Research progress on histone acetylation/methylation in oral diseases / 口腔疾病防治

@#Histone acetylation and methylation can affect chromatin conformation and regulate a variety of biological activities. Abnormal histone acetylation and methylation modifications are related to the occurrence and development of a variety of oral diseases. Histone acetylation and methylation increase or decrease in an orderly manner to regulate the development of teeth. Fluoride ions can destroy the balance between histone acetylation and methylation, which may be related to the occurrence of dental fluorosis. In addition, histone acetylation and methylation are involved in the regulation of oral inflammatory diseases. In the inflammatory microenvironment, the expression of histone acetyltransferase GCN5 decreases, and the expression of Dickkopf 1 (DKK1) decreases, activating the Wnt/β-catenin pathway and ultimately inhibiting the osteogenic differentiation of periodontal ligament stem cells. Enhancer of zeste homolog 2 (EZH2) and H3K27me3 levels were decreased in inflamed dental pulp tissues and cells. EZH2 inhibition inhibited the expression of interleukin (IL)-1b, IL-6 and IL-8 in human dental pulp cells under inflammatory stimulation. Histone acetylation/methylation modifications can interact with multiple signaling pathways to promote the occurrence and development of oral tumors and are related to the high invasiveness of salivary gland tumors. Small molecule drugs targeting histone acetylation and methylation-related enzymes can regulate the level of histone methylation/acetylation and have shown potential in the treatment of oral and maxillofacial diseases. For example, the histone deacetylase inhibitor vorinostat can inhibit the secretion of inflammation-related cytokines; it also promotes the maturation of odontoblasts and the formation of dentin-related matrix, demonstrating its potential in pulp preservation. Understanding the role of histone acetylation/methylation modifications in the occurrence and development of oral diseases will help promote research on epigenetic modifications in oral diseases and provide new perspectives for disease diagnosis and treatment.

Effectiveness of Scutellaria baicalensis Georgi root in pregnancy-related diseases: A review / 中西医结合学报

The root of Scutellaria baicalensis Georgi, also called Huangqin, is frequently used in traditional Chinese medicine. In ancient China, S. baicalensis root was used to clear heat, protect the fetus, and avoid a miscarriage for thousands of years. In modern times, pregnancy-related diseases can seriously affect maternal and fetal health, but few systematic studies have explored the mechanisms and potential targets of S. baicalensis root in the treatment of pregnancy-related diseases. Flavonoids (baicalein, wogonin and oroxylin A) and flavonoid glycosides (baicalin and wogonoside) are the main chemical components in the root of S. baicalensis. This study presents the current understanding of the major chemical components in the root of S. baicalensis, focusing on their traditional uses, potential therapeutic effects and ethnopharmacological relevance to pregnancy-related disorders. The mechanisms, potential targets and experimental models of S. baicalensis root for ameliorating pregnancy-related diseases, such as recurrent spontaneous abortion, preeclampsia, preterm birth, fetal growth restriction and gestational diabetes mellitus, are highlighted.

Research progress on in vivo models for stem cell-based pulp regeneration / 口腔疾病防治

@#Basic research on pulp regeneration requires in vivo experiments. The PubMed database was searched for in vivo models of stem cell-based pulp regeneration using the following keywords: "pulp regeneration", "stem cell" and "animal model". The retrieved models were classified into ectopic, semiorthotopic and orthotopic regeneration models and their characteristics and clinical values were reviewed. This literature review indicated that the ectopic regeneration model is the most widely used model for the simple steps. However, this model does not accurately capture clinical situations. The semiorthotopic regeneration model, which is an improvement of the ectopic regeneration model, can create a more realistic regeneration environment. The orthotopic regeneration model can simulate clinical procedures that more closely resemble application, but it is less commonly used for difficult operations and long experimental periods. The applicability of the above three animal models depend on the stage of the animal experiment: the ectopic regeneration model is suitable to test the regenerative effect and biocompatibility of the implant complex; the semiorthotopic regeneration model is suitable to more persuasively evaluate the regeneration effect of the implant complex; and the orthotopic regeneration model is suitable to confirm the regeneration effect and practicability of the regenerative implant complex prior to clinical study.

Research progress on multifunctional natural antioxidants in oral therapies / 口腔疾病防治

@#Oxidative stress is closely associated with the development of oral diseases such as caries, periodontitis and endodontitis. The accompanying oxidative stress during inflammation could aggravate tissue damage. However, numerous studies have shown that some dental materials, such as composite resins, bleach, drugs for root canal irrigation and dental implants, can give rise to abundant free radicals, which have adverse effects on peripheral tissues. Therefore, it is essential to supplement with extra antioxidants against free radicals. Plant-derived natural antioxidants have attracted great attention in biomedicine because of their excellent biocompatibility and easy access. This paper focused on the redox imbalance in the oral cavity and the application of natural antioxidants to oral therapy and their modification of dental materials. Current research shows that by constructing polyphenol-based metal organic nanoenzymes or adding vitamins and polyphenols to bionic hydrogels, the safety and utilization rate of antioxidants can be significantly improved. However, these polymer delivery systems have problems such as poor degradability, hepatotoxicity and nephrotoxicity, and the research is still in its infancy. In terms of material modification, it is crucial to choose the type and ratio of natural antioxidants and raw materials, as well as appropriate modification methods. A strong chemical bond between the antioxidant and the raw material may lead to the failure of antioxidant release from the modified composite, lowering the antioxidant activity. At the same time, the selection of polyphenols rich in pyrogallol functional groups can retain more free phenolic hydroxyl groups after chemical modification, which is conducive to greater antioxidant activity by the implant materials. Although research on natural antioxidants in oral therapy has made progress, there is a lack of data supporting clinical trials and long-term application effects, and further research is still needed.

Research on the color stability of Biodentine and MTA within the blood environment / 口腔疾病防治

Objective@#To compare the color stability of Biodentine and mineral trioxide aggregate (MTA) within the blood environment in vitro and to further investigate the underlying reasons for such color instability. @*Methods @#We first generated Biodentine and MTA discs with a diameter of 5 mm and a height of 3 mm. 24 discs of each material were randomly divided into two groups: the deionized water group and the defibrinated sheep blood group. Discs of each group were immersed for 1 day or 7 days before assessments. First, all discs were photographed to directly compare the discoloration of Biodentine and MTA. The color degree of the two materials was tested by a spectrophotometer. Then, the high-resolution morphological characteristics were observed by scanning electron microscopy. Finally, the chemical contents of each element in the material were measured by energy-dispersive spectroscopy.@*Results @#Compared to immediately after stripping, a change in the brightness of discs after immersion in defibrinated sheep blood for 1 day was observed only in MTA. On the 7th day after being immersed in blood, the colors of both the Biodentine and MTA discs darkened and turned deep red, but the darkness of the MTA discs increased significantly. The color change of MTA immersed in blood was measured on a spectrophotometer with a greater 7-day ∆E (21.257 ± 0.955) than the Biodentine 7-day ∆E (5.833 ± 0.501) (t=24.781, P < 0.001). MTA exhibits more discoloration as the immersion time goes on. A significant difference was noted between the 1-day ∆E(6.233 ± 0.888) and the 7-day ∆E(t=19.956, P < 0.001) of MTA immersed in blood. However, there was no statistically significant difference between the 1-day ∆E (6.790 ± 0.831) and the 7-day ∆E(t=1.707, P=0.163) of Biodentine immersed in blood. It was observed by scanning electron microscopy that after 7 days of immersion in the defibrinated sheep ablood, the surface porosity of MTA was larger than that of Biodentine, and the crystal edge of MTA became rounded and blunt. The analysis by energy-dispersive X-ray spectroscopy showed that the oxygen content decreased and the bismuth content increased in MTA after immersion in defibrinated sheep blood for 7 days. Zirconium was not detected in Biodentine due to its low radiodensity, but the contents of other elements were stable in Biodentine after immersion in defibrinated sheep blood for 7 days. @* Conclusion@#The color stability of Biodentine within the blood environment is better than that of MTA in vitro, which is mainly related to the low surface porosity and stable composition of the anti-radiation agent of Biodentine.

Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation

ABSTRACT Twelve known compounds, including eight alkaloids, three lignans and one gossypol derivative, were isolated from the branches of Polyalthia rumphii (Blume ex Hensch.) Merr., Annonaceae. The chemical structures were determined by spectroscopic methods and comparison with literature data. All the isolates were evaluated the cytotoxicity against three human cancer cell lines: Hela, MCF-7 and A549, the results showed that partial of isolates displayed weak cytotoxicities with the IC50 values ranging from 25 to 40 µg/ml.

The binding mechanisms of F VIII Trp1707Ser mutation-associated inhibitor / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the binding mechanisms of FVIII Trp1707Ser mutation-associated inhibitor.</p><p><b>METHODS</b>The APPT, PT, TT, Fg and FVIII:C were detected to make phenotypic diagnosis of haemophilia A. Inhibitors titer were measured by Bethesda method. Long distance-PCR (LD-PCR) and sequence-specific PCR were adopted for screening the intron 22 and intron 1 inversions respectively. FVIII coding and boundary sequences were analyzed by direct DNA sequencing. Inhibitor was reacted with different segments of FVIII, including heavy chain and its components A1 and A2, light chain and its components A3, C1 and C2. Corrected test was used to measure the remaining F VIII:C (% ) by adding pooled normal plasmas. After labeling purified inhibitors with biotin, western blot was performed to further confirm the binding reactions between inhibitors and segments.</p><p><b>RESULTS</b>The haemophilia A patient had mild deficiency of FVIII:C (1.1%) and had high FVIII inhibitor titer of 18.4 BU. A mutation c.97223C>G in exon 14 of F8 gene resulted to p.Trp1707Ser was identified by DNA sequencing. Corrected test showed that the remaining F VIII:C was increased when inhibitors reacted with heavy chain and light chain, especially with heavy chain. The remaining FVIII:C was also increased in the A2 and C2 domain reactions. No significant differences were seen in the A1, A3 and C1 domain reactions. Antigen-antibody reaction bands were confirmed by western blots when degenerated B-domain deleted recombinant FVIII, A2 and C2 were used as antigens.</p><p><b>CONCLUSION</b>The binding sites of FVIIITrp1707Ser mutation inhibitor were the A2 domain of heavy chain and C2 domain of light chain. The binding reaction with heavy chain was more intense.</p>

Role of GSK-3β in isoflurane-induced neuroinflammation and cognitive dysfunction in aged rats / 华中科技大学学报（医学）（英德文版）

This study investigated the role of glycogen synthase kinase-3β (GSK-3β) in isoflurane-induced neuroinflammation and cognitive dysfunction in aged rats. The hippocampi were dissected from aged rats which had been intraperitoneally administered lithium chloride (LiCl, 100 mg/kg) and then exposed to 1.4% isoflurane for 6 h. The expression of GSK-3β was detected by Western blotting. The mRNA and protein expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Morris water maze was employed to detect spatial memory ability of rats. The results revealed that the level of GSK-3β was upregulated after isofurane exposure. Real-time PCR analysis demonstrated that isoflurane anesthesia increased mRNA levels of TNF-α, IL-1β and IL-6, which was consistent with the ELISA results. However, these changes were reversed by prophylactic LiCl, a non-selective inhibitor of GSK-3β. Additionally, we discovered that LiCl alleviated isoflurane-induced cognitive impairment in aged rats. Furthermore, the role of GSK-3β in isoflurae-induced neuroinflammation and cognitive dysfunction was associated with acetylation of NF-κB p65 (Lys310). In conclusion, these results suggested that GSK-3β is associated with isoflurane-induced upregulation of proinflammatory cytokines and cognitive disorder in aged rats.

Functional study of abnormal fibrinogen caused by Arg275His mutation in fibrinogen γ chain / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees.</p><p><b>METHODS</b>Routine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity.</p><p><b>RESULTS</b>The coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876T>C (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time.</p><p><b>CONCLUSION</b>We first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.</p>

Role of GSK-3β in isoflurane-induced neuroinflammation and cognitive dysfunction in aged rats / 华中科技大学学报（医学）（英德文版）

This study investigated the role of glycogen synthase kinase-3β (GSK-3β) in isoflurane-induced neuroinflammation and cognitive dysfunction in aged rats. The hippocampi were dissected from aged rats which had been intraperitoneally administered lithium chloride (LiCl, 100 mg/kg) and then exposed to 1.4% isoflurane for 6 h. The expression of GSK-3β was detected by Western blotting. The mRNA and protein expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Morris water maze was employed to detect spatial memory ability of rats. The results revealed that the level of GSK-3β was upregulated after isofurane exposure. Real-time PCR analysis demonstrated that isoflurane anesthesia increased mRNA levels of TNF-α, IL-1β and IL-6, which was consistent with the ELISA results. However, these changes were reversed by prophylactic LiCl, a non-selective inhibitor of GSK-3β. Additionally, we discovered that LiCl alleviated isoflurane-induced cognitive impairment in aged rats. Furthermore, the role of GSK-3β in isoflurae-induced neuroinflammation and cognitive dysfunction was associated with acetylation of NF-κB p65 (Lys310). In conclusion, these results suggested that GSK-3β is associated with isoflurane-induced upregulation of proinflammatory cytokines and cognitive disorder in aged rats.

The molecular mechanism of haemophilia B caused by the Arg327Ile novel mutation in FIX gene in vitro expression / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg327Ile (R327I) in FIX gene.</p><p><b>METHODS</b>The R327I, R327Ala(A), R327Lys(K), R327Asn(N) and a replacement mutant (FIXβFVII), in which FIX β strand 324-329 was replaced by that of FVII 298-303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type (WT) FIX expression plasmid. The HEK293 cell was transiently transfected, then the activity of FIX (FIX:C) was assayed by one stage method in the conditioned medium, while the FIX:Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells.</p><p><b>RESULTS</b>FIX:C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced (CRMR). The viable cell fluorescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX:C of the R327A, R327K, R327N and FIXβFVII mutants was reduced compared to that of WT, the reduction of FIX:C of FIXβFVII was the most significantly amount among all the mutants in medium. FIX:Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium.</p><p><b>CONCLUSION</b>The abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as the β strand domain of R327 located play important roles of the specific function of FIX.</p>

Phenotype and genotype analysis of two Chinese pedigrees with type 3 von Willebrand diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype of two Chinese pedigrees with von Willebrand diseases, and to investigate the molecular pathogenesis.</p><p><b>METHODS</b>Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor-ristocetin cofactor (vWF:Rco), von Willebrand factor antigen (vWF:Ag), von Willebrand factor activity (vWF:A), von Willebrand factor collagen binding assay (vWF:CB) and multimer analysis were used for phenotype diagnosis. DNA was extracted. All of the 52 exons and exon-intron bounda ries of the VWF gene were amplified with polymerase chain reaction(PCR) and analyzed by direct sequencing.</p><p><b>RESULTS</b>APTT and BT were prolonged. Plasma RIPA, vWF:Rco, vWF:Ag, vWF:A and vWF:CB was significantly decreased. No VWF multimer can be found by plasma VWF multimer analysis. Homozygous insertional mutation g.82888_82889insCATG in exon 17 was found in proband A. Compound heterozygous mutations g.94865 G to A (Trp856stop) in exon 20 and g.110698_110699delinsG in exon 28 were found in proband B.</p><p><b>CONCLUSION</b>Homozygous insertional mutation g.82888_82889insCATG and compound heterozygous mutations g.94865G to A(Trp856X) and g.110698_110699delinsG probably have respectively induced type 3 von Willebrand diseases in the two probands.</p>

Genotype and function analyses of four inherited dysfibrinogenemia pedigree caused by Arg16 amino acid substitution in fibrinogen Aα chain / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype, genotype and function in four Chinese pedigrees with inherited dysfibrinogenemia.</p><p><b>METHODS</b>Routing tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), the activities of antithrombin (AT), protein C (PC) and protein S (PS) were detected in four pedigrees. The activity and antigen of plasma fibrinogen were analyzed by Clauss and immunoturbidimetry methods, respectively. The molecular weight of fibrinogen of four probands was assessed by Western blot. The function of abnormal fibrinogen was evaluated by fibrinogen clottability, fibrinogen dynamic polymerization and fibrinolysis velocity, respectively. The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>Four probands had prolonged TT and RT, reduced plasma fibrinogen activity levels and normal antigen levels. The assays of Western blot showed no abnormal molecular weight of fibrinogen. Function tests revealed reduced fibrinogen clottability, delayed and decreased fibrinogen dynamic polymerization and reduced fibrinolysis velocity. Aα chain Arg16His and Arg16Cys mutations were identified in the four probands, respectively.</p><p><b>CONCLUSION</b>The four probands with dysfibrinogenemia were caused by the mutations of Aα chain Arg16His or Arg16Cys. Mutation of the fibrinogen induced dysfunction of plasma fibrinogen.</p>

Molecular analysis of a patient with hemophilia A caused by FVIII His99Arg mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).</p><p><b>METHODS</b>FVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.</p><p><b>RESULTS</b>The proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.</p><p><b>CONCLUSION</b>Both the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.</p>

Two novel mutations in one pedigree with hereditary Factor VII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mutations of coagulation factor VII (FVII) gene in one pedigree with hereditary FVII deficiency, and to investigate the molecular mechanisms of FVII deficiency.</p><p><b>METHODS</b>FVII gene mutations were analysed in the pedigree by direct DNA sequencing. The mutated DNA fragments were cloned into pMD19-T simple TA vector, and sequenced to confirm their distribution on chromosome. The plasma activity of FVII of the probands and their family members was detected with coagulation assay. The antigen of FVII were identified with ELISA.</p><p><b>RESULTS</b>Three gene mutations were detected in the pedigree: A/G to C at 15386 resulting in Arg353Pro/Gln353Pro, A to T at 15274 resulting in Lys316Stop, all three mutations were heterozygotes. Three kinds of polymorphisms were identified in his father: A to G transition at position 15386 resulting in Arg353Gln, heterozygotic deletion of 2050 - 2059 cctatatcct in promoter and G to A mutation in intron 1a, the same polymorphisms were found in his grandfather. The three polymorphisms were located in the same chromosome of his father.</p><p><b>CONCLUSION</b>Two mutations were found in the pedigree with hereditary FVII deficiency. One is nonsense mutation (Lys316Stop), the other is missense one (Gln353Pro). Gln353Pro and Lys316Stop might be the molecular mechanisms of FVII deficiency. The two novel mutations were reported for the first time in the literature.</p>

Analysis of phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia.</p><p><b>METHODS</b>Laboratory tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), and the activities of antithrombin (AT:C), protein C (PC:C) and protein S(PS:C) were detected in three pedigrees. The activity and antigen of plasma fibrinogen (Fg) were analyzed by Clauss and immunoturbidimetry methods, respectively. The Fg of three probands was assessed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of all the exons and exon-intron boundaries of the three Fg genes FGA, GFB and FGG were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>Three probands had normal APTT, PT, PC:C, PS:C and AT:C, but prolonged TT and RT. The activity levels of the 3 probands's plasma Fg were reduced, but antigen levels were normal. Western blot and SDS-PAGE showed no abnormal molecular weight of Fg. The 3 heterozygous mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys were identified in the 3 probands, respectively.</p><p><b>CONCLUSION</b>The three probands with dysfibrinogenemia were caused by the mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys, respectively. Both Aα Pro18Leu and Aα Arg16Cys were first reported in Chinese population.</p>

Phenotype and genotype analysis of three Chinese pedigrees with von Willebrand disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism.</p><p><b>METHODS</b>Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor (vWF): ristocetin cofactor (RCof) (vWF:RCof), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB), vWF and Factor VIII (FVIII) binding assay (vWF:FVIII:B) and multimer analysis were used for phenotype diagnosis. Genomic DNA was extracted from the peripheral blood (PB). All the 52 exons and flanking sequences of the probands' vWF gene were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>APTT were prolonged in all three probands, while BT were normal excepting for proband 3. Plasma RIPA, vWF:RCo, vWF:Ag, vWF:A and vWF:CB were decreased in different extents. In multimer analysis, proband 3 lost the large and intermediate molecular weight multimers, while proband 1 and 2 were normal. Gene analysis in the three probands revealed three heterozygous missense mutations of 144067 G→A (R2287Q) in exon 39, 110374G→A (R1374H) and 110770C→T (S1506L) in exon 28 and heterozygous polymorphism 110667G→A (D1472H) in exon 28, respectively.</p><p><b>CONCLUSION</b>The three heterozygous mutations (R2287Q, R1374H and S1506L) and an heterozygous polymorphism (D1472H) are genetic defects of the hereditary vWD of the three pedigrees respectively. R2287Q is a novel mutation reported for the first time in the literature.</p>

Molecular mechanisms of recurrent venous thrombosis in two pedigrees with type I antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency.</p><p><b>METHODS</b>The routine coagulation screening tests were detected, thrombin generation tests was performed to evaluate the hypercoagulation. Anticardiolipin antibody (ACA) and lupus anticoagulant (LA) were detected with enzyme-linked immunosorbent assay (ELISA) and diluted viper venom time assay (DVVT), respectively. The activities of protein C, protein S and AT (PC:A, PS:A, AT:A) were tested with chromogenic substrate assay or clotting method. The antigen of AT (AT:Ag) was performed with immunoturbidimetry methods. Western blot was used to analyze the molecular weight (MW) and the plasma levels of AT:Ag. All 7 exons and the flanking sequences were amplified by PCR. The mutation of AT gene and thrombophilia associated gene polymorphisms were analyzed by direct DNA sequencing. The expression plasmid of Ala404Asp mutant was constructed with site-directed mutagenesis method based on the wild-type (WT) AT cDNA contained in pcDNA 3.1 vector, and transiently expression of AT WT and the Ala404Asp mutant was performed using HEK293T cells. Cultured supernatant and cell lysates were collected and measured for AT:Ag by ELISA and Western blot.</p><p><b>RESULTS</b>The results of routine coagulation tests in two probands were normal, thrombin generation tests indicated that proband 1 presented hypercoagulable state with 2.8 and 1.5 times higher of the endogenous thrombin potential (ETP) and peak height compared with that of normal, respectively. The levels of PC:A, PS:A, ACA and LA were normal. AT:A in proband 1 and proband 2 were 45% and 32%, and AT:Ag were almost half of the normal (121 mg/L and 158 mg/L), respectively. The results of Western blot showed that both probands' plasma levels of AT:Ag were lower than the normal pooled plasma and MW was normal. Two heterozygous mutations of g.3291C→T(Thr98Ile), g.13863C > A(Ala404Asp) were identified in the probands, respectively. No proband had venous thrombosis associated gene polymorphisms. Expression in vitro showed that AT:Ag in culture media and lysates of Ala404Asp are 4.8% and 60.6% of that of WT, respectively.</p><p><b>CONCLUSION</b>Thr98Ile and Ala404Asp mutation of AT gene significantly correlate with recurrent venous thrombosis in the two probands, respectively. Ala404Asp has not been described before. The mutant Ala404Asp protein can not be expressed due to impaired secretion and increased intracellular degradation, resulting in type I AT deficiency.</p>

Analysis of phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V (FV) deficiency.</p><p><b>METHODS</b>The tests of activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) were used for phenotype diagnosis. All the exons and exon-intron boundaries of F5 gene were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>The APTT and PT in each of the four probands were obviously prolonged, and both activity and antigen of FV in the four probands were extremely lower compared with that of normal mixed plasma. Sequencing of F5 gene in proband 1 identified a heterozygous mutation, G16088C (Asp68His), and four polymorphisms, T35788C (Met385Thr), A47295G (His1299Arg), A58668G (Met1736Val) and A74083G (Asp2194Gly), which were located in the same chromosome; proband 2 was homozygous for two mutations, C46253T (Arg952Cys) and C46724T(Gln1109stop); the F5 gene of proband 3 showed a homozygous missense mutation, C67793G(Pro2006Ala); and proband 4 was homozygous for one missense mutation, C74022T (Arg2174Cys).</p><p><b>CONCLUSION</b>Five mutations (Asp68His, Arg952Cys, Gln1109stop, Pro2006Ala and Arg2174Cys) and four polymorphisms (Met385Thr, His1299Arg, Met1736Val and Asp2194Gly) may lead to type I inherited FV deficiency for these four probands, respectively. Gln1109stop, Pro2006Ala and Arg2174Cys haven't been identified before.</p>

Two new mutations of AT gene in type I inherited antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the clinical phenotype and gene mutation in two kindreds with type I inherited antithrombin (AT) deficiency.</p><p><b>METHODS</b>The coagulation and anticoagulation testing and thrombophilia screening were used for phenotypic diagnosis and immunonephelometry and chromogenic assay for plasma level of AT antigen (AT:Ag) and AT activity (AT:A), respectively. All of the seven exons and intron-exon boundaries and untranslation regions of AT gene were amplified by PCR, and the PCR products analysis was by direct sequencing. The corresponding gene sites of the two family members and healthy individuals were detected according to the gene mutation sites.</p><p><b>RESULTS</b>The plasma levels of AT:Ag of proband 1 and proband 2 were 126 mg/L and 117 mg/L, and AT:A was 49% and 48%, respectively. Heterozygotic deletion of 3239-3240delCT in proband 1 and nonsense mutation 3206A-->T (K70Stop) in proband 2 were rchaacterized in exon 2 of AT gene. And some of their family members were also detected with the heterozygotic gene mutation.</p><p><b>CONCLUSION</b>Type I inherited antithrombin deficiency of the two probands were caused by AT gene mutation 3239-3240delCT and 3206A-->T (K70Stop).</p>