Efficacy of insulin targeted gene therapy for type 1 diabetes mellitus: A systematic review and meta-analysis of rodent studies.

Diabetes mellitus (DM) is a major worldwide public health challenge, for which gene therapy offers a potential therapeutic approach. To date, no systematic review or meta-analysis has been published in this area, so we examined all relevant published studies on rodents to elucidate the overall effects of gene therapy on bodyweight, intraperitoneal glucose tolerance test (IPGTT), fasting blood glucose, and insulin in animals with type 1 DM. The Cochrane Library, PubMed, Embase, ISI Web of Science, SCOPUS, and Google Scholar were systematically searched for potentially relevant studies. Mean±standard deviation (SD) was pooled using a random-effects model. After the primary search, out of 528 studies identified, 16 studies were in concordance with predefined criteria and selected for the final assessment. Of these, 12 studies used viral manipulation, and 4 employed non-viral vectors for gene delivery. The meta-analysis showed gene therapy with a viral vector decreased mean IPGTT (-12.69 mmol/l, P<0.001), fasting blood glucose (-13.51 mmol/l, P<0.001), insulin (398.28 pmol/l, P<0.001), and bodyweight (24.22 g, P<0.001), whereas non-viral vectors reduced fasting glucose (-29.95 mmol/l, P<0.001) and elevated insulin (114.92 pmol/l, P<0.001). Gene therapy has favorable effects on alleviating type 1 DM related factors in diabetic rodents.

TFPI2 and NDRG4 gene promoter methylation analysis in peripheral blood mononuclear cells are novel epigenetic noninvasive biomarkers for colorectal cancer diagnosis.

BACKGROUND: As a result of the growing prevalence of colorectal cancer (CRC), new screening and early detection methods are required. Among the novel biomarkers, DNA methylation has emerged as a high-potential diagnosis/screening molecular marker. The present study aimed to assess non-invasive early diagnosis of CRC by examining promoter methylation of TFPI2 and NDRG4 genes in peripheral blood mononuclear cells (PBMCs). METHODS: Fifty CRC patients and 50 normal controls were recruited to the present study. Quantitative methylation of the promoter region of the TFPI2 and NDRG4 genes was analyzed in DNA extracted from PBMCs of all cases and control subjects using a methylation-quantification endonuclease-resistant DNA (MethyQESD) method. RESULTS: The sensitivity and specificity of the TFPI2 gene for the diagnosis of CRC was 88% and 92%, respectively, and, for the NDRG4 gene, it was 86% and 92%, respectively. The methylation range for the TFPI2 gene was 43.93% and 11.56% in patients and controls, respectively, and, for the NDRG4 gene, it was 38.8% in CRC patients and 12.23% in healthy controls (p < 0.001). In addition, we observed that a higher percentage of methylation was correlated with the higher stage of CRC. CONCLUSIONS: The results of the present study reveal that PBMCs are reliable sources of methylation analysis for CRC screening. Furthermore, the TFPI2 and NDRG4 genes provide sufficiently high sensitivity and specificity to be nominated for use in a novel noninvasive CRC screening method in PBMCs.

Single nucleotide polymorphism rs10889677 in miRNAs Let-7e and Let-7f binding site of IL23R gene is a strong colorectal cancer determinant: Report and meta-analysis.

Single nucleotide polymorphisms (SNPs) in the recognition sites of microRNAs (miRNAs), located at 3' untranslated region (UTR) of mRNAs, interfere with posttranslational gene regulation. Deregulation of genes may contribute to some disease susceptibility including colorectal cancer (CRC). In the present study, in a case-control setup, 167 CRC patients and 161 control subjects were studied for allele and genotype frequency of rs10889677 polymorphism in miRNAs Let-7e and Let-7f binding sites at 3' UTR of IL23R gene using PCR-RFLP assay. Also, related articles were retrieved from MEDLINE, Cochrane review, Google Scholar and Scopus databases for meta-analysis study. According to our results, AA genotype of SNP rs10889677 was significantly correlated with increased risk of CRC (OR = 3.10; 95% CI [1.86-5.18]; P: < 0.001). In a meta-analysis on 10 risk estimates for the CC versus AA genotype, we found an inverse association between CC SNPs and risk of all cancer (OR = 0.59; 95% CI [0.49-0.71]; P < 0.001). In conclusion, our results demonstrate that rs10889677 polymorphism is significantly associated with CRC risk.

Novel High-Fat Diet Formulation and Streptozotocin Treatment for Induction of Prediabetes and Type 2 Diabetes in Rats.

BACKGROUND: The previously established methods for type 2 diabetes (T2D) have mainly concentrated on overt diabetes model development. Here, our intention was to create an animal model passing through distinct phases such as obesity with insulin resistance, prediabetes, and gradual progress to the overt diabetes stage. A high-fat high-carbohydrate diet formulation was prescribed combined with multiple low-dose streptozotocin (STZ) injections after obesity establishment. MATERIALS AND METHODS: Sixteen male Wistar rats were separated randomly into two groups and fed a normal diet for 1 week after which the body weight and biochemical indices of each rat were measured and recorded. Subsequently, one group (n = 8) switched to the high-fat high-carbohydrate diet formulated by us for 10 weeks, whereas the other group (n = 8) continued with the normal diet. Body weight and biochemical indices of the rats in the high-fat diet (HFD) group were measured at the end of 10 weeks, and each rat received 30 mg/kg intraperitoneal STZ injections with 1-week intervals in two steps and was continued on a high-fat high-carbohydrate diet. The differences between the groups were analyzed using the Student's t-test or one-way analysis of variance and by post hoc multiple comparisons. RESULTS: A significant change in weight, fasting blood glucose, and triglyceride was observed in rats fed with a HFD after 10 weeks. The HFD rats showed typical characteristics of T2D mellitus (T2DM) such as insulin resistance and hyperglycemia following 30 mg/kg STZ. CONCLUSIONS: The novel high-fat high-carbohydrate formulation we used, along with multiple low doses of STZ, can mimic peculiar characteristics of T2DM development.

Pharmacogenomics of Sulfonylureas Response in Relation to rs7754840 Polymorphisms in Cyclin-Dependent Kinase 5 Regulatory Subunit-associated Protein 1-like (CDKAL1) Gene in Iranian Type 2 Diabetes Patients.

BACKGROUND: Sulfonylureas are important drugs of choice for treatment of type 2 diabetes mellitus (T2DM). It is suggested that differential response to sulfonylureas from T2DM patients is under influence of single nucleotide polymorphisms in some of the target genes. In spite of favorable therapeutic effects, sulfonylureas are associated with some adverse side effects such as microvascular complications and stroke, especially in older patients. Therefore, for T2DM patients who are getting less benefit, sulfonylureas should be avoided. Cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like (CDKAL1) gene variation is reported to be associated with sulfonylureas effectiveness. Due to the inconsistency of available data regarding association of rs7754840 in CDKAL1 gene with sulfonylureas response in T2DM patients, the present study is conducted. MATERIALS AND METHODS: Fifty-one diabetic patients sensitive to sulfonylureas and 51 patients resistant to sulfonylureas treatment were recruited to this study. After extraction of DNA from patients' peripheral blood samples, rs7754840 single-nucleotide polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism assay using MaeII (Tail) restriction enzyme. RESULTS: Frequency of G allele in resistant group was more than sensitive group (71, 6% vs. 57, 8%). Regression analysis was shown significant association between GG genotype and higher risk of resistance to sulfonylureas treatment (odds ratio = 2.250 [95% confidential intervals: 1.010-5.012]; P = 0.046). CONCLUSION: Our data confirmed that genotypes of rs7754840 are significantly associated with sulfonylureas treatment response. rs7754840 in CDKAL1 gene in combination with other clinicopathological findings would help to move towards personalized therapy of T2DM patients.

Single nucleotide polymorphism rs4648298 in miRNAs hsa-miR21 and hsa-miR590 binding site of COX gene is a strong colorectal cancer determinant.

BACKGROUND: Genetic determinants are considered as driving forces in development colorectal cancer (CRC), a malignancy that ranks as the second cause of cancer death in the world. Single nucleotide polymorphisms (SNPs), are considered as the main genetic factor in cancers susceptibility. MicroRNAs are critical players in posttranslational gene regulation by binding to their specific recognition sequences located at 3' untranslated region (UTR) of mRNAs. In present study we have elucidated the role of 9,850 A > G (rs4648298), in development of sporadic CRC in Iranian population. METHODS: A case-control study using 88 CRC patients and 88 noncancerous counterparts was undertaken in order to determine rs4648298 genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Also, a meta-analysis was performed based on 9 articles accessed via the MEDLINE, Cochrane review, Google Scholar and Scopus databases. RESULTS: AA genotype was determined to be associated with significant decreased risk of CRC in our study population [odds ratio (OR) =0.14; 95% confidence interval (CI), 0.05-0.34; P<0.001]. In a meta-analysis on 6 risk estimates for the AG versus AA genotype, we found a significant inverse association between AG SNPs and risk of gastric adenocarcinoma, CRC, breast cancer and prostate cancer (OR =0.86; 95% CI, 0.76-0.98; P<0.02). CONCLUSIONS: Our results suggest significant correlation between rs4648298 polymorphism and CRC risk in Iranian population.

Micro R-410 Binding Site Single Nucleotide Polymorphism rs13702 in Lipoprotein Lipase Gene is Effective to Increase Susceptibility to Type 2 Diabetes in Iranian Population.

BACKGROUND: The relationship between dyslipidemia and type 2 diabetes mellitus (T2DM) has been frequently reported. Lipoprotein lipase (LPL) is considered to be an effective gene in regulating lipid profile. MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional regulation of gene expression. In the present study, we have evaluated rs13702 (C/T) polymorphism located in miRNA-410 binding site of LPL gene in subset of Iranian T2DM patients and their normal counterparts. MATERIALS AND METHODS: In this case-control study, 102 T2DM patients and 98 healthy controls were worked out for rs13702 single nucleotide polymorphism genotypes. High resolution meting (HRM) analysis was used for genotyping. RESULTS: C allele of rs13702 C/T polymorphism located in miRNA-410 binding site in LPL gene was detected to be significantly associated with T2DM (C allele; odds ratios (OR) = 1.729 (95% confidential intervals (CI) = 1.184-2.523); P = 0.005) also its CC genotype (OR = 3.28 (95% CI 8.68-1.24); P = 0.010) showed the same association. CONCLUSION: Correlation of rs13702 C allele with susceptibility to T2DM may be due to the higher level of LPL that leads to increased plasma fatty acids and its entry into peripheral tissues such as skeletal muscle, liver, and adipocytes causing development of insulin resistance and ultimately T2DM.

Single nucleotide polymorphism rs696 in miR449a binding site of NFKBIA gene is correlated with risk of colorectal cancer.

AIM: In present study we have elucidated the role of 2758 A>G (rs696), in the recognition site of miR449a in the 3' UTR of NFKB inhibitor alpha (NFKBIA) gene, in development of sporadic colorectal cancer. BACKGROUND: Colorectal cancer (CRC) is rated as second cause of cancer death. Genetic determinants are considered as driving forces in development of sporadic CRC. Single nucleotide polymorphisms (SNPs), are attributed as the main genetic factor in cancers susceptibility. MicroRNAs, are key players in post-translational gene regulation by binding to their specific recognition sequences located at 3' untranslated region (UTR) of mRNAs. METHODS: A case-control study using 143 CRC patients and 137 noncancerous counterparts were undertaken in order to determine rs696 genotypes using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: There was a significant difference for the genotype frequencies of rs696 between patients and controls. The frequencies of GG, AG, AA genotypes in the control group were 38.7, 45.3, and 16.1 %, respectively, and the genotype frequencies in case group were 19.6, 40.6, and 39.9 %, respectively. CONCLUSION: Our results suggest significant correlation between rs696 polymorphism and colorectal cancer risk.

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women.

Mouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples. Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.

Polymorphism (rs16917496) at the miR-502 Binding Site of the Lysine Methyltransferase 5A (SET8) and Its Correlation with Colorectal Cancer in Iranians.

BACKGROUND: One of the gene expression regulatory mechanisms is mediated by small noncoding RNAs called microRNA (miRNA). They interact with a recognition sequence located mostly in 3'-untranslated regions (3'-UTRs) of mRNAs. Polymorphisms in miRNAs recognition sequences could affect gene expression which in turn may alter disease susceptibility. SET8, a member of the SET domain-containing methyltransferase, acts in a variety of biological processes such as genomic stability. Here, we report correlation of rs16917496 polymorphism, located in the recognition sequence of miR-502 within 3'-UTR of SET8, with colorectal cancer (CRC) in Iranians. MATERIALS AND METHODS: One hundred and seventy CRC patients and 170 noncancer counterparts were recruited in this case-control study. Genotyping of rs16917496 was performed using polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: There was no significant association of rs16917496 with CRC in population under study (P value for genotype and allele distribution were >0.05). However, stratification analysis based on smoking status revealed that TT+TC genotypes of SET8 rs16917496 are strongly associated with increased risk of CRC (odds ratio: 5.8, 95% confidence interval: 1.37-24.34, P - 0.005) in smoker subgroup. CONCLUSION: Correlation of rs16917496 T allele with CRC in smokers is emphasizing the importance of individuals' genotype in the recruitment of adverse health hazards of smoking more profoundly for certain people compared to others.

Mutation in δ-Sg Gene in Familial Dilated Cardiomyopathy.

BACKGROUND: Mutations in different genes including dystrophin-associated glycoprotein complex caused familial dilated cardiomyopathy which is a genetically heterogeneous disease. The δ-SG gene contains nine exons spanning a 433-kb region of genomic DNA. It encodes a 35-kDa, singlepass, and type II transmembrane glycoprotein. MATERIALS AND METHODS: In this study for the first time in Iran we screened 6 patients of a large family that they had positive family history of MI or sudden death by next generation sequencing method. RESULTS: By employing NGS method we found missense mutation (p.R97Q) of δ-SG gene in 2 of 6 patients. CONCLUSIONS: The missense mutation (p.R97Q) in familial DCM patients is reported for the first time in Iranian patients with cardiac disease. Although this mutation is already known in other populations in Iran, it is not reported before.

Recent Advances in Therapeutic Applications of Induced Pluripotent Stem Cells.

Induced pluripotent stem (iPS) cells are generated by reprogramming of differentiated somatic cells. These cells are identical to human embryonic stem cells (hESCs) in gene expression pattern and the ability to differentiate. iPS cells can be used in in vitro modeling of diseases, testing drugs, assessing gene therapy methods, and cell therapy. Yet, the most important and promising application of iPS cells is in regenerative medicine. Regenerative medicine is a novel area in medicine aiming at the treatment of impaired or lost tissues by replacing them with functional and healthy ones. Currently, organ transplantation, which is considered the only treatment and cure for a number of diseases, is limited by shortage of organ donors and availability of the right match. Therefore, utilization of an alternative source of cells and tissues is critical in transplantation therapy. In this study, we review recent advances in therapeutic application of iPS cells in diseases where organ transplantation remains the only solution and will discuss the potential and usage of iPS cells in different areas of regenerative medicine. The primary theory of using iPS cells in regenerative medicine has brought lots of promises due to its potential for solving the immunological, social, and ethical problems of using ESCs. Nevertheless, several issues and problems have to be resolved before applying iPS cells in therapeutic applications.

Genetic polymorphism of 8 Y-STR loci in native population of Isfahan province in central part of Iran.

BACKGROUND: Y-chromosome short tandem repeats (Y-STRs) are genetic markers with practical applications in human identification and population studies. AIM: Here we present the allelic and haplotype frequencies of 8 Y-STR loci most commonly used in forensic medicine in 103 unrelated native males of Isfahan province, central part of Iran. SUBJECTS AND METHODS: The cases were selected on the basis of strict criteria to assure pure native populations of Isfahan origin. DNA extracted from peripheral blood samples and PCR amplified for each marker. Y-specific STR loci DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 were included in this study. RESULTS: The most common alleles for each locus were: DYS19, allele 12; DYS385, allele 12; DYS389I, allele 13; DYS389II, allele 29; DYS390, allele 24; DYS391, allele 10; DYS392, allele 11; and DYS393, allele 13. Gene diversity value was calculated from the allelic frequency for each locus. The average gene diversity was 0.6518. A total of 101 haplotypes were observed in eight Y-specific STR loci, the haplotype diversity was raised to 0.986. CONCLUSION: The results revealed that a set of eight Y-specific STR loci were able to discriminate most of the male individuals in the population studied. A search through the Y Haplotype Reference Database demonstrated 21 matched haplotypes to 160,693 haplotypes, exclusively with Eurasian-European, Eurasian, and Eurasian-Indo Iranian populations.

Genetic analysis of Iranian family with hereditary cardiac arrhythmias by next generation sequencing.

BACKGROUND: Cardiac arrhythmias are responsible for several cases of syncope and sudden cardiac death annually worldwide. Due to overlapping clinical symptoms in some cardiac arrhythmias genetic studies would help to confirm the primary clinical diagnosis made on the basis of solely clinical findings. In addition clinical management of the patient, family screening and provide appropriate counseling and risk assessment for the family members are other advantages of genetic study. MATERIALS AND METHODS: Totally nine patients from a family included in this study. The primary diagnosis on the basis of clinical findings was second-degree atrioventricular (AV) block for this family. Mutation in SCN5A gene is frequently reported for second-degree AV block and hence the gene was analyzed using whole gene sequencing but no mutation was detected. Subsequently, the samples were subjected to customized Ampliseq 77 gene panel using next generation sequencing to detect the underlying molecular defects. RESULTS: We found c. 5570T>A missense mutation in ANK2 gene for this family. Based on the Online Mendelian Inheritance in Man, ANK2 gene and the mutation detected correspond to long QT syndrome type 4. CONCLUSION: This mutation, although already known in other populations, but is reported for the first time in Iranian patients with cardiac arrhythmias. As the case with this family, genetic analysis of patients with cardiac arrhythmias would be helpful in reassessment of clinical diagnosis and therefore would help for patients' management and in some cases re-evaluation of ongoing treatment may be needed.

Genetic analysis of cardiac SCN5A Gene in Iranian patients with hereditary cardiac arrhythmias.

OBJECTIVE: SCN5A encodes alpha subunit of the major sodium channel (Nav1.5) in human cardiac tissue. Malfunction of this cardiac sodium channel is associated with a variety of cardiac arrhythmias and myocardial inherited diseases. METHODS: Fifty-three members from three families each diagnosed with long-QT syndrome type 3 (LQTS3), Brugada syndrome (BrS), or sick sinus syndrome (SSS) were included in this observational, cross-sectional study. In this study, we analyzed the sequences of coding region of the SCN5A gene. RESULTS: Eleven members of the LQTS family (39%) showed p.Gln1507-Lys1508-Pro1509del mutation, 8 of BrS family (50%) showed p.Arg222Ter nonsense mutation, and 5 of 9 SSS family members (55%) showed a novel p.Met1498Arg mutation in the SCN5A gene. CONCLUSION: p.Gln1507-Lys1508-Pro1509del mutation, p.Arg222Ter nonsense mutation, and p.Met1498Arg in LQTS, BrS, and SSS, respectively, are reported for the first time in the Iranian population. Information regarding underlying genetic defects would be necessary for verifying certain clinically diagnosed arrhythmia types, carrier screening in affected families, and more precise therapy of the patients are required.

Methylation pattern of ALX4 gene promoter as a potential biomarker for blood-based early detection of colorectal cancer.

BACKGROUND: To develop a non-invasive screening method for colorectal cancer, we evaluated the methylation of ALX4 gene promoter in serum samples from patients with colorectal cancer (CRC) and equal number of healthy individuals. MATERIALS AND METHODS: In serum samples from 25 patients with colorectal cancer and 25 healthy control subjects, isolated serum free-floating DNA was treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction (MSP) with primers specific for methylated or unmethylated promoter CpG island sequences of the ALX4 gene. RESULTS: Methylation of the ALX4 gene promoter was present in the serum DNA of patients with adenoma and colorectal cancer. A sensitivity of 68% and specificity of 88% were achieved in the detection of promoter methylation in colorectal neoplasia samples. The difference in methylation status of the ALX4 promoter between the patients with colorectal neoplasia and the control group was statistically highly significant (P < 0.001). CONCLUSIONS: The results indicate that this serum free DNA test of methylation of the ALX4 gene promoter is a sensitive and specific method. Therefore in combination with other useful markers it seems ALX4 has the potential of a clinically useful test for the early detection of colorectal cancer.

Effects of biosurfactant produced by Lactobacillus casei on gtfB, gtfC, and ftf gene expression level in S. mutans by real-time RT-PCR.

BACKGROUND: The Streptococci are the pioneer strains in plaque formation and Streptococcus mutans are the main etiological agent of dental plaque and caries. In general, biofilm formation is a step-wise process, which begins by adhesion of planktonic cells to the surfaces. Evidences show that expression of glucosyltransferase B and C (gtfB and gtfC) and fructosyltransferase (ftf) genes play critical role in initial adhesion of S. mutans to the tooth surface which results in formation of dental plaques and consequently caries and other periodontal disease. MATERIALS AND METHODS: The aim of this study was to determine the effect of biosurfactants produced by a probiotic strain; Lactobacillus casei (ATCC39392) on gene expression profile of gftB/C and tft of S. mutans (ATCC35668) using quantitative real-time PCR. RESULTS: The application of the prepared biosurfactant caused dramatic down regulation of all the three genes under study. The reduction in gene expression was statistically highly significant (for gtfB, P > 0.0002; for gtfC, P > 0.0063, and for ftf, P > 0.0057). CONCLUSION: Considerable downregulation of all three genes in the presence of the prepared biosurfactant comparing to untreated controls is indicative of successful inhibition of influential genes in bacterial adhesion phenomena. In view of the importance of glucosyltransferase gene products for S.mutans attachment to the tooth surface which is the initial important step in biofilm production and dental caries, further research in this field may lead to an applicable alternative for successful with least adverse side effects in dental caries prevention.

Effects of Lactobacillus reuteri-derived biosurfactant on the gene expression profile of essential adhesion genes (gtfB, gtfC and ftf) of Streptococcus mutans.

BACKGROUND: Streptococci are the main causative agents in plaque formation and mutans streptococci are the principle etiological agent of dental plaque and caries. The process of biofilm formation is a step-wise process, starting with adhesion of planktonic cells to the surfaces. It is now a well known fact that expression of glucosyltransferases (gtfs) and fructosyltransferase (ftf) genes play a critical role in the initial adhesion of Streptococcus mutans to the tooth surface, which results in the formation of dental plaques and consequently caries and other periodontal diseases. MATERIALS AND METHODS: In the present study, we have determined the effect of biosurfactants purified from Lactobacillus reuteri (DSM20016) culture on gene expression profile of gftB/C and fft of S. mutans (ATCC35668) using quantitative real-time polymerase chain reaction. RESULTS: The application of biosurfactant caused considerable down-regulation of the expression of all three genes under study. The reduction in gene expression was statistically very significant (P > 0.0001 for all three genes). CONCLUSIONS: Inhibition of these genes by the extracted L. reuteri biosurfactant shows the emergence of a powerful alternative to the presently practicing alternatives. In view of the importance of these gene products for S. mutans attachment to the tooth surface, which is the initial important step in biofilm production and dental caries, we believe that the biosurfactant prepared in this study could be considered as a step ahead in dental caries prevention.