BackgroundCoagulation Factor VII is a vitamin K-dependent serine protease which has a pivotal role in the initiation of the coagulation cascade. The congenital Factor VII deficiency is a recessive hemorrhagic disorder that occurs due to mutations of F7 gene. In the present study C91S (p.C91S) substitution was detected in a patient with FVII deficiency. This mutation has not been characterized by a functional study.ObjectivesIn this study, we aimed to evaluate the impact of C91S substitution on factor VII expression and function.Materials and MethodsThe F7 complete cDNA was isolated from HepG2 cell line and inserted into the pcDNA3.1 mammalian expression vector. The desired mutation was generated by the site-directed mutagenesis and the wild-type and mutated constructs were transfected into CHO-K1 cells. The protein activity and antigen level (antigen concentration) were validated in the culture medium and cell lysate of the transiently transformed cells. An immunocytochemistry procedure was also performed to evaluate the intracellular localization of the mutated and the wild-type FVII, as well.ResultsThe present in vitro study has demonstrated that C91S antigen expression was increased in the transfected CHO-K1 cells compared to the wild-type (WT) protein. Despite an increased protein secretion, the factor VII coagulant activity was diminished following C91S substitution when it was assessed by a standard one-stage analysis. In addition, the immunocytochemistry procedure revealed that there was no difference in the intracellular localization of the C91S mutated FVII compared to the WT protein.ConclusionsOur results present that C91S mutation has an effect on the coagulation activity, secretion, biosynthesis, and probably folding of the FVII leading to the FVII deficiency.
Factor VII (FVII) serves an essential role in the initiation of blood coagulation. Mutations in conserved residues within its serine protease domain may lead to dysregulated coagulation activity. The objective of the present study was to elucidate the impact of altering two conserved residues, H348R and S282R, on the functional properties of the FVII protein. The mutation‑harboring fragments were derived from genomic DNA of a FVII deficient patient. The fragments were integrated into a pcDNA vector containing FVII cDNA of HepG2 cells. The wild-type and mutated FVII constructs were transfected into CHO‑K1 cells as a mammalian cell model. The coagulation activity, antigen levels and intracellular localization of the recombinant proteins were studied in association with their pathological importance. Results indicated that FVII activity was not detectable in conditioned media of the cells transfected with the mutated constructs. The H348R mutation reduced the expression of intracellular and secreted forms of the FVII protein. Following S282R transfection, intracellular FVII expression showed no significant variation; however, extracellular protein was reduced. The pattern of intracellular localization of mutated FVII remained unaltered in comparison to the wild-type protein. In conclusion, the present study suggested that missense mutations within the serine protease domain of FVII affect extracellular levels in addition to the coagulation activity of FVII. These results may contribute to further understanding of the molecular pathogenesis of FVII deficiency and the development of pharmaceutical candidates with improved therapeutic properties.
Our results show that there is a spectrum of GATA4 mutations resulting in septal defects.
Coagulation factor VII (FVII) is a key enzyme of the extrinsic coagulation cascade that is predominantly produced by hepatocytes. The F7 gene mutations cause FVII deficiency with considerable molecular and phenotypic heterogeneity. We characterized the molecular alterations of the F7 gene and their corresponding mRNA transcripts in Iranian patients from eight unrelated families. The mutations were detected by polymerase chain reaction (PCR)-sequencing of all F7 gene exons, their flanking intronic sequences, as well as their corresponding cDNA fragments. Homozygous P303T, C91S and R304Q mutations were detected in patient 2, patient 5, and patient 6, respectively. Patient 7 was a compound heterozygote for S282R and H348R and patient 8 was a compound heterozygote for R304Q and IVS7+7A>G mutations. Furthermore, our investigation revealed three heterozygous individuals, patient 1 and patient 3 with the A244V mutation who were symptomatic and patient 4 with V(–39)I mutation who was also asymptomatic. The F7 mRNA expression analysis revealed that, except the transcript of V(–39)I, other mutation-harboring transcripts were expressed at detectable levels. In conclusion, this report reinforces the genetic and phenotypic heterogeneity of FVII deficiency. The findings of the mRNA study implied that decreased FVII protein activity subsequent to missense mutations does not completely reflect the degradation of mutation-harboring mRNA.
Hematological parameters are appraised routinely to determine overall human health and to diagnose and monitor certain diseases. In GWASs, more than 30 loci carrying common deoxyribonucleic acid (DNA) polymorphisms have been identified related to hematological traits. In this study, we investigated the contribution of ABO rs2073823 along with AQP1 rs1049305 and rs10244884 polymorphisms in hematological traits variation in a cohort of Iranian healthy individuals.Genomic DNA was extracted from peripheral blood of 168 healthy volunteer. Genotyping was performed by ARMS-PCR or PCR-RFLP and confirmed by DNA sequencing. Complete blood analyses were conducted for the participants.Significant association was observed between AQP1 rs1049305 and the hematological traits including hemoglobin, hematocrit, and platelet count (P = 0.012, 0.008, and 0.011, respectively). The AQP1 rs10244884 status was also significantly linked to hemoglobin and hematocrit levels in the study cohort (P = 0.015 and 0.041, respectively). Furthermore, ABO rs2073823 polymorphism was identified as a hemoglobin and hematocrit levels modifier (both with P = 0.004).AQP1 and ABO variants appear to predict hemoglobin and hematocrit levels but not other erythrocyte phenotype parameters including red blood cell counts and red blood cell indices.
Every second counts for patients with life-threatening injuries, and trauma centers deliver timely emergency care to patients with traumatic injuries. Quality assessment and improvement are some of the most fundamental concerns in trauma centers. In this study, a comprehensive organizational resilience approach is proposed to evaluate performance in trauma centers using the European Foundation for Quality Management as a fundamental and strategic approach. We propose a unique intelligent algorithm composed of parametric and non-parametric statistical methods to determine the type and the extent of influence within the organizational resilience and quality management perspectives. We use structural equation modeling to examine the reliability and validity of the input data. The efficiency of each trauma center is then measured using a machine learning method with genetic programming, support vector regression, and Gaussian process regression. The mean absolute percentage error is used to determine the optimal model, and a fuzzy data envelopment analysis model is used to verify and validate the results obtained from the optimal model. The results show that customer results, human capital results, and key performance results have the highest importance weights and positive influence on quality management. Cognitive resources, roles and responsibilities, and self-organization have the highest importance weights and positive influence on organizational resilience.
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