Clinical and molecular characteristics of a family with familial hypercholesterolemia with LDLRAP1 and ABCG8 gene abnormality / 中华检验医学杂志

Objective:To study the clinical and molecular characteristics of a family with familial hypercholesterolemia (FH) with LDLRAP1 and ABCG8 gene abnormality.Methods:In September 2020, one case of FH was included in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine; peripheral venous blood samples of members of the family were collected to detect serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) indicators; use high-performance liquid chromatography to detect serum stigmasterol and sitosterol content; perform second-generation gene sequencing to detect gene mutations in probands and family members; use Pymol software to detect gene mutations point for pathogenicity analysis, and use Uniprot Modelling software to perform protein structure modeling.Results:The patient presented with anemia, multiple xanthomas and early-onset acute coronary syndrome. The coronary angiography showed severe coronary artery lesions; abdominal ultrasound showed splenomegaly; blood smear showed shaped erythrocytes and large platelets. The level of serum TC, LDL-C, stigmasterol and sitosterol was 8.54 mmol/L (2.3-5.7 mmol/L), 4.84 mmol/L (range of normal value 1.3-4.3 mmol/L), 44 μmol/L (1.0-10 μmol/L), 28 μmol/L (1.0-15 μmol/L), respectively; LDLRAP1 gene mutation was found: exon4 c.415C>T:p.Q139X; the truncated protein formed by this homozygous mutation lost multiple stable protein structure regions, which can not have a normal function. At the same time, ABCG8 gene mutations were also found: exon13 c.1895T>C (p.V632A) and exon8 c.1199C>A:p.T400K . Two cases of family members had a mild increase in HDL-C (Ⅱ5: 2.33 mmol/L, Ⅱ6∶2.96 mmol/L), 3 cases carrying the ABCG8 gene mutations had a slight increase in stigmasterol (Ⅱ8: 23 μmol/L, Ⅱ7: 24 μmol/L, Ⅰ2: 18 μmol/L) and sitosterol (Ⅱ8: 41 μmol/L, Ⅱ7: 33 μmol/L, Ⅰ2: 45 μmol/L), suggesting that its association with the concentration of plant sterols. Conclusions:FH patients with LDLRAP1 and ABCG8 gene abnormalities may have abnormal plant sterol concentrations, and their clinical manifestations are more complicated. Therefore, family history, LDL-C, plant sterol levels, and genetic test results should be considered comprehensively.

Analysis of a pedigree affected with congenital dysfibrinogenemia due to heterozygous Gln195Arg mutation of fibrinogen γ chain gene / 中华检验医学杂志

Objective:To analyze the phenotype and genotype of a Chinese pedigree with congenital dysfibrinogenemia and investigate the molecular mechanism of the disease.Methods:Pedigree analysis. Peripheral blood samples were collected from 7 members of the pedigree and routine coagulation tests were conducted. The activity of fibrinogen was measured using Clauss method, and fibrinogen antigen was measured by immunoturbidimetry. All the exons and exon-intron boundaries of FGA, FGB and FGG genes were amplified using PCR, which was followed by direct sequencing. Electrophoretic and immunological analysis of fibrinogen, fibrinogen clottability measurement, fibrin polymerization measurement and scanning electron microscopy were used to investigate the pathogenesis of this disease. Results:The proband showed normal activated partial thromboplastin time (APTT) , prolonged prothrombin time(PT), thrombin time (TT),and reptilase time (RT).The antigen level of fibrinogen in the proband (1.6 g/L) decreased slightly, while the activity level of fibrinogen (0.7 g/L) decreased significantly. His father and grandmother showed normal APTT and PT, prolonged TT and RT. The antigen levels of fibrinogen in both of them were normal (2.0 g/L and 2.2 g/L, respectively), while the activity levels of fibrinogen were low (1.0 g/L and 1.1 g/L, respectively). The results of other members from the pedigree were all within the normal range. Genetic analysis revealed a heterozygous A>G mutation at nucleotide 4774 in exon 6 of FGG gene in the proband, which was predicated to be a novel Gln195Arg mutation. The mutation was also found in his father and grandmother.Western blot results showed that no abnormal bands of plasma fibrinogen were found in the proband, his father and grandmother. The fibrinogen clottability in the proband was 49.3%, while that in the heathy control was 98.9%. Both thrombin-induced fibrin polymerization and reptilase-induced fibrin polymerization were significantly impaired in the proband, compared to that in the heathy control. Scanning electron microscopy revealed that compared with the heathy control, the average fiber diameters of the fibrin clot in the proband increased significantly ( P<0.001), while the density of fibers decreased and the arrangement of fibers was sparse. Conclusions:The heterozygous Arg19Gly mutation, which probably damages functions of fibrinogen, should be responsible for the congenital dysfibrinogenemia in this pedigree. This mutation has not been reported.

Identification and molecular pathogenesis study of a case of inherited dysfibrinogenemia / 临床检验杂志

Objective@#To analyze the phenotype and genotype of a Chinese pedigree with inherited dysfibrinogenaemia and investigate the molecular mechanism of the disease. @*Methods@#Venous blood samples were collected from all family members, and routine coagulation tests were conducted. Functional fibrinogen in venous blood samples was measured by Clauss method, and the antigen level of fibrinogen in plasma was measured by immunoturbidimetry assay. All the exons and exon-intron boundaries of the three fibrinogen genes were analyzed by direct sequencing. Fibrinogen electrophoresis, fibrinogen clottability measurement, fibrin polymerisation measurement and electron microscopy scanning were also used to investigate the molecular characteristics and pathogenesis. @*Results@#The proband had normal activated partial thromboplastin time, prothrombin time and plasma fibrinogen antigen, but prolonged thrombin time, prolonged reptilase time and reduced fibrinogen activity level, which were also found in his father. The sequencing results of the proband revealed heterozygous A1211G in the exon 2 of FGA gene originating from his father, which caused Arg19Gly missense mutation. The western-blot results showed that no abnormal bands of plasma fibrinogen were found in the proband and his father. Both thrombin-induced fibrin polymerisation and reptilase induced fibrin polymerisation were significantly impaired compared to normal control. Fibrinogen clottability measurement showed that only about 20.8% molecules of plasma fibrinogen in the proband were involved in the clot formation. Scanning electron microscopy revealed that the proband′s average fibre diameters were found to be significantly thicker than that of the control(P＜0.001), and the density was smaller than that of normal control. @*Conclusion@#The Arg19Gly mutation should be responsible for the proband′s dysfibrinogenaemia and the relevant clinical symptoms.

Identification of genetic defects in a Chinese pedigree with factor XIII deficiency: case report and literature review / 中华血液学杂志

<p><b>OBJECTIVE</b>To perform phenotypic diagnosis, genetic diagnosis and prenatal diagnosis of inherited coagulation factor XIII (FXIII)deficiency in a Chinese family also provide a review of inherited coagulation F XIII deficiency.</p><p><b>METHODS</b>The activity levels of F XIII (F XIII:C) of proband and family members were measured by clot solubility test and REA-chrom F XIII kit. The antigen levels of F XIII(FXIII:Ag) were measured by enzyme-linked immunosorbent assay. Thrombelastography (TEG) test was used to make a comprehensive evaluation of coagulation status in the proband. All 15 exons and exon-intron boundaries of the F13A1 gene were amplified by PCR, and DNA sequencing was performed then. The mutation identified in the proband was screened in the family members. Furthermore, the related literatures were reviewed to provide a profile of clinical manifestation, gene mutations, the relationship between the mutations and phenotype, and treatments of inherited coagulation F XIII deficient cases.</p><p><b>RESULTS</b>The clot solubility test was positive in the proband. The FXIII:Ag level of the proband was less than 1% and the FXIII:C level was below the lower limit of detection (<3%). Two compound heterozygous missense mutations (p.Arg662* and p.Trp665*) were identified in the proband. Family study showed that the two mutations were both inherited from the parents. The fetus also carried two compound heterozygous mutations, the same as the proband, and was diagnosed with severe F XIII deficiency. As reported in the literatures, most mutations were missense mutations and nonsense mutations, and no hot spot was found. The clinical pattern of F XIII deficiency varied among patients, with potentially fatal consequences from severe bleeding complications.</p><p><b>CONCLUSION</b>Better understanding of F XIII biochemical properties and function and developing of FXIII laboratory assays and genetic detection could prevent missed diagnosis, and patients moght benefit from better care.</p>

Study on the impact of Trp1707Ser mutation on the binding mechanism of rF VIII light chain with VWF / 中华血液学杂志

<p><b>OBJECTIVE</b>To disclose the impact of Trp1707Ser mutation on the binding mechanism of rFVIII light chain (rFVIII LC) with VWF.</p><p><b>METHODS</b>Using long-chain PCR technique, we constructed rFVIII LC plasmids of both wild type and Trp1707Ser mutant type. BL21 competent cells were used for protein expression. Gradient renaturation was employed to refold protein. SDS-PAGE and Western blot were performed to identify the molecular weight of expressed protein. GST-Sefinose was used for protein purification and surface plasmon resonance (SPR) was employed to detect binding of B-domain-deleted rFVIII (BDD-rFVIII), wild and mutant rFVIII LC with VWF, respectively.</p><p><b>RESULTS</b>The results of SDS-PAGE and Western blot showed a molecular weight of 110×10(3) of expressed proteins, which were consistent with objective proteins. The expression quantity of wild type was higher than that of mutant type. A concentration-dependent combination of the 3 testing proteins with VWF was found. The KD value of BDDrFVIII (12.2) was lower than that of both rFVIII LCs (wild type 48.9 and mutant type 46.3), whereas there was no discrepancy between wild rFVIII LC and mutant rFVIII LC.</p><p><b>CONCLUSION</b>Trp1707Ser mutation didn't impact the binding of rFVIII LC expressed by BL21 competent cells with VWF. The heavy chain played a more important role in impacting the binding of FVIII with VWF.</p>

The functional study of antithrombin L99 mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the molecular mechanisms of inherited antithrombin (AT) deficiency caused by AT L99 mutation.</p><p><b>METHODS</b>Wild type (WT), L99V, L99A, L99I and L99S AT were purified from drosophila expression system. The binding capacity of AT and the low molecular weight heparin sodium was analyzed by the heparin binding assay. Surface plasmon resonance (SPR) was used to detect the binding ability of AT to thrombin (FIIa) or AT to coagulation factor Xa (FXa). The activity of AT(AT∶A)was detected by chromogenic assay.</p><p><b>RESULTS</b>The purified WT and mutant AT were at the same size. No additional band was observed by coomassie blue staining and western blot assay. Compared to the WT AT, the binding abilities of the low molecular weight heparin sodium to the AT L99V, L99A, L99I and L99S were (44.8±3.6)%, (118.9±14.0)%, (15.2±8.8)%, and(23.0±8.2)%, respectively. The binding abilities of FIIa to AT L99V, L99A, L99I and L99S were 13%, 57%, 3%, and 29%, while the binding of FXa to AT L99V, L99A, L99I and L99S were 7%, 51%, 1%, and 25%. The AT∶A of WT, L99V, L99A, L99I and L99S AT were 146.5%, 21.4%, 120.9%, 10.8%, and 39.0%, respectively.</p><p><b>CONCLUSION</b>The binding abilities of AT to heparin, FIIa and FXa were damaged by the L99 mutation, which resulted in decreased AT∶A and inherited AT deficiency.</p>

The phenotypic and genotypic diagnosis of three Chinese patients with von Willebrand disease / 中华内科杂志

Objective To analyze the phenotype and genotype of three patients with yon Willebrand disease (vWD),and to explore its molecular pathogenesis.Methods Bleeding time (BT),APTT,ristocetin induced platelet aggregation (RIPA),von Willebrand factor (vWF):ristocetin cofactor (Rco)(vWF∶ Rco),vWF antigen (vWF∶ Ag),vWF activity (vWF∶ A) test,vWF collagen binding assay (vWF∶ CB) and multimer analysis were detected for phenotype diagnosis.The dynamic process of blood coagulation was evaluated by using the thrombelastography.Genomic DNA was extracted from the peripheral blood.The vWF gene mutation was detected by sequencing.Results APTT,BT were prolonged in the three probands.Plasma vWF∶ Rco,vWF∶ Ag,vWF∶ A and vWF∶ CB were decreased in different degrees.RIPA was reduced in probands B and C.vWF multimer analysis found the lost of the large molecular weight multimers in proband B,while basically normal in probands A and C.The dynamic process of blood coagulation of proband C presented obvious hypocoagulability by using the thrombelastography.Heterozygous missense mutation g.106782G ＞ T resulting in Cys1130Phe in exon 26,g.110988G ＞ A resulting in Gly1579Arg in exon 28 and g.110373C ＞T resulting in Arg1374Cys in exon 28 were found in the probands A,B and C,respectively.Conclusion Three probands were diagnosed as type 1,type 2A or type 2MvWD by phenotype detection.Heterozygous missense mutation Cys1130Phe,Gly1579Arg and Arg1374Cys induced vWD of three probands,respectively.

Compound heterozygous mutations of a family with inherited hypofibrinogenemia / 中华检验医学杂志

Objective To analyze the phenotype and genotype of a Chinese family with inherited hypofibrinogenemia,and to investigate its molecular mechanism.Methods Peripheral blood was collected from seven people of this family and then plasma was separated.Activated partial thromboplastin time ( APTT),prothrombin time ( PT),thrombin time ( TT),reptilase time ( RT),the activities of antithrombin( AT∶ A ),protein C ( PC ∶ A ) and protein S ( PS ∶ A ) were tested.The activity and antigen of plasma fibrinogen were analyzed by Clauss method and immunoturbidimetry method,respectively.The fibrinogen peptide chain of the proband was semiquantitatively assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thrombin generation test was performed by calibrated automated thromhogram.The dynamic process of blood coagulation was evaluated by the thrombelastography (TEG).Genomic DNA was extracted from the peripheral blood.The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes FGA,FGB and FGG were amplified by polymerase chain reaction ( PCR ) and analyzed by direct sequen(c)ing.Results The activity and the antigen levels of the proband' s plasma fibrinogen were reduced to 0.48 g/L and 0.68 g/L,respectively.TT prolonged to 29.2 s and RT prolonged to 75.8 s.The assays of SDS-PAGE showed no abnormal molecular weight of fibrinogen.Peak height of thrombin generation was reduced to 249.93 nmol/L and endogenous thrombin potential was reduced to 1007.0 nmol · L-1 · min.Hypocoagulability state of the whole blood was found by TEG test.The coagulation index was - 8.6.The proband was diagnosed as inherited hypofibrinogenemia by phenotype analysis.Two mutations (Gln143Pro and g.4642delC) were found in the proband's fibrinogen Aa-chain gene,Gln143Pro came from her mother and g.4642delC came form her father.Conclusion Compound Heterozygous Mutations (Gln143Pro and g.4642delC ) of fibrinogen Aa-chain causes the proband congenital hypofibrinogenemia.

A pedigree analysis of pulmonary embolism caused by compound heterozygous mutations of protein C / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the molecular pathogenesis of protein C (PC) deficiency in a patient with pulmonary embolism and in his family members.</p><p><b>METHODS</b>Anticoagulated blood samples were collected from the proband and his family members to detect PC, PS and AT activities. PC antigen level was measured using ELISA. The genomic DNA was extracted to amplify all the 9 exons and their flanking sequences of PC gene using PCR, and the PCR products were sequenced. The mutated exons identified were amplified and sequenced for the other family members.</p><p><b>RESULTS</b>The proband and his parents and sister were identified as carriers of PC gene mutation, which led to type II PC deficiency. Sequencing of the proband's PC gene showed two heterozygous point mutations in exon 3 (G5540A) and exon 7 (C10230T) to cause compound heterozygous mutations of PC E29K and PC R147W, which were inherited from his father and mother, respectively. His sister was a heterozygote of PC R147W.</p><p><b>CONCLUSION</b>The proband is a compourd heterozygous mutations carrier of PC E29K and PC147W. PC E29K is a novel PC mutation, and PC R147W is a reported PC gene mutation seen in patients with type II hereditary PC deficiency and recurrent thrombosis.</p>

Studies on the mechanism of the Arg327Ile mutant causing defect in the function of factor Ⅸ / 中华检验医学杂志

Objective To investigate function of Arg327Ile (R327I) and Arg327Ala(R327A) FⅨ mutants and to study the molecular pathogenesis of haemophilia B(HB) caused by R3271 mutation.Methods Hygromycin-resistant cell line was screened and the secretion of FⅨ antigen into the medium was measured by ELISA.The cell line with appropriate expression levels of F Ⅸ antigen was selected for culture.Recombinant F Ⅸ (rF Ⅸ ) was purified from concentrated medium by two step methods of Q-Sepharose Fast Flow and anion exchange chromatography.The concentration and purity of rF Ⅸ were determined by ELISA and SDS-PAGE,respectively.The activation of wild-type ( WT),R327I and R327A of rFⅨ by FⅦa/TF/Ca2+ or FⅪa/Ca2+ was identified by Western blot in different time periods.The FⅨa and FⅧa complex formed by interaction with different concentrations of FⅧa was used to activate F X,the apparent dissociation constant (Kd) for FⅧa binding was calculated by the kinetic results.The kinetic data of the activation of FX by WT,R327I and R327A FⅨa with or without FⅧa were calculated.Results The amount of WT,R327I and R327A rFⅨ were 450,210,64 μg,and the purity of rFⅨ was confirmed by SDSPAGE.Both R3271 and R327A could be normally activated by FⅧa/TF/Ca2+ or FⅪa/Ca2+.Kd for FⅧa binding showed that the binding capacities of R327I and R327A were 4 and 5 times lower than WT,respectively.The catalytic efficiencies of R327I and R327A F Ⅸ a for F X were 6 and 8 times lower with FⅧa,and 3 and 7.4 times lower without F Ⅷ a,respectively.Conclusions R327I and R327A rF Ⅸ mutants impair their binding to the FⅧa.The site on R327 contributes to FⅧa binding.It is partly related to the activation of FX.The low FⅧa binding to R327I FⅨa may cause HB.

Study of molecular mechanisms of hereditary protein C deficiency in four Chinese families / 中华检验医学杂志

Objective To investigate the phenotype, genotype and molecular mechanisms in four Chinese pedigrees with venous thrombosis caused by hereditary PC deficiency. Methods The plasma activity of PC: A, TPS: A and FPS: A of the probands and their family members were detected with chromogenic and coagulation assay. The antigen of PC and FPS were identified with ELISA. Thrombin generation tests were applied to indicate the coagulation status. All of the nine exons and intron-exon boundaries of PC gene and PS gene were amplified by PCR and directly sequenced for mutaiton investigation. Results Compound heterozygous mutations of L-34P, K150del and A209V with 36% of PC: A and 57% of PC: Ag were identified in proband 1. PC: A was 46% , PC: Ag was 64. 4% while TPS: A, FPS: A and FPS: Ag were 36% , 19.5% and 20.9% respectively in proband 2. Two independent heterozygous mutations of R147W in PC gene inherited from his mother and T519stop in PS gene inherited from his father were identified. The anticoagulant activity of Proband 2 and his parents were declined in thrombin generation assay. In proband with PS defeciency and his father, the inhibition of thrombin generation capacity was decreased with exogenous APC, while his mother did not have significant difference. In Proband 3, PC: A was 32% while PC: Ag was 48.42% . Two independent mutations of R147W and R178W in Exon 7 were detected. Compound heterozygous mutations of R178W and D255H,with 21% of PC : A and 18. 36% of PC: Ag were identified in the Proband 4. Conclusions Hereditary PC deficiency or combined PC and PS deficiency result in venous thrombosis in four Chinese families. Mutants of L-34P, A209V, R178W, R147W and D255H might be the molecular mechanisms of PC deficiency.

Investigation on gene mutation from hereditary protein S deficiency pedigree / 中华检验医学杂志

Objective To identify the clinical phenotypic diagnosis and gene mutation detection of two kindreds with PS deficiency. MethodsPS: A was measured by chromogenic substrate method;TPS:Ag, FPS: Ag levels were measured by ELISA method; PS gene(PROS1 gene)was detected by amplifying 15 exons and flanking intron sequences from the propositus with PCR method. PCR products were purified and directly sequenced. Results For propositus 1,PS: A was 48.6% ,TPS: Ag was 136 mg/L, FPS : Ag was 41 mg/L, PROSI gene exon 2 was in c. Heterozygous base substitutions was detected in C121T locus, which led to Arg-1Cys (R-1C) heterozygous roissense mutation encoded in PS proteins. For propositus 2, PS: A was 29.2%, TPS: Ag was 83 mg/L, FPS: Ag was 26 mg/L, PROSI gene exon 14 was in c. Heterozygous base substitutions was identified in CI687T locus, in which Gln.522Stop heterozygous nonsense mutation was encoded in PS proteins. Conclusions c. C121T is a novel mutation locus detected in PROS1 gene. This heterozygous mutation could lead to type Ⅱ PS hereditary deficiency, while c. C1687T heterozygous mutation could bring about type Ⅰ PS hereditary deficiency.

Genetic diagnosis of four combined deficiency of factor Ⅴ and Ⅷ patients / 中华检验医学杂志

Objective To investigate the genetic diagnosis and molecular pathogenesis of four patients with combined deficiency of coagulation factor Ⅴ and Ⅷ and their family members. Methods The APPT, FT, FⅤ: C, FⅧ: C were detected for phenotypic diagnosis. Thrombin generation assay was applied to determine the generation condition of thrombin in patients and healthy controls. Cenomic DNA was extracted from peripheral blood using the TianGen RelaxCene Blood DNA System;amniotic fluid DNA was extracted with phenol-ethyl ether method. The LMAN1 and MCFD2 genes were analyzed by PCR. Gene mutations were detected with nucleotid sequences by using end-labeling dideoxy method. Results The APTT of Proband 1 was significantly prolonged to 88. 2s and her PT was prolonged to 19. 6 s. The combined deficiency was identified with FⅧ (FⅧ: C 24. 2% ) and FV(FⅤ: C 9. 1% ). Proband 2 and 3 were sisters. The coagulation studies revealed that both of them had prolonged APTT (71.6 s and 74.6 s respectively) and PT (22. 1 s and 18. 3 s respectively). The combined deficiency of FⅤ (FⅤ: C 7. 6% and 14. 5% respectively) and FⅧ( FⅧ: C 25% and 19.6% respectively) were identified. Proband 4 was detected to have the prolonged APTT (70.3 s),PT (18.2 s) and the deficiency of FⅤ(FⅤ: C 9. 4% ) and FⅧ (15. 7% ). The remaining phenotype indicators test of the 4 probands were normal. The diagnosis for the 4 probands was combined deficiency of factor Ⅴ and Ⅷ. The proband 1 was detected to have compound heterozygous mutations in LMAN1 gene while having the LMAN1 and MCFD2 direct gene sequencing. One mutation was a small insertion located on exon 8 [ nt912insA (X71661. 1)] that resulted in p. 305frameshiftX20 and her mother was detected to have the same heterozygous mutation on the the locus. The other mutation was located on exon 11: nt1366C > CT ( X71661. 1 ) , p. 456Arg > Stop which was inherited from her father. Amniocyte DNA was detected to have only one heterozygous mutaion [nt1366C > CT (X71661. 1) , 456Arg > Stop] inherited from the father. No mutation in MCFD2 gene was found in proband 1 and her parents. The analysis of the MCFD2 gene in proband 2 and 3 revealed a novel homozygous single base substitution (nt411T>C) in exon 4, which results in the exchange of the amino acid isoleucine by the amino acid threonine at amino acid position 136 (p. Ile136Thr). Sequencing of the whole LMAN1 gene showed that the proband 4 had one homozygous nonsence mutation in the exon 5 of the LMAN1 ( nt615C >T,p. 202 Arg> Stop). All of the 4 probands with combined deficiency of FⅤ and FⅧ showed declined endogenous thrombin potential in the thrombin generation tests. Conclusion The combined deficiency of FⅤ and FⅧ in the proband 1 results from the compound heterozygous mutations ( nt1366C > CT and nt912insA) in LMAN1 gene, which are inherited from her parents respectively. The prenatal genetic investigation for the patient mother with preganency indicates that the fetus is a female carrier with one mutation (nt1366C > CT) inherited from the father. The homozygous missence mutation ( nt411T > C, p. Ile136Thr) in the MCFD2 gene accounts for the proband 2 and 3. The daughter of the proband 2 is a carrier with a heterozygous mutation inherited from her mother. The homozygous nonsence mutation in the LMAN1 gene of the proband 4 results in the deficency of F Ⅴ and FⅧ.

Study on the molecular mechanism of the inherited factor X deficiency in three unrelated families / 中华检验医学杂志

Objective To identify the clinical features, the molecular diagnosis and the molecular mechanism of three unrelated factor X deficiency families. Methods Three probands were male and the diagnosis was validated by coagulant parameters. The F X coagulation activity ( F X∶ C ) and antigen (FX∶ Ag) were tested by clotting test and ELISA method. The cross-corrected test was used to rule out the inhibitor of FX in plasma. Thrombin generation test was evaluated. The antigen and the molecule weight of the FX in plasma were measured with western blotting. Gene mutations were analyzed in the probands and their family members with PCR and DNA sequencing. FX expression plasmids were constructed and transientby being transfected into 293T cells. FX: C and FX: Ag of the expression products were tested. Results APTT and PT in proband 1 were obviously prolonged, 113.4 s and 62.3 s, respectively. And there was no inhibitor in plasma. The thrombin generation was lower compared to normal reference. APTT and PT in proband 2 were 56. 5 s and 28.7 s. There was no inhibitor in the plasma. The thrombin generation was 1 101.5 nmol · min. APTT and PT in proband 3 were 117.3 s and 44. 3 s. The thrombin generation was 782.5 nmol · min. FX∶ C and FX∶ Ag in proband 1 were 1.4% and 3.6%, with a homozygous mutation in FX gene (Ser425→Pro). In vitro expression of the mutation showed a normal synthesis in the cell but secretion dysfuntion. In proband 2 F X: C and F X: Ag were 2. 2% and 5. 5%, with two heterozygous mutations in FX gene (Ala-29→Pro and Phe324→Leu). The Ala-29 → Pro mutation led to significantly reduced expressions of FX in both cell lysate and cell culture supernatants compared to wild-type plasmid,(41.32 ±5.21 )% and(6. 30 ± 1.84)% respectively. However Phe324→Leu mutation almost did not affect the FX synthesis. FX: C and FX: Ag in proband 3 were 2. 2% and 35%, with two heterozygous mutations in FX gene( Ala235→Thr and Arg347→Cys). The expressions of these two mutant FX proteins in cell lysate were similar to those of wild-type but obviously lower in the supernatant. Conclusions Five mutations of F X gene are found in this study. These mutations (Ser425Pro, Phe324Leu, Ala235Thr and Arg347Cys)can not affect F X protein synthesis. However Ala-29Pro mutation can reduce F X protein synthesis and cause secretion dysfunction.

Study on the molecular mechanism of two new mutations causing haemophilia B / 中华检验医学杂志

Objective To study two new factor Ⅸ mutations Cys82Ser and Ile288Ser in vitro and research the molecular mechanism of haemophilia B. Methods PcDNA3. 1 ( - ) FⅨwt expression plasmid was prepared. The mutated FⅨcDNA expression plasmids, PcDNA3.1 ( - ) FⅨM1 (Cys82Ser) and PcDNA3. 1 ( - ) F Ⅸ M2 (Ile288Ser) were constructed by megaprimer method respectively. Transient expression experiments were performed using HEK293 cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA. PcDNA3. 1 ( - ) was used as a blank control. The expression proteins were detected by ELISA, factor activity assay and flourescence stain. Results The results suggested that the two FⅨ gene mutations did not induce the reduction of the mutant FⅨ mRNA compared with the wild-type FⅨ mRNA. The FⅨ:Ag in culture media and cell lysate of wild type conduct were assigned as 100. 0%. The results of PcDNA3.1 ( - ) FⅨ M1 mutation protein were (27. 1 ± 5. 2)% and (99.4 ±4. 1)% respectively. For PcDNA3. 1( - )FⅨM2, the results were (5.3 ± 1.8)% and (31.7 ±2. 5)% respectively. The FⅨ: C in culture media of wild type conduct was also assigned as 100. 0%. Then the two types of mutant protein were ( 8. 5 ± 3.2 ) % and ＜ 1%, respectively. Immunofluorescence microscopy result suggested that the intensity of perinuclear spot was reduced in cells transfected with PcDNA3.1 ( - ) FⅨM2 while staining for PcDNA3. 1 ( - ) FⅨM1 was predominantely diffuse without perinnclear enhancement. Conclusions These results strongly suggest that the FⅨ Cys82Ser mutation protein is not been correctly folded, by any possibility. The mutation protein has secretion defect. The secretion dysfunction and the protein degradation intracellular are possiblely the molecular pathology of Ile288Ser mutant protein.

Mutation analysis of coagulation factor Ⅺ gene of inherited factor Ⅺ deficiency / 中华检验医学杂志

Ⅺ deficiency in Chinese Han population. Conclusion The 13 mutations of the F Ⅺ gene which were found in this study may unravel the molecular pathogenesis of the F Ⅺ deficiency in Chinese Han population.

Molecular analysis of the genotypes and phenotypes in three pedigrees with inherited antithrombin defidency / 中华检验医学杂志

Objective To investigate the clinical phenotype and genotype in three probands with antithmmbin(AT)deficiency and their families,and to identify the molecular mechanism of AT deficiency.Methods Chromogenic substrate method and immunoturbidimetry assay was used to detect the plasma levels of AT:A and AT:Ag,respectively.Genomic DNA was extracted from the peripheral blood.All 7 exons and the flanking sequences were amplified by PCR.and the abnormal mutant genes were analyzed by direct sequencing.Western blot was used to detect the AT levels and thrombin generation tests were used to detect coagulation status.Results The plasma levels of AT:A and AT:Ag of the three probands declined by 50%.G7386C(Trp225Cys)mutation in exon 4,C2591G(Ser36stop)in exon 2 and C9819T(Arg359stop)in exon 5 were characterized in the three prebands and they could result in W(Trp)225C(Cys)missense mutation,S(Set)36X(stop)nonsense mutation and R(Arg)359X(stop)nonsense mutation respectively,The testing results of phenotype and genotype from some of their family members showed consistent with results from the probands.Western blot results indicated that the Icyels of PC:Ag were lower compared with the normal pooled plasma.The hypercoagulative status was present in the probands using thrombin generation tests.Conclusions Type Ⅰ hereditary AT deficiency was found in these three families.The 3 heterozygous mutations.W225C,S36X and R359X are genetic defects of hereditary AT deficiency.W225C and S36X have not been described before.

Genetic diagnosis of two haemophilia families with recombination / 中华检验医学杂志

objective To make genetic diagnosis in two haemophilia families with recombination. Methods For hemophilia A(HA)family,screening of the F Ⅷ intron 22 and intron 1 inversion mutations was employed to identify the mutation. Linkage analysis with 8 polymorphic markers was adopted in the pedigree. For hemophilia B(HB)family,DNA sequencing of all coding regions of FⅨ gene Was used to detect the mutation directly. The muhifluorescent PCR method employing six FⅨ related STR was adopted in linkage analysis.Results In the HA family,the proband was positive in inversion 1 detection and the relative female was inversion 1 carrier. But linkage analysis with polymorphic markers showed contrary resuhs. Some markers certified that the female inherited the disease chromosome of the family while the others showed contrary results.In the HB family,it was unsuccessful in sequencing the exon 7 of the F Ⅸ gene in the proband and there was no mutation found in the other parts. The relative female and her amniocyte DNA were successful in sequencing the whole F Ⅸ gene and no mutation was detected.The linkage analysis of the family showed contrary results. Recombination occured in these two families. Conclusions Although the linkage analysis iS convenient and effective in carrier and prenatal diagnosis of hemophilia families. The recombination risk shouldn't be neglected especially when the polymorphic markers give inconsistent information for linkage analysis. It is necessary to find some high inforrnative markers intragenic or on the telomeric side to the gene in order to prevent the risk of recombination.

Analysis of phenotype and genotype in three Chinese pedigrees with glanzmann thrombasthenia / 中华检验医学杂志

Objective To identify the gene mutations of platelet membrane glycoprotein Ⅱ b,Ⅲa(GPⅡb/Ⅲa)in three Chinese pedigrees with Glanzmann thrombastIlenia.Methods All exons and exonintron boundaries of GP Ⅱ b/Ⅲ a gene were amplified by PCR analysis followed by DNA sequencing.DNA sequencing was used to exclude gene polymorphisms.Results The probands in the three pedigrees had a normal platelet count,coagulation profiles,scattered platelets on the blood film,a prolonged cutaneous bleeding time,and impaired or minimal ex vivo platelet aggregation in response to ADP,thrombin,collagen,adrenaline and arachidonic acid,but normal platelet aggregation in response to ristoeetin.Both FACS and Western blotting demonstrated trace content of αⅡb in the platelets from proband 1 and proband 3,who were classified as type Ⅰ GT,and a small amount of αⅡb in the platelets from proband 2,who was classified as type Ⅱ GT.Compound heterozygous mutations,T2255G(Leu721Arg)and C2671T(Gln860Stop)were identified in proband 1.The proband 2 had homozygous A2334C(Gln747Pro)missense mutation.Nonsense mutations C1750T (Arg584Stop)and 69-79 deletion mutation were identified in proband 3. Conclusions Compound heterozygous mutations T2255G and C2671T of αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 1. Homozygous mutation A2334C of αⅡb gene leads to type Ⅱ Glanzmann thrombasthenia for proband 2. Compound heterozygous mutations C1750T and 69-79del αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 3. T2255G,C1671T and 69-79del aye novel mutations for αⅡb gene.

Haemophilia A in a female with a novel INV22 mutation and skewed X-chromosome inactivation / 中华检验医学杂志

0bjective To make genetic and prenatal diagnosis of a female with Haemophilia A.Methotis The FⅧ:C.BT and VWF were detected to make phenotypic diagnosis.LD-PCR was adopted for screening the intron 22 inversion and PCR was adopted for the screening the intron 1 inversion.The coding and boundary sequences of FⅧgene were analyzed by PCR and DNA equencing.Eight combined polymorphie markers(Amelo,F8IVS13,CA22,DXS15,DXS9901,G6PD,DXS1073 and DXS1108)were applied for linkage analysis of the family by multiplex fluorescent PCR.The polymorphism of DXS52 (ST14)was analyzed by PCR and electrophoresis. Assessment of X inactivation was performed using an Hpa II-polymerase chain eaction (PCR)assay for the X-inked human androgen receptor gene(HUMARA). Results The female HA patient showed severe FW deficiency(FⅧ:C 2.1%)and other phenotypie tests were normal.Her family members showed normal in all tests.The female proposita was found to be a carrier of FW gene intron 22 inversion.But her family members as well as her etus showed negative results.Except this inversion,no other mutation Wag found then.The female inherited two X chromosomes from both her parents' and her fetus inherited the maternally derived X chromosome from the female proposita according to the linkage analysis.Furthermore.X-inactivation paRern of the female was unbalanced and her aternally derived X chromosome Wag inaetived mostly while the majority of her paternal derived one kept active.Conclusions The severe haemophilia A in the proposita resulted from the de novo Ⅷ intron 22 inversion which most probably arose in the paternal germ line.Associated with a skewing pattern of inactivation of the maternally derived X chromosome.Her etus is normal female.