[Evaluation of the two variants of p63 [d4TAp63 and delta Np73L] expression as molecular markers in the diagnosis and prognosis of breast cancer]

The TP63 gene, as the oldest homologue of TP53, encodes two main N-terminal variants by different promoters: the trans-activating variant, TAp63 with tumor suppressor activity and the amino terminal truncated variant, delta Np63 with oncogenic activity. As the result of the deletion of exon 4 in each of the variants, two further N-terminal variants have been reported for p63 in the last decade: d4TAp63 and delta Np73L, but the exact function of these variants have not been determined in normal and tumoral cells. In this study we evaluated the expression pattern of p63 variants and usefulness of d4TAp63 and delta Np73L as potential diagnostic molecular markers in breast cancer. In this study 30 tumoral and 20 non tumoral samples of marginal tissues were studied by use of Semi-quantitative Reverse Transcriptase-nested PCR [RT-nPCR] method. SPSS16 software was used for data analysis. In all cases with expression of TAp63 and delta Np63 variants, d4TAp63 and delta Np73L variants were always expressed with their long variants simultaneously. In tumoral and marginal samples delta Np73L mean expression level was significantly higher than the mean expression level of d4TAP63 [P = 0.009 and P=0.008 respectively]. delta Np63 expression levels in tumoral and marginal samples were also higher than those of TAp63, which had a statistically significant difference in tumoral samples [P=0.03], but no significant difference was detected in marginal samples [P = 0.11]. The results indicated dominant negative inhibitory activity of delta Np63 and its potential role as an oncogene in breast cancer. delta Np73L variant compared to d4TAp63, in tumoral and non tumoral breast tissues can act as dominant negative variant. Both variants [delta Np73L and d4TAp63] with similar expression patterns to their respective longer variants [delta Np63 and TAp63] and simultaneous expression with their variants are likely to be involved in strengthening the tumor suppressor activity in normal and tumoral breast cells. On the other hand, according to the expression patterns of delta Np73L and d4TAp63 variants, they cannot be considered as molecular markers in the diagnosis of breast tumors

[Detection of P53 gene exons 5 and 6 mutations among east azerbaijani women with breast cancer]

Breast cancer [BC] is the most common invasive malignancy affecting women worldwide. The tumor-suppressor P53 gene [P53] is frequently mutated in breast tumors. To use P53 as a target for therapy, it is important to accurately assess p53 mutation status in tumor samples. A total of 102 tumor samples were collected from breast cancer patients referred to Tabriz hospitals between the 2007-2009 period. DNA was extracted by Proteinase K- Isopropanol method and then performed amplification and sequencing of P53 from exons 5 and 6. Mutations in the P53 gene were detected in 17.6% of the patients. Including 7 polymorphisms [6.68%] and 11 mutations [10.78%]. Overall, 18.2% of the mutations were found in codons 160 [ATG>AAG] and 163 [ATC>AAG] in exon 5. Also 81.8% of the mutations observed in exon 6: codon 193[CAT>AAT], codon195 [ATC>TTC], codon 195 [ATC>AAC], codon 198[GAA>TAA], codon 220 [TAT>TGT], codon 213 [CGA>CTA], and codon 214 [CAT>CG]. No alteration observed in intron5 and all of polymorphism detected in 13399A>G nucleotide of exon 6. The majority of detected mutations are missense that located on DNA-binding domain of P53. This type of mutation usually leads to the production of a mutant protein with a compromised structure and altered DNA-binding capacity. This is the first report of its kind from the East Azarbaijan region. Our results indicate a rather high frequency of exon 6 mutations in P53 among patients with breast cancer. Furthermore, the mutation pattern appears differs from other regions. However, further studies are needed to determine the role of P53 mutations in breast cancer development

[Analysis of beta thalassemia mutations using the single strand conformation polymorphism [SSCP] technique]

beta-thalassemia [beta-thal] is one of the most prevalent hereditary diseases in Iran. There are more than two million carriers of beta-thal in Iran. Detection of the beta globin gene mutations is necessary for a definitive diagnostic and management plan such as prenatal diagnosis of beta-thalassemia. In our country, the PCR-Amplification Refractory Mutation System [PCR-ARMS] has been frequently used for detection of beta globin gene mutations. Here, we used the PCR-single strand conformation polymorphism [PCR-SSCP] assay for detection of mutations of beta globin gene. In the patients with confirmed mutations, we amplified 281base pairs containing exon of one of a beta globin gene by PCR. Based on SSCP technique 2.5 micro l of the reaction products appeared in polyacryamide gel electrophoresis and the bands were visualized by silver staining. Seven mutations and one polymorphism were evaluated by PCR SSCP assay. The results of this study demonstrated that the patterns of mobility of single strands were different from each other and those of control sample. Our study showed the PCR-SSCP technique can meet the need for direct genomic sequencing of DNA and could be applied in the developing countries where financial resources are limited but genetic hemoglobin disorders are highly prevalent

[Detection of beta-thalassemia mutations in Kurd patients of Kurdistan and West Azerbaijan provinces]

Beta-thalassemia is the most common autosomal recessive disorder. More than 200 different mutations in the beta-globin gene have been detected which can lead to decreased or absent beta-globin chain synthesis. Since the Iranian Population is a mixture of different ethnic groups, it is necessary to determine the frequency and distribution of these mutations in the different ethnic groups of our county. Therefore, in this study we determined the Spectrum and the frequency of beta-thalassemia mutations in the patients with beta-thalassemia major in the Kurd population of Kurdistan and West Azerbaijan provinces of Iran. To detect mutations, extracted DNA of 110 chromosomes from 55 unrelated patients, were studied by PCR-ARMS [Polymerase Chain Reaction-Amplification Refractory Mutation System] SSCP [Single Strand Conformation Polymorphism] and direct sequencing methods. The results of this study showed that IVS-II-1 [G-A] was the most common mutation with a frequency of 31%; FSC 8/9[+G] with a frequency of 19% was the second most prevalent mutation among all chromosomes. Other mutations were IVS-I-1[G-A] FSC8 [-AA] IVS-I-110[G-A] FSC36/37[-T] IVS-I-5[G-C], IVS-I-128[T-G] FSC44 [-C], FSC 5[-CT] and +22UTR [G-A] These mutations comprised 79% of beta-thalassemia mutations in this region and 21% of the mutations still remains to be explored. The results of this study showed that, there are similarities and differences between this region and other parts of Iran and also neighboring countries. Therefore, determination of beta-thalassemia mutations in this region seems to be necessary and beneficial for designing prenatal diagnosis programs

[Contamination of drinking water with arsenic and its various health effect in the village of Ghopuz]

Arsenicosis is a serious environmental disease caused by chronic exposure to arsenic- usually from drinking water. Signs and symptoms of chronic arsenic poisoning include hyperkeratosis, hyper- or hypopigmentation, and ulcers. Also, the incidence of cancer is increased in the exposed population. There is some evidence of high arsenic levels in drinking water in the village of Ghopuz, located in Hashtrud District, East Azerbaijan province. We evaluated the genetic and health effects of chronic arsenic exposure in the residents of Ghopuz. In this cross-sectional study we determined the prevalence of hyperkeratosis, hyperpigmentation and hypertension in Ghopuz village. The study involved 101 individuals in Ghopuz and 107 in the adjacent village of Mayan, who were all visited by a trained physician. A total of 46 blood samples were collected for kariotyping. The level of heavy metals in water was determined by the Inductively Coupled Plasma [ICP] method. We detected high arsenic levels in the drinking water at Ghopuz [mean concentration in water = 1.03 mg/L]. There were chromosomal defects in the exposed group. Mean systolic blood pressure at Ghopuz [137mmHg, 95% CI: 132-142] was significantly higher than in Mayan [107, 95% CI: 99.9-114]. Also, mean diastolic blood pressure at Ghopuz [82, 95% CI: 79-85] was significantly higher than in Mayan [71, 95% CI: 66-75]. Hyperkeratosis was 34 times more frequent in the exposed population [OR = 34, P< 0.001]. Also, hyperpigmentation was significantly more frequent in the exposed population [OR = 2.4, P < 0.007]. Water arsenic and nitrate levels at Ghopuz were higher than the maximum permissible levels. The prevalence of skin lesions and hypertension is increased at Ghopuz village due to arsenic exposure. There is also some evidence of chromosomal defects in the exposed group. Affected people need appropriate medical care, and safe drinking water should be provided to reduce arsenic exposure

[Detection of rare beta globin gene mutations in north western Iran]

Recent molecular studies on Iranian -thalassemia genes revealed the presence of eight common mutations associated with beta-thalassemia. Although these mutations are frequent, there are other rare and unknown mutations that can create large problems in designing preventive programs. We detected and explained the common mutations in north-western Iran previously and detection of the rare and unknown mutations could be useful in diagnosis and design of future preventive programs. In this study. 5ml peripheral blood from 20 Azari- beta-thalassemia patients whose mutation was not revealed in the previous study was collected and DNA extraction was done by isopropanol and proteinase k method. initially, samples were examined for the rare mutations: Codon6, Codon16, Codon4l/42, Codon36/37, - 88 and Codon22 by ARMS - PCR techniques and then the unknown cases were directly sequenced. According to our results, Codon15[TGG-TGA], Codon16[-C], Codon36/37[-T], lVSII-848[C-A], IVSII-745[C-G], -28[A-C] and Codon25/26[+T] were recognized and added to the spectrom of beta globin gene mutations in Azerbaijan and Iran. Also, we detected four SNP sites: 5'UTR+20[C-T], Codon2 [CAC-CAT] IVSII-16[C-G] and IVSII-666[T-C] in beta-thalassemia genes. Our results could be useful for developing molecular screening plans and help prenatal diagnosis of beta thalassemia in Azerbaijan, Iran and other neighboring countries