Xiuping Hao

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.

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.

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.

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.

Unique de-novo mutation of fibrinogen gene in a Chinese girl with hypofibrinogenemia.

Hypofibrinogenemia is characterized by low plasma fibrinogen concentrations (<1.5 g/l). It is usually caused by heterozygous mutations in one of the three fibrinogen genes. To the best of our knowledge, the vast majority of these mutations have been proven to be inherited from a parent. We studied here a Chinese family in which the proband had low functional and antigen fibrinogen levels, 0.77 and 0.90 g/l, respectively. DNA sequencing revealed a novel Asp316His mutation in the &ggr;D domain of fibrinogen. However, both parents were negative for the mutation. Modeling analysis indicated that the Asp316His mutation may destabilize the conformation of the &ggr;314-&ggr;318 loop and lead to hypofibrinogenemia. Haplotype analysis of single-nucleotide polymorphisms excluded the possibility of nonpaternity, indicating it is a de-novo event. Further investigation of her living environment suggested the Asp316His mutation might have been induced by exposure to chromate mutagens.