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.

Clinical Characterization and Molecular Analysis of Fourteen Chinese Patients with Factor V Deficiency.

INTRODUCTION: Coagulation factor V (FV) functions as a vital cofactor that performs procoagulant roles in the coagulation system. We investigated 14 unrelated patients whose plasma FV levels were all below the reference range. METHODS: FV activity (FV:C) and FV antigen were detected by one-stage clotting and ELISA, respectively. All 25 exons of the F5 gene in patients were amplified by the PCR, and they were sequenced directly. Haplotype analysis was performed with different polymorphisms on F5. Protein modeling was applied to analyze the potential molecular mechanisms. RESULTS: Of five patients with higher FV levels (FV:C > 10%), only one had minor bleeding symptoms. In contrast, of the remaining eight patients with lower FV levels (FV:C < 10%), six showed various bleeding manifestations. A total of 10 mutations were detected from 14 patients (6 were novel mutations). Interestingly, the homozygous p.Phe190Ser was found in five pedigrees, and haplotype analysis showed that they shared almost the same haplotype, indicating the common origin rather than a hotspot mutation. In silico analysis preliminarily investigated the potential pathogenic mechanism of the mutation. Modeling analysis showed that all six missense mutations would lead to conformational alterations in the FV protein. Among them, three (p.Gly1715Ser, p.Ser1753Arg, and p.Asp68His) would decrease hydrogen bonds. CONCLUSION: This is the largest genetic analysis of a single cohort of FV deficiency in Chinese. The study demonstrated that FV levels tended to be correlated with the probability of hemorrhage. The identification of a large number of unique FV-deficient pedigrees highlighted the screening for mutations in F5.

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.

Genetic Analysis of Hereditary Coagulation Factor V Deficiency in Two Chinese Families Caused by Compound Heterozygous Mutations.

OBJECTIVE: This study aims to provide a preliminary discussion of the molecular basis of FV deficiency caused by compound heterozygous mutations in two Chinese families. METHODS: Relative coagulation index was measured by the one-stage clotting method and the FV:Ag was measured by ELISA. All exons and flanking regions of the F5 gene were amplified by PCR and directly sequenced. ClustalX-2.1-win was used to analyze the conservation of mutations. The online software was used to predict the pathogenicity of mutations. PyMOL was used to analyze the variation in the spatial structure of the FV protein before and after mutations. Calibrated automated thrombogram was used to analyze the function of the mutant protein. RESULTS: Phenotyping suggested that both probands had a simultaneous decrease in FV:C and FV:Ag. Their genetic tests showed that proband A had a missense mutation p.Ser111Ile in exon 3 and a polymorphism p.Arg2222Gly in exon 25. At the same time, the proband B had a missense mutation p.Asp96His in exon 3 and a frame-shift mutation p.Pro798Leufs*13 in exon 13. Meanwhile, the p.Ser111Ile is conserved among homologous species. The bioinformatics and protein model analysis revealed that p.Ser111Ile and p.Pro798Leufs*13 were pathogenic and could affect the structure of the FV protein. The thrombin generation test revealed that the clotting function of proband A and B had been affected. CONCLUSION: These four mutations may be responsible for the reduction of FV levels in two Chinese families. Moreover, the p.Ser111Ile mutation is a novel pathogenic variant that has not been reported.

[Genetic analysis of two Chinese pedigrees affected with Hereditary hypofibrinemia due to missense variants].

OBJECTIVE: To retrospectively analyze the clinical phenotypes and genetic variants in two Chinese pedigrees affected with Hereditary hypofibrinemia (IFD) and explore their molecular pathogenesis. METHODS: Two probands and their pedigree members were admitted to the First Affiliated Hospital of Wenzhou Medical University on March 30, 2021 and May 27, 2021, respectively. Clinical phenotypes of the probands were collected, and blood clotting indexes of the probands and their pedigree members were determined. Variants of the FGA, FGB and FGG genes were analyzed by Sanger sequencing, and candidate variants were verified by sequence comparison. Bioinformatic software was used to analyze the conservation of the amino acids and pathogenicity of the proteins. Alteration in protein structure and intermolecular force before and after the variant was analyzed by simulating the protein model. RESULTS: Proband 1, a 18-year-old male, had significantly low plasma fibrinogen activity (Fg:C) and plasma fibrinogen antigen (Fg:Ag), respectively at 0.80 g/L and 1.00 g/L. Proband 2, a 43-year-old male, had slightly low Fg:C and Fg:Ag at 1.35 g/L and 1.30 g/L, respectively. The Fg:C and Fg:Ag of proband 1's father, proband 2's father and son were also below the normal level. Genetic testing showed that proband 1 had harbored a heterozygous missense variant of c.688T>G (p.Phe230Val) in exon 7 of the FGG gene, which was inherited from his father. Proband 2, his father and son all had harbored a heterozygous variant of c.2516A>C (p.Asn839Thr) in exon 6 of the FGA gene. Homology analysis showed that the Phe230 and Asn839 residues were highly conserved among homologous species. Bioinformatic analysis predicted that both p.Phe230Val and p.Asn839Thr were pathogenic variants. CONCLUSION: Analysis of protein simulation model showed that the p.Asn839Thr variant has changed the hydrogen bo`nd between the amino acids, thus affecting the stability of the protein structure. The heterozygous missense variants of p.Phe230Val and p.Asn839Thr probably underlay the IFD in the two pedigrees.

Gene Variants in Two Families with Inherited Coagulation Factor XI Deficiency and Identification of Mutations.

INTRODUCTION: Mutations in the F11 gene can cause factor XI (FXI) deficiency, leading to abnormal coagulation activity and injury-related bleeding tendency. Therefore, identifying F11 gene mutations and studying the molecular basis will help us understand the pathogenesis of FXI deficiency. METHODS: Coagulation tests and gene sequencing analysis of all members were performed. FXI wild-type and mutant expression plasmids were constructed and transfected into HEK293FT cells. The FXI protein expression level was evaluated by ELISA and Western blot. RESULTS: The FXI activity (FXI:C) and FXI antigen (FXI:Ag) of proband-1 were decreased to 2% and 5%, respectively. FXI:C and FXI:Ag of proband-2 were reduced to 15% and 32%, respectively. Four mutations were found in the two unrelated families, including c.536C>T (p.T179M), c.1556G>A (p.W519*), c.434A>G (p.H145R), and c.1325_1325delT (p.L442Cfs*8). In vitro studies in transiently transfected HEK293FT cells demonstrated that p.T179M, p.W519*, and p.L442Cfs*8 mutations significantly lowered the FXI levels in the culture media. The FXI levels in the culture media and cell lysates of p.H145R mutation were similar to the wild type. CONCLUSION: Our results confirm that the four mutations in the F11 gene are causative in the 2 FXI deficiency families. Moreover, the p.H145R mutation is a cross-reactive material (CRM)-positive phenotype. The other three mutations are CRM-negative phenotypes and lead to FXI protein secretion disorder.

Cross-talk between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells.

NOTCH and bone morphogenetic protein (BMP)/SMAD signaling play key regulatory roles in mammalian ovarian development. The study aimed to investigate interregulatory mechanisms between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells (GCs). The results showed that NOTCH2 silence reduced the mRNA expression of SMAD1, SMAD5, SMAD8 (also known as SMAD9) and Mg2+/Mn2+- dependent Protein Phosphatase 1A (PPM1A), which are effectors of BMP/SMAD signaling pathway (P < 0.01). Overexpressing NOTCH2 intracellular sequence increased the mRNA expression of BMPR1A, SMAD1, SMAD4, SMAD5, SMAD8 and PPM1A (P < 0.01). Meanwhile, treating GCs with BMP4 inhibited the mRNA expression of its downstream gene SMAD1 and steroidogenesis genes STAR and CYP11A1 in the presence of follicular stimulating hormone (FSH) (P < 0.01). Moreover, BMP4 inhibited the mRNA expression of NOTCH signaling pathway target gene HES1 (P < 0.05), while the increase in NOTCH2 may be due to negative feedback of HES1. By and large, these results indicated that NOTCH2 up-regulated key genes of BMP/SMAD signaling in bovine follicle GCs, while BMP4 inhibited its downstream signaling factors and NOTCH signaling pathway target gene HES1. This study suggests there are complex synergistic and antagonistic effects between the two signaling pathways, which jointly participate in regulating bovine follicular development through regulating follicular GCs.

[Corrigendum] Metformin reverses multidrug resistance in human hepatocellular carcinoma Bel­7402/5­fluorouracil cells.

Subsequently to the publication of the above paper, the authors have reviewed its content and the primary data, and have realized that the western blots selected to show the ß­actin experiments featured in Fig. 4A and Fig. 3C were the same blot, albeit with a different exposure time. The control blots correctly presented for Fig. 3C were inadvertently copied into Fig. 4A owing to an error made during the figure compilation process. The revised version of Fig. 4, containing the correct ß­actin blots for Fig. 4A, is shown below. Note that this error did not significantly affect the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors thank the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this corrigendum, and apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 10: 2891­2897, 2014; DOI: 10.3892/mmr.2014.2614].