Avicenna Journal Of Medical Biotechnology (20084625)7(4)pp. 173-178
Background: The risk of developing female infertility has been associated with gene polymorphisms that decrease the activity of enzymes involved in systemic Oxidative Stress (OS). In this study, PON1 L55M polymorphism for association with susceptibility to infertility was investigated among Iranian female population. Methods: Samples from 120 Iranian females [20 endometriosis; 30 Polycystic Ovary Syndrome (PCO); 70 controls] were analyzed and PCR-RFLP assay was used to determine the PON1 rs854560 (L55M) frequencies. The paraoxonase (PONase) and arilesterase (AREase) activities of PON1 enzyme were also assessed in order to investigate the association between serum PON1 activities, female infertility, and PON1 L55M polymorphism. Results: The women with a MM genotype (p=0.021; OR=2.55) showed more possibilities of experiencing infertility than those with a LM genotype (p=0.039; OR=1.91). According to LSD test, endometriosis subjects had significantly lower paraoxonase enzyme activity compared to control group (p=0.0024; CI=95%). No significant difference was found in women with PCOS for both PONase and AREase activity in comparison with control group (p=0.469; CI=95%). Furthermore, PON1 activities were the highest in LL genotype followed by LM and then MM genotype (MM
Objective: People are usually susceptible to carcinogenic aromatic amines, present in cigarrette smoke and polluted environment, which can cause DNA damage. Therefore, maintenance of genomic DNA integrity is a direct result of proper function of DNA repair enzymes. Polymorphic diversity could affect the function of repair enzymes and thus augment the risk of different cancers. Xeroderma pigmentosum group D (XPD) gene encodes one of the most prominent repair enzymes and the polymorphisms of this gene are thought to be of importance in lung cancer risk. This gene encodes the helicase, which is a component of transcription factor IIH and an important part of the nucleotide excision repair system. Studies reveal that individuals with Lys751Gln polymorphism of XPD gene have a low repairing capacity to delete the damages of ultraviolet light among other XPD polymorphisms.
Journal of Isfahan Medical School (10277595)31(264)
Background: Hydroxyurea is a chemotherapeutic agent for treatment of cancer. This drug induces globin-γ synthesis, so it could be used for treatment of thalassemia. Several studies have been shown that treatment with hydroxyurea increases Hb and HbF levels in patients with intermediate betathalassemia. However, the efficiency of hydroxyurea treatment in patients with beta-thalassemia is unclear. In the present study, clinical response of these patients to the drug was investigated. Methods: In this prospective study, the samples were patients with beta-thalassemia intermedia admitted to Sayed-al-Shohada hospital, Isfahan, Iran, during the years 2011-13. Efficiency of hydroxyurea in 46 patients was studied by determining the changes of Hb and HbF levels before and after one year of treatment with the drug. Treatment was performed using 500 mg capsule with dosage of 20 mg/day/kg. Patients were monitored for side effects, too. Findings: After treatment, the means of Hb and HbF levels increased at a rate of 0.47 ± 1.12 g/dl and 6.04 ± 1.43 percent, respectively; where the first was statistically significant, but the latter was not. Use of drug improved the quality of the patient's condition and there was no side effect. Conclusion: According to our results, it is suggested that treatment with hydroxyurea could be effective in majority of patients with intermediate beta-thalassemia.
Journal of Isfahan Medical School (10277595)32(279)pp. 359-367
Background: Fibroblast growth factor receptor 2 (FGFR2) is a receptor of tyrosine kinase with a pivotal role in the cell growth and differentiation. FGFR2 gene was identified as susceptibility gene for breast cancer by Genome-wide associated study. rs1219648 polymorphisms in intronic region are associated with breast cancer. FGFR2 gene is amplified in 15-10% of breast tumors. Single-nucleotide polymorphisms (SNPs) of this region are involved in FGFR2 amplification. In this study, the association of rs1219648 in intron 2 region of FGFR2 gene and breast cancer was assessed. Methods: In the present study, 80 cases of breast cancer and 100 healthy controls were studied. After DNA extraction from blood, specific sequence was amplified by tetra primer ARMS-PCR (amplification-refractory mutation system-polymerase chain reaction) technique and genotype of C/T polymorphism was determined by agarose gel electrophoresis. Findings: Individuals with G/G and A/G genotype were at a significantly higher risk of breast cancer (OR = 5.32, P = 0.018). G allele frequency in case patients were greater than controls but this increase did not show significant relationship with breast cancer (P = 0.230). Conclusion: Single nucleotide polymorphism of G/A in intron 2 of the FGFR2 tyrosine kinase receptor gene may play a role as a risk factor for breast cancer susceptibility.
Journal Of Kerman University Of Medical Sciences (20082843)20(5)pp. 460-469
Background & Aims: The most significant cause of infertility in men is the genetic deletion in the azoospermia factor (AZF) region that is caused by the process of intra- and inter-chromosomal homologous recombination in amplicons. Homologous recombination could also result in partial deletions in AZF region. The aim of this research was to determine the association between the partial AZFc deletions and infertility. Methods: The blood samples were taken from 100 infertile men' who referred to the Infertility Center of Isfahan' Iran. 100 healthy matched people were also selected as the control group. The five markers of sY1201' sY1206' sY1161' sY1291, and sY1191 were applied in order to study partial deletions. Partial deletions were analyzed in AZF region using the Multiplex-STS-PCR technique. The chi-square test was conducted to check the difference between pretest and posttest. Differences were considered significant if P < 0.05. Results: 9% of studied persons showed gr/gr deletion (in the patient group). Only one case of gr/gr deletion was observed in the control group. Five patients showed b2/b3 deletion. One b2/b3 deletion was seen in the control group. The b2/b4 deletion was observed in 3 patients. In conclusion' partial deletions were observed in 14% of the patients. The statistical analysis of the gr/gr deletion in the study indicates a meaningful difference, but b2/b3 deletion does not represent a meaningful difference. Conclusion: Our results suggest that gr/gr deletions are associated with spermatogenic failure, and there is no association between b2/b3 deletion and infertility.
Iranian Journal of Reproductive Medicine (20082177)10(4)pp. 315-320
Background: About 10% of infertilities with obstructive azoospermia are congenital and caused by CF gene mutations. M469I mutation was observed for the first time in Taiwanese patients. This mutation not only causes CF, but also may be the origin of infertility too. Objective: In this study, we aimed in designing a rapid, reliable RFLP-PCR procedure for detection of M469I mutation. The correlation and association between M469I mutation with infertility was investigated in this study. Materials and Methods: one hundred ten patients (90 non obstructive and 20 obstructive) and 60 normal individuals were considered in this study. M469I mutation was detected using RFLP-PCR. This technique was completely designed for M469I genotyping, for the first time in our study. Amplification of the region surrounding the mutation in exon 10 of CFTR gene was then performed. RFLP analysis was carried out using the NdeI restriction enzyme. Results: All genomic DNA samples were genotyped successfully. M469I mutation was observed only in patients group. Therefore, genotype containing mutant allele (GT) has been detected only in the patients group. There was no significant correlation between GT and TT genotypes with infertility (p=0.437). Conclusion: The M469I mutation has only been observed in Exon 10 CFTR gene of infertile patients, not in the control group. This mutation causes congenital bilateral absence of vaz deferens and finally infertility. This indicates a strong association between the M469I mutation and male infertility. Therefore, this is a CF-causing CFTR mutation that could be considered as a cause of infertility.
Journal Of Research In Medical Sciences (17357136)17(SUPPL.2)
BACKGROUND: Lung cancer has remained the most prevalent malignancy worldwide. It is the fifth leading cause of cancer death in Iran. Nevertheless, during last few years a gradual permanent increase in its incidence has been reported. Although the crucial role of tobacco smoke in lung cancer initiation has long been established, it is tempting to hypothesize that genetic polymorphisms may contribute to lung cancer predisposition. CYP1A1 gene encodes the main enzyme responsible for metabolic activation of several tobacco carcinogens. CYP1A1 MspI (6235T→C) polymorphism is the most studied variation within the CYP1A1, impacts on the basal levels of metabolism and is believed to be associated with elevated lung cancer risk, mainly in Asian population. METHODS: We investigated the frequency of this genetic variation in Iranian lung cancer patients through a cross-sectional study. 65 lung cancer cases and 80 healthy controls were recruited. RESULTS: The present findings confirmed the low frequency of the variant CYP1A1*2A allele in the control group. A significant increased risk for lung cancer was observed among those who possessed heterozygous (*1/*2A) genotype (Odds ratio = 2.79, 95% CI: 1.01-7.65). Adenocarcinoma was more frequent in non-smoker group (p = 0.00064); however, no significant increased risk was observed for squamous cell carcinoma and small cell carcinoma with respect to smoking. CONCLUSIONS: heterozygous (*1/*2A) genotype may increase the risk of lung cancer.
Journal of Isfahan Medical School (10277595)30(205)pp. 1393-1402
Background: Gelatinase B is not only involved in metastasis, but also alters and processes growth factors, growth factor receptors, angiogenic factors, and other proteinases and thus affects early carcinogenesis. In other words, it has a fundamental role in initiation and development of cancer. Genetic polymorphism in the promoter of gelatinase B has been reported to be associated with the risk of several cancers including lung cancer. Methods: Genotyping of gelatinase B was carried out by taking blood samples from 172 patients with lung cancer and 100 controls using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique. The observed numbers of each gelatinase B genotype were compared with that expected for a population in Hardy-Weinberg equilibrium by χ2 test. The significance of the differences of the observed alleles and genotypes between groups was tested using the odds ratio (OR) analysis. Findings: The percentage of smokers in patients was more than controls (57.5% vs. 30%). Distribution of gelatinase B genotype was significantly associated with the risk of lung cancer [OR = 2.56; 95% confidence interval (CI) = 0.06-23.82]. Conclusion: Our results indicated that smokers who carry TT and CT+TT genotypes have 4 (OR = 3.45; 95% CI = 1.28-9.24) and 15 (OR = 14.66; 95% CI = 3.95-53.47) fold risks of lung cancer, respectively.
Journal of Isfahan Medical School (10277595)30(203)
Background: Type IV collagenase gene is capable of degrading type IV collagen which is the major structural component of basement membrane. It also increased the bioavailability of pro-angiogenic factors including vascular endothelial growth factor and transforming growth factor-β. A cytosine (C) thymine (T) single nucleotide polymorphism (SNP) at position 1562 of the type IV collagenase promoter has been reported to affect gene expression. The purpose of this study was to investigate the association between the C(-1562)T polymorphism and risk of lung cancer in different age groups in Iran population. Methods: Genotyping of C(-1562)T polymorphism in type IV collagenase gene was performed by taking out genomic DNA from blood samples of 120 patients with lung cancer and 100 age-matched controls by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Chi-square test was used to calculate adjusted odds ratio (OR) and 95% confidence interval (95% CI). All analyses were performed in SPSS. Findings: Distribution of C(-1562)T genotype in type IV collagenase promoter was significantly associated with the risk of lung cancer in the age group of < 60 years (OR = 19.89; 95% CI = 3.21-120.60). Conclusion: Our results indicated that C(-1562)T polymorphism in type IV collagenase gene affects the risk of lung cancer in different age groups, i.e. aging less than 60 years was significantly related with initiation of lung cancer.
Journal Of Research In Medical Sciences (17357136)17(10)pp. 962-966
Background: Matrix metalloproteinases comprise a family of enzyme degrade components of extra cellular matrix. There are single nucleotide polymorphisms in the promoter regions of several genes with ability to influence cancer susceptibility. The aim of this study was to analyze association between MMP3 promoter polymorphisms and colorectal cancer occurrence and progression. Materials and Methods: In this case-control study 120 colorectal cancer patients and 100 controls were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on the genomic deoxyribonucleic acid (DNA). The patients group was divided into different subgroups: a subgroup without metastatic activity (M-) and a subgroup that had developed metastasis (M+). Results: There was a significant difference in frequency of the MMP-3 genotype between cases and controls (χ2 =16.17; P=0.0003). The 5A homozygote in patients and controls was significantly different. The frequency of the 5A allele among affected patients (67.91%) was significantly higher than among the healthy controls (49%; χ2 =16.17, P=0.00005). At the time of diagnosis, individual who was carrying the 5A allele was more represented in the M+ subgroup than in M- subgroup (χ2 =7.49; P=0.006, OR=3.86; 95% CI, 1.43-10.33). The difference between M- and controls did not observe statistically significant (χ2 =0.009; P=0.92). Conclusions: Our results suggest that the presence of 5A polymorphism at the MMP-3 promoter region may favor the growth and the metastasis process in colorectal cancer patients and could be looked at as a risk factor for a worse prognosis.
Journal of Isfahan Medical School (10277595)29(137)
Background: Colorectal cancer is the third cause of cancer death in western countries. Age, inadequate diet, obesity, inactivity and genetic changes are some of the risk factors of colorectal cancer. Corelation of genetic diversity in homologous recombination repair system with cancer was evaluated in many recent studies. This study was done to investigate the correlation of T241M polymorphism in Xrcc3 gene and colorectal cancers. Methods: In this cross-sectional study after collecting blood samples and extraction genomic DNA, genotype distribution of the polymorphism was determined by RFLP-PCR (Restriction fragment lengh-polymerase-Polymerase chain reaction) method. Finding: A significant corelation between T241M polymorphism with colorectal cancer was seen. Age and family history were also corelated with this cancer. Although, there was no statistically relationship between smoking status and colon cancer, but it showed correlation with rectum cancer and it has been also observed that the most occurance of metastatic activity is in the rectum. Conclusion: According to our study T241M polymorphism in Xrcc3 gene from homologous recombination repair systm could be a suitable factor for early diagnosis of colorectal cancer especially rectum and its co-operation with smoking status.
Journal of Isfahan Medical School (10277595)29(142)
Background: Infertility is one of the important human problems. One of the main genetic factors of infertility is the deletions in the chromosome Y' which is reported in 10-15% of men with severe azoospermia and oligospermia. Three regions in azoospermia factor (AZFa), (AZFb) and (AZFc) are specified as the spermatogenetic regions. In this study we investigated the effect of changes in Yq11.223 (DAZ) region in chromosome Y on infertility. Methods: In this study the blood samples of 100 infertile men who referred to the Infertility Center of Isfahan and 100 healthy people, as the control group were taken. The genomic DNA was extracted from blood samples. The analyses were performed by applying the polymerase chain reaction (PCR) technique in AZF region of Yq11.223. Finding: In this study 70 azoospermia and 30 oligospermia patients were investigated. The deletions in AZF region of Yq11.223 were recognized in 7 azoospermia patients but there were not detected in oligospermia patients and the control group. It was also observed that the intensity of bands were changed comparing between azoospermia patients. Conclusion: Deletion frequency in the region of Yq11.223 was observed in 7 of 100 (7%) infertile Oligospermia and Azoospermia men. Our results confirm the effect of DAZ in the man's infertility.
Journal of Mazandaran University of Medical Sciences (17359260)21(84)pp. 23-31
Background and purpose: Polymerase chain reaction (PCR) is a rather quick and accurate method employed for gene detection and isolation. Primer designing is an important issue in this technique and plays a critical role in considering both the genome properties and cloning of the isolated genes. Streptomycin antibiotic is produced by Streptomyces griseus using str gene cluster with more than 25 genes. This gene cluster contains StrR gene encoding a specific protein regulator of this cluster. The pathway specific transcriptional activator then induces transcription of other genes in the str gene cluster. In this study, the researchers aimed at isolating promoterless StrR2 gene and then cloning it. Materials and methods: To isolate promoterless StrR gene, a set of primers (StrR2) was designed. One pair of these primers (St Nes) detected the StrR from the genome. Moreover, Nested-PCR was then used to detect amplified StrR2 gene. Sites for BamHI and XbaI were designed in other primers (Str nP1and Str nP2). These primers not only amplified the StrR2 gene but also created restriction enzyme sites in the amplified fragments. Results: Using PCR, the promoterless StrR2 gene was amplified and its structure was confirmed. This gene was successfully cloned, too. The correct structure of the recombinant plasmids was confirmed using different techniques such as gel electrophoresis, PCR and restriction digestion analysis. Conclusion: Using this vector, one can subclone the promoterless StrR2 gene in the Streptomyces expression vectors containing inducible promoters.
Objective: The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). Materials and Methods: In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. Results: The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. Conclusion: The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assay.
Hemmati, S., Sadeghi, M., Bashi naeini, M.M., Sahebi, A., Vaise malekshahi, Z.
Journal of Biological Research (Greece) (1790045X)13pp. 113-118
Matrix metalloproteinase-9 (MMP-9) in blood is a promising new tumor marker. Previously, a correlation between C/T-1562 MMP-9 polymorphism and tumor progression of breast cancer was reported. In the present study, we examined the association between the C/T polymorphism and plasma MMP-9 level in breast cancer patients. This study included 124 breast cancer patients, 52 of which with initial breast cancer and 72 with progressive breast cancer. Different allelic genotypes of MMP-9 promoter polymorphism were determined by PCR-RFLP and the plasma MMP-9 concentrations were measured using ELISA. Plasma MMP-9 levels were significantly increased in breast cancer patients with the MMP-9 promoter T allele compared with patients with the MMP-9 promoter C allele (p < 0.001). The plasma MMP-9 levels were correlated with progression of breast cancer and lymph node involvement (p = 0.002). According to our findings, individuals with MMP-9 promoter T allele are at a risk of higher plasma MMP-9 and they are more susceptible to breast cancer.
Iranian Journal Of Biotechnology (23222921)7(2)pp. 112
The claR of Streptomyces clavuligerus in the clavulanic acid gene cluster encodes a transcriptional regulator that controls clavulanic acid biosynthesis. The main goal of this study was isolation and molecular detection of the claR gene and its cloning in the Streptomyces specific vector (pMA:: hyg). By cinsideration of the claR gene's start codon, the specific primers were designed. After genomic DNA extraction from S. clavuligerus, the claR gene was amplified by Polymerase Chain Reaction (PCR). The structure of the amplified claR was confirmed by nested-PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), and sequencing. A ligation mixture was prepared with the isolated claR gene and cut pMA::hyg vector. Escherichia coli competent cells were finally transformed with the ligation mixture. Presence of the recombinant vector in the transformed colonies was then confirmed by the colony-PCR procedure. The claR gene was also isolated from S. clavuligerus DSM41826, cloned and sequenced in the same manner. The pMA::hyg vector is a shuttle vector, which exists as a multicopy plasmid in E. coli, and as an integrative plasmid in Streptomyces. Therefore, the newly constructed vectors of this study can be regarded as an appropriate tool for site-directed mutagenesis and gene replacement strategies in S. clavuligerus.
Iranian Journal Of Biotechnology (23222921)6(1)pp. 45-49
In the human genome, chromosome 11 contains a cluster of matrix metalloproteinase (MMP) genes. Single nucleotide polymorphisms in the promoter region of MMP genes are important for MMP expression. A common adenine deletion polymorphism (5A) at position -1171 of the MMP-3 gene promoter (5′-AAAAAACCAT-3′ change to 5′-AAAAACCAT-3′) facilitates transcriptional factor binding and MMP-3 promoter activity. A case-control study was performed including 120 breast cancer patients (60 patients with metastatic activity and 60 patients without metastatic activity); and 60 healthy controls. Whole blood samples were obtained from patients and healthy controls. Genomic DNA was extracted from samples and the MMP-3 5A/6A genotypes were determined using PCR-RFLP. MMP-3 genotype distributions between patients and controls were similar (OR= 0.89, 95%CI, 0.43-1.84, P= 0.047). It was observed that the 5A allele was more frequent among patients with metastatic activity than controls (OR= 2.9, 95%CI, 0.94-8.9, P= 0.074). Therefore, the 5A polymorphism in the MMP-3 promoter showed correlation with the metastasis group than patients without metastasis; both at the time of diagnosis. However our results do not show evidence for correlation between 5A/6A polymorphism and breast cancer susceptibility.
