Clinical and molecular genetics association of polymorphisms in interleukin-17A genes with risk of Oral Lichen Planus (OLP) in an Azery population

Lichen Planus (LP) is a chronic disease that affects the skin and oral mucosa. Although the precise aetiology of LP is not known, it is thought to be comprised of, in majority, genetic and immunological factors. The objective of this study was to assess the association of interleukin-17A (IL-17A) with Oral LP (OLP). 22 adult patients with OLP and 40 healthy controls were genotyped by polymerase chain reaction (PCR) and DNA direct   sequence technology for the polymorphism of the IL-17A gene. The genotype frequencies of G1776A  (p.Arg29Ter rs139620979) and G3566A (rs7747909) in the IL-17A gene polymorphism were 9 and 13.6% in the OLP group and 0 and 40% in the controls, respectively. Although the proportion of detected polymorphisms did not differ between individuals, a higher prevalence of G3566A (rs7747909) homozygote polymorphism (4.5%) was observed in the OLP patients. Our results show no statistically significant difference in the IL-17A  genotype single nucleotide polymorphisms (SNPs) distribution amongst the two groups. Therefore, further  studies on a larger population and novel genetic variants are needed to better understand the pathobiology of OLP.Key words: Oral Lichen Planus (OLP), interleukin-17A (IL-17A) gene, single nucleotide polymorphisms (SNPs), direct sequencing

Microsatellite instability in colorectal cancer

Colorectal cancer (CRC) is a heterogeneous disease that is caused by the interaction of genetic and environmental factors. Although it is one of the most common cancers worldwide, CRC would be one of the most curable cancers if it is detected in the early stages. Molecular changes that occur in colorectal cancer may be categorized into three main groups: 1) Chromosomal Instability (CIN), 2) Microsatellite Instability (MSI), and 3) CpG Island Methylator phenotype (CIMP). Microsatellites, also known as Short Tandem Repeats (STRs) are small (1-6 base pairs) repeating stretches of DNA scattered throughout the entire genome and account for approximately 3 % of the human genome. Due to their repeated structure, microsatellites are prone to high mutation rate. Microsatellite instability (MSI) is a unique molecular alteration and hyper-mutable phenotype, which is the result of a defective DNA mismatch repair (MMR) system, and can be defined as the presence of alternate sized repetitive DNA sequences which are not present in the corresponding germ line DNA. The presence of MSI is found in sporadic colon, gastric, sporadic endometrial and the majority of other cancers. Approximately, 15-20 % of colorectal cancers display MSI. Determination of MSI status in CRC has prognostic and therapeutic implications. As well, detecting MSI is used diagnostically for tumor detection and classification. For these reasons, microsatellite instability analysis is becoming more and more important in colorectal cancer patients. The objective of this review is to provide the comprehensive summary of the update knowledge of colorectal cancer classification and diagnostic features of microsatellite instability

Detection of t(8;14) c-myc/IgH gene rearrangement by long-distance polymerase chain reaction in patients with diffuse large B-cell lymphoma

AbstractObjective/backgroundSpecific chromosomal translocations are found in human leukemias and lymphomas. These translocations are closely related to particular histological and immunological phenotypes. In Burkitt’s lymphoma, translocation t(8;14)(q24;q32), which involves the c-myc gene (8q24) and the immunoglobulin heavy-chain (IgH) locus (14q32), accounts for 90–95% of all chromosomal translocations. This translocation can be found in 2–5% of diffuse large B-cell lymphoma (DLBCL). Long-distance polymerase chain reaction (LD-PCR) assays, which can identify oncogene/Ig gene rearrangement, can detect these fusion genes. The objective of this study was to detect t(8;14) c-myc/IgH gene rearrangement by LD-PCR in patients with DLBCL.MethodsIn this study, 54 DLBCL cases were tested by LD-PCR with specific primers. LD-PCR was used for two breakpoints in both the IgH gene (joining region and γ switch region) and the myc gene (Exons 2 and 3).ResultsAs much as 1.85% of the samples were positive for the γ constant region and Exon 2 of the myc gene.ConclusionLD-PCR can be used for the detection of t(8;14) c-myc/IgH gene rearrangement in patients with DLBCL

Analysis of Methylation and Expression Profile of Foxp3 Gene in Patients with Behçet’s Syndrome

Forkhead box P3 (Foxp3) gene is an important means in the Treg cells function, in both maintenances of immune tolerance and regulation of response. Epigenetic modifications of the foxp3 gene at its regulatory regions control the chromatin accessibility for the transcription factors and other transcriptional regulators in order to control Foxp3 expression. In addition, the methylation status of CpG islands within the Foxp3 promoter and regulatory elements regulate the expression of Foxp3. This study was performed to assess the role of the foxp3 gene in patients with Behçet’s syndrome (BS). Venous blood samples were collected from all participants and peripheral blood mononuclear cells (PBMC) were extracted through Ficoll-Hypaque method. Genomic DNA was randomly sheared by sonication and immunoprecipitated with a monoclonal antibody. The status methylation of the foxp3 gene was estimated in 108 blood samples of active BS patients and healthy individuals (controls); using methylation DNA immunoprecipitation (MeDIP) technique. Expression analysis was carried out; using Real-time PCR. The expression of foxp3 gene in the patients' group (mean±SD: 1.79±1.12) was significantly lower than the healthy group (mean±SD: 2.73±1.33) (p<001). Also, the methylation levels of Foxp3 promoter showed that its level in patients (mean±SD: 2.3±1.16) was higher than the healthy group (mean±SD: 1.85±0.59). However, this increase was not statistically significant (p>0.05). Also, these results indicated that increasing the amount of methylation of the foxp3 gene by reducing its expression leads to an increase and intensifying of the disease. The decrease in Foxp3 expression is possibly associated with hypermethylation of the gene, and it can be considered as a risk factor for BS. Future studies may be needed to identify the capability of specific DNA methylation alterations in this syndrome

Evaluation of microsatellite instability in tumor and tumor marginal samples of sporadic colorectal cancer using mononucleotide markers

Microsatellite instability (MSI) is a unique molecular alteration that is due to a defective DNA mismatch repair (MMR) system. Approximately, 15-20 % of sporadic colorectal cancers (CRC) display MSI. Determination of MSI status in CRC has prognostic and predictive implications. Additionally, detecting MSI is used diagnostically for tumor detection and classification. The present study analyzed a panel of five mononucleotide markers, BAT- 25, BAT-26, NR-21, NR-22 and NR-27, amplified in a single multiplex PCR reaction to evaluate MSI status in CRC patients. Genomic DNA from 50 CRC and paired adjacent normal tissues was used for PCR-based MSI analysis. Our finding showed microsatellite instability in 36 % of specimens. Instability with differences in allele lengths was observed in the tumoral DNA compared to the tumor-free margin DNA sample. The frequency of instability in NR-21, BAT-26 and BAT-25 markers were more than others; their frequency were 35.48 %, 29.03 %, and 22.58 %, respectively. In conclusion, the NR-21, BAT-26, and BAT-25 were the most useful markers for discriminating cancer tissue from normal, therefore these markers have demonstrated promising potential for determining MSI status in patients with sporadic colorectal cancer

Therapeutic Effects of Myeloid Cell Leukemia-1 siRNA on Human Acute Myeloid Leukemia Cells

Purpose: Up-regulation of Mcl-1, a known anti-apoptotic protein, is associated with the survival and progression of various malignancies including leukemia. The aim of this study was to explore the effect of Mcl-1 small interference RNA (siRNA) on the proliferation and apoptosis of HL-60 acute myeloid leukemia (AML) cells. Methods: siRNA transfection was performed using Lipofectamine™2000 reagent. Relative mRNA and protein expressions were quantified by quantitative real-time PCR and Western blotting, respectively. Trypan blue assay was performed to assess tumor cell proliferation after siRNA transfection. The cytotoxic effect of Mcl-1 siRNA on leukemic cells was measured using MTT assay. Apoptosis was detected using ELISA cell death assay. Results: Mcl-1 siRNA clearly lowered both Mcl-1 mRNA and protein levels in a time-dependent manner, leading to marked inhibition of cell survival and proliferation. Furthermore, Mcl-1 down-regulation significantly enhanced the extent of HL-60 apoptotic cells. Conclusion: Our results suggest that the down-regulation of Mcl-1 by siRNA can effectively trigger apoptosis and inhibit the proliferation of leukemic cells. Therefore, Mcl-1 siRNA may be a potent adjuvant in AML therapy

Investigating the effect of Sclareol on IRE-1 and PERK genes the pathway of reticulandaplasmic system stress in gastric cancer cells MKN-45

Background & Aims:  Despite the decline in the prevalence of gastric cancer in recent years, it remains the fourth leading cause of death from cancer in the world. Common cancer treatments may reduce the size of the tumor, but it is transient and does not have a positive effect on the patient's survival and there is a possibility of recurrence of the disease. Strong induction of reticulom endoplasmic has been shown to increase the susceptibility to anti-cancer therapy. Regarding the importance of medicinal herbs in recent years and its low side effects after administration, compared with synthetic drugs, this study investigated the effects of salvia Sclareol purification from sage on the induction of reticulum endoplasmic system stress. Materials and Methods: The MKN-45 cell line from the Pasteur Institute of Iran was purchased and cultured in a complete culture medium of RPMI-1640 with cetacean embryos. Cells cultured with 0, 20, 40, 60, 80 and 100 μm concentrations of Sclareol treatment for 5 hours. The rate of expression of IRE-1 and PERK genes by quantitative real time -PCR and the level of proteins IRE-1 and PERK by western blotting method was investigated. Results: The rate of expression of IRE-1 in doses of 20, 40 and 60 μM Sclareol was significantly increased while decreasing in doses of 80 and 100 μM (p <0.0001). Also, the expression of PERK gene expression at doses of 20, 40 and 60 μM Sclareol was significantly increased, but no increase was observed in doses of 80 and 100 μM (p <0.0001). Also, the levels of IRE-1 and PERK proteins in doses of 20, 40 and 60 micromoles of Sclareol showed a high increase in doses of 80 and 100 μM. Conclusion: From the results of this study, it seems that doses between 20 and 60 μmol of can be Sclareol helpful in increasing the amount of reticuloendoplasmic stress, but doses higher than 60 milimoles do not have like this effect

A novel TNFRSF1A gene mutation in a patient with tumor necrosis factor receptor-associated periodic syndrome

© 2016 King Faisal Specialist Hospital & Research CentreTumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a periodic fever syndrome inherited in an autosomal dominant fashion. It stems from mutations in the TNFRSF1A (accession number: NM_001065) gene expressing the receptor for tumor necrosis factor α. A patient with TRAPS may present with prolonged episodes of fever attacks, abdominal pain, severe myalgia, and painful erythema on the trunk or extremities. Here, we report an 8-year-old boy with febrile attacks occurring every 1–2 months and continuing for 3–4 days. The patient experienced 40 °C-fever attacks without chills. Approximately 80% of fever attacks were accompanied by abdominal manifestations. Direct sequencing analysis was used to assess the genomic DNA of the patient, and a heterozygous R426L mutation in exon 10 of the TNFRSF1A gene in an autosomal dominant inheritance fashion was identified. Further genetic analyses were also carried out on his parents. Due to the fact that the mutation was not inherited from the parents, it was likely that R426L was a de novo and novel mutation in the TNFRSF1A gene, which can trigger TRAPS or TRAPS-like symptoms

Common Mutations of the Methylenetetrahydrofolate Reductase (MTHFR) Gene in Non-Syndromic Cleft Lips and Palates Children in North-West of Iran

Introduction: Cleft lips and cleft palates are common congenital abnormalities in children. Various chromosomal loci have been suggested to be responsible the development of these abnormalities. The present study was carried out to investigate the association between the suspected genes (methylenetetrahydrofolate reductase [MTHFR] A1298C and C677T) that might contribute into the etiology of these disorders through application of molecular methods.   Materials and Methods: This cross-sectional and explanatory study was carried out on a study population of 65 affected children, 130 respective parents and 50 healthy individuals between 2009 and 2012 at Tabriz University of Medical Sciences, IR Iran. After DNA extraction, amplification refractory mutation system–polymerase chain reaction (ARMS-PCR) and restriction fragment length polymorphism (RFLP)-PCR were used respectively to investigate the C677T and A1298C mutations for the MTHFR gene.   Results: There was a significant difference in the rates of the C677T mutation when affected patients and their fathers were compared with the control group (odds ratio [OR]=0.44) (OR=0.64). However, there was no significant difference observed in the rate of this mutation between the patients’ mothers and the control group (OR=1.35). In addition, the abnormality rate was higher in patients with the A1298C mutation and their parents, when compared with the control group. This abnormality rate was higher for the affected children and their fathers in comparison with their mothers (Fathers, OR=0.26; Mothers, OR=0.65; Children, OR=0.55). No significant difference was seen in the rate of the polymorphism C677T in its CC, when the affected children and their parents were compared with the control group. However, there was a significant difference in the A1298C mutation.   Conclusion:  An association was seen between the A1298C mutation and cleft lip and cleft palate abnormalities in Iran. However, there seems to be a stronger relationship between the C67TT mutation and these abnormalities in other countries, which could be explained by racial differences. Moreover, this association was more notable between the affected children and their fathers than their mothers. The findings in this study may be helpful in future studies and screening programs