Yanhui Jin

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.

Severe female hemophilia A patient caused by a nonsense mutation (p.Gln1686X) of F8 gene combined with skewed X-chromosome inactivation.

Hemophilia A is a congenital coagulation disorder with a worldwide prevalence of approximately 1 in 5000 males, which results from a deficiency or dysfunction of coagulation factor VIII (FVIII, F8) [1]. Clinical severity is inversely related to residual factor VIII activity (FVIII:C), and hemophilia A is classified as severe, moderate, or mild, depending on patients with FVIII:C less than 1%, 1–5%, and 5–30%, respectively. The gene encoding F8 is located on the long arm of chromosome X (Xq28) and consists of 26 exons [2]; thus it is an X-chromosomelinked recessive bleeding disorder. It typically occurs in male individuals, and female patients with hemophilia A have rarely been reported [3].

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.

Diagnostic Error of a Patient with Combined Inherited Factor VII and Factor X Deficiency due to Accidental Ingestion of a Diphacinone Rodenticide

BACKGROUND To explore the characteristics of laboratory examination and confirm the diagnosis of a patient with combined inherited FVII and FX deficiency after he ingested diphacinone rodenticide accidentally. METHODS The coagulant parameter screening tests and coagulation factor activities were tested many times in the patient due to accidental ingestion of a diphacinone rodenticide. After the patient was treated for more than one year, gene analysis of correlated coagulation factors was analyzed in the patient and other family members by DNA direct sequencing. 106 persons were selected as controls from routine health examinations. RESULTS After the patient was admitted to hospital, routine coagulation screening tests revealed the prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) and low levels of vitamin K-dependent coagulation factors (FII, FVII, FIX, FX) activity, which was 102.4 seconds, 88.5 seconds, 7%, 3%, 8%, and 2%, respectively. During more than one year of treatment, the value of PT and APTT still showed significantly prolonged activity and FVII and FX activity levels were about 5%. While FII and FIX activity levels were in the normal range after 12 weeks of treatment. Two homozygous mutations, g.11267C>T of F7 gene resulting in the substitution Arg277Cys and g.28139G>T of F10 gene leading to the substitution Val384Phe, were identified in the patient. The patients parents and sister was heterozygous for Arg277Cys and Val384Phe mutations. FVII and FX antigen levels in the patient were 7% and 30%, respectively. CONCLUSIONS There were many similarities in the characteristics of laboratory examination between combined inherited FVII and FX deficiency and acquired vitamin K deficiency. The best way to identify them was gene analysis.

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.

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.

Congenital hypofibrinogenemia associated with a novel mutation in FGG gene

OBJECTIVE To identify the genetic mutation underlying congenital hypofibrinogenamia in a Chinese pedigree. METHODS Standard coagulation tests including the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), plasminogen activity (PLG:A), D-Dimer (DD) and fibrin degradation products (FDP) were tested with fresh plasma using a STA-R analyzer. The activity of fibrinogen (Fg:C) and fibrinogen antigen (Fg:Ag) were measured respectively with the Clauss method and immunoturbidimetry. All exons and exon-intron boundaries of the fibrinogen Aα-, Bβ-, and γ-chain genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Suspected mutation was confirmed by reverse sequencing and analyzed with a Swiss-PdbViewer. RESULTS The PT level in the proband was normal, while the APTT and TT were slightly prolonged. The functional and antigen fibrinogen levels were both significantly reduced (0.91 g/L and 0.95 g/L, respectively). Similar abnormalities were also found in her father, elder sister, son and niece. The coagulant parameters of her mother were all within the normal range. Genetic analysis has reveled a heterozygous A>C change at nucleotide 5864 in exon 7 of γ gene in the proband, predicting a novel Lys232Thr mutation. The probands father, elder sister, son and niece were all carriers of the same mutation. Protein model analysis indicated that the Lys232Thr mutation did not disrupt the native network of hydrogen bonds, but has changed the mutual electrostatic forces, resulting in increased instability of the protein. CONCLUSION The heterozygous Lys232Thr mutation identified in the FGG gene probably underlies the hypofibrinogenemia in this pedigree.

Genetic analysis of a pedigree with hereditary coagulation factor VII deficiency

OBJECTIVE To identify potential mutations in a family affected with inherited factor Ⅶ (FⅦ) deficiency. METHODS Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FⅦ activity (FⅦ:C) and other coagulant parameters of the proband and 15 family members were measured. Potential mutations were screened in the pedigree by polymerase chain reaction and direct DNA sequencing. RESULTS The PT of the proband and his younger brother was significantly prolonged to 39.0 s and 30.1 s, respectively. FⅦ:C of the proband and his younger brother was obviously reduced to 2% and 3%, respectively. FⅦ:C of his grandmother, maternal grandmother, aunt, father, mother, maternal uncle and maternal aunt was all below the normal range (80%-108%), which measured 68%, 54%, 71%, 73%, 62%, 72% and 59%, respectively. The other coagulant parameters were in the normal range. Two heterozygous mutations, g.11349G>A and g.11482T>G, both reside in exon 8 of the F7 gene, have resulted in p.Arg304Gln and p.His348Gln substitutions, were identified in the proband. The same mutations were also found in the probands younger brother. Four maternal members in this family (grandmother, mother, maternal uncle and maternal aunt of the proband) were heterozygous for the p.Arg304Gln mutation, while three paternal members (grandmother, aunt and father of the proband) were heterozygous for the p.His348Gln mutation. CONCLUSION The proband had inherited two independent mutations of the F7 gene including g.11349G>A and g.11482T>G from his mother and father, respectively. The compound heterozygous mutation probably explains the low FⅦ concentrations in this pedigree.

Identification of a novel heterozygous mutation in a pedigree with hereditary coagulation factor XII deficiency

OBJECTIVE To identify potential mutation underlying hereditary coagulation factor XII (FXII) deficiency in a pedigree and explore its molecular pathogenesis. METHODS Activated partial thromboplastin time (APTT), FXII activity (FXII:C) and FXII antigen(FXII:Ag) and other coagulant parameters of the proband and 5 family members were measured. Potential mutations in the 14 exons and intron-exon boundaries of the FXII gene were screened with polymerase chain reaction (PCR) and direct DNA sequencing. Suspected mutations were confirmed with reverse sequencing. Corresponding PCR fragments from other family members were also sequenced. RESULTS APPT of the proband and his son were significantly prolonged to 121.5 s and 98.5 s, respectively. FXII:C and FXII:Ag of the proband and his son have reduced to 5%, 6.8% and 9%, 12.2%, respectively. Plasma plasminogen activity (PLG:A) in both individuals was slightly higher than the normal reference range. FXII:C of his second daughter and grandson were slightly reduced to 64% and 60%. FXII:C of the other family members were all in the normal range (72%-113%). A heterozygous missense mutation, g.8597G>A, was identified in exon 13 of the FXII gene in the proband, which resulted in an p.Asp538Asn substitution. For the promoter regions of the FXII gene, the genotype of the proband was 46TT. The same mutations and 46T/T were also found in the probands son but not in other members of the family. The genotypes of the probands spouse, eldest daughter and grandson were 46CT, and his second daughter was 46TT. CONCLUSION The heterozygous mutation of g.8597G>A identified in exon 13 of FXII gene is a novel mutation. Heterozygous p.Asp538Asn mutation and 46TT in the FXII gene can cause hereditary FXII deficiency, which was probably responsible for the low FXII concentrations in this pedigree.