Association of CDH1 and TERT Single-Nucleotide Polymorphisms with Susceptibility to Familial Breast Cancer Risk.

Breast cancer (BC) is a multistep disease that is thought to result from an interaction between genetic background and environmental factors. In Iran, one of the strongest risk factors for developing BC is a positive family history of the disease. Recently, various polymorphisms of E-cadherin (CDH1) and TERT have been found to be associated with increased BC risk worldwide. This study aimed to analyze the association of CDH1 and TERT single-nucleotide polymorphisms with susceptibility to familial BC (FBC) risk in the Iranian patients. One hundred five patients with FBC and 110 non-FBC (NFBC) were genotyped to elucidate the potential association between CDH1 rs5030625 polymorphism and TERT rs2736098 polymorphism by polymerase chain reaction-restriction fragment length polymorphism. Then, results were evaluated by electrophoresis and Epi Info(™) 2012 software. A significant association was found between CDH1 rs5030625 GAGA genotype and FBC risk. Compared with the control group, the FBC patients had a lower frequency of GG genotype (69% vs. 85%) and a higher frequency of GAGA (5% vs. 2%, P < 0.02). Furthermore, the patients with FBC had a lower frequency of TERT rs2736098 GG genotype (38% vs. 49%, P = 0.001) and a higher frequency of rs2736098 AA genotype (12% vs. 5%, P = 0.001) compared with the NFBC. In contrast, the TERT rs2736098 GG genotype potentially increased the recurring risk of FBC (odds ratio = 3.17, P < 0.01). Allele genotypic frequencies in the FBC patients differed from those of the controls. Interestingly, tumors in FBC patients with rs2736098 GG genotype and rs5030625 GAGA exhibited higher mitotic activity, higher grade, lower estrogen receptor, and progesterone receptor than the other genotypes. In conclusion, CDH1 rs5030625 GAGA genotype and TERT rs2736098 GG genotype in combination with clinical parameters may be prognostic factors rather than susceptibility factors during the progression of FBC.

Overproduction of Erythromycin by Ultraviolet Mutagenesis and Expression of ermE Gene in Saccharopolyspora erythraea.

Erythromycin is a macrolide antibiotic with broad-spectrum activity against gram-positive bacteria that stops protein synthesis by binding to 50s ribosomal subunit. Classical and recombinant strain improvement, such as application of ultraviolet (UV) mutagenesis and selection of overproduction mutant, is the most important and convenient method in enhancement of antibiotic production. In the present study, Saccharopolyspora erythraea was mutagenized using UV lights and selection by tylosin resistance mutant to improve yield of erythromycin. In other sides, to improve the erythromycin yield in mutant, effects of various parameters such as carbon concentration and ermE gene expression were analyzed. In primary selection, high erythromycin producing strains and high erythromycin producer mutant were isolated by plaque agar, and an increase of 87% was observed in tylosin resistance mutant compared to wild-type strain. In secondary selection, a mutant strain (RHU233) with a production of 1.39 mg erythromycin per mL was isolated in fermentation process, which was 20 times more productive than the wild type. In contrast, it was found that glycerol can be used as an alternate carbon source in enhancement of erythromycin production. Comparison of ermE gene expression in mutants RHU233 high producer mutant RHU233 and wild type in Escherichia coli detected in accumulation of soluble hexahistidine-ermE was up to 45% of total cell protein after 18 h in mutants RHU233. Metal-chelation chromatography yielded 126 mg of hexahistidine-ermE per liter of culture with a purity slightly >95% in mutants RHU233. Finally, these optimized conditions could be used for the commercial production of this unique antibiotic.

Association of Mannose-Binding Lectin rs1800450 and Tumor Necrotic Factor-α rs1800620 Polymorphism with Helicobacter pylori in Type II Diabetes Mellitus.

Type II diabetes mellitus (T2DM) is the prevalent type of diabetes, including 90% of the cases world-wide. Helicobacter pylori plays a pathogenic role in the development of T2DM. The host genetic factors have a significant impact on the clinical outcome and anatomical distribution of H. pylori infection and polymorphisms in several genes such as tumor necrotic factor (TNF)-α and mannose-binding lectin (MBL) and are considered to increase the risk for the development of T2DM. In this study, we investigate the prevalence rate of H. pylori infection and its relationship to MBL rs1800450 and TNF-α rs1800620 polymorphism in T2DM. In this case-control study, 174 patients with type II diabetes and 185 healthy controls were studied. Also, demographics, physical, and biochemical parameters were performed in all patients. The DNA extracted from blood specimens was amplified by H. pylori cagA-specific primers. The MBL rs1800450 and TNF-α rs1800620 genotyping were detected by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The results show that H. pylori cagA positivity was detected in 42.82% of the diabetic patients and in 22.16% of the control group, and H. pylori infection was closely correlated with MBL rs1800450 AA genotype and TNF-α rs1800620 GG genotype when compared with healthy controls. Furthermore, these two genotypes were strongly associated with H. pylori cagA(+) samples when compared with cagA(-) samples. In addition, the presence of H. pylori cagA(+) infection was significantly associated with the elevated serum levels of total cholesterol and low-density lipoprotein cholesterol. In general, it can be concluded that molecular analysis of MBL rs1800450 AA genotype and TNF-α rs1800620 AA genotype is important in the early detection and treatment of T2DM with H. pylori cagA(+) infection.

Investigation of Methylenetetrahydrofolate Reductase C677T Polymorphism and Human Papilloma Virus Genotypes in Iranian Breast Cancer.

Breast cancer (BC) is the leading cause of death among Iranian women. Development of BC is a multistep process, arising from genetic changes such as methylenetetrahydrofolate reductase (MTHFR) polymorphism and infection with human papillomavirus (HPV). In this study, we investigated HPV genotypes associated with BCs and its relation with MTHFR C677T polymorphism for early detection of familial BCs. A total of 84 archival BC samples from Iran were collected. Verification of each cancer reported in a relative was sought through the pathology reports of the hospital records. Then, DNA was extracted from all samples by standard methods and HPV genotypes and MTHFR C677T polymorphism genotypes were analyzed using multiplex polymerase chain reaction (PCR) and amplification-refractory mutation system (ARMS)-PCR. Finally, data analysis was performed using version 7 of the Epi Info™ 2012 software and test chi-square (x2) for trend. The frequencies of the CC, TC, and TT genotypes of MTHFR (C677T) were 0.53, 0.38, and 0.09 in familial BC patients, and 0.46, 0.51, and 0.03, respectively, in nonfamilial BC patients. Furthermore, HPV DNA typing identified 29 infections and C677T TT genotype was significantly associated with an increased risk of familial BC in the study population. Our results demonstrate that infection with HPV was prevalent among Iranian women with familial BC. Finally, the testing of C677T GG genotype in combination with HPV genotyping as molecular markers can be helpful in the early diagnosis of Iranian familial BCs by PCR.

Cloning and Expression of L1 Protein Human Papillomavirus Type 31 Isolated from Iranian Patients in Escherichia coli.

Human papillomavirus (HPV), a major pathogen of human cervical cancer, contains a full-length L1 gene encoding its surface capsid protein. One group of potential vaccine candidates against this virus in Iranian patients is based on surface protein components such as HPV31 L1 protein that can make virus-like particles (VLPs). The high immunity response stimulation of this effecter VLP was observed in host, suggesting that the individual characteristics of a particular effecter may require empirical testing for vaccination. In the present study, we decided to clone and express HPV31 L1 protein to investigate its use as a subunit vaccine and furthermore to insert the gene into an Escherichia coli background so as to analyze production of this recombinant protein. We report the presentation of HPV31 in 100 cervical lesion tissue samples based on polymerase chain reaction (PCR). Type of lesion, age, and other characteristics were reviewed and confirmed by a pathologist. The sequence from L1 genes of HPV was selected using special primers. The gene encoding the major capsid protein L1 was used for subcloning in pTG19-T and pET-32a plasmid. The recombinant protein expression was confirmed by RT-PCR using L1 primers and detected by absorption sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot testing. The results presented here offer new insights into the in vivo response of HPV31 in Iranian patients and European models. On the other hand, the use of recombinant L1 protein for Iranian patient protection as well as vaccination studies will permit testing of this antigen protection rate and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical cervical cancers.

Detection of Human Papillomavirus Genotypes and Major BRCA Mutations in Familial Breast Cancer.

Breast cancer is a multistep disease and infection with a DNA virus could play a role in one or more of the steps in this pathogenic process. High-risk human papillomaviruses (HPVs) are the causative agents of several cancers. In this study, we investigated HPV genotypes associated with breast cancer and its relationship with BRCA mutation for the detection of familial breast cancer. We analyzed 84 formalin-fixed, paraffin-embedded tissue blocks from 38 familial breast cancer and 46 nonfamilial breast cancer samples by multiplex polymerase chain reaction and clinical parameters. Overall prevalence of HPV infection was 27 of 84: 10 (37.03%) HPV-16, 9 (29.62%) HPV-18, 4 (14.81%) HPV-11, 1 (3.7%) HPV-31, 1 (3.7%) HPV-33, and 2 (7.4%) HPV35. Furthermore, 17 mtDNA4977 deletions and 5 5382insC mutations were detected from 38 familial breast cancer samples. Our results demonstrate that infection with HPV was prevalent among Iranian women with familial breast cancer and the testing of mtDNA4977 deletions and 5382insC mutations in combination with clinical parameters as major risk factors can serve in the identification of familial breast cancer.

Investigation of hFVIII production in mammary glands of transgenic mice.

Hemophilia A is an X-linked disorder affecting 1 in 10,000 males. The disease is caused by a defect or mutation of factor 8 or 9. Human factor 8 gene (hFVIII) is a relatively large gene consisting of 26 exons and approximately 2,351 amino acids with a length of 9 Kb mRNA. Expression of hFVIII in mammalian milk is becoming a widespread strategy for high-level production of hFVIII because of the most complex post-translational modifications. The aim of this study was the cloning and expression of hFVIII in mammary glands of two transgenic mice. To obtain a recombinant plasmid, first a plasmid carrying an FVIII gene fragment (pCMV6-hFVIII) was digested by EcoRI-SalI restriction enzymes and then the fragment was purified from agarose gel and inserted into a pUCWAP7 vector carrying a tissue-specific promoter (mWAP 4.1 kbp). After that, it was isolated by agarose gel and transferred into the murine zygotes by standard microinjection methods. Methods for expression of recombinant FVIII RT-PCR and ELISA were studied. The results show the successful expression of factor FVIII gene and its product in the mouse mammary glands.