Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients

BACKGROUND: Studying whole blood DNA methylation as a risk marker has valuable applications in either diagnosis or staging of breast cancer. We investigated whole blood DNA methylation status of VIM, CXCR4, DOK7, and SPDEF genes in breast cancer patients in comparison to healthy control subjects. MATERIALS AND METHODS: 60 patients with breast cancer and 40 healthy controls were examined. Genomic DNA isolated from peripheral blood and restriction enzyme polymerase chain reaction (REP) method was applied for analysis. Real-time PCR was used to confirm methylation status of the aforementioned genes and therefore to find out the methylation differences between normal and breast cancer subjects. RESULTS: Level of DOK7 promoter hypomethylation in normal and breast cancer samples was significant (P-value = 0.001). The study, also, showed that hypomethylation of VIM and CXCR4 genes are significant in patients compared with normal cases (P-value < 0.05). Furthermore, SPDEF promoter hypomethylation was not significantly differed between normal and breast cancer samples (P-value = 0.2). CONCLUSIONS: Hypermethylation of DOK7 gene in DNA from patients affected with breast cancer offers a biomarker for diagnosis of the breast cancer. This study indicates that methylation status of VIM and CXCR4 genes changes in breast cancer; so, they can be used as molecular biomarkers in breast cancer prognosis.

Down-regulatory effects of mir-211 on long non-coding rna sox2ot and sox2 genes in esophageal squamous cell carcinoma

Objective: MicroRNAs [miRNAs] are a class of non-coding RNAs [ncRNAs] that tran-scriptionally or post-transcriptionally regulate gene expression through degradation of their mRNA targets and/or translational suppression. However, there are a few reports on miRNA-mediated expression regulation of long ncRNAs [lncRNAs]. We have previ-ously reported a significant upregulation of the lncRNA SOX2OT and its intronic coding gene, SOX2, in esophageal squamous cell carcinoma [ESCC] tissue samples. In this study, we aimed to evaluate the effect of induced overexpression of miR-211 on SOX2OT and SOX2 expression in vitro

Materials and Methods: In this experimental study, we performed both bioinformatic and experimental analyses to examine whether these transcripts are regulated by miRNAs. From the list of potential candidate miRNAs, miR-211 was found to have complementary sequences to SOX2OT and SOX2 transcripts. To validate our finding experimentally, we transfected the NT-2 pluripotent cell line [an embryonal carcinoma stem cell] with an expression vector overexpressing miR-211. The expression changes of miR-211, SOX2OT, and SOX2 were then quantified by a real-time polymerase chain reaction [RT-PCR] approach

Results: Compared with mock-transfected cells, overexpression of miR-211 caused a significant down-regulation of both genes [P<0.05]. Furthermore, flow-cytometry analysis revealed a significant elevation in sub-G1 cell population following ectopic expression of miR-211 in NT-2 cells

Conclusion: We report here, for the first time, the down-regulation of SOX2OT and SOX2 genes by an miRNA. Considering the vital role of SOX2OT and SOX2 genes in pluripotency and tumorigenesis, our data suggest an important and inhibitory role for miR-211 in the aforementioned processes

Association of the MTHFR C677T polymorphism and fragile X syndrome in an Iranian population

Background & Objective: Fragile X syndrome is one of the most common causes of inherited mental retardation in males after Down syndrome. To date less attention was to study secondary genetic factor that may play role in fragile X neuropathology. In central nervous system, folic acid derivatives participate in different process such as neural development and function, synthesis of neurotransmitters and DNA methylation. This study aimed to assess the possible association of three common polymorphisms of folate pathway key enzymes, methylentetra hydrofolate reductase, MTHFR (C677T and A1298C), and methionine synthase reductase, MTRR (A66G), polymorphisms with the development of fragile X syndrome. Methods: Genetic polymorphisms were examined in a case-control study of 38 unrelated male patients and 60 age/sex matched unrelated controls using PCR-RFLP method. Allele frequencies and genotypes were calculated by chi-square test. Results: signifi cantly increased frequencies of the 677T allele and the 677CT genotype in patients were observed compared to control (p=0.010; OR=2.459 for T allele frequency; p=0.028; OR=2.608 for CT genotype frequency). The statistical analysis demonstrated the signifi cant correlation between C677T MTHFR polymorphism and fragile X syndrome in Iranian population (p=0.018). However, no signifi cant case-control differences were found for A1298C MTHFR and A66G MTRR polymorphisms. Conclusions: The association between C677T MTHFR polymorphism and fragile X syndrome has been demonstrated for the fi rst time in Iran population. This study may be important in better understanding of molecular pathology of fragile X syndrome. Further studies need to be undertaken to evaluate these preliminary results in other populations.

Evaluation of two DNA extraction methods from maternal plasma for using in non-invasive bovine fetus gender determination

Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8[th] week of pregnancy, by maternal blood sample testing. The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Maternal blood samples were taken from 40 pregnant cows during the 8[th]-38[th] weeks of gestation. DNA was extracted from 350 micro l of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A[260] and purity [A[260]/A[280]] of extracted DNA were detected by ultraviolet spectrophotometer. Three micro l of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 [p>0.05, p=0.3549], but the difference between mean purity [A260/A280] of DNA extracted by phenol-chloroform method and salting-out method was significant [p<0.001]. X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma

low-cost efficient multiplex PCR for prenatal sex determination in bovine fetus using free fetal DNA in maternal plasma

In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction [PCR] analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 micro l maternal plasma. Two primer pairs for bovine amelogenin gene [bAML] and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses

Association study of six SNP[s] in PRM1, PRM2 and TNP2 genes in Iranian infertile men with idiopathic azoospermia

Histones are replaced by protamines to condensate and package DNA into the sperm head during mammalian spermatogenesis. Protamine genes defects have been reported to cause sperm DNA damage and male infertility. In this study relationship among some protamines genes family SNPs include PRM1 [C321A], PRM2 [C248T] and TNP2 [T1019C], [G1272C], [G del in 1036 and 1046 bp] were studied in 96 idiopathic infertile men with azoospermia or oligospermia and 100 normal control men. Analysis of SNPs was performed using restriction fragment length polymorphism [PCR-RFLP], single strand conformational polymorphism [PCR-SSCP] and PCR sequencing. No polymorphisms were found for tested SNPs except for PRM1 [C321A] and TNP2 [G1272C] in which frequency of altered AA and GG genotypes were slightly higher in infertile case group. Statistical analysis showed no significant association related to PRM1 [C321A] p=0.805 and TNP2 [G1272C] loci p=0.654. These results are consistent with previous studies and indicating that all tested SNPs was not associated with oligospermia and azospermia and idiopatic male infertility in Iranian population

Heme oxygenase-2 gene mutations and blood bilirubin level in Iranian patients with premature atherosclerosis

Heme oxygenase-2 [HO-2] is a critical antioxidative stress enzyme found in endothelial cells and adventitial nerves. This enzyme in conjunction with other HOs [1 and 3] metabolize heme molecule into ferrous iron, carbon monoxide [CO], and biliverdin which is further converted to bilirubin. Both biliverdin and bilirubin are potent antioxidants, reducing the risk of atherosclerosis. HO-2 also induces endothelial relaxation by synthesizing CO. This is the first study to evaluate the association of HO-2 gene mutation in patients affected with atherosclerosis. Blood samples from patients [n=137] and normal controls [n=100] were collected. Three pairs of primers were designed to amplify exons 2 to 4 related to human HO-2 gene. The PCR products were analyzed by SSCP and sequencing to find out mutations. Iron and bilirubins [Total, Direct and Indirect] levels were determined in patients and controls. Two nucleotide substitutions were found among 10% of patients, consisted of a newly reported transversion mutation, C to A substitution in codon A70D [GCC to GAC] [Ala to Asp] and a previously reported transition mutation, A to G substitution in codon K89E [AAG to GAG] [Leu to Glu]. Significant associations were obtained between risk of atherosclerosis and A437G substitution in codon K89E of HO-2 gene [P<0.006 and X[2] >6.82] and reduced level of total [P<0.016 and X [2] >6.01], and indirect [P< 0.016 and X [2] >5.99] bilirubins with no significant association with serum iron and direct bilirubin. No significant associations were observed among C381A substitution in codon [A70D, P< 0.11 and ?[2] >2.97], level of serum iron, bilirubin and risk of atherosclerosis. These findings indicate the importance of A437G substitution in the development of atherosclerosis. Further studies are required to study the association of HO-2 gene mutations with atherosclerosis in other populations

Use of D11S2179 and D11S1343 markers as informative markers for prenatal diagnosis in Iranian ataxia-telangiectasia patients

Ataxia-telangiectasia is an autosomal recessive disorder affecting 1/40000 to 1/100000 of reported populations. There is 25% possibility for having an affected child when parents are carriers for ATM gene mutation. There is no cure available for this disease and prenatal testing is strongly recommended to prevent this disease. Although preferred method is the direct mutation analysis of ATM gene, but large size of the ATM gene with 63 exons and the large number of possible mutations in patients considerably limit efficiency of mutation analysis as a choice in diagnosis. Indirect method is a better tool when parents are not carriers of founder mutation and pass different mutations to their children. Indirect molecular diagnosis using ATM related molecular markers facilitates prenatal diagnosis of AT children. In this study, four molecular markers: D11S2179, D11S1787, D11S535, D11S1343 are genotypes in 12 unrelated families [19 patients] from different regions of Iran. Those markers are amplified using extracted sequence primers from Gene Bank with their described PCR conditions. The amplified products were separated using denaturing PAGE gels, and the data were analyzed to detect their pattern of inheritance in each family. In all families, segregation of alleles were according to mandelian inheritance and affected chromosomes were distinguishable from unaffected ones. All carriers and affected patients were diagnosed accurately. Thus, this method is effectively usable in prenatal diagnosis of ataxia telangiectasia

Methylenetetrahydrofolate Reductase [MTHFR] Gene C677T Polymorphism Is Associated with Coronary Atherosclerosis Disease in a Sample of Iranian Patients

Several studies showed that elevated plasma homocysteine level is a risk factor for coronary artery disease [CAD]. A common polymorphism C677T of methylenetetrahydrofolate reductase [MTHFR] gene is reported to be associated with decreased enzyme activity and increased blood homocysteine level. This study evaluated the association between C677T polymorphism and blood homocysteine level with CAD in 100 patients compared to 100 normal controls. Higher prevalence of the C677T polymorphism as well as elevated level in blood homosysteine were observed in Iranian CAD cases compared to the normal control. The C677T MTHFR common polymorphism was significantly associated with CAD, supported by a P value 0.032 and Chi-square equal to 6.87. The TT genotype of MTHFR gene was attributed to increased blood homocysteine level in patients compared to T/C and C/C genotypes in studied Iranian cases. This study shows the advantage of testing C677T polymorphism in affected patients as a risk factor for coronary artery disease

ATM gene mutations detection in Iranian Ataxia-Telangiectasia patients

Ataxia-Telangiectasia [AT] is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition. The ATM gene on human chromosome 11q22.3 has recently been identified as the gene responsible for ataxia-telangiectasia [AT]. The gene mutated in AT, which has been designated as the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. More than 100 mutations are broadly distributed throughout the ATM gene. The large size of the ATM gene [66 exons spanning approximate 150kb of genomic DNA] together with the diversity and broad distribution of mutations in AT patients, greatly limits the utility of direct mutation screening as a diagnostic tool. In this study, 20 families with at least one affected child clinically suspected to have ataxia-telagiectasia were examined and their DNA was extracted and amplified with standard methods. Sequencing methods were used to detect the new point mutation. Four exons which were hot spots for point mutations in ATM gene were detected by PCR-SSCP or PCR-RFLP