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<p><b>OBJECTIVE</b>To explore the genetic basis for a Chinese pedigree affected with congenital hypofibrinogenamia.</p><p><b>METHODS</b>Peripheral blood samples were collected from 9 members from the pedigree. Routine coagulation tests including activated partial thromboplastin time (APTT), thrombin time (TT), the prothrombin time (PT) were carried out. The activity of fibrinogen (Fg: C) was measured using Clauss method, and fibrinogen antigen (Fg: Ag) was measured with immunoturbidimetry. All exons and exon-intron boundaries of the fibrinogen Aα, Bβ and γ chain genes were amplified using PCR, which was followed by direct sequencing. Suspected mutation was confirmed by reverse sequencing. The mutant fibrinogen was analyzed with Swiss-PdbViewer.</p><p><b>RESULTS</b>The proband showed prolonged APTT, PT and TT. Her functional fibrinogen (Fg: C) and antigen fibrinogen (Fg: Ag) levels were reduced to 0.69 g/L and 0.72 g/L, respectively. Her mother and grandmother also had a low levels of fibrinogen, which were 0.99 g/L and 0.83 g/L for Fg: C, 1.02 g/L and 0.87 g/L for Fg: Ag, respectively. The results of other members from the pedigree were all within the normal range. Genetic analysis reveled a heterozygous G>T mutation at nucleotide 7590 in exon 8 of γ gene in the proband, which was predicted to be a novel Ser313Ile mutation. The mutation was also found in her mother and grandmother. Model analysis showed that the Ser313Ile mutation disturbed the hydrogen bonds between Ser313, Asn319 and Asp320. Moreover, the mutation also altered the mutual electrostatic force and affected the folding and instability of the mutant fibrinogen.</p><p><b>CONCLUSION</b>The heterozygous Ser313Ile mutation probably underlies the hypofibrinogenemia in this pedigree.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Afibrinogenemia , Genetics , Fibrinogen , Chemistry , Genetics , Heterozygote , Mutation , PedigreeABSTRACT
Objective To analyze the mutations of F12 gene in one pedigree with congenital factor FⅫ(FⅫ)deficiency, and investigate the molecular mechanisms of FⅫ deficiency.Methods Pedigree investigation.In February 2015,a patient with hereditary FⅫdeficiency was admitted to the Third Clinical College of Wenzhou Medical University.Activated partial thromboplastin time(APTT), prothrombin time (PT),FⅫactivity(FⅫ:C),FⅫ antigen(FⅫ:Ag)and other coagulant parameters were tested in the proband and his family members.5′and 3′UTR, all exons and their exon-intron boundaries of F12 gene were analyzed by direct sequencing.The detected mutations were confirmed by reverse sequencing.The conserved amino acids were analyzed by ClustalX-2.1-win software, and four bioinformatics softwares (PolyPhen-2,PROVEAN,SIFT and MutationTaster)were also used to analyze the effect of mutations on protein function.Results The proband and her younger brother showed a markedly prolonged APTT which were 116.4 s and 101.3 s, while her father had slightly prolonged APTT, and other family members were normal.The FⅫ:C and FⅫ:Ag of family members were also decreased(the proband,2.0% and 1.0%;her younger brother,2.0% and 1.0%; her father,18.0% and 13.0%).The phenotype of all members was consistent with cross-reactive material(CRM)negative.Nucleotide sequencing analysis showed that the proband and her younger brother had missense mutations in the F 12 gene, including one homozygous mutation c.1681G>A(p.Gly542Ser)and a commonly reported single nucleotide polymorphism site within the promoter region of the F12 gene(46T/T).Sequencing results from the proband's parents and son demonstrated them as carriers of a heterozygous missense mutation.The proband's husband was normal and with 46C/C in the promoter region.The ClustalX-2.1-win results indicated that the Gly542 was highly conserved among the homologousspecies.The predicting outcomes of the four bioinformatics softwares were the same,the PolyPhen-2(score 1.000)and PROVEAN(score -4.975)both declared p.Gly542Ser was a harmful mutation.The SIFT(score 0.00)and the MutationTaster(score 0.999)manifested the mutation could affect the protein funtion.Conclusions c.1681G>A(p.Gly542Ser)in exon 14 and 46T/T were related with the significant decrease of the FⅫlevel of this pedigree of hereditary FⅫ deficiency.
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Objective To explore the correlation between B lymphocyte activation and CD154 expression and lupus anticoagulants (LAC) in patients with systemic lupus erythematosus (SLE). Methods Sixty newly diagnosed SLE patients (SLE group) and 32 healthy controls (control group) were involved. The expressions of CD27 and CD154 in peripheral blood were determined by flow cytometry, and the positive expression rates were computed. The LAC was determined by activated partial thromboplastin time, and the LAC ratio > 1.20 was positive. Results The positive rate of CD27, expression intensity of CD27, positive rate of CD154 and expression intensity of CD154 in SLE group were significantly higher than those in control group: 0.047 ± 0.021 vs. 0.035 ± 0.014, 0.387 ± 0.185 vs. 0.214 ± 0.091, 0.191 ± 0.108 vs. 0.101 ± 0.081 and 0.049 ± 0.018 vs. 0.022 ± 0.012, and there were statistical difference (P<0.05 or <0.01). Among patients with SLE, the LAC positive was in 28 cases, and the LAC negative was in 32 cases. The positive rate of CD27, expression intensity of CD27, positive rate of CD154 and expression intensity of CD154 in SLE patients with LAC positive were significantly higher than those in SLE patients with LAC negative: 0.055 ± 0.023 vs. 0.037 ± 0.014, 0.444 ± 0.179 vs. 0.329 ± 0.123, 0.218 ± 0.101 vs. 0.158 ± 0.044 and 0.058 ± 0.035 vs. 0.020 ± 0.009, and there were statistical differences (P<0.05). Conclusions The B lymphocyte activation and CD154 abnormal expression correlates with generation of LAC in patients with SLE. It provides a basis for the further study on intervening the generation of LAC and reducing the risk of thromboembolism.
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Objective To investigate the effect of high iodine traditional Chinese medicine in the treatment of subclinical hypothyroidism in serum VB12,Hcy and thyroid function.Methods 84 patients of subclinical hypothyroidism from August 2014 to May 2016 in our hospital randomly divided into two groups,the control group of 42 cases were treated with levothyroxine sodium tablets treatment,42 cases in the experimental group received more with high iodine traditional Chinese medicine.The changes of serum VB12,Hcy and thyroid function were observed before and after treatment in two groups. Results Compared with before treatment, levels of blood lipid,Hcy and TSH in two groups significantly decreased,levels of VB12 increased(P<0.05);compared with the control group after treatment,levels of blood lipid,Hcy and TSH in experimental group were significantly lower than the control group, the level of VB12 was higher than the control group,the differences were statistically significant (P <0.05).Conclusion High iodine can effectively reduce blood lipids in patients with subclinical hypothyroidism,levels of Hcy and TSH in pregnancy,increased the levels of VB12,which has good clinical curative effect.
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Objective To study the Val247Leu and Trp316Ser polymorphisms of β2-glycoprotein Ⅰ(β2GPⅠ) in systemic lupus erythematosus (SLE) patients and their associations with antiphospholipid antibodies and thrombotic complications.Methods We used DNA sequencing to detect the polymorphisms of Val247Leu and Trp316Ser in 378 SLE patients and 240 normal controls.Anti-β2GP Ⅰ antibodies and anticardiolipin (ACA) were tested by enzyme linked immunosorbent assay (ELISA).Lupus-type anticoagulants(LAC) was performed by diluted Russell's Viper Venom Test.Then the patient group was further analyzed according to APLs (Anti-β2GP Ⅰ antibody,LAC and ACA),thrombosis and obstetrical complications using Logistic regression analysis to confirm whether there are associations between β2GPⅠpolymorphism and those factors.Results For Va1247Leu,the predominant genotype was LL in healthy controls which accounted for 57.08％,while it was VL in SLE patients which accounted for 59.5％ (x2=45.01,P=0.000).Frequency of VV genotype was significantly higher in patients with thrombosis,anti-β2GP Ⅰ,ACA and obstetrical complications (OR=6.79,3.75,2.12 and 3.85,respectively;P=0.000,0.001,0.044 and 0.017,respectively).Those patients with VL genotype tended to have positive anti-β2GPI,LAC,ACA,thrombosis and also obstetrical complications (OR=2.95,1.88,2.47,2.97 and 2.74,respectively;P=0.000,0.007,0.000,0.001 and 0.016,respectively) than those negative ones.The predominant genotype of Trp316Ser was TT,then TS.No correlations could be found between Trp316Ser polymorphism and APLs,neither relation to thrombosis complications.Conclusion The polymorphism of Val247Leu is significantly associated with the presence of APLs,thrombosis and obstetrical complications.Both VV and VL genotype are risk factors for the generation of APLs,occurrence of thrombosis and obstetrical complications.The VV genotype is a high risk factor for thrombosis.Trp316Ser polymorphism does not contribute to the APLs production and also have no correlations with thrombotic complication.
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<p><b>OBJECTIVE</b>To explore the molecular pathogenesis and clinical phenotypes in 10 probands with inherited fibrinogen (Fg) deficiency.</p><p><b>METHODS</b>The diagnosis of hereditary Fg deficiency was validated by prothrombin time (PT), thrombin time (TT), Fg activity (Fg:C) and Fg antigen (Fg:Ag) in plasma. All of the exons and their flanking sequences of the Fg gene were analyzed by direct sequencing. Detected mutations were confirmed by reverse sequencing.</p><p><b>RESULTS</b>The ranges of Fg:C and Fg:Ag in the 10 probands were 0.52-0.91 g/L and 0.62-2.98 g/L, respectively. Five of the probands had type I disorders, and 5 had type II disorders. Seven point mutations were identified, among which 6 have located in the D region. γThr277Arg, γAsp316His, γTrp208Leu and Lys232Thr were novel mutations, and αArg19Ser was first reported in Chinese. Four probands had the same mutation site (γArg275). As to the clinical manifestation, probands with type I disorders were asymptomatic or with mild or medium symptoms, while those belonged to type II disorders had moderate or serious symptoms. Two probands have carried an Arg275Cys mutation but had different clinical manifestations.</p><p><b>CONCLUSION</b>Mutations of the Fg gene seem to aggregate to the D region of FGG in our region, and Arg275 is a common mutation. However, no correlation has been found between the mutation site and clinical manifestations.</p>