME targeted drugs for esophageal carcinoma.

Research Progress on Anti-tumor Effect and Mechanism of Cucurbitacins / 中国实验方剂学杂志

Cancer is a major global public health problem. Statistics from the national cancer center of China also show that cancer has become a major disease threatening human health with increasing morbidity and mortality. The occurrence and development mechanism of cancer is complex， involving multiple stages， multiple genes and multiple signaling pathways. Conventional chemoradiotherapy and emerging targeted therapy methods are the main methods in treatment of tumor. However， the quality of life of patients as well as the sustained and effective therapeutic effect are seriously affected due to the toxic side effects and drug resistance. Therefore， it is the global focus to find safe and effective anti-cancer drugs. The research and development and application of anti-cancer herbal medicines such as paclitaxel， vinblastine， podophyllin， ginsenoside and ginseng polysaccharide have brought new hope for the treatment of cancer. Cucurbitacine from Chinese medicine cucurbitaceae plantsare is a kind of highly oxidized tetracyclic triterpene compound with extensive pharmacological effects and complex mechanism. In the family of cucurbitacines， cucurbitin B， D， E and I have been studied most frequently on anticancer effect， and in a large number of studies， they have been found to play an important role in tumor diseases of the digestive system， respiratory system， reproductive system， blood system， urinary system and so on. With significant effect in inhibiting tumor cells proliferation， blocking the cell cycle， inducing apoptosis and autophagy death， inhibiting cell migration and invasion， inhibiting tumor angiogenesis， regulating the levels of reactive oxygen species and regulating immune system， such cucurbitacins are expected to be developed as a new kind of anti-cancer drugs. The authors of this study aim to provide reference for the further research and development of new anti-cancer drugs about cucurbitines by summarizing the anti-tumor effect and mechanism of the cucurbitacins.

Chinese Medicine Injections in Treatment of Esophageal Cancer： A Review / 中国实验方剂学杂志

Esophageal cancer has a high morbidity and mortality worldwide， and the burden of esophageal cancer is still heavy in China. The conventional treatments such as radiotherapy and chemotherapy fail to yield a satisfactory outcome， implying that the safe and effective treatments are not available for the majority of patients. At present， esophageal cancer is mainly treated by Chinese medicine combined with radiotherapy and chemotherapy. The retrieval of related papers in Chinese and English reveals that Kang'ai injection， Compound Kushen injection， Kanglaite injection， Xiaoaiping injection， Aidi injection， Bruceae Fructus Oil Emulsion， Cinobufotalin injection， and Shenmai injection have been commonly used in esophageal cancer treatment. These eight injections are either single medicinal or compound preparations， involving 11 Chinese medicinals， which synergize the effects of radiotherapy and chemotherapy and reduce their adverse reactions in the treatment of esophageal cancer. Due to the complexity of Chinese medicinal components， a series of adverse reactions such as phlebitis， phlebosclerosis， and hemolysis may be caused by irrational dosage， administration speed， and compatibility in the use of injection. There are many clinical studies on Chinese medicine injections for anti-esophageal cancer but few studies on their active components and molecular mechanism. This paper reviewed the active components， effectiveness， safety， and mechanism of Chinese medicine injections frequently employed for treating esophageal cancer， in order to provide reference for their clinical application as well as the development of new Chinese drugs.

Effect of Chinese Medicine Regulating Protein Phosphorylation on Tumorigenesis and Development / 中国实验方剂学杂志

Protein phosphorylation is one of the main ways to activate protein bioactivity and make it participate in cell life activities. Researches have shown that approximately 30% of proteins in the human body are modified by phosphorylation at different levels and at different sites. If the protein phosphorylation modification level or site is abnormal, it will cause the occurrence and development of malignant tumors. Malignant tumors have always been a kind of diseases that endanger human life and health. According to statistics, as many as 18 million malignant tumors and more than 9.6 million deaths occur every year worldwide. With the continuous recognition on the abnormality of protein phosphorylation modification in tumorigenesis and development, the research and development of drugs for abnormality of protein phosphorylation modification has become a focus in the field of tumor therapy at present. Each traditional Chinese medicine(TCM) can be regarded as a natural molecular library, and it participates in the regulation of protein phosphorylation modification level with the advantages of multiple components and multiple targets, with slight side effect and low drug resistance, so TCM is one of the main sources of drug development for regulating protein phosphorylation modification levels. Through the search of multiple databases at home and abroad, it was found that certain monomers, parts extracted from TCM, single-TCM and TCM compounds can affect the tumor progression by regulating the level of protein phosphorylation and exert better anti-tumor effect. Based on the current research status of protein phosphorylation regulation by TCM at home and abroad, we found that TCM can inhibit tumor cell proliferation, invasion and metastasis, angiogenesis, and maintenance of stem cell stemness by regulating protein phosphorylation levels, and exert antitumor effects by promoting apoptosis. In order to clarify the molecular mechanism of TCM in regulating protein phosphorylation level and exerting antitumor effect, and provide evidences for the development and clinical application of antitumor TCM, the authors reviewed the mechanism of TCM in regulating protein phosphorylation level and exerting their antitumor effect from different ways in this paper.

Synergistic anti-tumor effect of metformin and dichloroacetate in MFCW-ampIified neuroblastoma BE-2C cells / 中国药理学通报

To investigate the effect of metformin production of combined group compared with alone combined with dichloroacetate on the proliferation of A/FC/V-amplified neuroblastoma BE-2C cells and its mechanisms. Methods The inhibitory effects of metformin and dichloroacetate alone or in combination on BE-2C cells was measured by CCK-8 assay; the concentrations of glucose and lactic acid in the medium were measured by a glucose test kit and L-lactic acid assay kit; cell apoptosis was determined by flow cytometry (FCM) with Annexin V-FITC conjugated propidium iodide (PI) staining; the expressions of apoptosis related protein were detected by Western blot. Results Metformin and dichloroacetate showed significant proliferation inhibition activity on BE-2C cells; there was obviously decreased glucose uptake and lactic acid production of combined group compared with alone group (P <0. 01) ; the apoptotic rate was significantly higher in combined group compared with that in alone group (P <0. 01) ; Bax and cleaved caspasce-3 protein expression markedly increased in combined group, but Bcl-2 protein expression significantly increased compared with alone group (P <0. 01). Conclusions Metformin combined with dichloroacetate shows a significant synergistic anti-tumor effect against BE-2C cells by reducing the accumulation of lactic acid caused by metformin and inducing apoptosis.

Correlation between PPARgamma and VEGF-C expression in extrahepatic cholangioadenocarcinoma (EHCAC) and their prognostic significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the expression of vascular endothelial growth factor C (VEGF-C) and peroxisome proliferators-activated receptors (PPARgamma) in extrahepatic cholangioadenocarcinoma (EHCAC) and to elucidate its correlation with clinicopathological factors and their significance in prognosis.</p><p><b>METHODS</b>The expressions of PPARgamma and VEGF-C were detected by immunohistochemistry in 69 cases of EHCAC, 12 cases of non-tumor bile duct epithelium, and their relationship to clinicopathological parameters and follow-up were analyzed.</p><p><b>RESULTS</b>The positive rate of PPARgamma expression in 69 cases of EHCAC was 59.4%, significantly higher than that in 12 cases of non-tumor bile duct epithelium (0%), (P < 0.01). The positive rate of VEGF-C in 69 cases of EHCAC was 84.1%, also significantly higher than 16.7% in 12 cases of benign bile duct epithelium (P < 0.05). PPARgamma expression was associated with clinical TNM stage and lymph node metastasis. VEGF-C expression was associated with lymph node metastasis. Cox analysis results showed that portal vein and/or hepatic artery invasion, lymph node metastasis and VEGF-C expression were independent prognostic factors of EHCAC (P < 0.05).</p><p><b>CONCLUSION</b>PPARgamma expression may play an important role during tumorigenesis of extrahepatic cholangioadenocarcinoma. The expressions of PPARgamma and VEGF-C are significantly correlated with the clinicopathological characteristics and biological behavior of EHCAC. Expression of VEGF-C is an independent prognosis factors in EHCAC. The detection of PPARgamma and VEGF-C is valuable for evaluation of prognosis of EHCAC.</p>

Surgical procedure and prognotic analysis of extrahepatic cholangiocarcinoma: A report of 161 cases / 肿瘤

Objective: To explore the impact of clinical features and surgical procedure on survival of extrahepatic cholangiocarcinoma (EHCC) and the prognostic factors in patients with EHCC after curative resection. Methods: From 1995 to 2006, 161 EHCC patients undergoing operations were included in this study. The clinical features, diagnosis, surgical procedure and follow-up data were retrospectively analyzed. Factors influencing postoperative survival were analyzed using COX multiple stepwise regression model. Results: Of the 161 patients, 110 underwent radical resection and 32 underwent palliative resection, 19 underwent drainage or laparotomy. The overall 1-, 2-, 3-,and 5-year survival rates were 74.9%, 45.3%, 36.5%, and 11.1%, respectively. COX analysis showed hepatic infiltration, portal vein and/or hepatic artery invasion, and lymph node metastasis were independent prognostic factors after radical resection (P < 0.05). Conclusion: The radical resection is the key factor for increasing the long-term survival rate and improving the life qualities of EHCC patients. Partial hepatectomy or/and pancreatoduodenectomy in combination with skeletonization may be helpful for increasing radical resection rate and long-term efficacy.

Role of amino acid residues at positions 322 and 329 of hemagglutinin in virulence of H5N1 avian influenza virus / 病毒学报

Two H5N1 avian influenza viruses (AIV), A/mallard/Huadong/S/2005 (S, IVPI = 2.65, in mallard) and A/mallard/Huadong/Y/2003 (Y, IVPI = 0, in mallard), were capable of distinct in pathogenicity to non-immunized mallards (Anas platyrhynchos). There were two amino acid residues difference in the HA cleavage site between two viruses, 322 (S, Leu; Y, Gln) and 329 (S, deletion; Y, Lys). Based on the variation, a series of recombinant viruses carrying HA gene either from S or Y virus with mutation at 322 and/or 329 were constructed via reverse genetics system to explore the influence of the two amino acid residues on viral pathogenicity in mallards. Recombinant viruses with S virus backbone were completely attenuated in terms of their virulence to ducks when position 322 (L322Q) and/or position 329 (-329K) of HA gene had been mutated. The critical role that L322 and -329 of HA protein from S virus play in the high virulence to ducks were influenced by the entire background of that protein because the recombinant virus with HA gene from Y and other seven genes from S were completely attenuated even if Q322L and K329- mutations of HA gene had been achieved. Recombinant viruses with Y virus backbone significantly increased their virulence to ducks when position 322 (Q322L) and/or position 329 (K329-) of HA gene had been mutated. All recombinant viruses carrying HA gene from Y with Q322L and/or K329-mutations and other seven genes from S were completely attenuated in terms of virulence to ducks whereas all recombinant viruses carrying HA gene from Y with same mutations and other seven genes from Y gained significant virulence. It seems that the compatibility among eight genes might be an important factor for HA to exert its functions. Results indicated that the mutation at amino acid position 322 and deletion at 329 in HA cleavage site significantly influence the pathogenicity of S and Y viruses in mallard, the compatibility among eight genes also contribute to the pathogenicity of both viruses in mallard.

Analysis of phenotype and genotype in two Chinese pedigrees with hereditary protein C deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the phenotype and gene mutation in two Chinese pedigrees with hereditary protein C deficiency.</p><p><b>METHODS</b>The plasma level of protein C activity (PC: A) , protein C antigen (PC: Ag), protein S activity (PS: A), and antithrombin activity (AT: A) of the probands and their family members were detected using chromogenic assay and ELISA, respectively. All of the nine exons and intron-exon boundaries of protein C gene were amplified by PCR and analyzed by direct sequencing of the probands. Restriction enzyme site analysis was used to confirm the mutation.</p><p><b>RESULTS</b>The plasma PC: A and PC: Ag for proband 1 was 1.2% and 0, respectively. Compound heterozygous mutations, C(TGC)64W (TGG) and F(TTC) 139V(GTC) , were identified in her, the former being inherited from the maternal side and the later the paternal side. Further genetic analysis showed that her husband ( II 8) had the heterozygous deletion mutation (K150 or 151 Del) in exon 7, her daughter had the same heterozygous deletion mutation and a F139V. The plasma PC: A and PC: Ag for proband 2 was 50. 3% and 1.9 mg/L, respectively. He had the heterozygous Lys150 or Lys151 deletion mutation, which was inherited from his father. Polymorphisms of C/T at position - 1654, A/G at - 1641 , and A/T at - 1476A/T in the promoter region of protein C were confirmed in all members of the two pedigrees, of which, proband 2 had homozygous CC/GG/TT. The F139V mutation was confirmed by restriction enzyme site analysis and polymorphism for this mutation was excluded. PS: A and AT: A were in normal range for all members.</p><p><b>CONCLUSION</b>Compound heterozygous mutation C64W and F139V of protein C gene lead to type I hereditary protein C deficiency for proband 1. K150 or 151 deletion mutation and polymorphism of CC/GG/TT might lead to type I hereditary protein C deficiency for proband 2. C64W is a novel mutation for protein C gene. F139V and K150 or 151 deletion mutation are reported for the first time in China.</p>

Molecular mechanisms of protein C deficiency caused by C64W and F139V mutations / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the molecular mechanisms of protein C (PC) deficiency caused by PC gene mutations of C64W, F139V and K150 deletion (K150d).</p><p><b>METHODS</b>Wild-type and mutant PC cDNA expression plasmids (PCwt, PC C64W, PC F139V, PC K150d) were constructed and transfected into COS-7 cells or CHO cells respectively for in vitro expression study and immunofluorescent assay. Fluorescent real-time PCR was used to detect the expression of PC mRNA, protein degradation inhibition and endo-beta-N-acetylglucosaminidase H (Endo H) digestion experiments to explain the mutant protein degradation pathway and its localizations inside the cells.</p><p><b>RESULTS</b>PC C64W was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC F139V, most of the protein molecule was not secreted and degraded intracellularly. Mutant PC K150d was secreted normally from the cells. Fluorescent realtime PCR analysis of total mRNA from transfected cells showed no reduction of the mutant PC mRNA expression compared with that of wild-type PC mRNA. Protein degradation inhibition experiments showed that mutants PC C64W and PC F139V were degraded intracellularly through the proteasome pathway. Endo H digestion experiments and immunofluorescence results suggested that mutant PC molecules were located mainly in pre-Golgi apparatus.</p><p><b>CONCLUSIONS</b>Impaired secretion and degradation intracellularly of the mutants might be the molecular mechanisms of PC deficiency caused by C64W and F139V mutations. K150 deletion mutation might not affect the secretion of the mutant.</p>

Inherited afibrinogenemia caused by compound heterozygous mutations in the beta beta-chain of fibrinogen / 中国实验血液学杂志

Congenital afibrinogenemia is a rare autosomal recessive disorder, characterized by the complete absence or extremely reduced level of fibrinogen. To analyze the phenotype and genotype of a family with inherited afibrinogenemia, laboratory studies including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were tested in the proband and 9 family members. Fibrinogen (Fg) in plasma were measured by both functional and immunoturbidimetry assay. All the exons, exon-intron boundaries and promoter regions of three Fg genes were analyzed by direct sequencing. 102 healthy blood donors were used as normal control. The results showed that phenotype of the proband was diagnosed as afibrinogenemia. Compound heterozygous mutations in Fg FGB gene were detected in the proband. One was a nonsense mutation (Arg17stop) in exon 2, traced back to the proband's mother. The other was a missense mutation (Gly347Arg) in exon 7, which was from the proband' s father. It is concluded that afibrinogenemia is caused by the compound heterozygous mutations Arg17stop and Gly347Arg in the Beta beta-chain of fibrinogen.

A heterozygous point mutation G13328A in antithrombin gene causes thrombosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the phenotype and the gene mutation in a kindred with antithrombin (AT) deficiency.</p><p><b>METHODS</b>Immuno-nephelometry and chromogenic assay were used to detect the plasma level of AT antigen (AT: Ag) and activity (AT: A), respectively. All the seven exons and intron-exon boundaries of AT gene from the propositus were amplified by PCR and direct sequencing of the PCR pro-ducts was performed. Corresponding PCR fragments from the kindred were also sequenced directly. Megaprimer method was used to construct the mutant AT cDNA expressing vector from normal plasmid pCRII AT cDNA. The normal and mutant AT plasmid were transiently transfected into Cos-7 cells and AT: Ag was detected in supernatant and lysate of transfected cell with ELISA.</p><p><b>RESULTS</b>The plasma level of AT: Ag and AT: A for the propositus were 179 mg/L and 42.3%, respectively. A heterozygous G13328A missense mutation in exon 6 was identified, which led to the substitution of Thr (ACC) 404 for Ala (GCC). The sequencing results from the pedigree suggested that three other members also had the mutation. The level of AT:Ag in supernatant and lysate from cells transfected with mutant AT cDNA was 40% and 68% of that of normal AT cDNA transfected cells.</p><p><b>CONCLUSION</b>This is an unreported AT gene mutation in China, which causes type I hereditary antithrombin deficiency and thrombosis in the proposita.</p>

Severe hereditary coagulation factor V deficiency caused by two novel heterozygous mutations / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify gene mutations of a pedigree with inherited factor V (FV) deficiency.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) tests were performed for phenotypic diagnosis. The genomic DNA was extracted from the peripheral blood of the proband and all the 25 exons and their flanks of FV gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations were further confirmed by restriction enzyme digestion.</p><p><b>RESULTS</b>APTT, PT, TT, FV:C, FV:Ag of the proband were 249.2 s, 46.6 s, 17.9 s, 0.1% and 1.5%, respectively. FII, FVII, FVIII, FIX, FX activities, vWF and Fg were within normal ranges. Taking the GenBank Z99572 sequence as the reference, four mutations were identified in FV gene of the proband. They were a heterozygous two bases deletion in exon 13 (2238 approximately 2239delAG) introducing a frameshift and a premature stop at codon 689, and a heterozygous missense mutation in exon 23 (G6410T) resulting in the substitution of Gly for Val at codon 2079, respectively. The proband's father and mother were heterozygous for G6410T and for 2238 approximately 2239delAG, respectively.</p><p><b>CONCLUSION</b>The severe FV deficiency of the proband is caused by a frameshift mutation of 2238 approximately 2239delAG and a missense mutation of G6410T, which haven't been identified before.</p>

The inherited coagulation factor XI deficiency caused by intronic mutation IVS J-4delgttg / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify gene defect in a Chinese pedigree of hereditary coagulation factor XI (FXI) deficiency.</p><p><b>METHODS</b>The peripheral blood samples were collected from the proband and her family members. The plasma PT, APTT, FXI:C and FXI:Ag were assayed. The FXI gene exons and exon-intron boundaries of the proband were amplified by PCR and then sequenced directly. The mRNA of FXI in the peripheral blood was analyzed with RT-PCR.</p><p><b>RESULTS</b>The proband and some of her family members had prolonged APTT. The plasma FXI:C and FXI:Ag of the proband, her brother and her parents were lower than 10% and 50% of the normal values, respectively. Nucleotide sequence analysis revealed that the proband and her brother had a homozygous mutation of IVS J-4delgttg in FXI gene. The mutation was inherited from her parents who were heterozygotes. The mutation was not found in 60 normal subjects. No FXI mRNA was detected in peripheral blood sample of the proband.</p><p><b>CONCLUSION</b>The IVS J-4delgttg is a novel mutation causing FXI deficiency, which may interfere with mRNA splicing.</p>

Study on the molecular mechanism of antithrombin gene C2759T (Leu99Phe) mutation causing antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the molecular mechanism of antithrombin (AT) gene C2759T (Leu99Phe) mutation causing AT deficiency.</p><p><b>METHODS</b>A mutated AT cDNA expression plasmid ATM2759 was constructed by mega-primer method. ATM2759 and wild type AT cDNA expression plasmid ATN were transfected into COS7 cells or CHO cells by using Superfect reagent respectively for in vitro expression study and immunofluorescence assay.</p><p><b>RESULTS</b>The antigen levels of AT (AT:Ag) in the cell lysate of ATM2759 transfected COS7 cells and the cell culture supernatant were 174.97% and 35.63% of that of ATN transfected COS7 cells respectively, whereas the AT activity in the cell culture supernatant was 47.73% of the control's. Immunofluorescence analysis showed that the fluorescence intensity was significantly higher in ATM2759 transfected CHO cells than in those transfected with ATN.</p><p><b>CONCLUSIONS</b>Leu99Phe substitution may not affect the binding capacity of AT with heparin. Secretion defect and intracellular accumulation of the mutated AT protein might be the mechanisms of this mutation causing AT deficiency.</p>

FXI gene mutations in two pedigrees of congenital clotting factor XI deficiency / 中华血液学杂志

<p><b>OBJECTIVES</b>To identify the FXI gene mutations in two Chinese pedigrees of congenital factor XI deficiency.</p><p><b>METHODS</b>The peripheral blood samples were collected from the probands and their family members and the plasma FXI:C and FXI:Ag were determined. All the exons and exon-intron boundries of FXI gene were amplified with PCR and sequenced thereafter.</p><p><b>RESULTS</b>A nonsense mutation Trp228stop and two missense mutations Glu323Lys and Leu172Pro were disclosed in the two pedigrees. All mutations existed in a heterozygous state.</p><p><b>CONCLUSION</b>The FXI gene mutations Trp228stop, Glu323Lys and Leu172Pro attribute to the pathogenesis of the congenital factor XI deficiency in Chinese. The Leu172Pro is identified for the first time.</p>

Inherited coagulation factor X deficiency caused by two novel mutations in factor X gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanisms involved in a pedigree with inherited coagulation factor X (FX) deficiency.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT), prothrombin time (PT), FX activity (FX:C) and FX antigen (FX:Ag) test were adopted for phenotype diagnosis. All the 8 exons, intron/exon boundaries and the 5'untranslated regions (UTR) of the FX gene were amplified by polymerase chain reaction (PCR) from the genomic DNA extracted from the peripheral blood of the propositus. The PCR products were screened by direct sequencing. The mutation was confirmed by allele specific PCR (ASPCR).</p><p><b>RESULTS</b>The phenotype of the propositus was identified as FX deficiency (type II). Two novel FX gene mutations were detected in the propositus: one was a donor site splice mutation in intron 1 (IVS1 + 1G-->A), another was a missense mutation 1185G-->A in exon 8 (Arg347His).</p><p><b>CONCLUSION</b>The FX deficiency of the propositus is caused by double heterozygous mutations IVS1 + 1G-->A and Arg347His.</p>

Hereditary hemorrhagic telangiectasia resulted from a nonsense mutation Arg479 Stop in the ALK-1 gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the gene mutations in a pedigree with hereditary hemorrhagic telangiectasia.</p><p><b>METHODS</b>Genomic DNA was extracted from the peripheral blood of the propositus. All of the exons, intron/exon boundaries and the 5' untranslation regions (UTR) of the ALK-1 and endoglin gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing.</p><p><b>RESULTS</b>The mutation is a C1437T substitution in exon 10 of the ALK-1 gene, resulting in Arg 479 Stop.</p><p><b>CONCLUSION</b>The hereditary hemorrhagic telangiectasia propositus is caused by a heterozygous Arg 479 Stop mutation in the ALK-1 gene which has not been identified previously.</p>

Biochemical activity and gene analysis of inherited protein C and antithrombin deficiency in two Chinese pedigrees / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We identified the gene mutations in two Chinese pedigree of type I hereditary protein C deficiency and type I hereditary antithrombin deficiency.</p><p><b>METHODS</b>The plasma level of protein C activity (PC:A), protein C antigen (PC:Ag), protein S activity, antithrombin activity (AT:A) and antithrombin antigen (AT:Ag) of propositi and two family members were detected using ELISA and chromogenic assay, respectively. All exons and intron-exon boundaries of protein C gene and antithrombin gene were analyzed by direct sequencing of the corresponding amplified PCR products in DNA from the propositus.</p><p><b>RESULTS</b>The plasma PC:A and PC:Ag of propositus 1 was 26% and 1.43 mg/dl, respectively. The PC:Ag and PC:A of his father were normal. The decreased PC:A level was seen in his mother and 4 of his maternal pedigree. PS:A and AT:A were all normal in pedigree 1 members. A C5498T heterozygous mutation in exon 3 of protein C gene, resulting in the substitution of Arg for Trp at the 15th amino acid, was identified in propositus 1 and 8 of his relatives. The plasma AT:A and AT:Ag of propositus 2 was 48.6% and 10.4 mg/dl, respectively. The reduced AT:A and AT:Ag levels were found in his father and 5 of paternal pedigree. PC:A, PC:Ag and PS:A were all in normal range. A heterozygous 13387-9G deletion in exon 6 of antithrombin gene was identified in propositus 2. This mutation introduced a frameshift and a premature stop at codon 426 and existed in 6 members of pedigree 2.</p><p><b>CONCLUSION</b>The C5498T heterozygous mutation in exon 3 of protein C gene, first reported in China, leads to type I hereditary protein C deficiency. The 13387-9G deletion, a novel mutation, can cause antithrombin deficiency and thrombosis.</p>

Prothrombin deficiency resulted from a homozygous Glu29 to Gly mutation in the prothrombin gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the gene mutations in a pedigree with inherited prothrombin (FII) deficiency.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT), prothrombin time (PT), FII activity (FII:C) and FII antigen (FII:Ag) test were used for phenotype diagnosis. The genomic DNA was extracted from the peripheral blood of the propositus. All the 14 exons, intron/exon boundaries and the 5' and 3' untranslated regions (UTR) of the prothrombin gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations detected were further confirmed by restricted enzyme digestion. One hundred and three healthy blood donors were used as controls.</p><p><b>RESULTS</b>The phenotype of the propositus was prothrombin deficiency (type I). With reference to the prothrombin nucleotide sequence published by Degen & Dacie, three variations were found in the FII gene of the propositus. Among them, the novel mutation was a homozygous A601G subtitution in exon 2.</p><p><b>CONCLUSION</b>The prothrombin deficiency of the propositus is caused by a homozygous Glu29 to Gly mutation in the prothrombin gene.</p>