Antithrombin deficiency caused by SERPINC1 gene mutations predispose to venous thrombotic events / 中华检验医学杂志

Objective:To analyze 12 antithrombins (AT) gene mutations that cause AT deficiency and discuss the relationship between the SERPINC1 gene. mutations and venous thrombotic events.Methods:This study belongs to case series of observational studies. Collected the clinical data of 12 AT deficiency cases in the First Affiliated Hospital of Wenzhou Medical University from April 2014 to April 2021 and collected the blood samples before treatment. The AT activity (AT: A) and AT antigen (AT: Ag) was detected by chromogenic substrate and immunoturbidimetry, respectively. The 7 exons and flanking sequences of the SERPINC1 gene were sequenced directly by PCR, the suspected mutations were validated by reverse sequencing. Analyzed the correlation between the SERPINC1 gene. mutations and venous thrombotic events and figured out the proportion.Results:The AT: A of the 12 patients all decreased significantly, ranging from 30% to 66%, and the AT: Ag of the 7 patients decreased accordingly, showing type Ⅰ AT deficiency, and the AT: Ag of the other 5 patients were normal, presented type Ⅱ AT deficiency. 12 mutations were found including 6 heterozygous mutations which were discovered for the first time: c.456_458delCTT(p.phe121del), c.318_319insT(p.Asn75stop), c.922G>T(p.Gly276Cys), c.938T>C (p.Met281Thr), c.1346T>A(p.Leu417Gln)and c.851T>C(p.Met252Thr). All 12 patients had venous thrombosis, and 3 cases including 2 compound heterozygotes and 1 single heterozygote all suffered from deep venous thrombosis (DVT) when they were younger without obvious triggers. The other 9 patients all combined with the other thrombotic factors including old age, hypertensive, smoking, pregnancy, and prolonged immobilization.Conclusion:Patients with AT deficiency caused by SERPINC1 gene defects are prone to venous thrombosis, especially combined with other thrombotic factors.

Identification of compound heterozygous variants of F12 gene in a pedigree affected with inherited coagulation factor XII deficiency / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular pathogenesis for a pedigree affected with hereditary coagulation factor XII (FXII) deficiency.@*METHODS@#Potential variant of the F12 gene was analyzed by PCR and Sanger sequencing. Expression plasmids were constructed by site-directed mutagenesis based on the wild-type and transiently transfected into 293T cells. FXII:C and FXII:Ag of the expression products were determined in the supernatant and cell lysate. Western blotting was used to verify the identify of the protein.@*RESULTS@#Gene sequencing revealed that the proband has carried 46TT genetype and heterozygous p.Glu502Lys variants in exon 13, and a heterozygous p.Gly542Ser variant in exon 14 of the F12 gene. Transfection experiment suggested that the FXII:C and FXII:Ag of p.Glu502Lys variant in the supernatant were 28% and 24%, compared with the wild-type (100%) and FXII:Ag of cell lysates was 39% compared to the wild-type (100%). The FXII:C and FXII:Ag of p. Gly542Ser variant in the supernatant were 32% and 17% and the FXII:Ag of cell lysates was 59%.@*CONCLUSION@#The 46TT genetype, p.Glu502Lys and p.Gly542Ser variants of the F12 gene probably underlie the low FXII level in the proband. As shown by in vitro experiment, the p.Glu502Lys and p.Gly542Ser variants can both inhibit the synthesis and secrection of the FXII protein.

Analysis of a Chinese pedigree affected with hereditary factor VII deficiency caused by compound heterozygous variants of F7 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for a Chinese pedigree affected with hereditary coagulation factor VII (FVII) deficiency.@*METHODS@#The coding regions of F7 gene were amplified by PCR and sequenced. Suspected variants were confirmed by reverse sequencing and validated in other members from the pedigree. Pathogenicity of the variants was analyzed with multiple bioinformatic tools.@*RESULTS@#Genetic analysis revealed that the proband has carried compound heterozygous c.985T>C (p.Ser329Pro) and c.1091G>A (p.Arg364Gln) variants in exon 8 of the F7 gene. Her mother, brother and son were heterozygous for c.985T>C (p.Ser329Pro), while her father was heterozygous for c.1091G>A (p.Arg364Gln). Phylogenetic analysis suggested that both p.Ser329 and p.Arg364 are highly conserved among homologous species. Online bioinformatic software predicted both variants to be deleterious. Protein model analysis suggested that the Pro329 side chain may form a new hydrogen bond with Leu333. The Pro benzene ring may clash with Glu325 in the p.Ser329Pro variant model. The p.Arg364Gln variant have two additional hydrogen bonds compared with wild type Arg364. Both variants may lead to alteration of the protein structure.@*CONCLUSION@#The p.Ser329Pro and p.Arg364Gln variants in exon 8 of the F7 gene probably account for the reduced FVII in this pedigree.

Clinical characteristics and genetic analysis of a combined inherited antithrombin and factor Ⅶ deficiency pedigree / 中华检验医学杂志

Objective:To study the clinical characteristics and gene mutations in a family with combined inherited antithrombin (AT) and factor Ⅶ (FⅦ) deficiency, and explore the relationship between AT gene, F7 gene mutations and diseases. Methods:Pedigree investigation. Blood samplesand clinical dataswere collected fromthe proband and her family members (a total of 16 people in 3 generations) who admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018. The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), antithrombin activity (AT: A), antithrombin antigen (AT: Ag), protein C activity (PC: A), protein S activity (PS: A), FⅦ activity (FⅦ: C), FⅦ antigen (FⅦ: Ag) and other indicators were detectedto confirm the diagnosis. DNA direct sequencing analysis of all exons, flanking sequences, 5′ and 3′ untranslated regions of AT genes and F7 genes, and the mutation sites were confirmed by clone sequencingor reverse sequencing. Results:The AT: A and AT: Ag of the proband were 46% and 135 mg/L, respectively (reference range: 250-360 mg/L), some of her family members′ s (father, aunt, two cousins, younger brother and nephew) AT: A and AT: Ag were reduced to 50% of normal range. Her father (Ⅰ 2), aunt (Ⅰ 4), elder brother (Ⅱ 7), younger brother (Ⅱ 8), and nephew (Ⅲ 3)′s FⅦ: C were 45%, 50%, 48%, 47% and 48%, respectively; and their FⅦ:Ag was within the normal range. Genetic analysis revealed that the proband(Ⅱ 6) and some of her family members (father, aunt, two cousins, younger brother and nephew) took rs3138521 polymorphism in the 5′ untranslated region of AT gene. Her father (Ⅰ 2), aunt (Ⅰ 4), elder brother (Ⅱ 7), younger brother (Ⅱ 8), nephew (Ⅲ 3) took c.1091G>A heterozygous missense mutationin exon 8 of F7 gene, resulting in p.Arg304Gln. Conclusion:The rs3138521 in AT gene and c.1091G>A in F7 gene, which may be the molecular mechanism leading to combined inherited AT and FⅦ deficiency in this family.

Homozygous missense mutation p.Val298Met of F10 gene causing hereditary coagulation factor X deficiency in a Chinese pedigree / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutation underlying coagulation factor X (FX) deficiency in a consanguineous Chinese pedigree.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 proband's 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.</p><p><b>CONCLUSION</b>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.</p>

Association of polymorphism in the promoter region of PCA3 gene with risk of prosate cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism in the promoter region of PCA3 gene and its relationship with risk of prostate cancer (PCa).</p><p><b>METHODS</b>The promoter region of PCA3 gene of the DNA of peripheral blood mononuclear cells was detected by sequence analysis in the 186 PCa and 141 BPH patients and 135 healthy control individuals. If the samples were detected with polymorphism of insection/deletion, clone sequence analysis was used with pBS-T carrier to verify it.</p><p><b>RESULTS</b>There were 5 polymorphisms. TAAA repeat times: 4, 5, 6, 7, 8, and 8 genotypes (TAAA 4/5, TAAA 4/6, TAAA 5/5, TAAA 5/6, TAAA 5/7, TAAA 5/8, TAAA 6/6, and TAAA 6/7) were detected in the promoter region of PCA3 gene. The eight genotypes were divided into three groups: ≤10TAAA, 11TAAA, ≥12TAAA. Unconditional logistic regression analysis models were used to analyze the relationship between different genotypes and cancer risks adjusted by sex and age. The type 11TAAA and ≥12TAAA was associated with higher relative risk for prostate cancer than the group ≤10TAAA [OR=1.74, 95% CI=1.06-2.87 (for type 11TAAA); OR=5.63, 95% CI=1.85-17.19 (for type ≥12TAAA)]. In the 186 PCa patients, there was 62.4% allele of PCA3 gene with AG/CA mutation found in the promoter 18-19 bp region of PCA3 gene and it had a close relation with the development of prostate cancer.</p><p><b>CONCLUSIONS</b>Short tandem repeats are found in the promoter region of the PCA3 gene in PCa patients, and the increase of TAAA repeat sequences highly enhance the relative risk of prostate cancer development. The occurrence of such STR might be related to the mutations in their upstream loci.</p>

Analysis of molecular pathogenesis and clinical phenotypes in 10 probands with inherited fibrinogen deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular pathogenesis and clinical phenotypes in 10 probands with inherited fibrinogen (Fg) deficiency.</p><p><b>METHODS</b>The diagnosis of hereditary Fg deficiency was validated by prothrombin time (PT), thrombin time (TT), Fg activity (Fg:C) and Fg antigen (Fg:Ag) in plasma. All of the exons and their flanking sequences of the Fg gene were analyzed by direct sequencing. Detected mutations were confirmed by reverse sequencing.</p><p><b>RESULTS</b>The ranges of Fg:C and Fg:Ag in the 10 probands were 0.52-0.91 g/L and 0.62-2.98 g/L, respectively. Five of the probands had type I disorders, and 5 had type II disorders. Seven point mutations were identified, among which 6 have located in the D region. γThr277Arg, γAsp316His, γTrp208Leu and Lys232Thr were novel mutations, and αArg19Ser was first reported in Chinese. Four probands had the same mutation site (γArg275). As to the clinical manifestation, probands with type I disorders were asymptomatic or with mild or medium symptoms, while those belonged to type II disorders had moderate or serious symptoms. Two probands have carried an Arg275Cys mutation but had different clinical manifestations.</p><p><b>CONCLUSION</b>Mutations of the Fg gene seem to aggregate to the D region of FGG in our region, and Arg275 is a common mutation. However, no correlation has been found between the mutation site and clinical manifestations.</p>

Genetic analysis of a pedigree with hereditary coagulation factor Ⅶ deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutations in a family affected with inherited factor Ⅶ (FⅦ) deficiency.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 proband's 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.</p><p><b>CONCLUSION</b>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.</p>

Identification of a novel heterozygous mutation in a pedigree with hereditary coagulation factor XII deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutation underlying hereditary coagulation factor XII (FXII) deficiency in a pedigree and explore its molecular pathogenesis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 proband's son but not in other members of the family. The genotypes of the proband's spouse, eldest daughter and grandson were 46CT, and his second daughter was 46TT.</p><p><b>CONCLUSION</b>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.</p>

Phenotype and genotype analysis for a consanguineous pedigree with combined coagulation factor VII and X deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutations and explore the molecular mechanism underlying combined inherited coagulation factors VII(FVII) and X(FX) deficiency for a family featuring consanguineous marriage between maternal cousins.</p><p><b>METHODS</b>Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FVII activity (FVII:C), FX activity (FX:C), FVII antigen (FVII:Ag), FX antigen (FX:Ag) and other coagulant parameters of the proband and 5 family members were measured. Potential mutations in exons, exon-intron boundaries and 5', 3' untranslated sequences of F7 and F10 genes were screened by polymerase chain reaction and direct sequencing. Suspected mutations were confirmed by sequencing the opposite strand.</p><p><b>RESULTS</b>PT and APTT of the proband were obviously prolonged to become 76.4 s and 60.2 s, respectively. FVII:C, FVII:Ag,FX:C and FX:Ag of the proband were obviously reduced to become 4%, 6%, 6% and 33%, respectively. Both PT and APTT of her grandmother, father, mother and daughter were slightly prolonged, which have measured 16.4 s, 15.8 s,16.9 s, 16.5 s, and 44.0 s, 42.1 s, 41.1 s, 43.5 s, respectively. And their FVII:C (34%, 39%, 31%, 40%, respectively), FX:C (50%, 58%, 47%, 42%, respectively) and FX:Ag (51%, 54%, 58%, 47%, respectively) were slightly reduced, while FVII:Ag was in the normal range. The coagulant parameters of her younger brother were within normal range. Two homozygous mutations, g.11267C to T in exon 8 of F7 gene, which resulted in an Arg277Cys substitution, and g.28139G to T in exon 8 of F10 gene which led to a Val384Phe substitution, were identified in the proband. The proband's grandmother, parents and daughter were heterozygous for both Arg277Cys and Val384Phe mutationss. Wild-type alleles of both F7 and F10 genes were also found in the younger brother.</p><p><b>CONCLUSION</b>A homozygous Arg277Cys mutation and a Val384Phe mutation have been respectively identified in the F7 and F10 genes, which can explain the low levels of FVII and FX in this family. The former has been inherited from the consanguineous parents.</p>

Relationship between the valine/leucine247 polymorphism of apolipoprotein H gene and generation of antiphospholipid antibodies in patients of Han nationality with systemic lupus erythematosus in Wenzhou region / 中华皮肤科杂志

Objective To study the relationship between the valine/leucine247 (817G/T) polymorphism in exon 7 of apolipoprotein H (apoH) gene and the generation of antiphospholipid (APL)antibodies in patients of Han nationality with systemic lupus erythematosus (SLE) in Wenzhou region.Methods This study included 165 patients with SLE and 160 healthy controls of Han nationality in Wenzhou region.Venous blood samples were obtained from all of the subjects followed by the isolation of blood plasma,sera and white blood cells.PCR and DNA sequencing were carried out to assess the Leu/Va1247 polymorphism in apoH gene.Lupus anticoagulant (LAC) was detected by Russell viper venom time (RVVT) assay.Enzyme-linked immunosorbent assay (ELISA) was carried out to quantify the serum levels of anti-β2-glycoprotein Ⅰ (GPI) antibodies and anticardiolipin antibodies (ACA).Chi-square test was carried out to compare the 817G/T polymorphism between the patients and controls,and Logistic regression analysis to evaluate the correlation between the 817G/T polymorphism and production of antiphospholipid antibodies.Results There were significant differences between the patients and controls in the genotype distribution and allele frequency at position 817 of apoH gene (both P ＜ 0.01).The TT,GT genotypes and T allele were more frequent,while GG genotype and G allele were less frequent,in the patients than in the controls.The GT genotype at position 817 was a risk factor for the production of LAC (P＜ 0.05,OR =2.33,95％CI =1.18-4.59),anti-β2GPl antibodies(P＜ 0.01,OR =5.92,95％CI =2.61-13.46) and ACA(P＜ 0.05,OR =2.52,95％CI =1.22-5.24),and the TT genotype was associated with an increased frequency of anti-β2GPI antibodies (P ＜ 0.01,OR =5.84,95％CI =1.69-20.20).Conclusions The 817G/T(Leu/Va1247) polymorphism in exon 7 of apoH gene is associated with the generation of APL antibodies in patients of Han nationality with SLE in Wenzhou region.The TT and GT genotypes at position 817 of apoH gene appear to be a risk factor for the production of APL antibodies.

Identification of a novel mutation of factor XII gene in a family with coagulation FXII deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation and molecular pathogenesis in a family affected with inherited coagulation factor XII(FXII) deficiency.</p><p><b>METHODS</b>Activated partial thromboplastin time (APTT), FXII procoagulant activity (FXII:C), FXII antigen (FXII:Ag) and other coagulants were measured. For affected members of the family, exons 1-14 and flanking intronic regions of the FXII gene were amplified with polymerase chain reaction (PCR) and sequenced thereafter. Expression plasmids containing mutant FXII cDNA was constructed and transfected into COS7 cells transiently. Expressions of FXII:Ag and FXII:C were analyzed.</p><p><b>RESULTS</b>The proband has manifested a prolonged APTT of 108.1 s (reference range: 27.0-41.0 s). Her husband has a normal APTT. Other members of the family had slightly increased APTT. The FXII:C and FXII:Ag of the proband have both dropped to about 0.01 (reference range: 0.72-1.13). The FXII:C levels of her husband, son, daughter and grandchild were 0.57, 0.24, 0.14, 0.16, respectively. And the FXII:Ag levels in her husband, son, daughter and grandchild were 0.55, 0.27, 0.15, 0.21, respectively. The proband and her daughter have both carried a heterozygous deletional mutation 6800-6808delAGCTGGGAG (6800-6808del9bp) in exon 9. For the promoter region of the FXII gene, the genotypes of the proband, her son, daughter and grandchild was TT, whilst that of her husband was CT. Expression study has shown that, whilst the mutant FXII protein has accumulated in the cells similar to wild-type protein, its secretion has reduced approximately by half.</p><p><b>CONCLUSION</b>A novel deletional mutation 6800-6808del9bp has been identified in the FXII gene. Although mutant FXII protein can still accumulate in cells, its secretion has become insufficient. The 6800-6808del9bp mutation and 46T/T have both contributed to the pathogenesis of FXII deficiency in the family, but may have not been the sole cause.</p>

Molecular mechanism of a pedigree with inherited coagulation factor Ⅶ deficiency / 中华检验医学杂志

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.