The identification of R152Q and IVS6+1G→T double heterozygous mutation in a Chinese family with inherited F VII deficiency
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Objective To identify the gene mutations of an inherited coagulation factor Ⅶ deficiency pedigree.Methods PCR and DNA sequencing were used to identify the FⅦ gene mutations in the proband.The identified mutations were validated by PCR followed by restriction fragment length polymorphism technique or DNA sequencing.100 healthy volunteers were chosen randomly as controls. Results R1S2Q and IVS6+1G→T double heterozygous mutations were discovered in the Droband.The pedigree analysis showed that R152Q missense mutation inherited from his father,and IVS6+1G→Twas from his mother. The R1S2Q missense mutation in exon 6 was not found in 100 healthy volunteers. Conclusion The congenital deficiency of F Ⅶ in the proband might be caused by the coinheritance of the R152Q missense mutation in exon 6 and the splicing donor site mutation ( ⅣS6+1G→T)in intron 6.
Key words:
Factor Ⅶ deficiency; Factor Ⅶ; Pedigree; Mutation; Polymerase chain reactionKeywords:
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Objective To investigate the clinical phenotype and gene mutation for a pedigree with inherited antithrombin(AT) deficiency.Methods Immunonephelometry and chromogenic assay were used for detecting plasma level of AT antigen(AT:Ag) and AT activity(AT:A),respectively.All of the seven exons and their intron-exon boundaries of AT gene(SERPINC1)were amplified by PCR using the DNA extracted from the proband's peripheral blood,and PCR products were analyzed by direct sequencing.The corresponding gene fragment with mutations which found in proband was amplified from the DNA of family members and 100 healthy individuals and sequenced for pedigree analysis and polymorphism exclusion,respectively.Results The plasma levels of AT:Ag and AT:A of the proband were 114 mg/L and 54.8%,respectively.Two heterozygous missense mutations in exon 2 of AT gene(c.134GA and c.342TG) were found in the proband and some of his family members but were not found in 100 healthy individuals.Pedigree analyzed results revealed that the two mutations inherited from one allele.Conclusion The compound heterozygous mutations from one allele was the molecular mechanism of this pedigree with Type Ⅰ inherited antithrombin deficiency.
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Compound heterozygosity
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To identify the genetic mutations of a severe inherited coagulation factor VII (FVII) deficiency pedigree.The diagnosis was validated by coagulant and haemostatic parameters. FVII gene mutations were screened in the propositus and his family members by DNA direct sequencing and confirmed by digestions of the restriction enzymes of the PCR production.Two heterozygous missense mutations were found in the propositus of the pedigree: a G to T transversion at position 9482 in exon 6 and a C to T mutation at position 11348 in exon 8 resulting in the amino acid substitution of Arg152 with Leu and Arg304 with Trp, respectively. A heterozygous single nucleotide deletion (C) at position 11487-11489(CCC) within exon 8 was identified, which predicted the frameshift mutation at position His351 followed by the changes of six corresponding amino acids and appearance of a premature protein caused by stop codon. The heterozygous mutations identified in the proband were derived from his father (Arg152 to Leu) and his mother (Arg304 to Trp mutation) and a heterozygous deletion (C) at position 11487-9(CCC). By tracing the other pedigree members, it was found that his grandmother had a heterozygous mutation of Arg304Trp and a heterozygous polymorphism of Arg353Gln and his grandfather had a heterozygous Arg152Leu mutation.Three heterozygous mutations were found in a pedigree with hereditary coagulation factor VII deficiency. Arg152Leu and deletion C at position 11487-9(CCC) were novel mutations.
Transversion
Proband
Compound heterozygosity
Factor IX
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Objective To identify the mutations of coagulation factor Ⅷ in a female with hemophilia A. Methods The plasma FⅧ :C et al was determined used the peripheral blood samples. Intron 22 inversion was excluded by LD-PCR,then all the exons and exon-intron boundaries of factor Ⅷ gene were analyzed by PCR and direct DNA sequencing. Results FⅧ:C of the proband and his daughter were 6. 9% and 3. 4% , respectively, each of them three had no intron 22 inversion. Double heterozygote mutation were detected in the female;a spontaneous heterozygous A insertion in a sequence of 9 As ( codons 1191-1194) within exon 14, and a heterozygous missense mutation in exon 18 (131252T→C) resulting in Leu1975Pro,which was the only mutation in all the exons and exon-intron boundaries in the proband. Proband' s wife had no mutations in these 2 site. Conclusion The FⅧ deficiency of the female are propably caused by the framshift of (112183-112191)insA and the missense mutation of 131252T→C which is a novel mutation in F8 gene.
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Compound heterozygosity
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To discover the mutations of human blood coagulation factor V (FV) gene in a Chinese family with congenital factor V deficiency, and to explore the molecular mechanism associated with the congenital factor V deficiency.PCR and DNA sequencing were used to look for the FV gene mutations in the proband. And the novel mutation were testified by PCR restriction fragment length polymorphism technique or reverse DNA sequencing. One hundred healthy volunteers were chosen as controls at random.Two novel mutations were discovered in the FV gene of proband, which were the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16 (G-->T). The pedigree analysis showed that the two mutations inherited from his parents respectively: the A1763C came from his father, and the G-->T from his mother. The A1763C missense mutation in exon 11 was not found in each of 100 healthy volunteers.The congenital deficiency of FV in the proband might be caused by the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16, which jointly caused the proband to be a double heterozygote.
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Splice site mutation
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Objective To identify gene mutations and explore the molecular mechanism of a pedigree with inherited coagulation F Ⅶ deficiency. Methods The levels of F Ⅶ: Ag in the proband and other family members were measured by ELISA assay. The values of PT, F Ⅶ: C and other coagulant parameters were determined by one-stage clotting for laboratory phenotype diagnosis. All the exons,exon-intron boundaries and 5',3' untranslated sequences of F7 gene were amplified by direct sequencing. The detected mutations were further confirmed by sequencing the other stand. The CLC Protein Workbench software was used to analyze the species conservation of the mutated site and the protein secondary structure. 100 healthy individuals were selected to exclude gene polymorphism. Results PT, FⅦ∶C and FⅦ: Ag in the proband and his sister were abnormal, which were 36. 3 s, 5.0%, 40. 7% and 33.4 s,5. 0%, 37.4%, respectively. Both PT and FⅦ∶C in the proband's father, mother, daughter and niece were slightly abnormal, which were 14.9 s, 14. 6 s, 15.5 s, 14. 6 s and 70%, 85%, 59%, 79%, respectively.The heterozygous mutations c. 784T ＞ C and c. 964T ＞ G in exon 8 of F7 gene were found in the proband,resulting in the substitutions of Ser269Pro and Cys329Gly respectively. Compound heterozygous mutations c. 784T ＞ C and c. 964T ＞ G were found in the proband's sister. The proband's mother was heterozygous for c. 784T ＞ C. His father, daughter and niece were heterozygous for c. 964T ＞ G. The protein biological characteristics analysis revealed that the Cys329Gly caused the change of spatial configuration, and Ser269Pro led to the change of amino acid polarity and hydrophobicity. Conclusion Compound heterozygous mutations of Cys329Gly and Ser269 Pro in F7 gene may be the underlying molecule mechanism of FⅦ deficiency in this pedigree.
Key words:
Factor Ⅶ deficiency; Blood coagulation disorders,inherited; Mutation; Polymerase chain reaction
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Compound heterozygosity
Daughter
Sanger sequencing
Protein C deficiency
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To investigate the pathogenesis of inherited coagulation factor VII (FVII) deficiency.The diagnosis was validated by coagulant parameter assay. FVII gene mutations were analysed in the proband by DNA direct sequencing of PCR products of all exons, exon-intron boundaries and the 3', 5'untranslated sequences. The mutations were confirmed by reverse sequencing. The ectopic transcripts of RT-PCR were used to confirm the characteristics of the mutation in non-canonical splice site (IVS1a + 5g > a).Double heterozygous mutations in the propositus were identified: a T to G mutation at position 10961, resulting in His348Gln substitution, a non-canonical splice site (IVS1a + 5g > a) mutation, causing the new model of splice and frameshift mutation.Double heterozygous mutations of His348Gln and IVS1a + 5g > a were identified in a propositus, the splicing pattern of the IVS1a + 5g > a mutation was reported for the first time.
splice
Splice site mutation
Proband
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Objective: To identify the gene mutation and explore the molecular pathogenesis of an inherited factor VII deficiency pedigree. Methods: Prothrombin time(PT), activated partial thromboplastin time(APTT), fibrinogen(Fg), F II activity(F II:C), F V activity(F V:C), F VII activity(F VII:C), F X activity(F X:C) and F VII antigen(F VII:Ag) were measured for phenotype diagnosis. All the exons, exon-intron boundaries and 5', 3'untranslated sequences of F7 gene of the proband and other family members were analyzed by direct sequencing. The detected mutations were confirmed by sequencing the complementary strand. Results: The value of PT in the proband was extended to 22.4 s. and the F VII:C and F VII: Ag of the proband were obviously reduced, which were 7% and 10% respectively. The PT of his father, mother, sister and son were either slightly prolonged or normal, their F VII:C(63%, 54%, 57% and 46%, respectively) and F VII:Ag(67%, 53%, 61% and 49%, respectively) were reduced. The APTT and other coagulant parameters of the proband and other family members were within normal range. Genetic analysis revealed T to G transition at 11482 and G to A transition at 11496 in the exon 8 of F7 gene of the proband(which resulted in His348Gln missense mutation and Arg353Gln polymorphism, respectively), both of them were heterozygous. The heterozygous polymorphism(Arg353Gln) was identified in his father, while the same heterozygous mutation(His348Gln) was identified in other family members. Conclusion: The heterozygous mutation of His348Gln combined with heterozygous polymorphism of Arg353Gln in F7 gene detected from the proband relates to the F VII deficiency.
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Summary. To investigate the molecular defects in two Chinese pedigrees with inherited factor V (FV) deficiency. A 37‐year‐old male (proband 1) and an 18‐month‐old boy (proband 2) were diagnosed as inherited coagulation FV deficiency by severely reduced plasma levels of FV activity and antigen. All 25 exons and their flanking sequence of F5 gene were amplified by polymerase chain reaction (PCR) for both probands and the PCR products were directly sequenced. Total RNA was extracted from the peripheral lymphocytes of proband 1 for detecting the changes at mRNA level. The homozygous deletion IVS8 −2A>G was identified in the F5 gene of proband 1 and complementary DNA (cDNA) analysis revealed the abolishment of the canonical splicing site by the mutation and the activation of the cryptic acceptor site 24 bp upstream instead. The insertion introduced eight additional amino acids (AA) into the FV protein. Two heterozygous mutations of F5 gene were discovered in proband 2. The 2238‐9del AG in exon 13 introduced a premature termination code at 689 AA and the substitution of G6410 by T in exon 23 lead to the missense mutation Gly2079Val. Three F5 gene mutations, IVS8 −2A>G, 2238‐9del AG and G6410T, have been identified in two Chinese pedigree with congenital FV deficiency, respectively.
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Pedigree chart
Compound heterozygosity
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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 FⅫ deficiency was admitted to the Third Clinical College of Wenzhou Medical University.Activated partial thromboplastin time (APTT), prothrombin time (PT), FⅫ activity (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 F12 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 FⅫ level of this pedigree of hereditary FⅫ deficiency.(Chin J Lab Med, 2018, 41: 214-218)
Key words:
Factor Ⅻ deficiency; Pedigree; Homozygote; Mutation
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Mutation Testing
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Objective To identify the F11 gene mutation responsible for congenital coagulation factor Ⅺ deficiency.Methods The 50 years old female proband diagnosed by preoperative tests including hemostasis test revealed prolonged activated partial thromboplastin time (APTT) and normal prothrombin time (PT). The genomic DNA was extracted from the peripheral blood of the proband and family members. The FⅪ:C was assayed and all the exons and boundaries of F11 were sequenced after amplified by polymerase chain reaction (PCR).Results The reduced FⅪ:C of the proband with only 2.1% of normal range thus further verified the diagnosis. The homozygous missense mutation Gly400Val (GGC→GTC) in the exon 11 was identified in the proband. The FⅪ:C of the family members who were heterozygous for the mutation being only about 27.0%～48.4% of the normal, showed decrease in all.Conclusion The missense mutation Gly400Val is the cause of FⅪ deficiency in both European and Chinese. The FⅪ level of heterozygotes may further decrease via a dominant negative mechanism.
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Compound heterozygosity
Heterozygote advantage
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