Rapid and simultaneous genotypic detection of Rifampin-Isoniazid and Ethambutol resistant Mycobacterium tuberculosis by use of MAS-PCR

AbstractAims and objectivesThis study aims to identify common mutations leading to Isoniazid (INH), Rifampin (RMP) and Etambutol (EMB) resistance using Multiplex Allele-Specific Polymerase Chain Reaction (MAS-PCR).MethodIn a cross-sectional study during 2012–2013, 257 patients with smear-positive pulmonary tuberculosis residing in five frontier west and north-west provinces of Iran were evaluated in respect of common point mutations leading to resistance to tree first-line drugs.ResultsThe overall frequency of mutations was 37 out of which 8 mutations were related to katG 315, 26 mutations pertained to rpoB 516, 526 and 531 and 3 mutations related to emb B. The rpoB single, double and triple mutations were found in 45.3%, 42.3% and 15.4% of rpoB, respectively. Frequency of patients with mutation to katG and at least one rpoB codon was 7cases (2.7%) at the same time. In this study 60.0% of INH-resistant and 83.3% of RMP-resistant isolates were detected by MAS-PCR technique. Mutation odds were higher in females and in patients with a history of anti-TB drug use.ConclusionThe MAS-PCR is a relatively rapid, sustainable, efficient and accurate technique for detection of drug resistance in tuberculosis. This highlights also the role of mutation at inhA, ahp and oxy R genes in the creation of IHN resistance which may be the causative factor in the remainder of cases

Interleukin-6 G-174C gene polymorphism and susceptibility to upper respiratory tract infection among endurance athletes

The aim of this study was to investigate the influence of interleukin (IL)-6 gene polymorphisms on upper respiratory tract infection (URTI) incidence. To this end, 100 healthy elite male athletes participating in the study were classified as either healthy or prone to frequent URTI. Blood samples and DNA isolation, multiplex polymerase chain reaction, and Taqman real-time polymerase chain reaction were carried out. Genomic DNA was extracted from peripheral leukocytes of whole blood samples using the QIAmp DNA Blood Mini Kit according to the manufacturer's protocols. For comparison of the distribution of genotypes between the two groups and for estimating odds ratios for URTI susceptibility in relation to the IL-6 polymorphism, Pearson's χ2 and logistic regression methods were used, respectively. The IL-6-174 genotype distribution differed between athletes with URTI and healthy athletes (χ2 = 11.68, p = 0.003). The IL-6 low-expression genotype (CC), relative to the other two genotypes combined (GC + GG), was associated with a tendency for an increased likelihood of frequent URTI (odds ratio: 3.33, 95% confidence interval: 1.40–7.92; p = 0.006). In conclusion, findings from this study have identified a potential role of genetic variation in influencing the risk for URTI in athletic populations and single nucleotide polymorphisms in the IL-6 genes were associated with an altered risk profile. These measures may have a predictive value in the identification of individuals who are more likely to experience recurrent infections when exposed to high physical stress in the areas of athletic endeavor

Sex determination using free fetal DNA in early pregnancy: With the approach to sex linked recessive disorders

Introduction: Prenatal diagnosis is testing for detection of diseases or conditions in a fetus or embryo before it is born. Most of prenatal diagnostic (PD) techniques are invasive and done in late stages of pregnancy. Using fetal DNA in maternal blood for fetal sex determination in early pregnancy might help in management of X-linked genetic diseases. This study aimed to investigate the accuracy of sex determination using fetal DNA in maternal blood at 8-12 weeks of gestation. Methods: In this cross-sectional study, 30 pregnant women at 8-12 weeks of gestation were enrolled. The sex-determining region Y (SRY) gene expression with the internal control (IC) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was investigated with quantitative real-time polymerase chain reaction (PCR) using specific primers and probes. Results: Accuracy of sex determination with SRY gene expression in 8-12 weeks of pregnancy were 85%, 85%, 90% and 100% respectively. Conclusion: It seems that fetal sex determining using fetal DNA in maternal blood is a reliable method for early stage of pregnancy

MLL-AF4 fusion transcripts in Acute Lymphocytic Leukemia patientsin Children hospital of Tabriz

Background: MLL-AF4 positive Leukemias comprise about 50-70% of acute lymphoid leukemias in children and about 5% of adolescents and adults Despite recent advances in the treatment of hematologic malignancies of children with ALL in particular, but it seems that poor results are obtained from treating this type of malignancy. Perhaps it is due to the lack of enough knowledge about the expression pattern of the fusion gene induced by chromosomal translocations. This study aims to consider several aspects of the common chromosomal disorder, t (4 11): due to lack of accurate statistical results for this type of translocation in our country, acceptable results are provided Sprevalence of isoforms of recombinant genes involved in MLL-AF4 are explained. Materials and methods: Of 36 patients with ALL between 4 months -11 years of age, peripheral blood sampling was done and total RNA extracted and cDNA was made. Then cDNA was amplified in two steps with the PCR and Nested PCR reactions. After electrophoresis the products were compared and analyzed in comparison with the internal control. Results: The results showed that MLL-AF4 recombinant gene expression in the age between 4 to 12 months range is maximum in the second stage by Nested PCR. Also the highest frequency of fusion isoforms of the gene involved in the same age range is e11-e4 isoform with the frequency of 0.13. Conclusion: It seems that investigation of translocation and chromosomal abnormalities using molecular techniques is one of the most accurate and suitable methods for identifying chromosomal characteristics in patients with acute leukemia, particularly ALL

Genomic analysis of Mycobacterium tuberculosis in respiratory positive-smear patients using PGRS-RFLP in northwest and west provinces of Iran

Aims and objectives: Clustering of and determining Mycobacterium tuberculosis (MTB) strains is of great concern in control programs of tuberculosis (TB). Identification of transmission type and tracking the infection source is also highly necessary. The aim of the present study is to track and determine the type of MTB infection, as well as its relationship with demographic factors using PGRS-RFLP. Methods: In this cross-sectional study, 84 smear-positive patients from 5 frontier provinces (East Azerbaijan, West Azerbaijan, Ardebil, Kurdistan, and Kermanshah) were investigated according to PGRS-RFLP. Demographic data were collected using a questionnaire. The results were analyzed by SPSS-18 and G-Box. Result: Based on clustering, recent transmission was 66%. Most clusters were obtained from Kurdistan and Kermanshah. Vaccination record (p = 0.49) and treatment group (without previous treatment) (p = 0.004) had a significant relationship with clustering. Other demographic factors including age, gender, religion, drug abuse, smoking, history of migration, and marital status did not show a significant relationship with clustering. Conclusion: Genetic variation of MTB is high in this region. The rate of recent transmission based on clustering was unexpected (global average is 30–40%). Recent transmission was more dynamic in west Iran than northwest Iran. The strong relationship between treatment group 1 (without previous treatment) and clustering based on PGRS-RFLP can demonstrate the high correlation between molecular and classic information. In addition, the significant relationship between vaccination record and clustering highlights the necessity to conduct more extensive studies

Frequency of null allele of Human Leukocyte Antigen-G (HLA-G) locus in subjects to recurrent miscarriage

Background: Human leukocyte antigen-G (HLA-G) is a non-classical class I molecule highly expressed by extravillous cytotrophoblast cells. Due to a single base pair deletion, its function can be compensated by other isoforms. Investigating the frequency of null allele in Recurrent Miscarriage (RM) subjects could be useful in understanding the relationship between frequency of this allele and RM in a given population. Objective: This study aimed to determine the frequency of HLA-G*0105N null allele and its potential association with down-regulation of HLA-G in subjects with RM. Materials and Methods: Western blotting was used to assess the level of HLA-G protein expression. For investigating the frequency of HLA-G*0105N null allele in RM subjects, PCR-RFLP method was used. Exon 3 of HLA-G gene was amplified by polymerase chain reaction (PCR). Subsequently, PpuM-1 enzyme was employed to digest the PCR products and fragments were analyzed using gel electrophoresis. Results: Digestion using restriction enzyme showed the presence of heterozygous HLA-G*0105N null allele in 10% of the test population. Western blotting results confirmed the decrease in expression of HLA-G in the placental tissue of subjects with RM compared to subjects who could give normal birth. Conclusion: The frequency of heterozygous HLA-G*0105N null allele was high to some extent in subjects with RM. The mutation rate in subjects suggested that there is a significant association between RM and frequency of mutations in this allele

ACE insertion/deletion polymorphism and restenosis events after coronary stent placement in East Azerbaijan Province of Iran

Renin-angiotensin system is thought to play a role in coronary thrombosis and restenosis. Plasma angiotensin I-converting enzyme (ACE) activity is associated with an insertion/deletion polymorphism in the gene coding for ACE. The association among restenosis within coronary stents, the D/I polymorphism is analyzed in the present study. We are selected 50 patients who had undergone successful percutaneous transluminal coronary angioplasty, and angiography during 1 year after it for some reason. The I/D alleles were identified on the basis of polymerase chain reaction (PCR) amplification of the respective fragments from intron 16 of ACE gene. The differences between groups were tested by Chi-square test or Fisher exact test. Result from chi-square test and Fisher exact test shows the lack of differences between two groups with respect to sex, age, diabetes, hypertension, cholesterol, hyperlipidemia, stent type, length of stented segment, stent diameter and target vessel. In the second set of analysis, the relationship between genotypes and the outcome variable was tested. These analyses indicated that there were no statistically significant differences the three groups (χ2 (2) = 1.40, p=0 .47). The ACE D/D genotype or D allele does not influence the clinical and angiographic outcome of patients undergoing coronary stent placement. These data suggest that routine determination of the ACE genotype may not help identify patients who are at a higher risk of thrombotic and restenosis events after coronary stent placement. [Med-Science 2016; 5(2.000): 519-28

Genetic Variations of Tumor Necrosis Factor –α-308 and Lymphtoxin-α+252 in Non-Hodgkin Lymphoma and Acute Lymphoblastic Leukemia Patients

Objective(s): Non- Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL) are two main hematological malignances which have been driven from lymphoid tissue. Genetic polymorphisms in tumor necrosis factor-α (TNF-α) -308 and lymphotoxin-α (LT-α) +252 may affect their transcription and expression which leads to their high plasma level. The frequency of the TNF-α (-308) and LT-α (+ 252) polymorphisms are different for NHL and ALL cases in various populations with different ethnicity. This research is designed to investigate the prevalence and association of TNF-α (-308) and LT-α (+ 252) polymorphisms from NHL and ALL in Azarian patients and healthy individuals from Northwestern part of Iran.   Materials and Methods: Seventy subjects with ALL and 68 NHL, along with another 130 healthy subjects as control group took part in this study. Genomic DNA was extracted, then genetic polymorphisms in TNF-α and LT-α genes were analyzed with the PCR-RFLP and NCOI as restriction enzyme. A statistical analysis was performed by chi-square test using SPSS software. A P-value o

Enhancing radiosensitivity of TE1, TE8, and TE 11 esophageal squamous carcinoma cell lines by Hdm2-siRNA targeted gene therapy in vitro

Introduction: Human double minute2 (hdm2) level increases in most human malignancies. Therefore, inhibition of tumor growth and also induction of radiosensitivity may be provided by hdm2 inhibitors. The effects of hdm2-siRNA on hdm2 protein expression, cell apoptosis rate, and radiosensitivity of human esophageal squamous cell carcinoma (ESCC) were studied. Methods: The hdm2 gene was silenced in TE1, TE8, and TE11 ESCC cell lines using 200nM siRNA by liposomal transfection method followed by irradiation with 0.5, 1, 2, 4, and 6 Gy γ-rays in vitro. The gene expression levels were evaluated by real time PCR and Western Blotting methods. MTT, TUNEL, and also colony forming assays were used to compare the radiosensitivity of the cell lines before and after the treatments. Results: Hdm2-siRNA reduced the hdm2 protein as compared to the vehicle control and scrambled groups, and also increased the radiation-induced apoptosis especially in TE11 cells. The related dose reduction factors (DRFs) for the silenced TE1, TE8, and TE11 cells calculated to be 1.20, 1.30, and 2.75, respectively. Conclusion: Increasing radiosensitivity of tumor cells may be provided by silencing the oncogenes