Mitochondrial DNA G13708A variation and multiple sclerosis: Is there an association?

BACKGROUND: Multiple sclerosis (MS) is considered a pathogenetic enigma. Recently, efforts to implicate genetics in human susceptibility to MS have identified an important role of mitochondrial DNA (mtDNA). G13708A is a common mtDNA variation associated with MS in specific populations. This study tested the hypothesis that the mtDNA G13708A variation is associated with MS in an Iranian population. MATERIALS AND METHODS: Blood samples were collected from 100 MS patients and 100 unrelated healthy controls. DNA was extracted using a salting-out method, followed by polymerase chain reaction (PCR) amplification. For assessment of restriction fragment length polymorphism (RFLP), PCR products were restricted by restriction enzyme Mva I. Thereafter, the restriction products were assessed by means of an ultraviolet (UV) transilluminator following electrophoresis with 3% agarose gel. Accuracy of the genotyping procedure was assessed by direct sequencing. RESULTS: The mtDNA G13708A variation was found in 17 cases (17%) and 19 controls (19%) (P=0.7, OR: 0.8, 95% CI: 0.3-1.9). CONCLUSION: The findings of the present study fail to support the hypothesis that the G13708A mtDNA variation is associated with MS in the selected Iranian population.

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.

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.

MicroRNA-221: biogenesis, function and signatures in human cancers.

MicroRNAs are small non-coding RNAs of 18-25 nucleotides that regulate gene expression at the post-transcriptional level through binding to the 3´-UTR of mRNAs and block mRNA transcription or regulate its resistance. Increasing evidence indicates that dysregulation of miRNA is a hallmark of cancer. The miRNAs have an essential role in the regulation of oncogenes or tumor suppressor genes in cell signaling pathways. MiR-221 and miR-222 are two homologous microRNAs, the high expression levels of which have been commonly demonstrated in multiple human cancer types. The miR-221/miR-222 functions have been verified as oncogenes or tumor suppressors. Here, we reviewed the roles of miR-221/miR-222 in various kinds of cancer progression and development: controlling proliferative signaling pathways, avoiding cell deaths resulted from tumor suppressors, monitoring angiogenesis and even supporting epithelial-mesenchymal transition. We discussed that miR-221/miR-222 act as promising biomarkers for detection of human cancer types and suggested a new pathway for molecular targeted cancer therapy.

Comparison of gene-expression profiles in parallel bone marrow and peripheral blood samples in acute myeloid leukaemia by real-time polymerase chain reaction.

BACKGROUND: Gene signatures (Indicator genes) in bone marrow that provide more precise prognostication in haematological malignancy have been identified by microarray expression studies. It would be beneficial to measure these diagnostic signatures in peripheral blood. AIMS: To determine the degree of correspondence of gene expression for a set of Indicator genes between bone marrow and peripheral blood in acute myeloid leukaemia (AML). METHODS: Parallel bone marrow aspirate and peripheral blood samples were obtained from 19 patients diagnosed with AML and mononuclear cells isolated from both sample types. mRNA was globally amplified by polyadenylated real-time polymerase chain reaction (polyA RT-PCR); the expression of 15 AML Indicator genes, identified from previous microarray studies, was measured by RT-PCR. All values were normalised to the mean expression of three housekeeping genes (IF2-beta, GAP and RbS9) and were statistically compared using SPSS software. RESULTS: No significant difference in expression between bone marrow and peripheral blood was observed for 10 of the genes (leptin receptor, CD33, adipsin, proteoglycan 1, MB-1, cyclin D3, hSNF2b, proteasome iota, HkrT-1 and E2A), indicating its possible use in monitoring disease activity in peripheral blood samples, whereas c-myb, HOXA9, LYN, cystatin c and LTC4s showed significantly different expression between bone marrow and peripheral blood samples. CONCLUSION: These results indicate a possible use for the method in monitoring AML in peripheral blood by RT-PCR measurement of Indicator genes. In addition, the initial use of polyA PCR facilitates translation to very small clinical samples, including fractionated cell populations, of particular importance for monitoring haematological malignancy.

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.

Evaluation diagnostic usefulness of immunoglobulin light chains (Igκ, Igλ) and incomplete IGH D-J clonal gene rearrangements in patients with B-cell non-Hodgkin lymphomas using BIOMED-2 protocol.

PURPOSE: Evaluation diagnostic usefulness of immunoglobulin light chains (Igκ, Igλ) and incomplete IGH D-J clonal gene rearrangements in formalin-fixed, paraffin-embedded (FFPE) tissue of patients with B-cell non-Hodgkin lymphomas (B-NHL). MATERIALS AND METHODS: This study was performed on samples from 70 patients with B-NHL, including two cases of follicular lymphoma (FL), 20 cases of diffuse large B-cell lymphoma (DLBCL), one case of mantle cell lymphoma (MCL), and 47 cases of B-cell neoplasm (non-classified), which had been previously assessed for complete IGH clonality, and failure to clarify gene rearrangements. We used a gold standard multiplex PCR protocol provided by European Biomedicine and Health (BIOMED-2) Concerted Action Project BMH4-CT98-3936 for improvement of diagnosis and analysis of clonality gene rearrangement in lymphoma malignancies. RESULTS: Our results revealed a total positive monoclonality of 89 % (62/70) in Igκ, Igλ, and 11.4 % (8/70) polyclonality in gene rearrangements assay. The samples with positive clonality consisting (Igκ: 45 %, Igλ: 55 %) in DLBCL, (Igκ: 100 %) in FL, (Igλ: 100 %) in MCL, and (Igκ: 47 %, Igλ: 36 %) in B-cell neoplasm non-classified. None of the incomplete IGH D-J immunoglobulin gene families (0 %) showed monoclonality, and all samples demonstrated polyclonality pattern. CONCLUSIONS: Our findings on FFPE tissue revealed that immunoglobulin light chains clonality gene rearrangements assays using BIOMED-2 protocol, could be considered a valuable and reliable method for clonality detection, particularly in cases of failure of complete IGH gene rearrangements analysis. Clonal Ig gene rearrangements assay is applicable for routine diagnostic testing of lymphoproliferative disorders and as a reliable method for differentiating between malignant and benign lymphoma disorders.

Human papilloma virus in head and neck squamous cell cancer.

BACKGROUND: Epidemiologic and molecular evidences have established a strong link between high risk types of Human Papilloma Virus and a subgroup of Head and Neck Squamous Cell Carcinomas (HNSCC). We evaluated the frequency of HPV positivity in HNSCC and its relationship to demographic and some risk factor variables in an open case- control study. METHODS: Fourteen recently diagnosed patients with squamous cell cancer of oropharynx, hypopharynx and larynx aged 18-50 years were examined from 2008-2010 in Tabriz, Iran. HPV DNA was extracted from paraffin-embedded blocks of each patient's sample for PCR evaluation. Saliva samples of 94 control cancer-free subjects were collected for DNA analysis. Multivariable logistic regression method was used to calculate odds ratio for case-control comparisons. RESULTS: High risk HPV was detected in 6(42.8%) patients, and 6(5.3%) control subjects which was statistically significant (p<0.0001). HPV-18 was the most frequent type both in the cases and controls. HPV-16 DNA was detected in two patients of the case group, but it was not detected in any of the controls. The relation between demographic and risk factor variables was not statistically significant. CONCLUSION: HPV infection has a significant impact on HNSCC. Despite HPV-16 stronger impact, HPV-18 is more likely to cause malignant degeneration in such cancers amongst some communities. It is vital to introduce and conduct immunization schedules in health care systems to protect communities to some extent.

Gene expression analysis of myeloid and lymphoid lineage markers during mouse haematopoiesis.

Expression profiling of haematopoietic cells is hampered by the heterogeneous nature of haematopoietic tissues and the absolute rarity of early unrestricted progenitors. To overcome this, the expression profile of lymphoid and myeloid-associated genes (LEF1, EBF, CD19, Sox-4, B29, CD45, C-fms, lysozyme, PU.1 and CD5) were investigated in 40 mouse myeloid haematopoietic precursors covering the entire haematopoietic hierarchy from multipotential to committed single lineages. The lineage-specific expression seen in single-cell studies was confirmed by examining fractionated bone marrow, whole tissues and differentiation of the multipotent cell line FDCP (Factor Dependent Cell Paterson) mix. Analysis of the 40 single myeloid precursors failed to detect expression of lymphoid-associated genes, LEF1, EBF, CD19 and CD5, despite detection in lymphoid cell controls. Surprisingly, the lymphoid-associated genes, Sox-4 and B29 were detected in the single myeloid precursors, which was confirmed in bone marrow and a multipotential myeloid cell line. The pattern of Sox-4 and B29, is consistent with a potential role in the commitment of bipotential granulocytic/macrophage precursors towards the granulocyte or macrophage lineage. In addition to providing baseline values for myeloid and lymphoid lineage markers during mouse haematopoiesis, these results highlight the importance of single-cell analysis in the study of complex tissues.