Involvement of haptoglobin phenotypes and genotypes in non-muscle invasive bladder cancer: A possible prognostic marker for risk stratification.

The association of haptoglobin (Hp) with various cancers has been reported and also it has been documented that the Hp phenotypes/genotypes have different functional ability. So, we examined phenotypes/genotypes of Hp in newly diagnosed, untreated non-muscle invasive bladder cancer (NMIBC) patients and investigated its prognostic value for risk stratification of the cancer. In eighty NMIBC patients and 80 healthy individuals the Hp genotypes and phenotypes were analyzed using polymerase chain reaction (PCR) and two-dimensional gel electrophoresis (2D-GE), respectively. Besides, the presence of the Hpα1, α2, and ß chains in the sera was confirmed by Mass Spectrometry (MS). The frequencies of the 1-1 and 2-2 genotypes/phenotypes were respectively higher and lower in healthy subjects compared to the patients. Our results revealed that the 2-2 genotype/phenotype could increase the risk of NMIBC. There was a positive association between the 2-2 genotype/phenotype with the T category/grade of cancer (p<0.05). The present study implied a strong association between the Hp phenotypes and genotypes with NMIBC. It was found that the 2-2 genotype and phenotype could be a risk factor for NMIBC incidence, as well as, progression. This study introduced Hp genotyping as a possible cost-effective and precise method for prognosis of individuals at the risk of NMIBC.

Functional and Molecular Characterization of C91S Mutation in the Second Epidermal Growth Factor-Like Domain of Factor VII.

BACKGROUND: Coagulation 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. OBJECTIVES: In this study, we aimed to evaluate the impact of C91S substitution on factor VII expression and function. MATERIALS AND METHODS: The 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Novel mutation of GATA4 gene in Kurdish population of Iran with nonsyndromic congenital heart septals defects.

BACKGROUND: The mutations in GATA4 gene induce inherited atrial and ventricular septation defects, which is the most frequent forms of congenital heart defects (CHDs) constituting about half of all cases. METHOD: We have performed High resolution melting (HRM) mutation scanning of GATA4 coding exons of nonsyndrome 100 patients as a case group including 39 atrial septal defects (ASD), 57 ventricular septal defects (VSD) and four patients with both above defects and 50 healthy individuals as a control group. Our samples are categorized according to their HRM graph. The genome sequencing has been done for 15 control samples and 25 samples of patients whose HRM analysis were similar to healthy subjects for each exon. The PolyPhen-2 and MUpro have been used to determine the causative possibility and structural stability prediction of GATA4 sequence variation. RESULTS: The HRM curve analysis exhibit that 21 patients and 3 normal samples have deviated curves for GATA4 coding exons. Sequencing analysis has revealed 12 nonsynonymous mutations while all of them resulted in stability structure of protein 10 of them are pathogenic and 2 of them are benign. Also we found two nucleotide deletions which one of them was novel and one new indel mutation resulting in frame shift mutation, and 4 synonymous variations or polymorphism in 6 of patients and 3 of normal individuals. Six or about 50% of these nonsynonymous mutations have not been previously reported. CONCLUSION: Our results show that there is a spectrum of GATA4 mutations resulting in septal defects.

In vitro expression of mutant factor VII proteins and characterization of their clinical significance.

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 mutation­harboring 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 CHO­K1 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.

Association of ABO and Colton Blood Group Gene Polymorphisms With Hematological Traits Variation.

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.