Sequence variants of BRCA1 and BRCA2 genes in four Iranian families with breast and ovarian cancer

BRCA1 and BRCA2 genes have been recognized to be responsible for 20-30% of hereditary breast cancers and approximately 50% of familial breast and ovarian cancers. Therefore, the demand for BRCA1 and BRCA2 mutation screening is rapidly increasing as their identification will affect medical management of people at increased risk. Because of high costs involved in analysis of BRCA1 and 2 genes, contribution of different mutation types in BRCA1 and 2 and not knowing who should be tested has hampered wide spread use of molecular testing of high -risk families. There is a need to identify the genes and types of mutations involved in breast or ovarian cancers at different age of onsets and polymorphism and polymorphic variations in our population. Twenty-seven patients with either early onset breast cancer [at age

[Allele frequency of 12 microsatellites in chromosome 8, 11, 12 and 16 in Tehran lipid and glucose study]

Most linkage and population genetic studies that use microsatellites assume that the polymorphism observed at these loci is due simply to variation in the number of units of a single repeat. These variations guide us in the study of the genetic patterns of some disease. This study reports the allele frequency of 12 microsatellites in Tehranians. Five hundred and ninety-one subjects with an average age of 39 +/- 19 years, with and without metabolic syndrome, were selected for investigation of the allele frequency of 12 microsatellites, the D8S1132, D8S1779, D8S514, D8S1743, D11S1998, D11S1304, D11S934, D12S96, D12S1632, D12S329, D16S2624 and D16S3096 using and fragment analysis technique. There are different repeats in the chromosomes studied. Chromosome 8: 10-26.2bp repeats; chromosome 11: 8.1-35bp; chromosome 12: 2-32bp and chromosome 16: 9.2-28bp. The most hetrozygote marker is D8S1132 and the least is D12S96. The most significant findings of this study is reporting allele frequency of some Short Tandem Repeats for the first time in Tehran. In this study some new alleles were found in Iranian subjects, the presence of which may be a tool for genetic association studies in the future

Relationship between DNA polymorphisms at the BCL11A and HBS1L-MYB loci in beta-Thalassemia patients with increased fetal hemoglobin levels

High fetal hemoglobin [HbF] levels have a major impact on the hemoglobin disorders, i.e. beta -Thalassemia. Increased HbF production ameliorates the disease severity. Three loci-HBS1L-MYB intergenic region on chromosome 6q23, BCL11A on chromosome 2pl6, and the chi-globin gene on chromosome 11 account for up to 50% of the variations in HbF levels in patients with sickle cell anemia, thalassemia and healthy adults. In the present study, we evaluated the relationship between some polymorphisms on HBS1L-MYB BCL11A loci and increased HbF levels in thalassemia patients and normal subjects. In this case-control study, three common polymorphisms among 50 beta-thalassemia patients with increased HbF and 47 healthy individuals with normal HbF by using PCR-RFLP were genotyped: rs4895441, rsl 1886868, and rs28384513. Enzymatic digestion was performed by Rsal, MboII, and BstXI, respectively. Correlations with high levels of HbF were performed with a Chi-square test by using SPSS 16 and SNP analyzer2. Mutant allelic frequencies were 0.245, 0.521 and 0.309 in healthy and 0.3, 0.52 and 0.28 in patient for rs4895441, rsl 1886868 and rs28384513, respectively. Significant relationship was not observed among three polymorphisms studied in healthy volunteers and beta-Thalassemia major patients with increased HbF levels and P-value allelic and genotypic was higher than 0.05 at three SNPs. In spite of previous reports, evaluation of polymorphisms at the BCL11A and HBS1L-MYB loci in this study did not show up a significant correlation with increased HbF. Other polymorphisms might have a role in increasing HbF in our population

study of relationships between beta-globin gene mutations in beta-thalassaemia patients, in response to hydroxyurea treatment

Beta-thalassaemia is the most frequent inherited disorder in the world, especially in Iran and Mazandaran Province. It is caused by mulation in beta-globin gene on chromosome 11 with more than 150 different mulations causing beta-thalassaemia, has been identified in the beta-globin gene to date. Hydroxyurea, is one of the drugs used in Thalassemia patient's treatment, however, it is not effective in all patients. The mechanisms of the hydroxyuea effect in not clear yet. This study compared different beta-globin gene mutations in beta-thalassaemia patients who were referred to the Thalassemia Research Center in Sari in two groups, good responder and non-responder, to the hydroxyurea. This was a case-control study, comparing two groups of 30 thalassaemic patients who received hydroxyurea. Two groups were included, 30 good responders to hydroxyurea treatment [control] and 30 who did not respond to the treatment [case]. First, DNA was extracted from peripheral blood. Then, two different methods for mutation detection were used. In the Thalassaemia Research Center in Sari, mutations in 60 patients were identified using ARMS-PCR. Also the results were confirmed in Genetic laboratory of Amirkola, using two mutation detection methods, reverse-dot blot hybridization and ARMS-PCR. In the group of good responder [control], the average patient's age were 28/1 +/- 7/78 years, and the average age at the onset of blood transfusion was reported to be 8/5 +/- 8/56 year. In this group, the mean comparison of the hemoglobin level and red blood size [MCV] prior and after drug consumption were statistically significant. In the group of non-responder [case], the mean age was 21.3 +/- 6.43, the mean age starting blood transfusions was 3.3 +/- 3.75, and the mean of drug consumption was 2.3 +/- 0.8 months. From the mutations identified, IVSII-1G>A was the most common type in both case and control group, while of 30 of control group, 22 individuals were homozygous, and 7 individuals were heterozygous for this mutation [frequency% 42.5]. For the 30 case patients, 11 individuals were homozygous, while 11 were heterozygous [frequency% 27.5]. Comparison between two groups, case and control group, were statistically significance [P < 0.008]. The correlations of IVSII-1G>A mutation in good responder patients to hydroxyurea as compared to the non responder group, is significant and similar to the previous findings

Characterization of beta globin gene mutations in Zanjan province: An introduction to prenatal diagnosis of Thalassemia

B-thalassemia is an autosomal recessive disease characterized by reduction or complete absence of b-globin gene expression. It has been estimated that more than 2,000,000 carriers as well as 20,000 patients affected with b-thalassemia are living in Iran, a country with more than 70 million population and great ethnic diversity. In this study we aimed to find out the b-globin gene frequency and determine the spectrum of b-globin gene mutations in Zanjan province [northwest region] of Iran. 5527 individuals who were referred for pre-marriage tests to Zanjan clinic as well as 27 thalassemia patients were studied. Altogether one hundred and five chromosomes from 78 unrelated Bthalassemia patients or carriers were examined for b-globin gene mutations by ARMS-PCR and direct gene sequencing. Based on the previous information on common mutations in Mediterranean populations 24 sites were analyzed. It was found that the b-thalassemia frequency is 1.2 for Zanjan region. Using the above techniques, the mutations for 90/105 [86.7] of b-thalassemia chromosomes [13 different mutations] were identified. Fifty eight percent of the mutations were of common .Mediterranean. type. Of which, IVS-I 110 mutation showed the highest frequency [29.5] followed by IVS-II-1 [13.3], IVS-I-1 [12.4] and IVS-I-6 [2.9]. 10.5% of mutations were of common Asian Indian mutations [Fr 8/9, 6.7% and IVS-I-5, 3.8] respectively. CD5 and CD30 and CD36-37 mutations accounted for 13.3% of the mutations. [5.7%, 0.95% and 6.7% respectively] Mutations in 14 chromosomes [13.3] remained uncharacterized. These data suggests that the spectrum of mutations in Zanjan province differs from those reported from other parts of Iran, but Mediterranean type of mutations are more frequent in Zanjan region. Therefore, in order to save the time and cost, it is recommended that for prenatal diagnosis of thalassemia in Zanjan province analysis of Mediterranean mutations should be considered as a front line screening strategy

Cloning of protective antigen gene from Bacillus anthracis into Bacillus subtilis

Protective antigen [PA] of Bacillus anthracis is used as anthrax vaccine. Cloning and expression of the PA gene in various strain such as E.coli and Bacillus subtilis was reported that most expression was in B. subtilis up to 160 microg/ml. The objectives of this study were: to clone the gene of PA in an expression vector [pWB980] and then transformation into the B. subtilis WB600 strain. The pXOl plasmid was separated from the strain stern of B. anthracis with alcalin method and the PA gene with 2.4kb sequence amplified by PCR. Then the amplified fragment was directly cloned into pTZ57R plasmid as T-vector and transferred into E.coli DH5 alpha using CaC12 method. After that the gene was separated from the T-vector by enzymatic digestion [Sa1I and KpnI]. Ligation between the purified gene fragment of the PA and the vector was carried out. Then it was transferred into B. subtilis WB600 by electroporation method in 1000 V. In this study we isolated PA gene from B. anthracis strain stern with PCR and was cloned into pTZ57R plasmid. The Presence of the gene was confirmed by restriction analysis, PCR and sequencing. Then the PA gene was cloned into pWB980 and B. subtilis and the presence of the gene in two kanamycin resistant colonies [AMN1 and AMN3] was confirmed by restriction analysis and PCR. We may conclude that by making modification in the methods used and using pWB980 expression vector, we were able to clone the PA gene into B. subtilis. This is the first research project in Iran that the PA gene is isolated and cloned and B. subtilis is used as host

Development of transgenic mice harboring ovine beta lactoglobulin-calcitonin transgene

Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. We have attempted in this project to develop transgenic mice harboring a transgene driving mammary gland expression of hybrid human salmon calcitonin. A milk-specific ovine beta-lactoglobulin [oBLG] promoter was used to drive expression of recombinant calcitonin in mouse milk. A gene construct was generated, consisting of 10.7kbp of the oBLG gene including its promoter and 3 flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. The gene construct was purified using CsCl gradient, released from vector, and gel-purified. After appropriate dilution, it was microinjected into recently-fertilized mouse oocytes. These oocytes then were transferred to pseudo-pregnant foster mice. Forty one pups were born from foster mice, which were genotyped using PCR, slot blotting, and Southern blotting. Among 9 mice which showed positive PCR results, 6 mice resulted in pups with positive PCR tests. All six families transmitted the transgene to first and second generation. As the main criteria for considering a mouse as transgenic is transgene transmission to the next generation, all 6 mice which stably transmitted their transgene to progeny are considered as transgenic founders and constitute independent transgenic lines

Effect of different glucose concentrations on PC12 cells apoptosis: role of bax and Bcl[2] proteins

In diabetics, hyperglycemia is associated with neuropathy, nephropathy, and retinopathy. However, direct toxic effects of glucose on neurons are still largely unknown. Firstly, cellular vital capacity was determined by MTT. Then, effects of different glucose concentrations and the role of Bax protein-induced apoptosis in PC12 cells was examined by Hoechst staining and western blotting techniques, respectively. During MTT, cells revealed to have a meaningful apoptosis at hours 48, 72, and 96 when compared with controls [p<0.01]. Apoptosis was induced suitably at a glucose concentration of 13.5mg/dl after 72 hours. Western blotting showed a significant expression of Bax protein in PC12 cells treated with increased glucose concentrations for 72 hours. Increment in glucose concentration may induce apoptosis in PC12 cells. This process occurred under the intense influence of Bax protein