Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp

Background & objectives: Rapid and accurate diagnosis and identification of Leishmania sp causing cutaneous leishmaniasis is crucial in control and therapeutic programs. The problem of diagnosis with traditional methods is that they have a low sensitivity or time consuming but molecular techniques would be an alternative method for rapid and accurate diagnosis. In this work, tryparedoxine peroxidase gene-based real-time PCR was used for accurate identification of Leishmania spp causing Old-World cutaneous leishmaniasis. Methods: In this study, biopsies of specimens were taken from the ulcerative sites in 100 patients and used for direct microscopy, culture in NNN or fixed in alcohol for identification of Leishmania spp using tryparedoxin peroxidase gene-based realtime PCR (qPCR). Results: Using direct microscopy and culture method, Leishmania parasites were isolated from 68 out of 100 patient samples. However, 13 patients with negative finding on traditional tests, had positive results on RT-PCR test. After melting curve analysis of PCR product, Leishmania major in 75 and L. tropica in 4 cases were identified. The sensitivity and specificity of RT-PCR for diagnosis of cutaneous leishmaniasis was 98.7 and 59.8%, respectively. Conclusion: Results of this study showed that RT-PCR was the most sensitive diagnostic test for cutaneous leishmaniasis and represents a tool for rapid species identification

Environmental Risk Factors Associated with Sporadic Colorectal Cancer in Isfahan, Iran

Background: Records from the cancer registry system of Iran indicate that colorectal cancer is the third most common cancer in Iranian men and fourth most common among Iranian women. In this study we have investigated the environmental factors associated with colorectal cancer in Isfahan, Iran. Methods: In this case-control study, we randomly selected 187 patients with colorectal cancer who had positive results by colonoscopy and pathology (case group) and 250 persons who had negative colonoscopy results (control group) from the Colonoscopy Unit of Al Zahra Hospital and Colorectal Cancer Center of Seyed Al Shohada Hospital from 2014 to mid-2015. This study aimed to find the risk factors for sporadic colorectal cancer; therefore, we excluded patients with positive family history. Participants completed a self-administered questionnaire that asked about sex, age, body mass index, smoking status, job-related physical activity, and nonsteroidal antiinflammatory drug consumption. Results: This study enrolled 187 colorectal cancer patients (98 males and 89 females) and 250 individuals without colorectal cancer (107 males and 143 females). Multiple analysis demonstrated a significant association of age (odds ratio: 1.04; 95% confidence interval: 1.02, 1.06) and body mass index (odds ratio: 1.09; 95% confidence interval: 1.03, 1.15) with colorectal cancer risk. Men had an almost two-fold risk compared with women (odds ratio: 1.85; 95% confidence interval: 1.14, 2.99). Subjects who did not use nonsteroidal anti-inflammatory drugs had an almost three-fold risk compared with nonsteroidal anti-inflammatory drug consumers (odds ratio: 0.34; 95% confidence interval: 0.19, 0.62). Analysis for job-related physical activity, also indicated an association between the no/low active group with colorectal cancer (odds ratio no activity: 36.09; 95% confidence interval: 10.94, 119 and odds ratio low activity: 2.96; 95% confidence interval: 1.43, 6.13). Conclusion: Knowledge of the risk factors involved in colorectal cancer incidence makes it possible to identify people at risk and begin risk reduction strategies as well as screening programs

Cell-free Fetal Nucleic Acid Identifier Markers in Maternal Circulation

From the discovery of cell-free fetal (cff)-DNA in 1997 so far, many studies have been performed on various aspects of cff-nucleic acid. It is undoubted that currently, invasive prenatal diagnosis progresses to the noninvasive test. However, there are many problems. One of the most challenging issues in this field is differentiation and detection of the small amount of cff-nucleic acid in maternal plasma. Many markers and methods have been used for this purpose. This review makes an attempt to review and compare the studies in the field. Six identifier markers including Y-specific sequence, polymorphisms, epigenetic difference, DNA size difference, fetal mRNA, and microRNA as well as the advantages and disadvantages of each marker are discussed. This review provides a relatively perfect set on cff-nucleic acid biomarkers in various physiological and pathological status of pregnancy, helping to review and compare the prior obtained results, and improving designation in future studies

Simple and Easy to Perform Preimplantation Genetic Diagnosis for β-thalassemia Major Using Combination of Conventional and Fluorescent Polymerase Chain Reaction

Background: Thalassemias are the most common monogenic disorders in many countries throughout the world. The best practice to control the prevalence of the disease is prenatal diagnosis (PND) services. Extensive practicing of PND proved effective in reducing new cases but on the other side of this success high abortion rate is hided, which ethically unfair and for many couples, especially with a previous experience of a therapeutic abortion, or moral concerns, is not a suitable choice. Preimplantation genetic diagnosis (PGD) is a strong alternative to conventional PND. At present PGD is the only abortion free fetal diagnostic process. Considering the fact that there are more than 6000 single gene disorders affecting approximately 1 in 300 live-births, the medical need for PGD services is significant. Materials and Methods: In the present study development of a PGD protocol for a thalassemia trait couple using nested multiplex fluorescent polymerase chain reaction (PCR) for the combination of polymorphic linked short tandem repeat (STR) markers and thalassemia mutations is described. Restriction fragment length polymorphism used to discriminate between wild and mutated alleles. Results: In PGD clinical cycle, paternal and maternal alleles for D11S988 and D11S1338 STR markers were segregated as it was expected. PCR product for IVSII-1 mutation was subsequently digested with BtscI restriction enzyme to differentiate normal allele from the mutant allele. The mother's mutation, being a comparatively large deletion, was detectable through size differences on agarose gel. Conclusion: The optimized single cell protocol developed and evaluated in this study is a feasible approach for preimplantation diagnosis of β-thalassemia in our patients

Heterozygosity analysis of polycystic kidney disease 1 gene microsatellite markers for linkage analysis of autosomal dominant polycystic kidney disease type 1 in the iranian population

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease. Although imaging techniques are a means of accurate diagnosis when the cysts appear in the third or fourth decades of the patient's life, they are of little value for early diagnosis. Genetic tests are required for preimplantation genetic diagnosis, decision-making for kidney donation to an affected relative. Although mutation of the polycystic kidney disease (PKD1) gene is solely responsible for the most cases of ADPKD, direct genetic testing is limited by the large size of this gene and the presence of many mutations without hot spots. Therefore, indirect diagnosis with linkage analysis using informative microsatellite markers has been suggested. Materials and Methods: In this study, we assessed the informativeness of the PKD1 gene markers D16S475, D16S291, and D16S3252 in Iranian population. Using specific primers, fluorescent polymerase chain reaction (PCR) was performed on genomic DNA extracted from fifty unrelated individuals. PCR products were analyzed by the ALFexpress DNA sequencer system, and the number and frequency of alleles were determined to calculate the heterozygosity (HET) and polymorphism information content (PIC) values. Results: We found that the HET and PIC values for the D16S475 marker are 0.92 and 0.91, respectively. These two values are 0.82 and 0.80 for D16S291 and 0.50 and 0.47 for D16S3252, respectively. Conclusion: Based on this data, D16S475 and D16S291 are highly and D16S3252 is moderately informative for indirect genetic diagnosis of PKD1 mutations in this population

Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp

Background & objectives: Rapid and accurate diagnosis and identification of Leishmania sp causing cutaneousleishmaniasis is crucial in control and therapeutic programs. The problem of diagnosis with traditional methodsis that they have a low sensitivity or time consuming but molecular techniques would be an alternative methodfor rapid and accurate diagnosis. In this work, tryparedoxine peroxidase gene-based real-time PCR was used foraccurate identification of Leishmania spp causing Old-World cutaneous leishmaniasis.Methods: In this study, biopsies of specimens were taken from the ulcerative sites in 100 patients and used fordirect microscopy, culture in NNN or fixed in alcohol for identification of Leishmania spp using tryparedoxinperoxidase gene-based realtime PCR (qPCR).Results: Using direct microscopy and culture method, Leishmania parasites were isolated from 68 out of 100patient samples. However, 13 patients with negative finding on traditional tests, had positive results on RT-PCRtest. After melting curve analysis of PCR product, Leishmania major in 75 and L. tropica in 4 cases wereidentified. The sensitivity and specificity of RT-PCR for diagnosis of cutaneous leishmaniasis was 98.7 and59.8%, respectively.Conclusion: Results of this study showed that RT-PCR was the most sensitive diagnostic test for cutaneousleishmaniasis and represents a tool for rapid species identification

Micro R-410 Binding Site Single Nucleotide Polymorphism rs13702 in Lipoprotein Lipase Gene is Effective to Increase Susceptibility to Type 2 Diabetes in Iranian Population

Background: The relationship between dyslipidemia and type 2 diabetes mellitus (T2DM) has been frequently reported. Lipoprotein lipase (LPL) is considered to be an effective gene in regulating lipid profile. MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional regulation of gene expression. In the present study, we have evaluated rs13702 (C/T) polymorphism located in miRNA-410 binding site of LPL gene in subset of Iranian T2DM patients and their normal counterparts. Materials and Methods: In this case–control study, 102 T2DM patients and 98 healthy controls were worked out for rs13702 single nucleotide polymorphism genotypes. High resolution meting (HRM) analysis was used for genotyping. Results: C allele of rs13702 C/T polymorphism located in miRNA-410 binding site in LPL gene was detected to be significantly associated with T2DM (C allele; odds ratios (OR) = 1.729 (95% confidential intervals (CI) = 1.184–2.523); P = 0.005) also its CC genotype (OR = 3.28 (95% CI 8.68–1.24); P = 0.010) showed the same association. Conclusion: Correlation of rs13702 C allele with susceptibility to T2DM may be due to the higher level of LPL that leads to increased plasma fatty acids and its entry into peripheral tissues such as skeletal muscle, liver, and adipocytes causing development of insulin resistance and ultimately T2DM

Genetic polymorphism of 8 Y-STR loci in native population of Isfahan province in central part of Iran

Background: Y-chromosome short tandem repeats (Y-STRs) are genetic markers with practical applications in human identification and population studies. Aim: Here we present the allelic and haplotype frequencies of 8 Y-STR loci most commonly used in forensic medicine in 103 unrelated native males of Isfahan province, central part of Iran. Subjects and methods: The cases were selected on the basis of strict criteria to assure pure native populations of Isfahan origin. DNA extracted from peripheral blood samples and PCR amplified for each marker. Y-specific STR loci DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 were included in this study. Results: The most common alleles for each locus were: DYS19, allele 12; DYS385, allele 12; DYS389I, allele 13; DYS389II, allele 29; DYS390, allele 24; DYS391, allele 10; DYS392, allele 11; and DYS393, allele 13. Gene diversity value was calculated from the allelic frequency for each locus. The average gene diversity was 0.6518. A total of 101 haplotypes were observed in eight Y-specific STR loci, the haplotype diversity was raised to 0.986. Conclusion: The results revealed that a set of eight Y-specific STR loci were able to discriminate most of the male individuals in the population studied. A search through the Y Haplotype Reference Database demonstrated 21 matched haplotypes to 160,693 haplotypes, exclusively with Eurasian-European, Eurasian, and Eurasian-Indo Iranian populations

Novel High-Fat Diet Formulation and Streptozotocin Treatment for Induction of Prediabetes and Type 2 Diabetes in Rats

Background: The previously established methods for type 2 diabetes (T2D) have mainly concentrated on overt diabetes model development. Here, our intention was to create an animal model passing through distinct phases such as obesity with insulin resistance, prediabetes, and gradual progress to the overt diabetes stage. A high-fat high-carbohydrate diet formulation was prescribed combined with multiple low-dose streptozotocin (STZ) injections after obesity establishment. Materials and Methods: Sixteen male Wistar rats were separated randomly into two groups and fed a normal diet for 1 week after which the body weight and biochemical indices of each rat were measured and recorded. Subsequently, one group (n = 8) switched to the high-fat high-carbohydrate diet formulated by us for 10 weeks, whereas the other group (n = 8) continued with the normal diet. Body weight and biochemical indices of the rats in the high-fat diet (HFD) group were measured at the end of 10 weeks, and each rat received 30 mg/kg intraperitoneal STZ injections with 1-week intervals in two steps and was continued on a high-fat high-carbohydrate diet. The differences between the groups were analyzed using the Student's t-test or one-way analysis of variance and by post hoc multiple comparisons. Results: A significant change in weight, fasting blood glucose, and triglyceride was observed in rats fed with a HFD after 10 weeks. The HFD rats showed typical characteristics of T2D mellitus (T2DM) such as insulin resistance and hyperglycemia following 30 mg/kg STZ. Conclusions: The novel high-fat high-carbohydrate formulation we used, along with multiple low doses of STZ, can mimic peculiar characteristics of T2DM development