A comprehensive overview on the crosstalk between microRNAs and viral pathogenesis and infection.

Infections caused by viruses as the smallest infectious agents, pose a major threat to global public health. Viral infections utilize different host mechanisms to facilitate their own propagation and pathogenesis. MicroRNAs (miRNAs), as small noncoding RNA molecules, play important regulatory roles in different diseases, including viral infections. They can promote or inhibit viral infection and have a pro-viral or antiviral role. Also, viral infections can modulate the expression of host miRNAs. Furthermore, viruses from different families evade the host immune response by producing their own miRNAs called viral miRNAs (v-miRNAs). Understanding the replication cycle of viruses and their relation with host miRNAs and v-miRNAs can help to find new treatments against viral infections. In this review, we aim to outline the structure, genome, and replication cycle of various viruses including hepatitis B, hepatitis C, influenza A virus, coronavirus, human immunodeficiency virus, human papillomavirus, herpes simplex virus, Epstein-Barr virus, Dengue virus, Zika virus, and Ebola virus. We also discuss the role of different host miRNAs and v-miRNAs and their role in the pathogenesis of these viral infections.

Combined Genotype Effects of TP53 and PAI-1 Polymorphisms in Breast Cancer Susceptibility: Multifactor Dimensionality Reduction and in silico Analysis.

INTRODUCTION: Breast cancer is a heterogeneous and multifactorial disease. TP53 and PAI-1 as important tumor suppressor genes are involved in the development, invasion, and metastasis of many cancers. This study's objective was to demonstrate the combined genotype effects of these 2 genes by investigating their single nucleotide polymorphisms. METHODS: In this case-control study, 200 individuals with breast cancer and 179 healthy individuals were studied. The genotypes were determined using the tetra-ARMS method. For data analysis, MDR, online javstat statistics package, and SPSS v.24 software were used. Also, in silico studies on the estimated effects of each of these polymorphisms were performed. RESULTS: We showed a novel gene-gene interaction of these 2 genes and demonstrated a strong synergistic interaction for TP53/PAI-1, moderate synergistic interaction for PAI-1/age, and correlation for TP53/age. On the other hand, there was no association between the allelic and genotype frequency alone and in combination, with case-control status, using the parametric method, between TP53 and PAI-1. DISCUSSION/CONCLUSION: Our findings suggest that the polymorphism of codon 72 of the TP53 gene was significantly associated with tumor stage (p < 0.023). In conclusion, we showed a gene-gene interaction between TP53 and PAI-1, in combination, using the MDR method.

Expression Patterns of CXCL12 and its Receptor in Colorectal Carcinoma.

BACKGROUND: Stromal cell-derived factor-1 (also called CXCL12) and its receptor, CXCR4, have a key role in the pathogenesis and tumorigenesis of various cancers. The aims of the current study were to quantitatively examine the expression of CXCR4 and CXCL12 genes in colorectal cancer and to correlate their expression degree with clinicopathological features. METHODS: Tumor tissue samples were collected from 47 patients with CRC. Total RNA was isolated from resection tissues and real-time PCR analysis was performed to examine mRNA levels of CXCL12 and CXCR4 genes. RESULTS: No significant differences were observed for both CXCL12 and CXCR4 between tumor tissues and the adjacent non-affected tissues, although a borderline significant correlation (p = 0.052) were detected between gene expression of CXCL12 and CXCR4 in tumor tissues. Our results also indicated that there was no significant correlation between expression pattern of CXCL12/CXCR4 and clinicopathological variables. CONCLUSIONS: Our data showed that CXCL12 and CXCR4 are expressed simultaneously in colorectal carcinoma tissues, suggesting that expression of these chemokines and corresponding receptors may play a pivotal role in colorectal tumorigenesis, although it cannot be as a predictive factor for disease progression.

Analysis of the Association between MDM4 rs4245739 Single Nucleotide Polymorphism and Breast Cancer Susceptibility.

BACKGROUND: MDM4 is a negative regulator of the p53 tumor suppression pathway. Recent studies have revealed that the rs4245739 A>C polymorphism of MDM4 in the 3-untranslated region makes it a miR-191 target site which leads to lower MDM4 expression. This study is aimed to detect if rs4245739 single nucleotide polymorphism (SNP) of the MDM4 gene influences the breast cancer development in Iranian-Azeri women. METHODS: Blood samples were taken from 260 healthy controls and 220 breast cancer women with ethnicity of Iranian-Azeri. Genotyping was done using Tetra-ARMS PCR. RESULTS: Alleles of MDM4 rs4245739 SNP had no significant different frequency between patients and controls (p > 0.05). Additionally, genotypes of MDM4 rs4245739 SNP did not increase or decrease breast cancer risk in patients when compared to healthy women. Also, there was no significant association between the alleles of MDM4 rs4245739 SNP and clinicopathological factors (p > 0.05). CONCLUSIONS: Considering the lack of association between MDM4 rs4245739 polymorphism and breast cancer, rs4245739 polymorphism of this gene seems to have no significant role in the pathophysiology of the disease.

STC1 and NF-κB p65 (Rel A) is Constitutively Activated in Colorectal Cancer.

BACKGROUND: Stanniocalcin-1 (STC1) and nuclear factor (NF)-κB subunit p65 transcription factor are involved in various types of human malignancies. The roles of STC1 and NFκB-p65 in colorectal cancer (CRC) are still not fully understood. We investigated expression levels of NF-κB p65 and STC1 and also correlations between STC1 and NF-κB p65 expression and clinicopathological features in CRC. METHODS: Tumor tissue samples were collected from 48 patients with CRC. RT-PCR and Real-time PCR analysis was performed to examine mRNA levels of STC1 and NF-κB p65. RESULTS: The relative mRNA levels of STC1 and NF-κB p65 were significantly higher in tumor tissues than in adjacent mucosa (p = 0.025 and p = 0.044, respectively). The data also showed that STC1 and NF-κB p65 mRNA levels were not significantly associated with clinicopathological characteristics. In addition, there was no association between expression levels of STC1 and NF-κB p65 in tumor samples. CONCLUSIONS: Our data indicate that STC1 and NF-κBp65 is activated constitutively in colorectal carcinoma tissues, suggesting that activation of these factors might play an important role in colorectal tumorigenesis. Future studies should examine STC1 and NF-κBp65 as a molecular target for the treatment of CRC.

The evaluation of the possibility of Li-Fraumeni syndrome in cancer patients in East Azarbaijan Province of Iran.

INTRODUCTION: In 1969, Li-Fraumeni syndrome (LFS), which is a rare cancer predisposition syndrome, was reported for the first time. The main problem in LFS is the mutation in the TP53 gene, which is a crucial tumor suppressor gene in the cell cycle. A hereditary syndrome is inherited in an autosomal dominant pattern. There is a significant correlation between this syndrome and various cancers such as sarcoma, breast cancer, brain tumors, and different other types of malignancies. This study aimed to identify the possibility of LFS in cancer patients in the East Azarbaijan, Iran. METHODS: In this experimental study, 45 children with cancer in the Northwest of Iran were investigated for LFS. DNA was extracted from the whole blood cells using the salting-out method. The region within the exons 5-8 of the TP53 gene has been replicated via Polymerase Chain Reaction (PCR) method. The PCR products were sent for Sanger sequencing, and finally, the data were analyzed by Chromas software. RESULTS: In the studied probands, in 12 (26.67%) cases, polymorphisms in Exon 6 and Introns 6 and Intron 7 were identified, and no mutation was observed in exons 5-8 of the TP53 gene. CONCLUSION: Our results show that there were no mutations in exons 5-8 of the TP53 gene as an indication of LFS possibility in these families. Further studies are needed to be done in a bigger population, and Next-Generation Sequencing (NGS) needs to be done to evaluate the whole genome of these patients to complete our data.

Let-7a-3p overexpression increases chemosensitivity to carmustine and synergistically promotes autophagy and suppresses cell survival in U87MG glioblastoma cancer cells.

In terms of primary brain tumors, glioblastoma is one of the most aggressive and common brain tumors. The high resistance of glioblastoma to chemotherapy has made it vital to find alternative treatments and biological mechanisms to reduce the survival of cancer cells. Given that, the objective of the present research was to explore the potential of let-7a-3p when used in combination with carmustine in human glioblastoma cancer cells. Based on previous studies, the expression of let-7a is downregulated in the U87MG cell line. Let-7a-3p transfected into U87MG glioblastoma cells. Cell viability of the cells was assessed by MTT assay. The apoptotic induction in U87MG cancerous cells was determined through the utilization of DAPI and Annexin V/PI staining techniques. Moreover, the induction of autophagy and cell cycle arrest was evaluated by flow cytometry. Furthermore, cell migration was evaluated by the wound healing assay while colony formation assay was conducted to evaluate colony formation. Also, the expression of the relevant genes was evaluated using qRT-PCR. Transfection of let-7a-3p mimic in U87MG cells increased the expression of the miRNA and also increased the sensitivity of U87MG cells to carmustine. Let-7a-3p and carmustine induced sub-G1 and S phase cell cycle arrest, respectively. Combination treatment of let-7a-3p and carmustine synergistically increased arrested cells and induced apoptosis through regulating involved genes including P53, caspase-3, Bcl-2, and Bax. Combined treatment with let-7a-3p and carmustine also induced autophagy and increased the expression of the ATG5 and Beclin 1 (ATG6). Furthermore, let-7a-3p combined with carmustine inhibited cell migration via decreasing the expression of MMP-2. Moreover, the combination therapy decreased the ability of U87MG to form colonies through downregulating CD-44. In conclusion, our work suggests that combining let-7a-3p replacement therapy with carmustine treatment could be considered a promising strategy in treatment and can increase efficiency of glioblastoma chemotherapy.

Evaluating the association of rs6983267 polymorphism of the CCAT2 gene with thyroid cancer susceptibility in the Iranian Azeri population.

Thyroid cancer is the most common malignancy of the endocrine system. LncRNAs play critical role in various cellular processes and are associated with several diseases. CCAT2 is a lncRNA molecule overexpressed in thyroid cancer. Single nucleotide polymorphisms in CCAT2 gene can cause changes in the structure and function of CCAT2 transcripts and susceptibility to several diseases. This study aimed to evaluate the association of rs6983267 in CCAT2 gene with thyroid cancer susceptibility in the Azeri population of Iran. In this "case-control" study, genomic DNA was extracted from peripheral blood of 102 individuals affected by thyroid cancer and 103 healthy individuals as controls. Genotyping was performed using TETRA-ARMS polymerase chain reaction. Statistical analysis showed no significant association between genotypes and/or alleles with the occurrence of thyroid cancer in the studied population, patients' gender, and tumor type. Nevertheless, we found that the allelic and genotypic distribution of this SNP was associated with the size of thyroid tumors in patients. It is assumed that investigating more individuals from both case and control group may further determine the genotypic and allelic frequencies of this SNP locus in Iranian-Azeri population.

An overview on display systems (phage, bacterial, and yeast display) for production of anticancer antibodies; advantages and disadvantages.

Antibodies as ideal therapeutic and diagnostic molecules are among the top-selling drugs providing considerable efficacy in disease treatment, especially in cancer therapy. Limitations of the hybridoma technology as routine antibody generation method in conjunction with numerous developments in molecular biology led to the development of alternative approaches for the streamlined identification of most effective antibodies. In this regard, display selection technologies such as phage display, bacterial display, and yeast display have been widely promoted over the past three decades as ideal alternatives to traditional methods. The display of antibodies on phages is probably the most widespread of these methods, although surface display on bacteria or yeast have been employed successfully, as well. These methods using various sizes of combinatorial antibody libraries and different selection strategies possessing benefits in screening potency, generating, and isolation of high affinity antibodies with low risk of immunogenicity. Knowing the basics of each method assists in the design and retrieval process of antibodies suitable for different diseases, including cancer. In this review, we aim to outline the basics of each library construction and its display method, screening and selection steps. The advantages and disadvantages in comparison to alternative methods, and their applications in antibody engineering will be explained. Finally, we will review approved or non-approved therapeutic antibodies developed by employing these methods, which may serve as therapeutic antibodies in cancer therapy.

Investigating the ACE2 polymorphisms in COVID-19 susceptibility: An in silico analysis.

BACKGROUND: Novel coronavirus (SARS-CoV-2) became an epidemic disease and lead to a pneumonia outbreak first in December 2019 in Wuhan, China. The symptoms related to coronavirus disease-19 (COVID-19) were different ranging from mild to severe lung injury and multi-organ failure symptoms, eventually leading to death, especially in older patients with other co-morbidities. The receptor of this virus in the human cell is angiotensin-converting enzyme 2 (ACE2). METHODS: In this paper, we aimed to perform an in silico analysis of the frequently studied variants of the ACE2 gene and determine the effects of the variants in mRNA secondary structure and binding affinity of cellular factors. Fourteen single-nucleotide polymorphisms were selected based on previous studies and investigated. RESULTS: All of the variants were analyzed in the RNAsnp database and three revealed a significant p-value. The spliceAid2 database prediction showed that 7 out of 14 SNPs caused an alteration in a way that only the wild or mutated form was able to bind to proteins. The latter database also reported that three SNPs produces a dual form in which different specific proteins can bind to the sequence in a specific form (either wild or mutated form). CONCLUSION: Altogether, these estimations revealed the potential of variants in manipulation of the final stable form of ACE2 that can lead to different COVID-19 susceptibility.

Arsenic exposure, dermatological lesions, hypertension, and chromosomal abnormalities among people in a rural community of northwest Iran.

Chronic exposure to arsenic compounds is one of the major public-health problems in many developing and some developed countries. The aim of this study was to investigate the effects of chronic exposure to arsenic on dermatological lesions, hypertension, and chromosomal abnormalities among people in a community in the northwest of Iran. The occurrence of dermatological lesions, hypertension, and chromosomal abnormalities was investigated in two groups: Ghopuz village, including 101 subjects with chronic exposure to arsenic in drinking-water and Mayan village, including 107 subjects with no exposure. Daily/yearly absorbed amounts of arsenic were calculated for all subjects. Cumulative arsenic index for each individual was then estimated on the basis of age, water consumption, and location of residence. Arsenic concentration in drinking-water sources in Ghopuz and Mayan villages was 1031 +/- 1103 microg/L and non-detectable respectively. The mean systolic blood pressure in the exposure group [n=137, 95% confidence interval (CI 132-142)] was significantly higher than that in the control group (n=107, 95% CI 99.9-114). A similar significant difference was observed for diastolic blood pressure (exposed: n=82, 95% CI 79-85 vs non-exposed: n=71, 95% CI 66-75). The incidence of hyperkeratosis was 34 times higher among the exposure group compared to the control subjects [odds ratio (OR)=34, p<0.001)]. A significant difference was also observed in the occurrence of skin-pigmentation between the two groups (OR=2.4, p<0.007). Location and severity of the pigmentations were statistically different between the two groups. Twenty-five percent of the subjects in the exposure group showed chromosomal abnormalities (p=0.05). Arsenic exposure was a serious health problem in the region. More studies are needed to investigate the long-term effects and dose-response relationship of arsenic in the region and similar areas. Wide-ranging monitoring programmes for drinking-water sources should be implemented by public-health authorities.

beta-Thalassemia mutations in the Iranian Kurdish population of Kurdistan and West Azerbaijan provinces.

The aim of this study was to investigate the prevalence and spectrum of beta-thalassemia (beta-thal) mutations in the population of Sunni Muslim Kurds in western Iran and to set up a prenatal diagnostic laboratory. Sixty unrelated Kurdish beta-thal patients identified in hematology clinics from different cities were studied. The mutations in 120 chromosomes were studied by polymerase chain reaction-amplification refractory mutation system and direct sequencing methods. We found fifteen beta-thal mutations, and IVS-II-1 (G>A) was the most frequent, comprising 35% of all mutations. Other common mutations were frameshift codons 8/9 (+G) 15.7%, IVS-I-1 (G>A) 8%, FSC 5 (-CT) 6.7%, FSC 8 (-AA) 6.7%, and IVS-I-110 (G>A) 6%. This is the first comprehensive study in this region and could provide a reference for prenatal testing and genetic counseling in this population.

Homozygosity for a rare beta 0-thalassemia mutation [frameshift codons 25/26 (+T)] causes beta-thalassemia intermedia in an Iranian family.

The severity of beta-thalassemia (beta-thal) is remarkable for its variability in different populations, even in different patients. We studied a family from Azerbaijan Province, Northwestern Iran, who had a rare beta(0)-thal mutation, namely the frameshift codons (FSC) 25/26 (+T), originally reported in Tunisia. Unlike the Tunisian family, in our family the mutation was a beta(0) type and the affected members were dependent and independent of blood transfusions. This mutation was linked to the -158 (C>T) polymorphism on the (G)gamma-globin gene (XmnI marker) and two other polymorphisms in the (A)gamma-globin promoter at position +25 (G>A) and -588 (G>A). Deletions in the alpha- and beta-globin gene clusters were excluded in all samples. This is the first description of the FSC 25/26 mutation in Iran. The results of this study emphasize the complexity of genetic interactions that underlie the phenotype of beta-thal intermedia and highlight the importance of the regulation of hemoglobin (Hb) F production in the beta-thal syndromes. Simultaneous inheritance of some loci that interfere with the elevation of Hb F probably caused them to have high levels of total Hb and to be transfusion independent.

Is the frameshift codons 8/9 (+G) [FSC 8/9 (+G)] beta-thalassemia mutation, detected by the polymerase chain reaction-amplification refractory mutation system, really FSC 8/9 (+G)?

There are several polymerase chain reaction (PCR)-based approaches for the analysis of known mutations. The PCR-amplification refractory mutation system (PCR-ARMS) is one of the best known and frequently used for the detection of beta-thalassemia (beta-thal) mutations. However, there is an important point to be considered when searching for the frameshift codon (FSC) 8 (-AA) and FSC 8/9 (+G) mutations. Whereas the primer is specific for the FSC 8 mutation only, the primer for the FSC 8/9 mutation does not discriminate between the FSC 8/9 and FSC 8 mutations. Thus, the high number of FSC8/9 mutations reported in countries like India, Pakistan and some regions of Iran may be due to the use of the FSC 8/9 primer, without taking the FSC 8 mutation into account. It is thus advisable to test for FSC 8 before the FSC 8/9 mutation whenever PCR-ARMS is the method of investigation for these two mutations.

Haplotype and linkage disequilibrium of TP53-WRAP53 locus in Iranian-Azeri women with breast cancer.

Among the cancer susceptibility genes, TP53 is one of the crucial genes involved in cell cycle regulations and, therefore, it greatly affects breast cancer initiation and progression. In addition, WRAP53-a natural antisense transcript-regulates TP53 transcription and, as a protein, modulates the normal cell cycle, which results in breast cancer susceptibility. In this study, we aimed to analyze a haplotype comprising four SNPs, including rs1042522, rs17878362, rs2287499, and rs2287498, which are located at 5' regions of the TP53 and WRAP53 genes, in 118 patients and 110 healthy controls of the Iranian-Azeri population. In silico studies were conducted using the SIFT, Polyphen2, Fanthmm, RNAsnp, and SNP&GO online servers. Linkage disequilibrium (LD) and D' for each combination of the markers were calculated via the Haploview program. Our results showed that the GA1CC haplotype was the most frequent in the studied population. Additionally, no significant LD between any pairwise haplotypes was observed. The GA1CC and CA2GC haplotypes were significantly associated with breast cancer susceptibility. Moreover, the in silico analysis revealed the negative effects of rs2287499 and rs1042522 on WRAP53 and P53, respectively. In conclusion, the CA1GC haplotype was strongly identified as a breast cancer risk factor, and the GA1CC haplotype was assumed to be a protective factor against breast cancer risk. Hence, these markers may potentially be used as molecular prognostic and predictive biomarkers for breast cancer.

Dysregulated KDR and FLT1 Gene Expression in Colorectal Cancer Patients.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly-diagnosed malignancies throughout the world and the fourth-leading cause of cancer deaths globally. Angiogenesis and the resultant tumor neovascularization is a well-known cancer hallmark. Here we investigated the expression of FLT1 and KDR, the influential genes in angiogenesis regulation, in CRC patients. METHODS: We assessed FLT1 and KDR mRNA expression in 47 CRC samples and matched adjacent noncancerous tissues (ANCT) by quantitative real-time PCR. The Spearmen correlation coefficient and receiver operating characteristic (ROC) curves were also examined. RESULTS: Both genes were expressed at significantly greater levels in CRC tissues than in ANCT (p < 0.05). A significant association was found between KDR expression and disease stage and lymph status in CRC patients. Furthermore, the Spearman correlation demonstrated a moderate correlation between FLT1 and KDR expression in CRC samples. Finally, ROC curve analysis demonstrated that FLT1 had the greatest sensitivity (85.1%), while the greatest specificity was achieved by a combination of the two genes. CONCLUSION: The dysregulated FLT1 and KDR expression, in addition to the observed correlation and ROC curve results, indicate the critical importance of angiogenesis among the cancer pathways in CRC. These data can broaden our current knowledge of angiogenesis in CRC to improve disease diagnosis and patient treatment.

Molecular spectrum of beta-thalassemia mutations in Northwestern Iran.

Beta-thalassemia (beta-thal) is a hereditary autosomal disorder with decreased or absent beta-globin chain synthesis. This study was designed to identify the common and rare beta-thal mutations in the Azerbaijan provinces, Northwestern Iran, and to set up a prenatal diagnostic laboratory. One hundred unrelated patients with known beta-thal major and intermedia, registered with the thalassemia clinics in the provincial capitals of Tabriz and Ardebil, were included. Mutations were studied in 200 chromosomes, by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) and direct sequencing methods. We found 17 beta-thal mutations in this region of Iran. The results showed that IVS-II-1 (-->GA) was the most frequent mutation, comprising 21% of all mutations. Other common mutations were IVS-I-110 (-->GA) 18%, frameshift codons (FSC) 8/9 (+G) 14.5%, FSC 8 (-AA) 8% and IVS-I-1 (GA) 7.5%. This is the first comprehensive study in this region and could be useful for developing a beta-thal molecular screening in Azerbaijan-Iran.

Lack of Associations of the MDM4 rs4245739 Polymorphism with Risk of Thyroid Cancer among Iranian-Azeri Patients: a Case-Control Study

Background and Aim: MDM4, a negative regulator of the p53 tumor suppression pathway, has been demonstrated to be overexpressed in a variety of human cancers. Research has revealed that the rs4245739 A>C polymorphism of MDM4 in the 3'-untranslated region makes it a miR-191 target site, leading to lower MDM4 expression. This study aimed to detect if the rs4245739 single nucleotide polymorphism (SNP) impacts on thyroid cancer (TC) development in Iranian-Azeri patients. Materials and Method: Blood samples were taken from 232 healthy controls and 130 TC patients of Iranian-Azeri ethnicity. For genotyping, Tetra-ARMS PCR was performed. SPSS for Windows (version 22.0, IBM SPSS Inc., USA) and the SHEsis online software were used for data analysis. Results: Alleles of MDM4 rs4245739 SNP demonstrated no significant different in frequencies between patients and controls (p>0.05). Additionally, genotypes of MDM4 rs4245739 SNP did not increase or decrease TC risk in patients compared with healthy subjects. Conclusion: Considering the lack of any observed association between the MDM4 rs4245739 polymorphism and TC, we conclude no significant role in the pathophysiology of the disease.

mRNA expression of nuclear factor of activated T-cells, cytoplasmic 2 (NFATc2) and peroxisome proliferator-activated receptor gamma (PPARG) transcription factors in colorectal carcinoma.

Transcription factors are involved in cell cycle and apoptosis regulation and thus have a key role in the carcinogenesis of different tumors. Nuclear factor of activated T-cells, cytoplasmic 2 (NFATc2) and peroxisome proliferator-activated receptor gamma (PPARG) transcription factors are important in the carcinogenesis of colorectal cancer (CRC). In this study, we examined whether the expression of NFATc2 and PPARG genes is significantly altered during the carcinogenesis of CRC. A total of 47 tumor samples and matched normal tissue margins were collected during surgery from patients with CRC. In addition, three CRC cell lines (HCT119, SW480, and HT29) and healthy cell line were used. After total RNA extraction and cDNA synthesis, mRNA expression levels of NFATc2 and PPARG were examined by real-time polymerase chain reaction. The results showed that NFATc2 is overexpressed in the tumor tissues compared with normal tissue margins (p ≤ 0.05). However, the mRNA expression levels of PPARG were not significantly different between the tumor tissues and tissue margins. Our results indicate that NFATc2 may be used as an early diagnostic or predictive biomarker for CRC as well as a therapeutic target, providing that upcoming studies confirm these results.

Association of endothelial nitric oxide synthase gene variants (-786 T>C, intron 4 b/a VNTR and 894 G>T) with idiopathic recurrent pregnancy loss: A case-control study with haplotype and in silico analysis.

OBJECTIVE(S): Many lines of evidence suggest that reduced production of nitric oxide (NO) due to single nucleotide polymorphisms in endothelial nitric oxide synthase (eNOS) gene may affect the implantation and maintenance of pregnancy. Accordingly, our objective was to investigate whether the eNOS polymorphisms (-786 T>C, intron 4 b/a VNTR and 894 G>T) and haplotypes may be associated with increased susceptibility to recurrent pregnancy loss (RPL). STUDY DESIGN: A total of 130 women with a history of two or more unexplained consecutive first trimester miscarriages and 110 ethnically matched women with at least two normal pregnancies and no history of pregnancy loss were included in the study as cases and controls, respectively. To identify the genotypes, we used polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods In addition, an in silico analysis was conducted to predict the possible effects of the eNOS 894 G>T polymorphism on the structure and function of eNOS mRNA and protein using prediction servers. RESULTS: Our findings revealed that the prevalence of eNOS -786 T>C polymorphism, eNOS -786C allele and TC+CC genotype in cases were significantly higher than those in healthy controls (p<0.05). Also, the combination genotypes -786TT/4b4a and -786TT/894GG were significantly associated with reduced risk of RPL. We also found that the C-4a-G haplotype of the eNOS gene studied polymorphisms was significantly associated with a predisposition to RPL (odds ratio, 3.219; 95% confidence interval, 1.649-6.282; p=0.0003). The in silico analysis showed that the eNOS 894 G>T polymorphism couldn't affects eNOS mRNA and protein significantly. CONCLUSION: Our findings provide evidence to support the hypothesis that eNOS -786 T>C polymorphism and the -786C-4a-894G haplotype are associated with the high risk of RPL.