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 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.

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 study the GPⅡb/Ⅲa gene mutations of eight glanzmann thrombasthenia(GT) pedigrees. Methods Responses of eight probands to different agonists were observed by platelet aggregation test and the amount of αⅡb and β3 was determined by flow cytometry. All the exons of Ⅱb and Ⅲ a genes were amplified by PCR followed by sequencing for mutational screening. Further analysis of the normal population excluded the possibility of mutational sites as a polymorphism. Results Eight probands showed normal PLT counts, dispersion of the platelet particles without aggregation, prolonged bleeding time and severely reduced platelet aggregation in response to the physiological agonists- ADP, epinephrine, and collagen, but relatively normal aggregation of PLT in response to ristocetin. Flow cytometry showed that all probande were Ⅰ type GT, except that proband 2 was Ⅲ type GT and proband 6 was Ⅱ type GT. The sequencing results showed that twelve different types mutations were present in eight probands, including GIOA, Gl412T, GII99A, 1525deiC, G2223T, C2671T, 2930delG, IVSI5 (-1) delG, A2334C, C1750T, 69-79del and CA70A. We were not able to detected any mutations in GP Ⅱb/Ⅲa gone on proband 3. Conclusions GT is mainly caused by GPⅡb/Ⅲa gene mutations. G10A, 69-79del, C470A,G1412T, G2223T, C2671T and 1525delC were the novel mutations causing GT.

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 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.

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.

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.

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.