Lihong Yang

Identification of Genetic Defects Underlying FXII Deficiency in Four Unrelated Chinese Patients.

Congenital factor XII (FXII) deficiency is a rare autosomal recessive disorder, characterized by a great variability in its clinical manifestations. In this study, we screened for mutations in the F12 gene of 4 unrelated patients with FXII coagulant activity <10% of that of normal human plasma. To investigate the molecular defects in these FXII-deficient patients, we performed FXII mutation screening. By sequencing all coding exons as well as flanking intronic regions of the F12 gene, 6 different mutations, including 3 missense mutations (Gly341Arg, Glu502Lys and Gly542 Ser), 1 insertion (7142insertC) and 2 deletions (5741-5742 delCA and 6753-6755delACA), were identified on the F12 gene. Three of them (Gly341Arg, 5741-5742delCA and 6753-6755delACA) are reported here for the first time. Computer-based algorithms predicted these missense mutations to be deleterious. This study has increased our knowledge of the mutational spectrum underlying FXII deficiency.

The significance of F139V mutation on thrombotic events in compound heterozygous and homozygous protein C deficiency.

Both compound heterozygous and homozygous protein C deficiencies (PCDs) can cause lethal thrombotic events in children. This study investigated the significance of F139V mutation in activation of protein C in heterozygous and biallelic PCD. Two pedigrees with three probands were recruited, including heterozygous, compound heterozygous, and homozygous PCD and non-PCD families. The plasma levels of protein C activity (PC:A), protein C antigen (PC:Ag), factor V:C, factor VIII:C, fibrinogen (FIB), and D-dimer (D-D) were measured. Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were also detected. All nine exons of protein C gene (PROC) were sequenced. Protein C mutation, T>G at site 6128 (exon 7) resulting in F139V, was identified in both pedigrees. Heterozygous missense mutation F139V (n = 10) had 56.4% lower levels of PC:A and PC:Ag compared with members with wild-type PROC (n = 6). Biallelic compound heterozygous and homozygous PCDs with F139V (n = 3) significantly decreased the levels of PC:A and PC:Ag compared with heterozygous members (P < 0.05); however, these were not lethal. Heterozygous F139V mutations of PRO caused mild reduction of protein C function, which might be the reason for survival of compound heterozygous or homozygous PCD with F139V in adults.

A novel fibrinogen mutation (γ Thr277Arg) causes hereditary hypofibrinogenemia in a Chinese family.

Congenital hypofibrinogenemia is a rare disorder caused by heterozygous mutations in one of the three fibrinogen genes – fibrinogen &agr;-chain (FGA), fibrinogen &bgr;-chain (FGB) and fibrinogen &ggr;-chain (FGG) – which code for the A&agr;, B&bgr; and &ggr; chains, respectively. In this study, we identified a genetic defect in the FGG underlying the hypofibrinogenemia. The proposita had a prolonged blood clotting time (thrombin time 24.5 s, prothrombin time 16.8 s) and a low level of plasma fibrinogen (0.71 g/l by Clauss method and 0.79 g/l by immunoturbidimetry). DNA screening of the whole fibrinogen gene revealed a heterozygous GC mutation at nucleotide 7482 in her FGG gene. Her father and her half-brother are also heterozygous for this mutation. This mutation contributes to Thr277 → Arg in the &ggr; chain of fibrinogen. To the best of our knowledge, this is the first report of such a mutation that is associated with hypofibrinogenemia.

Genetic analysis of a hereditary factor XII deficiency pedigree of a consanguineous marriage due to a homozygous F12 gene mutation: Gly341Arg

ABSTRACT Objective and Importance: To study the gene mutations of factor XII (FXII) in a Chinese family of consanguineous marriage with FXII deficiency and illuminate the possible molecular pathogenic mechanism. It will contribute to our comprehension of the pathogenesis of the disease. Clinical presentation: The proband was a 26-year-old Chinese pregnant woman who was discovered, in a pregnancy test, with a prolonged activated partial thromboplastin time (APTT) at 61.6s (reference range, 29.0–43.0s). Techniques: The FXII activity (FXII:C) and FXII antigen (FXII:Ag) were tested with clotting assay and ELISA, respectively. The FXII gene (F12) was amplified by PCR with direct sequencing. A ClustalX-2.1-win and four online bioinformatics software (PolyPhen-2, PROVEAN, SIFT, and Mutation Taster) were used to study the conservatism and harm of the mutation. The reference range of each test indicator in our laboratory was established with 150 healthy subjects. Conclusion headings: The FXII:C and FXII:Ag of the proband were 12% and 10% (normal range, 72–113%), respectively. Gene sequencing detected a homozygous c.1078G > A point mutation in exon 10 resulting in a substitution of glycine 341 by arginine (Gly341Arg) in the proline-rich domain of FXII. Family study showed that her elder brother had the same phenotype and genotype with her. In addition, there were another six heterozygous members in her family. Both conservatism and bioinformatics results indicated the mutation probably had affected the function of the protein. We thought the Gly341Arg mutation was responsible for the decreased activity of FXII:C and FXII:Ag. And in vitro expression experiment is performed to elucidate the precise pathological mechanism of the mutation.

Homozygous missense mutation p.Val298Met of F10 gene causing hereditary coagulation factor X deficiency in a Chinese pedigree

OBJECTIVE To identify potential mutation underlying coagulation factor X (FX) deficiency in a consanguineous Chinese pedigree. METHODS Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FX activity (FX:C) and other coagulant parameters were determined with a one-stage clotting assay. The FX antigen (FX:Ag) was determined with an ELISA assay. All coding exons and exon-intron boundaries of the F10 gene were amplified with PCR and subjected to direct sequencing. Suspected mutation was confirmed by reverse sequencing and analyzed with CLC Genomics Workbench 7.5 software. RESULTS The PT and APTT in the proband were prolonged to 67.2 s and 102.9 s, respectively. Further study showed that her FX:C and FX:Ag were reduced by 1% and 8%, respectively. The PT of her father, mother, and little brother were slightly prolonged to 14.5 s, 14.4 s and 14.4 s, respectively. The FX:C and FX:Ag in her father, mother and little brother were all slightly reduced. Genetic analysis of the proband has revealed a homozygous G>A change at nucleotide 27881 in exon 8 of the F10 gene, which predicted a p.Val298Met substitution. The probands father, mother, and little brother were all heterozygous for the p.Val298Met mutation. The proband has inherited the homozygous mutation from her parents by consanguineous marriage. Other family members were all normal. Bioinformatics analysis has indicated that this mutation may result in changes in the secondary structure of the FX protein. CONCLUSION A homozygous mutation g.27881G>A(p.Val298Met) of the F10 gene has been identified, which probably accounts for the low FX concentrations in this pedigree.

A protein C and plasminogen compound heterozygous mutation and a compound heterozygote of protein C in two related Chinese families.

Hereditary protein C (PC) deficiency and congenital plasminogen (PLG) deficiency are both factors of thrombophilia which were caused by PC and PLG gene mutations with the characteristics of activity and antigen decreasing inconsonantly. To illustrate the connection between gene mutations and the corresponding deficiencies of PC and PLG, we studied two related cases. The proband 1 showed a cranial venous sinus thrombosis with reduced activities of PC and PLG, 55 and 56%, respectively. And the proband 2 was his asymptomatic nephew who had a reduced PC activity of 27%. All the PC genes (PROC) and PLG genes were amplified by PCR with direct sequencing. Then these detected mutations were analyzed by conservation, bioinformatics, and model. Genetic analysis detected two compound heterozygous missense mutations: the proband 1 carried a p.Gly86Asp in PC and a p.Ala601Thr in PLG, whereas the proband 2 took two mutations of PC (p.Gly86Asp and p.Arg147Trp). All conservation, bioinformatics prediction, and model analysis results indicated that these mutations probably affected the structures and stabilities of the matching proteins. We speculated that the three mutations are responsible for the reduction of PC activity and PLG activity. Furthermore, the p.Gly86Asp of PC has been detected for the first time in the world.

Severe coagulation factor VII deficiency caused by a novel homozygous mutation (p. Trp284Gly) in loop 140s.

Congenital coagulation factor VII (FVII) deficiency is a rare disorder caused by mutation in F7 gene. Herein, we reported a patient who had unexplained hematuria and vertigo with consanguineous parents. He has been diagnosed as having FVII deficiency based on the results of reduced FVII activity (2.0%) and antigen (12.8%). The thrombin generation tests verified that the proband has obstacles in producing thrombin. Direct sequencing analysis revealed a novel homozygous missense mutation p.Trp284Gly. Also noteworthy is the fact that the mutational residue belongs to structurally conserved loop 140s, which majorly undergo rearrangement after FVII activation. Model analysis indicated that the substitution disrupts these native hydrophobic interactions, which are of great importance to the conformation in the activation domain of FVIIa.

A novel gene insertion combined with a missense mutation causing factor VII deficiency in two unrelated Chinese families.

Hereditary coagulation factor VII (FVII) deficiency is a rare bleeding disorder characterized by reduced FVII activity (FVII:C) and inconsistent FVII antigen (FVII:Ag). In our study, two pregnant probands were diagnosed with FVII deficiency, based on the tests that FVII:C were both 3% and FVII:Ag were less than 7.5%. Gene sequencing revealed the same compound mutations, a recurrent missense mutation p.Arg277Cys and a novel insertion mutation g.11520–11521insT. What is more, haplotype analysis of SNPs excluded the possibility of consanguinity between the two families. According to the model, we speculated that although the insertion mutation was close to the carboxy-terminal, it induced the protein extension and affected the 3′ untranslated region of F7 gene, which is significant to posttranscriptional regulation. Hypothetically, the stability or translational efficiency of mRNA may be influenced, resulting in reducing FVII:C.

Double heterozygous mutations Gln100Leu and His348Gln of the F7 gene in a patient with factor VII deficiency.

A 25-year-old Chinese woman who had a history of easy bruising was admitted to hospital due to uncontrolled epistaxis. She showed factor VII activity level of 2% and factor VII antigen level of 4% of the normal value. We detected a novel missense mutation g.8355 A>T (p.Gln100Leu) in the second epidermal growth factor-like (EGF) domain and a g.11482 T>G (p.His348Gln) in the catalytic domain. Although the Gln100 residue is close to the junction of EGF-2 domain with the serine protease domain, we infer that the substitution of polar negatively charged Gln residue at the position 100 with introduction of nonpolar Leu residue may be likely to perturb proper folding, resulting in decreasing factor VII activity.

Analysis of an hereditary protein C deficiency pedigree with compound heterozygous gene mutations

OBJECTIVE To analyze genetic mutations and explore its molecular pathogenesis for an hereditary protein C (PC) deficiency pedigree. METHODS The pedigree has included 15 individuals from 4 generations. Plasma levels of PC activity (PC:A), PC antigen (PC:Ag) and other coagulant parameters were determined for members of the family. The 9 exons and intron-exon boundaries of protein C gene (PROC) of the proband were amplified with PCR and analyzed with direct sequencing. Detected mutations were confirmed with reverse sequencing. Corresponding PCR fragments from the family members were also directly sequenced. RESULTS Plasma PC:A and PC:Ag for the proband was 26% and 18.60%, respectively, both being lower than normal references. Seven members from the pedigree also had lower PC:A, six had lower PC:Ag. A compound heterozygous missense mutation, including a T to G transition at position 6128 of exon 7, which results in Phe139Val, and a G to C transition at position 8478 in exon 9, which results in Asp255His, were identified in the proband. The paternal grandma, father and two aunts were heterozygous for g.6128 T to G, whilst the mother, the second uncle, sister and son were heterozygous for g.8478 G to C. There were lower PC:A in family members with g.8478 G to C. CONCLUSION The proband had inherited two independent mutations of the PROC gene including g.6128 T to G in exon 7 and g.8478 G to C in exon 9 from her father and mother, respectively. The resulting compound heterozygous mutation has caused a serious hereditary protein C deficiency.