Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of ß-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common ß-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and ß-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis.

Vitamin D receptor gene polymorphisms in Iranian Azary patients with Behçet's disease.

OBJECTIVES: The aim of our study was to investigate the association of four polymorphisms of the VDR gene (FokI, BsmI, TaqI, and ApaI) with their susceptibility to Behçet's disease (BD) and their clinical manifestations with respect to the Iranian Azari population. METHOD: In this cross-sectional study we considered the BsmI, FokI, ApaI, and TaqI polymorphisms in 50 Iranian Azary patients with BD and 50 healthy controls, with the use of polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). RESULTS: A significant difference was found for the FokI polymorphism between the case and control groups. The f allele frequency of 26% was present in BD patients, compared to only 13% in the control group. In addition, the f/f genotype was significantly associated with BD. We found no significant differences between the BD and control groups regarding the distribution of ApaI, BsmI, and TaqI genotype frequencies. We found no association between VDR polymorphisms and the clinical manifestations of BD. CONCLUSIONS: The VDR f allele and f/f genotype are associated with BD in the Iranian Azari population.

Mutation analysis of the CYP21A2 gene in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is an inherited autosomal recessive enzymatic disorder involving the synthesis of adrenal corticosteroids. 21-Hydroxylase deficiency (21-OHD) is the most common form of the disease which is observed in more than 90% of patients with CAH. Early identification of mutations in the genes involved in this disease is critical. A marker of the disease, errors in the CYP21A2 gene, is thought to be part of the pathophysiology of CAH. Therefore, the identification of gene mutations would be very beneficial in the early detection of CAH. This research was a descriptive epidemiological study conducted on individuals elected by the inclusion criteria whom were referred to the Genetic Diagnosis Center of Tabriz during 2012 to 2013. After sampling and DNA extraction, PCR for the detection of mutations in the CYP21A2 gene was performed followed by sequencing. For data analysis, the results of sequencing were compared with the reference gene by blast, Gene Runner and MEGA-5 software. Obtained changes were compared with NCBI databases. The analysis of the sequencing determined the mutations located in Exons 6, 7, 8 and 10. The most frequent findings were Q318X (53%) and R356W (28%). Exon 6 cluster (7%), E431k (4%), V237E (2%), V281L (2%), E351K (2%), R426C (2%) were also frequent in our patients. The most frequent genotype was compound heterozygote, Q318X/R356W. Three rare mutations in our study were E431K, E351K and R426C. Observed mutation frequencies in this study were much higher than those reported in previous studies in Iranian populations. Thus, it seems that it is necessary to follow-up screening programs and use sequencing methods to better identify mutations in the development of the disease.

MicroRNA profiling during germline differentiation of mouse embryonic stem cells.

MicroRNAs are new classes of small non—coding regulatory RNAs which control degradation or suppress translation of its target mRNAs by sequence complementarity. Mature microRNAs are enriched in embryonic stem cells and play important roles in controlling stem cell self—renewal as well as control of differentiation. There is significant evidence that microRNAs are involved in the regulation of stem cell differentiation. The male mouse Embryonic Stem Cell line C57BL6/J with normal karyotype 46, XY was used for profiling microRNA expression in undifferentiated mouse embryonic stem cells (mESCs) and mESCs which were differentiated to germ line cells to determine and compare differences in microRNA expression before and after differentiation. Also, testis tissue samples of a 5—day—old mouse and a mature mouse was used as in vivo control. Profiling was performed by quantitative real—time PCR using locked nucleic acid microRNA—specific LNATM—enhanced primers. After data analysis and comparison of results profiled microRNAs expression, three microRNAs, mmu—miR—21, mmu—miR—21* and mmu—miR—16 showed 50.31, 43.76 and 46.77—fold change increase of expression, respectively, in differentiated mESCs in comparison with undifferentiated state with significant p—value (Average p—value p<0.001 for each members of microRNAs). Expression of Let—7 microRNA family increased in differentiated state when compared with undifferentiated mESCs (Average p—value<0.0001 for each members of family). The levels of expression all other profiled microRNAs were significantly higher in undifferentiated in comparison with differentiated mESCs and their expression was down regulated after differentiation. (Average p—value <0.003 for each members of microRNAs).

siRNA-mediated silencing of MDR1 reverses the resistance to oxaliplatin in SW480/OxR colon cancer cells.

One of the most challenging aspects of colon cancer therapy is rapid acquisition of multidrug resistant phenotype. The multidrug resistance gene 1 (MDR1) product, p—glycoprotein (P—gp), pump out a variety of anticancer agents from the cell, giving rise to a general drug resistance against chemotherapeutic agents. The aim of this study was to investigate the effect of a specific MDR1 small interference RNA (siRNA) on sensitivity of oxaliplatin—resistant SW480 human colon cancer cell line (SW480/OxR) to the chemotherapeutic drug oxaliplatin. SW480 cells were made resistant by continuous incubation with stepwise serially increased concentrations of oxaliplatin over a 6—months period. Resistance cell were subsequently transfected with specific MDR1 siRNA. Relative MDR1 mRNA expression was measured by Quantitative real—time PCR. Western blot analysis was performed to determine the protein levels of P—gp. The cytotoxic effects of oxaliplatin and MDR1 siRNA, alone and in combination were assessed using MTT and the number of apoptotic cells was determined with the TUNEL assay. MDR1 siRNA effectively reduced MDR1 expression in both mRNA and protein levels. MDR1 down—regulation synergistically increased the cytotoxic effects of oxaliplatin and spontaneous apoptosis SW480/OxR. Our data demonstrates that RNA interference could down regulate MDR1 gene expression and reduce the P—gp level, and partially reverse the drug resistance in SW480/OxR cells in vitro. Therefore, the results could suggest that MDR1 silencing may be a potent adjuvant in human colon chemotherapy.

Detection of immunoglobulin IGH gene rearrangements on formalin-fixed, paraffin embedded tissue in lymphoid malignancies.

Human lymphomas are aggressive malignant diseases, which can be categorized based on their B and T cell lineage. B-cell lymphomas form around 90% of the total lymphoma cases, the remnants of malignancies arise from the T cell branch. Lymphomas are mostly characterized as clonal proliferations of specific tumor cells. The detection of malignant lymphomas are extensively investigated by their morphological features, immunohistochemistry and flowcytometric immunophenotyping, but in some of cases remained unknown. The BIOMED-2 protocols were used to determine the clonality of IGH gene rearrangements in patients with lymphoma. PCR amplification was performed on FFPE of 50 patients with B-cell lymphoma, which consisted of 11 cases with HLs, 25 cases of B-NHLs and 14 cases of B-LPD (lymphoproliferative disorders) that diagnosed as unclassifiable lymphoma. The rate of positive clonality was detected in 96% (24/25) of B-NHLs, whereas in 4% (1/25) of cases clonality was showed in a polyclonal pattern. In B-HLs, 82% (9/11) of cases showed clonality and 18% (2/11) of the cases showed polyclonality. The rate of positive clonality observed in 64.3% (9/14) of cases with B-LPD and 35.7% (5/14) of cases clonality was not detected in any of immunoglobulin gene family (FR1, FR2, FR3). In groups with DLBCL, clonality was detected in 95% (19/20) of the cases. In patients diagnosed with FL and MALTs 100% cases showed clonality for complete IGH. Our study revealed that EuroClonality BIOMED-2 protocols could be considered as a valuable and reliable method for clonality detection, especially in IGH analysis.

MYCN gene amplification in patients with neuroblastic tumors.

Although neuroblastic tumors are the most prevalent solid tumors, little is known about the genetic basis underlying their progression. The prognostic role for the MYCN gene in neuroblastic tumors is irrefutable. The aim of this study is to identify the frequency of MYCN gene amplification and its relationship with clinicopathological and prognostic factors in 40 patients with neuroblastic tumors by using real-time quantitative PCR. There was significant association between the age of older than 18 months and the high number of metastasis. 83.3% of metastatic neuroblastic tumors in patients aged more than 18 months were in stage 4, while it was about 12.5% in patients aged less than 18 months. We found an amplification of MYCN in 19 out of 40 patients. Also, we found MYCN gene amplification in 64% of neuroblastoma (NB) and 8% of gangelioneuroblastoma (GNB) cases. There was a significant association between the histological type of samples with MYCN gene amplification. Neuroblastic tumors have a varied range of MYCN gene amplification depend on histopathology types. No significant associations have been found between MYCN gene amplification and tumor evaluation, CNS involvement, metastasis, stage of disease and patients outcome.

Expression profiling of microarray gene signatures in acute and chronic myeloid leukaemia in human bone marrow.

BACKGROUND: Classification of cancer subtypes by means of microarray signatures is becoming increasingly difficult to ignore as a potential to transform pathological diagnosis; nonetheless, measurement of Indicator genes in routine practice appears to be arduous. In a preceding published study, we utilized real-time PCR measurement of Indicator genes in acute lymphoid leukaemia (ALL) and acute myeloid leukaemia (AML) as a way of application of microarray gene signatures. More to the point, the specificity of such genes for this distinction was investigated by their measurement in cases afflicted with chronic myeloid leukaemia (CML) and with normal bone marrow (BM). MATERIAL AND METHOD: Mononuclear cells were sorted into unselected (total), CD34+ve, and CD34-ve fractions, mRNA globally amplified by using PolyA PCR. Moreover, the level of expression of 17 Indicator genes was identified by using real-time PCR. RESULTS: No statistically significant difference was observed in expression for any gene among CML cases. Cyclin D3 (p≤0.04) was exclusively upregulated in CML in the CD34+ fraction, notwithstanding upregulation of HkrT-1 (p≤0.02) and fumarylacetoacetate (p≤0.03) in AML. HOXA9 experienced a non-significant upregulation in AML; however, in combination with proteoglycan 1 distinguished between AML and normal samples in the CD34- fraction in unsupervised clustering. Unsupervised clustering distinguished among AML and the other diagnostic groups. CONCLUSION: The evidence from the present study suggests that the genes discriminatory between ALL and AML are uninformative in the context of CML and normal BM, excepting for distinction with AML.