The spectrum of factor XI deficiency in Southeast China: four recurrent variants can explain most of the deficiencies.

BACKGROUND: Factor XI (FXI) deficiency is an autosomal hemorrhagic disorder characterized by reduced plasma FXI levels. Multiple ancestral variants in the F11 gene have been identified in Ashkenazi Jews and other selected European populations. However, there are few reports of predominant variants in Chinese and/or East Asian populations. The aim of this study is to characterize the genotypes and phenotypes of FXI deficiency and identify the predominant variants. RESULTS: Of the 41 FXI-deficient patients, 39 exhibited severe FXI defects, considerably more than those with partial defects. The APTT levels showed a negative correlation with FXI activity levels (coefficient=-0.584, P < .001). Only nine patients experienced mild bleeding, including one partially defective patient and eight severely defective patients. The majority of patients were referred for preoperative screenings (n = 22) and checkups (n = 14). Genetic analysis revealed that 90% of the patients had genetic defects, with 2, 16, and 19 cases of heterozygous, homozygous, and compound heterozygous patients, respectively. Seventeen variants were detected in the F11 gene (6 novel), including eleven missense variants, four nonsense variants, and two small deletions scattered throughout the F11. Of the 11 missense variants, six have not yet been studied for in vitro expression. Protein modeling analyses indicated that all of these variants disrupted local structural stability by altering side-chain orientation and hydrogen bonds. Nine variants, consisting of three missense and six null variants, were detected with a frequency of two or more. The highest allele frequency was observed in p.Q281* (21.25%), p.W246* (17.50%), p.Y369* (12.50%), and p.L442Cfs*8 (12.50%). The former two were variants specific to East Asia, while the remaining two were southeast China-specific variants. CONCLUSION: Our population-based cohort demonstrated that no correlation between the level of FXI activity and the bleeding severity in FXI deficiency. Additionally, the prevalence of FXI deficiency may have been underestimated. The nonsense p.Q281* was the most common variant in southeast China, suggesting a possible founder effect.

PAHs removal by soil washing with thiacalix[4]arene tetrasulfonate.

Remediating soil contaminated with polycyclic aromatic hydrocarbons (PAHs) presents a significant environmental challenge due to their toxic and carcinogenic properties. Traditional PAHs remediation methods-chemical, thermal, and bioremediation-along with conventional soil-washing agents like surfactants and cyclodextrins face challenges of cost, ecological harm, and inefficiency. Here we show an effective and environmentally friendly calixarene derivative for PAHs removal through soil washing. Thiacalix[4]arene tetrasulfonate (TCAS) has a unique molecular structure of a sulfonate group and a sulfur atom, which enhances its solubility and facilitates selective binding with PAHs. It forms host-guest complexes with PAHs through π-π stacking, OH-π interactions, hydrogen bonding, van der Waals forces, and electrostatic interactions. These interactions enable partial encapsulation of PAH molecules, aiding their desorption from the soil matrix. Our results show that a 0.7% solution of TCAS can extract approximately 50% of PAHs from contaminated soil while preserving soil nutrients and minimizing adverse environmental effects. This research unveils the pioneering application of TCAS in removing PAHs from contaminated soil, marking a transformative advancement in resource-efficient and sustainable soil remediation strategies.

Clinical and functional characterization of rare compound heterozygous mutations in the SERPINC1 gene causing severe thrombophilia.

INTRODUCTION: Hereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder with significant clinical heterogeneity. In the study, we identified a patient with AT deficiency caused by compound heterozygous mutations in the SERPINC1 gene. METHODS: A total of 9 individuals from three generations were investigated. The mutations were identified by direct sequencing of SERPINC1. Multiple in silico tools were programmed to predict the conservation of mutations and the effect on the AT structure. The coagulation state was evaluated by the thrombin generation assay. Recombinant AT was overexpressed in HEK293T cells; the mRNA level was determined using RT-qPCR. Western blotting, ELISA, and immunocytofluorescence were applied to characterize the recombinant AT protein. RESULTS: The proband was a 26-year-old male who experienced recurrent venous thrombosis. He presented the type I deficiency with 33 % AT activity and a synchronized decrease in AT antigen. Genetic screening revealed that he carried a heterozygous c.318_319insT (p.Asn107*) in exon 2 and a heterozygous c.922G > T (p.Gly308Cys) in exon 5, both of which were completely conserved in homologous species and resulted in enhanced thrombin generation capability. Hydrophobicity analysis suggested that the p.Gly308Cys mutation may interfere with the hydrophobic state of residues 307-313. In vitro expression studies indicated that the levels of the recombinant protein AT-G308C decreased to 46.98 % ± 2.94 % and 41.35 % ± 1.48 % in transfected cell lysates and media, respectively. After treatment with a proteasome inhibitor (MG132), the quantity of AT-G308C protein in the cytoplasm was replenished to a level comparable to that of the wild type. The mRNA level of AT-N107* was significantly reduced and the recombinant protein AT-N107* was not detected in either the lysate or the culture media. CONCLUSION: These two mutations were responsible for the AT defects and clinical phenotypes of the proband. The p.Gly308Cys mutation could lead to proteasome-dependent degradation of the AT protein in the cytoplasm by altering local residue hydrophobicity. The c.318_319insT could eliminate aberrant transcripts by triggering nonsense-mediated mRNA degradation. Both mutations resulted in type I AT deficiency.

Molecular and clinical characterization of two unrelated families with factor V deficiency, including a novel nonsense variant (p.Gln1532*).

BACKGROUND: Factor V (FV) is an essential cofactor in the coagulation cascade. The characterization of novel mutations is advantageous for the clinical management of FV-deficient patients. METHODS: Coagulation screening and thrombin generation assay were performed with the plate-poor plasma. All 25 exons of the F5 gene were amplified and sequenced. The ClustalX-2.1 software was applied to the multiple sequence alignment. The possible adverse effects of mutations were investigated with online bioinformatics software and protein modeling. RESULTS: Two unrelated families with FV deficiency were under investigation. Proband A was an 18-year-old youth with recurrent epistaxis. Proband B was a 29-year-old woman who did not present with any bleeding symptoms. Three heterozygous mutations (p.Gln1532*, p.Phe218Ser, and p.Asp2222Gly) were detected. Interestingly, they were compound heterozygotes and both contained the p.Asp2222Gly, a polymorphism. The thrombin generation assay showed that both patients had impaired ability of thrombin generation, and in particular, proband A was more severe. Conservation, pathogenicity and protein modeling studies all indicated that these three mutations could cause deleterious effects on the function and structure of FV. CONCLUSION: These three mutations are responsible for the FV-deficient in two pedigrees. Moreover, the nonsense variant p.Gln1532* is first reported in the world.

Analysis of an Inherited FXII Deficiency Pedigree Associated with Double Heterozygous Mutations in the F12 Gene.

This article is intended to identify the potential mutations of the FXII gene (F12) in an inherited FXII deficiency pedigree and illuminate the pathogenesis of the disease. The coagulation FXII activity (FXII:C) and FXII antigen (FXII:Ag) were inspected by one-stage clotting assay and enzyme-linked immunosorbent assay(ELISA), respectively. Polymerase chain reaction amplification (PCR) was performed and the F12 gene was sequenced directly. A molecular model of FXIIprotein was established for further analysis. ClustalX-2.1-win and online bioinformatic software were used to estimate the conservatism and possible impact of the protein change. The proband presented prolonged APTT(180 s) and extreme low FXII:C and FXII:Ag (both < 1%, reference range:72-113%). A compound heterozygous were found by the direct sequencing of the F12 gene. One was a deletion mutation c.1792_1796delGTCTA, which is a novel mutation; the other was an insertion mutation, c.1092_1093insC. Bioinformatic and modeling analyses indicated that the the two frameshift mutations may be deleterious and possibly alter the structure and the function of the protein. The mutations c.1792_1796delGTCTA and c.1092_1093insC could be the main causes of reducing FXII in this pedigree, and c.1792_1796delGTCTA mutation was the first report in the world.

A Novel Fibrinogen Mutation p.BßAla68Asp Causes an Inherited Dysfibrinogenemia.

OBJECTIVE: Our study aimed to analyze the phenotype and genotype of a pedigree with inherited dysfibrinogenemia, and preliminarily elucidate the probable pathogenesis. METHODS: The one-stage clotting method was used to test the fibrinogen activity (FIB:C), whereas immunoturbidimetry was performed to quantify the fibrinogen antigen (FIB:Ag). Furthermore, DNA sequence analysis was conducted to confirm the site of mutation. Conservation analysis and protein model analysis were performed using online bioinformatics software. RESULTS: The FIB:C and FIB:Ag of the proband were 1.28 and 2.20 g/L, respectively. Gene analysis revealed a heterozygous c.293C > A (p.BßAla68Asp) mutation in FGB. Bioinformatics and modeling analysis suggested that the missense mutation could potentially have a deleterious effect on fibrinogen. CONCLUSION: The BßAla68Asp mutation in exon 2 of FGB may account for the reduced FIB:C levels observed in the pedigree. To our knowledge, this point mutation is the first report in the world.

A Novel Homozygous Missense Mutation (Ile583Asn) in a Consanguineous Marriage Family with Hereditary Factor XII Deficiency: A Case Report.

BACKGROUND: Hereditary coagulation factor XII (FXII) deficiency is an autosomal recessive disorder. At present, the contribution of severe FXII deficiency to the development of thromboembolism is still undetermined. There are limited reports on the relationship between the FXII defect and thromboembolism. CASE PRESENTATION: A 27-year-old woman came to our hospital for the treatment of shoulder trauma and cervical disc herniation caused by a car accident. The shoulder trauma was treated with five stitches. After physical examination, imaging examination, and routine coagulation examination, cervical disc herniation was treated conservatively. Combined with the examination results, the patient was diagnosed with FXII deficiency. Unfortunately, the patient was readmitted 10 days after the trauma with edema in the lower limbs and secondary varicose veins. The D-dimer increased to 6.22 mg/L. Thrombus in the inferior vena cava and right common iliac was shown by lower limb venography. According to the patient's medical history, the F12 gene was analyzed by direct sequencing. The patient was also screened for other thrombotic risk factors. Genetic analysis showed that the patient had a c.1748T > A (p.Ile583Asn) homozygous missense mutation in exon 14 of the F12 gene. No other hereditary thrombophilia risk factors screened were positive in the patient. CONCLUSION: The p.Ile583Asn missense mutation in exon 14 of the F12 gene might be responsible for the reduction of the FXII level in the patient.

A novel F13A1 gene mutation (Arg208Pro) in a Chinese patient with factor XIII deficiency.

The objective of the study was to analyse a novel F13A1 gene mutation in a Chinese patient with factor XIII (FXIII) deficiency and explore the molecular mechanism. Pedigree investigation, clinical diagnosis, phenotypic and genetic analysis were conducted. The F13A1 gene was amplified by PCR and directly sequenced. Online bioinformatics software was needed to analyse the mutation. A novel mutation c.515G>C (p.Arg208Pro) in exon 4 was found in the proband. Protein Arg208 is conserved highly among homologous species. Bioinformatics software showed that Arg208Pro mutation might affect the protein function. We preliminarily believed the mutation Arg208Pro was responsible for the decrease FXIII level. We reported a novel mutation in the F13A1 gene, which can flesh out the mutant library.

[Analysis of the molecular pathogenesis of hereditary protein C deficiency due to a p.Gly86Asp variant of the PROC gene].

OBJECTIVE: To explore the molecular pathogenesis of hereditary protein C (PC) deficiency due to a p.Gly86Asp variant of the PROC gene through in vitro expression experiment. METHODS: Wild type and Gly86Asp mutant expression plasmids of PC were constructed and respectively transfected into HEK 293FT cells. Total RNA was extracted from the transfected cells, and the expression of PROC gene was determined by quantitative real-time PCR (qRT-PCR). PC antigen (PC:Ag) in the supernatant of cell culture and cell lysate was determined by enzyme-linked immunosorbent assay (ELISA), and the level of PC protein was detected by Western blotting. RESULTS: qRT-PCR has detected no significant difference in the transcription level of wild-type and mutant-type PC. Compared with the wild type, the level of mutant PC:Ag in the supernatant and cell lysate were 81.3%±2.6% and 110.0%±2.8%, respectively. No difference was detected in the molecular weight between the wild-type and mutant-type PC by Western blotting. The PC content of mutant type was higher than wild-type in cell lysate, while the opposite was found with the cell culture supernatant. CONCLUSION: The impaired secretion by mutant PC may be the molecular mechanism of PC deficiency caused by the p.Gly86Asp variant.

[Analysis of genetic variant in a Chinese pedigree with hereditary factor VIII deficiency].

OBJECTIVE: To explore the genetic basis for a patient with factor VIII deficiency. METHODS: All exons of the F13A1 and F13B genes were amplified by PCR and sequenced directly. The sequencing was performed with a reverse primer if a variant was found. Conservation of variant site was analyzed by the ClustalX software. Four online bioinformatic software including Mutation Taster, PolyPhen-2, PROVEAN and SIFT were used to predict the function of the mutation site. The Swiss-PdbViewer software was applied to analyze the changes in the protein model and intermolecular force. RESULTS: The proband was found to harbor a novel c.515G>C (p.Arg171Pro) variant of the F13A1 gene. The corresponding amino acid Arg171 is conserved among homologous species. Bioinformatic analysis indicated that Arg171Pro variant may affect the protein function. Protein model analysis showed that in the wild-type, there is one hydrogen bond between Arg171 and Pro27; one hydrogen bond between Arg171 and Thr28; two hydrogen bonds between Arg171 and Glu102. When Arg171 was mutated to Pro171, the three hydrogen bonds between Arg171 and Pro27, Glu102 are all disappeared and formed a new benzene ring which might affect the stability of the protein structure. No variant was found in the F13B gene. CONCLUSION: The Arg171Pro variant may account for the decreased FVIII level. Above finding has enriched the spectrum of F13A1 gene variants.

Sustainable production of 4-hydroxyisoleucine with minimised carbon loss by simultaneously utilising glucose and xylose in engineered Escherichia coli.

4-Hydroxyisoleucine is a promising drug for diabetes therapy; however, microbial production of 4-hydroxyisoleucine is not economically efficient because of the carbon loss in the form of CO2. This study aims to achieve de novo synthesis of 4-hydroxyisoleucine with minimised carbon loss in engineered Escherichia coli. Initially, an L-isoleucine-producing strain, ILE-5, was established, and the 4-hydroxyisoleucine synthesis pathway was introduced. The flux toward α-ketoglutarate was enhanced by reinforcing the anaplerotic pathway and disrupting competitive pathways. Subsequently, the metabolic flux for 4-hydroxyisoleucine synthesis was redistributed by dynamically modulating the α-ketoglutarate dehydrogenase complex activity, achieving a 4-hydroxyisoleucine production of 16.53 g/L. Finally, carbon loss was minimised by employing the Weimberg pathway, resulting in a 24.5% decrease in sugar consumption and a 31.6% yield increase. The 4-hydroxyisoleucine production by strain IEOH-11 reached 29.16 g/L in a 5-L fermenter. The 4-hydroxyisoleucine yield (0.29 mol/mol sugar) and productivity (0.91 g/(L⋅h)) were higher than those previously reported.

Genetic analysis of compound heterozygous pathogenic variants of the F11 gene in two Chinese patients with hereditary factor XI deficiency.

The aim of this study was to explore the molecular pathogenesis of two families with compound heterozygous hereditary factor XI deficiency. All the exons, flanking sequences, 5' and 3' untranslated regions of the F11 gene were analysed by direct DNA sequencing. The suspected variants were confirmed by reverse sequencing. Bioinformatics softwares were used to analysis the conservation and the possible impact of these pathogenic variants. Genetic analysis revealed c.689G > T and c.1556G > A variants of F11 gene in family A; c.1107C > A and c.1557G > C variants of F11 gene in family B. These variants sites were highly conserved among homologous species. Bioinformatics and structural analysis demonstrated these variants were pathogenic and could affect the protein structure. The c.689G > T, c.1556G > A, c.1107C > A and the c.1557G > C heterozygous variants might be responsible for the decrease of FXI levels in probands. In addition, c.689G > T (NM_000128.4) is a novel pathogenic variant site, which has not been reported.

A novel mutation (Leu60Pro) in a Chinese pedigree with hereditary factor XI deficiency.

To analyse F11 gene mutations in a Chinese pedigree with hereditary factor XI (FXI) deficiency and investigate the molecular mechanism. The plasma FXI activity and FXI antigen of the proband and the family members were detected by clotting assay and ELISA, respectively. The F11 gene was amplified by PCR and sequenced directly. Online bioinformatics software were needed to analyse the mutations. The proband showed a prolonged activated partial thromboplastin time (93.3 s), whose FXI activity and FXI antigen were low to 2, 4.5%, respectively. A novel mutation c.233T>C (p.Leu60Pro) in exon 4 and a previously described mutation c.1253G>T (Gly400Val) were found in the proband. Protein Leu60 is conserved highly among homologous species. Bioinformatics software indicated that Leu60Pro mutation might affect the protein function. Other coagulation abnormalities were not found. We preliminarily considered the mutations Leu60Pro and Gly400Val were responsible for the decrease FXI level in the family. Leu60Pro mutation in the F11 gene has not been described elsewhere.

[Analysis of Phenotype and Genotype of A Family with Hereditary Coagulation Factor â ¤ Deficiency Caused by A Compound Heterozygous Mutation].

OBJECTIVE: To analyze the molecular pathogenesis by analysis of phenotype and gene mutation in families with hereditary coagulation factor V (FⅤ) defect caused by complex heterozygous mutation. METHODS: Plasma pro-thrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), FⅤ procoagulant activity (FⅤ∶C), FⅤ antigen (FⅤ∶Ag), and other related coagulation indexes were detected in the proband and his family members (3 generations 10 people). Using DNA direct sequencing to analyze all exons, flanks, 5' and 3' untranslated regions of F5 genes and the corresponding mutation site regions of family members, the mutation site was confirmed by reverse sequencing．The conservation of mutant amino acids was analyzed by ClustalX-2.1-win software. The PROVEAN and MutationTaster online bioinformatics software were used to predict the effect of mutation on protein function. Protein model and amino acid interaction at mutation sites was analyzed by Swiss-pdbviewer software. RESULTS: The PT and APTT of the proband were significantly prolonged compared with healthy controls (34.2 vs 13.2 s and 119.3 vs 36.0 s), while FⅤ∶C and FⅤ∶Ag extremely reduced (3% and 6%). The PT and APTT of the second-born, the third son, daughter, and grandson of the proband were slightly prolonged, and the FⅤ∶C and FⅤ∶Ag decreased to varying degrees. The related coagulant parameters of other family members were within normal range. Genetic analysis revealed that the proband had a c.911G>A heterozygous missense mutation on the exon 6 lead to p.Gly276Glu, and a c.5343C>G heterozygous missense mutation on the exon 16 lead to p.Ser1781Arg of the proband. The second-born, the third son, and grandson of the proband carry p.Gly276Glu heterozygotes, and the daughter carries p.Ser1781Arg heterozygotes, while the other family members were wild-type. The results of conservative analysis indicated that p.Gly276 and p.Ser1781 were highly conserved in homologous species. The two bioinformatics software predicted the same results, PROVEAN (score -6.214 and -12.79) indicated that the compound heterozygous mutation was a harmful mutation; MutationTaster (score 0.976 and 0.999) suggested that these mutations might cause corresponding disease. p.Gly276Glu protein model analysis showed that, the Glu side chain was prolonged and the molecular weight became larger, which would increase the steric hindrance between it and the surrounding amino acids, affect the normal local folding of the FⅤ protein, and eventually lead to the decrease of protein activity and content. This paper can not provide analysis of the spatial structure of p.Ser1781Arg mutant protein because of the lack of X ray 3 D structure file of FⅤ exon 16. CONCLUSION: The new compound heterozygous mutations (p.Gly276Glu and p.Ser1781Arg) identified in this study are the main reasons for the decrease in the FⅤ level of the family, among which p.Ser1781Arg is rarely reported at home and abroad.

[Analysis of a Chinese pedigree affected with hereditary factor VII deficiency caused by compound heterozygous variants of F7 gene].

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.

[Identification of compound heterozygous variants of F12 gene in a pedigree affected with inherited coagulation factor XII deficiency].

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.

Two Novel Mutations Cause Hereditary Antithrombin Deficiency in a Chinese Family.

OBJECTIVE: To study the molecular basis of hereditary antithrombin (AT) deficiency in a Chinese family. It will help us understand the pathogenesis of this type of disease. METHOD: AT activity (AT:A) and the AT antigen (AT:Ag) level were tested by chromogenic substrate and immunoturbidimetry, respectively. To identify the novel mutations, SERPINC1 gene sequencing was carried out. The possible impact of the mutations was analyzed by model and bioinformatic analyses. RESULTS: AT:A and the AT:Ag level of the proband were 43% and 113 mg/L (normal range: 98-119% and 250-360 mg/L), respectively. Sequencing analysis revealed compound heterozygous mutations, including a frameshift mutation (c.318_319insT) resulting in Asn75stop and a missense mutation (c.922G>T) resulting in Gly276Cys. The bioinformatic and model analyses indicated that these mutations may disrupt the function and structure of the AT protein. CONCLUSION: We detected 2 novel heterozygous mutations (c.318_319insT and c.922G>T) in the proband, and these were associated with decreased AT:A.