Objects To establish a simple,rapid carrier detection and prenatal diagnosis system for hemophilia.Methods Thirty-eight HA families were tested for the intron 22 and 1 inversions in factor VIII gene by LD-PCR and PCR.The remaining inversion negative families,but with family history,were screened using linkage analysis with 8 combined polymorphic markers,including St14,F8IVS13,CA22,DXS15,DXS9901,G6PD,DXS1073,and DXS1108.For sporadic families,the whole gene sequencing was applied directly to detect the mutation.For HB families,linkage analysis with 6 STRs,including DXS1192,DXS1211,DXS8094,DXS8013,DXS1227 and DXS102,was applied to get quick diagnostic information.The whole gene sequencing was used to get the final diagnosis.The rapid fluorescent PCR combined with polymorphism markers were applied for linkage analysis in HA and HB families,respectively.As soon as the pregnancy was identified,additional Amelo site detection would be performed.Results In 38 HA families,introns 22 and 1 inversions were found in 10 and 1 probands,respectively.The diagnostic rates of St14,F8IVS13,CA22,DXS15,DXS9901,G6PD,DXS1073 and DXS1108 were 61.11%,76.67%,71.43%,70.59%,62.50%,10.00%,75.00% and 50.00%,respectively.Combining inversion detection and linkage analysis,the diagnostic rate of carrier detection and prenatal diagnosis of families with HA family history were both 100%.One intron 22 inversion and 3 mutations were detected in 4 sporadic families.The total diagnostic rate of 38 HA families was 94.81%.And 10 mutations were detected in the 12 HB families.Combined with the linkage analysis,the total diagnostic rate was 96.88%.Conclusions Introns 22 and 1 inversion screening combined with the linkage analysis,using the highly informative polymorphic markers,can be used for carrier detection and prenatal diagnosis in Chinese HA families.The direct sequencing of FⅨ with the linkage analysis can be successfully applied for carrier detection and prenatal diagnosis of HB families.

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.

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.

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.

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.

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.

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.

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.

Anemia, Sideroblastic ; diagnosis ; genetics ; Asian Continental Ancestry Group ; Genetic Diseases, X-Linked ; diagnosis ; genetics ; Humans ; Pedigree

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.