Mingshan Wang

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.

Novel mutations (γTrp208Leu and γLys232Thr) leading to congenital hypofibrinogenemia in two unrelated Chinese families.

Congenital hypofibrinogenemia is a rare disorder caused by heterozygous mutations in the fibrinogen genes. The aim of this study was to elucidate the molecular defects in two unrelated families with hypofibrinogenemia. The proband from family A was a 19-year-old Chinese boy who was suffering from cervical lymphadenitis. A low plasma fibrinogen concentration (0.63 g/l by Clauss method and 0.77 g/l by immunoturbidimetry) was found in routine clotting tests. Further gene analysis revealed a heterozygous g.5792 G>T mutation in exon 7 of the FGG, leading to a novel Trp208Leu change in the &ggr; D domain. This mutation was also found in other family members with low fibrinogen levels. The proposita from family B was a 37-year-old female who suffered from recurrent shoulder pain for 7 years. Routine clotting studies revealed that her prothrombin time was 15.5 s (normal range: 11.8−14.8 s) and thrombin time was 22.8 s (normal range: 14.0–20.0 s), and the fibrinogen concentration in her plasma was only 0.64 g/l by Clauss method and 0.79 g/l by immunoturbidimetry. A heterozygous A>C transition at nucleotide 5864 of FGG was found in the &ggr; chain, causing a Lys232Thr substitution in the fibrinogen. Further sequencing established that her mother, son, brother and nephew were also heterozygous for the mutation.

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.

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.

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.