Amplification Refractory Mutation System PCR Research Articles

TGF-β1 promoter functional gene polymorphism -509 C/T in the maternal susceptibility to recurrent pregnancy loss in South Indian women

Transforming growth factor beta 1 (TGF-β1) is an anti-inflammatory pleiotropic cytokine that regulates implantation and adhesion of trophoblasts to the extracellular matrix. It regulates the balance of Th1/Th2 cytokines and the generation of anti-inflammatory peripheral regulatory T cells (FOXP3 + Tregs), which is necessary for a healthy pregnancy. Single nucleotide polymorphisms (SNP) affecting TGF-β1 production/function may predispose to pregnancy loss. The aim of this study is to evaluate the association between the TGF-β1 -509C/T polymorphism, its serum concentrations, and recurrent pregnancy loss. The study comprised 150 RPL cases and 150 healthy controls for genotyping TGF-β1 -509C/T polymorphism using the Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). Serum concentrations of TGF-β1 protein were estimated by enzyme-linked immunosorbent assay. From our study, women with CT genotype exhibited a protective role against RPL [OR (95 % CI) = 0.48(0.30–0.76), p = 0.002], while women with TT genotype had a significantly higher risk among RPL patients and exhibited twofold increased risk [OR (95 % CI) = 2.34(1.37–4.01), p = 0.002]. Observation of patient’s follow-up data revealed a significant relation with CT genotype of unfavorable pregnancy outcome (CT vs. CC + TT, OR 2.46, 95 % C.I: 1.11–5.43, p = 0.039). Further, variant allele T was significantly elevated in women with unfavorable pregnancy compared with favorable pregnancy outcome, indicating considerable association of T allele in unfavorable pregnancy outcome (T vs. C, OR: 2.68, 95 % C.I: 1.52–4.74, p = <0.001). Serum concentrations of TGF-β1 have shown significant variation between patients and controls, as well as among RPL patients with favorable and unfavorable pregnancy outcome in correspondence to each genotype CC, CT, and TT (p < 0.05). Furthermore, genotype-dependent variation was observed within both the patient and control groups; however, this variation was not observed among RPL women with favorable and unfavorable pregnancy outcome. Our investigation revealed a significant influence of TGF-β1C-509 T polymorphism on RPL risk in South Indian women.

Genetic Variation in Microrna-423 and Microrna 137 Genes among Patients with Breast Cancer

Aim MicroRNAs (miRNAs) are a family of non-coding RNAs that have been suggested as novel markers or therapeutic targets for breast cancer. In current study, we aimed to investigate the prevalence of microRNA-423 rs6505162C/T and the VNTR length of the MIR 137 gene (rs58335419) with regards to breast cancer susceptibility in Iraqi women. Methods: In this study, 190 individuals took part, out of which 110 were breast cancer patients and 80 were healthy controls. The amplification refractory mutation system PCR method (ARMS-PCR) was used to determine the polymorphism in the microRNA-423 (rs6505162) gene. While the VNTR length of the MIR 137 gene (rs58335419) was identified using PCR. The Graph Pad Prism 9 software was used to conduct the statistical analysis. Results: the allele frequency of T in the microRNA-423 rs6505162 was significantly associated with breast cancer (odds ratio [95% confidence interval] 2.333 [1.519-3.600], Risk factor [95% confidence interval]) 1.400 [1.184-1.655], p &lt; 0.0001). On the other hand, all the allele frequencies in the MIR 137 gene (rs58335419) were not found to be associated with breast cancer. Conclusion: Based on the results, the study suggests that microRNA-423 rs6505162C/T could be useful markers for diagnosing breast cancer, whereas the MIR 137 gene (rs58335419) is not useful for diagnosing breast cancer.

Prospective analysis of the effects of RAS mutation on local tumor progression and overall survival after radiofrequency ablation for colorectal liver metastases

Objectives To investigate the effects of RAS mutation on local tumor progression (LTP) and overall survival (OS) after radiofrequency ablation (RFA) for colorectal liver metastases (CRLMs). Methods This prospective study consecutively enrolled patients with CRLM who underwent ultrasound-guided RFA between June 2020 and July 2022. RAS mutation status was analyzed via the amplification refractory mutation system PCR method. The safety margin was measured via an anatomic landmark-based manual assessment method. Factors associated with time-to-LTP and OS were assessed via univariate and multivariate analyses. Results A total of 163 patients with 284 ablated tumors were included. Sixty-nine patients (42.3%) had RAS mutations. Compared with the RAS-wild-type group, the RAS-mutant group had a higher 2-year cumulative LTP rate (20.0% vs. 9.8%, p = 0.016) and a worse 2-year OS rate (55.7% vs. 79.8%, p < 0.001). Compared with RAS-wild-type tumors, RAS-mutant tumors had a significantly greater 2-year cumulative LTP rate in the subgroups with safety margins <5 mm (47.6% vs. 24.9%, p = 0.034) and ≥5 mm (8.6% vs. 1.9%, p = 0.045). In the multivariate Fine–Gray analysis, RAS mutation (SHR, 2.42; p = 0.007) and a safety margin <5 mm (SHR, 7.94; p < 0.001) were found to be independent predictors of the time-to-LTP. According to the multivariate Cox analysis, RAS mutation (HR, 2.13; p = 0.010), CEA >5 ng/mL (HR, 2.29; p = 0.017), and extrahepatic metastases (HR, 2.04; p = 0.016) were independent predictors of shorter OS. Conclusions RAS mutation was an independent predictor of LTP and OS after RFA for patients with CRLM. RAS-mutant tumors require wider safety margins.

Genetic Polymorphism of Interferon-Gamma +874T/A as a Risk Factor for Pulmonary Tuberculosis in Cirebon, Indonesia

Background: Tuberculosis (TB) is one of the leading causes of death globally, caused by Mycobacterium tuberculosis. With 10% of all cases worldwide in 2022, Indonesia is the second-largest contributor of tuberculosis cases. IFN-γ gene polymorphism is one of the factors that have been studied extensively for its association with TB. Aims: To analyze IFN-γ +874T/A gene polymorphism as a risk factor for pulmonary tuberculosis in Cirebon. Methods: Observational analysis with case control design was used in this study. Thirty-two tuberculosis patients as cases and 32 healthy controls at RSUD Waled were collected and performed DNA extraction to evaluate the polymorphism by using Amplification-refractory mutation system–polymerase chain reaction (ARMS–PCR). Statistical comparison was performed by using Pearson Chi-square and Kruskal Wallis test. Mann-Whitney U test was done for post hoc test. Odds ratio was calculated to see the risk of the assessed variables, including genotype, allele frequency, and the presence of polymorphism. Results: In the case group, the frequency of TT genotype was 3 (9.4%), TA genotype was 26 (81.3%), AA genotype was 3 (9.4%). In the control group, the frequency of TT genotype was 12 (37.5%), TA genotype was 17 (53.1%), AA genotype was 3 (9.4%). A significant difference (p=0.034) was found among 3 genotype groups. Post hoc test revealed that TT and TA was the pair with significant difference (p=0.007). In addition, TA polymorphism was significantly associated (p=0.004) with tuberculosis (OR=6.614; CI95% = 1.660-26.349). Conclusion: IFN-γ +874 TA gene polymorphism is associated with pulmonary tuberculosis in the population of Cirebon, West Java, Indonesia. Received: 25 September 2024 | Reviewed: 23 October 2024 | Revised: 10 November 2024 | Accepted: 30 November 2024

Biochemical Investigation of the Association of Apolipoprotein E Gene Allele Variations with Insulin Resistance and Amyloid-β Aggregation in Cardiovascular Disease.

This article investigates the intricate associations between apolipoprotein E (APOE) gene alleles variation, insulin resistance (IR) and amyloid-β aggregation in cardiovascular disease (CVD) patients. A cohort of 250 patients exhibiting the symptoms of CVD and 50 control subjects participated in this study. After applying the stringent inclusion and exclusion criteria, the diseased group was further stratified into three categories: CVD+ (Alzheimer's disease) AD, CVD + (diabetes mellitus) DM and CVD + DM + AD. Blood samples were collected from all recruited participants for the biochemical analyses of lipid profile, glycaemic status, liver function enzymes, inflammatory and oxidative stress biomarkers. Tetra amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) was employed for APOE gene analysis. Biochemical evaluations revealed the significant elevations in the serum levels of glucose, liver enzymes, interleukin-6 (IL-6), cholesterol, low-density lipoproteins (LDL), triglycerides (TG) and malondialdehyde (MDA) in CVD + DM + AD group. Conversely, the serum levels of insulin, HDL and hexokinase decreased in CVD + DM + AD group compared to the controls and other CVD groups. Tetra ARMS-PCR results indicated a higher percentage of the risk allele in CVD + DM + AD group when compared with the other groups. Our study elucidates the multifaceted cardiovascular risk factors contributing to IR and AD in CVD patients. Age-related risk factors, prevalence of APOE risk alleles and the impact of statin use on AD incidences were identified. These findings underscore the need for tailored preventive measures, particularly in APOEε4 and ε3/ε4 carriers with CVD. Further studies should delve into the knowledge-based protocols to comprehend the underlying mechanisms. Focusing on the therapeutic targets to prevent or delay DM and AD progression in CVDs, especially in APOEε4 carriers, is essential.

The Association of Interleukin 6 Receptor Gene Variant (Rs4129267) with Susceptibility to Ankylosing Spondylitis and Soluble IL-6 Receptor Level in Iranian Patients

Objectives: Ankylosing spondylitis (AS) is an axial skeleton-related auto-inflammatory arthritis. Interleukin 6 (IL-6) and its receptor (IL-6R) play a role in the etiopathology of AS. The recent genome-wide association studies (GWASs) revealed that rs4129267; an intrinsic variant of IL6-R is associated with the risk of AS. We aimed to study the rs4129267 IL6R variant in Iranian AS patients and the correlation of this variant with the soluble interleukin-6 receptor (sIL-6R) serum level. Materials and Methods: In this case-control study, we genotyped rs4129267 IL6-R variant in extracted DNA from 498 AS patients and 495 matched healthy controls by Amplification-Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR). The serum level of sIL-6R and IL-6 was also analysed in 39 controls and 42 AS patients by the ELISA method. The correlation between rs4129267 IL-6R variants and patients’ clinical manifestations and sIL-6R level was also investigated. Results: In comparison to the AS individuals, C allele were significantly less prevalent in the healthy control group (OR = 1.23 [1.01 –1.48], P value = .034). Patients had significantly elevated levels of IL-6 serum levels. There was a significant correlation between the presence of the rs4129267 T allele and increased sIL-6R level. Conclusions: Our study confirmed the significant association between the rs4129267 T variant and the protection against AS disease susceptibility. Increased IL-6 level and reduced frequency of sIL-6R rs4129267 T allele in patients which contributes to reduced sIL-6R serum level, highlights the significant role of IL-6 signalling pathway in AS pathogenesis.

Association of ANRIL Gene Polymorphisms with Gastric Cancer Risk: A Case-Control Study.

Background: Gastric cancer's (GC) cause is unknown, but its complexity indicates that, in addition to environmental factors, it may have genetic origins. Scientists are studying single-nucleotide polymorphisms (SNPs) in the antisense noncoding RNA in the INK4 locus (ANRIL) gene, which encodes a long noncoding RNA molecule. They found a link between the ANRIL gene product and some polymorphisms and GC, suggesting genetic changes may lead to precancerous conditions. Methods: In a case-control research that included 250 patients with GC and 210 controls who were age- and gender-matched, four SNPs within the ANRIL gene were genotyped. These SNPs were rs1333049, rs496892, rs2383207, and rs2151280. Tetra-primer amplification refractory mutation system-PCR was utilized to carry out the process of genotyping. Results: It was found that the chance of developing GC was connected with three SNPs rs2151280, rs1333049, and rs496892. Nevertheless, rs2383207 did not demonstrate any meaningful connection. In addition, whereas CCTC and TTCC haplotypes were shown to be less common, certain haplotypes that contained these SNPs (TTCG, TCTC, and TTTC) displayed a considerably higher prevalence in the cancer group in comparison to the control group. Conclusion: This study showed novel associations between specific ANRIL gene polymorphisms (SNPs) and the risk of GC. These findings shed light on the potential role of ANRIL SNPs in GC risk and highlight the need for additional research to clarify the underlying functional processes. Understanding these functional processes might lead to developing novel diagnostic or treatment approaches for this cancer.

Association between microsatellite instability status, clinicopathological features and mitochondrial DNA amplification in patients with colorectal cancer.

The relationship between BRAF-V600E mutations, mitochondrial DNA amplification and microsatellite instability-high (MSI-H) in colorectal cancer (CRC) has yet to be fully elucidated. The aim of the present study was to assess the association between the MSI status and BRAF-V600E gene mutations/clinicopathological features/mitochondrial DNA amplification in CRC. A non-interventional study analysis was performed using the clinicopathological features of 455 patients with CRC. Immunohistochemistry was used to evaluate four mismatch repair proteins (MutS homolog 2, MutS homolog 6, MutL homolog 1 and postmeiotic segregation increased 2), Ki-67 index, and programmed cell death protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) expression. Additionally, PCR coupled with capillary electrophoresis were used to ascertain the MSI status. Moreover, amplification refractory mutation system-PCR was used to detect BRAF-V600E gene mutation and fluorescence in situ hybridization analysis was used to assess mitochondrial DNA. A total of 455 patients were divided into the MSI high (MSI-H) group (n=52) and microsatellite stability (MSS) group (n=403) based on their MSI status. Compared with the results of immunohistochemistry of four mismatch repair proteins, the consistency rate between mismatch repair protein deficiency and MSI was 94.23%. There were significant differences in PD-L1, primary tumor site, clinical stage, degree of differentiation, tumor size, lymph node metastasis and the occurrence of multiple primary tumors between the MSI-H group and MSS group (P<0.05 or P<0.001). However, there were no significant differences for sex, age, PD-1, Ki-67 expression and BRAF-V600E. The 24-60-month survival rate of the patients in the MSI-H group was significantly higher than that of those in the MSS group (P<0.05). Furthermore, the number of mitochondrial DNA was significantly amplified in the MSI-H group. In conclusion, the present study demonstrated that the combined detection of PD-L1 and MSI in patients with CRC can provide more accurate and effective guidance for personalized treatment.

Driver gene alterations in NSCLC patients in southern China and their correlation with clinicopathologic characteristics.

In this study, we aimed to explore the relationship between clinicopathological features and driver gene changes in Chinese NSCLC patients. Amplification refractory mutation system PCR was used to detect the aberrations of 10 driver oncogenes in 851 Chinese NSCLC patients, and their correlation with clinicopathological characteristics was also analyzed. Moreover, three models of logistic regression were used to analyze the association between histopathology and EGFR or KRAS mutations. The top two most frequently aberrant target oncogenes were EGFR (48.06%) and KRAS (9.51%). These were followed by ALK (5.41%), HER2 (2.35%), MET (2.23%), RET (2.11%), ROS1 (1.88%), BRAF (0.47%), NRAS (0.24%), and PIK3CA (0.12%). Additionally, 11 (1.29%) patients had synchronous gene alterations in two genes. The main EGFR mutations were exon 21 L858R and exon 19-Del, which accounted for 45.97% and 42.79% of all EGFR mutations, respectively. Logistic regression analysis showed that the frequency of EGFR mutations was positively correlated with women, non-smokers, lung adenocarcinoma, and invasive non-mucinous adenocarcinoma (IA), and negatively correlated with solid nodule, micro-invasive adenocarcinoma, and solid-predominant adenocarcinoma. KRAS mutations were positively associated with men and longer tumor long diameters and negatively correlated with lung adenocarcinoma (P < 0.05 for all). Our findings suggest that the EGFR mutation frequency was higher in women, non-smokers, lung adenocarcinoma, and the IA subtype in lung adenocarcinoma patients, while the KRAS mutation rate was higher in men and patients with longer tumor long diameter and lower in lung adenocarcinoma patients.

T315I- a gatekeeper point mutation and its impact on the prognosis of chronic myeloid leukemia.

BCR-ABL kinase domain mutations are an important cause of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukaemia (CML) of which T315I is the most treatment-resilient. This study aimed to observe the frequency of T315I and its impact on disease prognosis in terms of progression and survival. Patients with a response which categorized them into warning zone/or who failed to respond to their TKI treatment completely as per the European LeukemiaNet (ELN) were labeled as non-responders. They were assessed for T315I mutation using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and validated via sequencing. Patients were then longitudinally followed for 96months for the prognostic impact of the mutation. Of the 102 non-responders, T315I mutation was detected in 21.6 % of patients with a female preponderance. Almost 59 % of mutation-harbouring patients were labelled as low Sokal risk at baseline. The disease progression into the blastic phase was reported in 58.8 % of mutation-harbouring patients. Overall survival (study period: 96months) was 81.8 % in patients harbouring T315I mutation. Patients in the blastic phase had significant odds of harbouring T315I mutation. Sub-optimal response or failure to TKI treatment indicates the development of resistance due to the presence of T315I mutation or other mutation(s). Early identification will help redirect the patient's treatment.

Analysis of PIK3CA mutations in the primary and recurrent tumors of hormone receptor positive/human epidermal growth factor receptor 2 negative breast cancer.

Our aim was to compare the PIK3CA mutation status in matched primary and recurrent tumors of hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2-) breast cancer (BC) to gain insight into the optimization of patient selection and detection time for PIK3CA-targeted therapy. The data were from 3035 patients with BC diagnosed at the Breast Disease Center, Peking University First Hospital, between January 2008 and December 2017. Matched primary and recurrent samples were profiled using amplification-refractory mutation system-polymerase chain reaction covering 11 mutational hotspots in PIK3CA. PIK3CA mutations were detected in 54.3% primary tumors and 48.6% corresponding recurrences. PIK3CA mutation was detected in 37.5% cases in the locoregional recurrent group and 40.0% of distant metastasis, without a statistical difference. Besides, PIK3CA mutations were concordant in 88.6% of the matched pairs. For patients treated with neoadjuvant chemotherapy, 100% concordance was observed. However, PIK3CA mutation was neither correlated with clinicopathological features nor associated with clinical outcomes. Mutations in PIK3CA in HR+/HER2- BC generally progressed to recurrent tumors. The high concordance rate of PIK3CA mutation status between primary tumors and corresponding recurrences suggests that the detection of primary tumors could be a substitute approach when recurrent samples are not easily obtainable.

Association between Mir-499, Mir-27a, and Mir-146a polymorphisms and their susceptibility to recurrent spontaneous abortion; in silico analysis.

Recurrent spontaneous abortion (RSA) is defined as two or more pregnancy losses before 24 gestational weeks, accounting for 1-3% of fertile couples. A vast majority of single-nucleotide polymorphisms (SNPs) in some microRNA (miRNA) genes can change the miRNA-mRNA interaction and are associated with the risk of RSA. This study was designed to better elucidate the association between miR-27a, miR-499, and miR-146a polymorphisms and RSA risk. SNP genotyping of miR-27a (rs895819), miR-499 (rs3746444), and miR-146a (rs2910164) was performed using polymerase chain reaction (PCR)-restriction fragment length polymorphism and tetra amplification-refractory mutation system PCR in 98 patients with RSA and 105 healthy subjects. Our results showed that the miR-499 rs3746444 and miR-27a rs895819 polymorphisms were significantly associated with RSA risk, whereas no significant differences were observed between the rs2910164 polymorphism and RSA susceptibility. We proposed that the miR-499 rs3746444 and miR-27a rs895819 polymorphisms were correlated with RSA in our population, but the miR-146a rs2910164 variant was not associated with the risk of RSA.

Genetic polymorphisms in FABP2, CYP2E1, and TP53 genes are potentially associated with colorectal cancer susceptibility

Colorectal cancer (CRC) is among the most prevalent cancers with a high mortality rate. Both genetic and environmental factors contribute to CRC development. This study aimed to assess the association of single nucleotide polymorphisms (SNPs) in the fatty acid binding protein-2 (rs1799883), Cytochrome P450 2E1 (rs3813865), TP53 (rs1042522), and Murine double minute 2 (rs1042522) genes with CRC. A cross-sectional case–control study was conducted at the Institute of Molecular Biology and Biotechnology from May 2020 to March 2021, involving CRC patients (N = 100) and controls (N = 100) recruited from the Multan district in Pakistan. Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were employed to investigate the studied SNPs. The association of SNPs in all genes with CRC was examined either individually or in various combinations. Genotypes at three SNPs, rs1799883 in FABP2, rs3813865 in CYP2E1, and rs1042522 in TP53, were found to be associated with the development of CRC, while rs1042522 in MDM2 was not. Patients who were married, smoked, lacked exercise habits or had a family history of CRC were at a greater risk of acquiring the disease. FABP2 gene rs1799883, CYP2E1 gene rs3813865, and TP53 gene rs1042522 polymorphisms are significant in the development of CRC in Pakistani participants.

Association of adiponectin gene single nucleotide polymorphisms with environmental risk factors in type 2 diabetes mellitus: An updated evidence of haplotype-based analysis study

Background and aimAdiponectin (ADIPOQ) gene is considered to be one of the promising players in deciphering the genetic bases of type 2 diabetes. This study investigated the associations between haplotype combinations of three single nucleotide polymorphisms (SNPs) of the ADIPOQ gene and two SNPs of the adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) genes with environmental risk factors for the prediction of T2DM disorder susceptibility in the Iranian population. MethodsThis case-control and cross-sectional study was conducted on 182 patients with T2DM and 155 healthy controls. Genotyping was performed using amplification refractory mutation system-PCR (ARMS-PCR) for rs17300539G/A, rs2241766T/G, and rs1501299G/T of the ADIPOQ gene, rs1342387C/T of the AdipoR1 gene, and rs10773989T/C of the AdipoR2 gene. ResultsAll polymorphisms met the Hardy-Weinberg equilibrium (p> 0.05). The studied SNPs; rs17300539, rs2241766 of the ADIPOQ gene and rs10773989 of the AdipoR2 gene, were significantly associated with an increased risk of T2DM. Two-way ANOVA analysis indicated that GG carriers of rs2241766T/G had a significantly lower waist-to-hip ratio (P= 0.049) and body mass index (P= 0.011) and higher HbA1c (P= 0.048) compared to TT carriers, while TT genotype carriers of rs2241766T/G showed the higher plasma adiponectin concentration compared to TG and GG carriers (P= 0.009 and P= 0.013, respectively). CC carriers of rs10773989T/C displayed a significantly higher LDL level compared to the TT genotype carries (P= 0.036). Also plasma adiponectin concentrations were significantly lower in AA genotype carriers of rs17300539G/A compared to GG and GA genotypes carriers in the control group only (P= 0.005 and P= 0.016, respectively). According to Combined Haplotype ([rs17300539, rs2241766, rs1501299]/[rs17300539, rs2241766, rs1501299]) analysis, GTT-homozygote carriers displayed the highest plasma adiponectin concentration and in contrast, GGG/GTG, ATG/GTG, and GGG/GGG showed the lowest plasma adiponectin concentration in the controls (p> 0.05). ConclusionThe adiponectin gene haplotype combinations were associated with plasma adiponectin concentration in healthy individuals. In T2DM, adiponectin genetic variants displayed less effect on adiponectin plasma concentration.

Association of VEGFA polymorphisms with the risk of oesophageal cancer in Punjab, India: A case-control study.

Background & objectives Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors which stimulates tumour progression induction of endothelial cell migration and division, inhibition of the apoptosis of endothelial cells, induction of serine protease activity and enhancement of vascular permeability. This study aimed to investigate the correlation of VEGF+405G/C,-7C/T and+936C/T polymorphisms with oesophageal cancer risk. Methods DNA samples of 464 subjects (231 sporadic oesophageal cancer affected individuals and 233 controls) were genotyped forVEGF+936C/T,+405G/C and-7C/T polymorphisms. VEGF+936C/T and +405G/C polymorphisms were genotyped by PCR-RFLP method whereas VEGF-7C/T polymorphism was genotyped using Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Results CT genotype of VEGF-7C/T polymorphism was significantly associated with reduced risk of oesophageal cancer. VEGF-7C/T polymorphism was significantly associated with reduced risk of oesophageal cancer underdominant, co-dominant, over dominant and log-additive genetic models in total patients and in the female group. C+936G+405T-7 haplotype was significantly associated with decreased risk (P=0.01)of oesophageal cancer in total patients and also in the male group (P=0.02). Interpretation & conclusions In future, replication of the findings of the present study in a larger sample from different ethnic groups, along with functional analysis, may be insightful for the role of VEGFA polymorphisms in the pathogenesis of oesophageal cancer. Identification of the correlation of VEGF variants with specific therapy in oesophageal cancer may help in better selection of patients and monitoring treatment response in VEGF-therapy.

Potential Association of The Pathogenic Kruppel-like Factor 14 (KLF14) and Adiponectin (ADIPOQ) SNVs with Susceptibility to T2DM.

To evaluate the associations of the pathogenic variants in Kruppel-like Factor 14 (KLF 14) and Adiponectin (ADIPOQ) with susceptibility to type 2 diabetes mellitus (T2DM). Type 2 diabetes mellitus (T2DM) is a pandemic metabolic disease characterized by increased blood sugar and caused by resistance to insulin in peripheral tissues and damage to pancreatic beta cells. Kruppel-like Factor 14 (KLF-14) is proposed to be a regulator of metabolic diseases, such as diabetes mellitus (DM) and obesity. Adiponectin (ADIPOQ) is an adipocytokine produced by the adipocytes and other tissues and was reported to be involved in T2DM. To study the possible association of the KLF-14 rs972283 and ADIPOQ-rs266729 with the risk of T2DM in the Saudi population. We have evaluated the association of KLF-14 rs972283 C>T and ADIPOQ-rs266729 C>G SNV with the risk to T2D in the Saudi population using the Amplification Refractory Mutation System PCR (ARMS-PCR), and blood biochemistry analysis. For the KLF-14 rs972283 C>T SNV we included 115 cases and 116 healthy controls, and ADIPOQ-rs266729 C>G SNV, 103 cases and 104 healthy controls were included. Results indicated that the KLF-14 rs972283 GA genotype and A allele were associated with T2D risk with OR=2.14, p-value= 0.014 and OR=1.99, p-value=0.0003, respectively. Results also ADIPOQ-rs266729 CG genotype and C allele were associated with an elevated T2D risk with an OR=2.53, p=0.003 and OR=1.66, p-value =0.012, respectively. We conclude that SNVs in KLF-14 and ADIPOQ are potential loci for T2D risk. Future large-scale studies to verify these findings are recommended. These results need further verifications in protein functional and large-scale case control studies before being introduced for genetic testing.

Association between Methylenetetrahydrofolate Reductase (MTHFR) and 5-Methyltetrahydrofolate-Homocysteine Methyltransferase Reductase (MTRR) Polymorphisms in Iraqi Patients with COVID-19.

The methylenetetrahydrofolate reductase (MTHFR) gene is an essential gene in the metabolism of folate-homocysteine. Recently, the level of homocysteine was found to be a significant marker in the follow-up of COVID-19 infection. Thus, this study aimed to detect the effect of genetic polymorphisms for single nucleotide polymorphisms (SNPs) (c.66A>G, c.1298A>C, and c.677CT) on COVID-19 infection. Blood samples were collected from 270 patients with COVID-19 in the medical center of Al-Shifa (Baghdad, Iraq) from November 2020 to March 2021. Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique was used for the detection of genotypes of SNPs. The odds ratio (OR) was used to detect the relationship between SNPs and COVID-19 infections. Haplotype analysis was performed by SHEsis software. There was a significant difference between mild/moderate cases and severe/critical cases for ages (35-45), (46-55), and (56-65) years (P<0.0001, P=0.01, and P=0.006, respectively). The results showed significant differences in the T allele for SNP c.677>C (P<0.0001 and OR=4.58). The C allele for SNP c.1298A>C indicated significant differences (P<0.001 and OR=3.15). Besides, the G allele for SNP c.677C>T showed significant differences (P<0.001 and OR=6.64). Consequently, these SNPs showed a predisposition to the development of COVID-19 infection. With regard to the C-A-A, T-A-A and T-C-G haplotypes indicated significant differences between the control and patient groups. The C-A-A was related to a decreased risk and indicated a protective effect against COVID-19 infection development (P<0.0001 and OR=0.218). The increased risk was associated with T-A-A and T-C-G haplotypes and indicated the risk impact on COVID-19 infection development (P<0.0001, P=0.004, and OR=15.5, OR=6.772, respectively). Furthermore, the linkage disequilibrium (LD) for SNPs was studied, and the complete D' value was 99. The genetic polymorphisms of SNPs (c.66A>G, c.1298A>C, and c.677C>T) in the Iraqi population were associated with COVID-19 infection.

Association of polymorphisms within P2RX4 with type 2 diabetes mellitus: a preliminary case-control study

Objective Type 2 diabetes mellitus (T2DM) is a complex heterogenic metabolic with a wide range of etiology. Purinergic receptors have pivotal roles in different processes and are hypothesized to have roles in the pathogenesis of T2DM. Materials and Methods Three hundred subjects affected by T2DM and 300 healthy subjects were genotyped by amplification refractory mutation system polymerase chain reaction (ARMS-PCR). SPSS V16.0 was recruited for statistical analysis. Results The findings showed that the G allele of rs25644A > G increases the risk of T2DM in our population statistically (OR = 1.51, 95% CI = 1.14–1.99, p = 0.003). This allele in some genotype models, including the dominant model, caused an increase in the risk of T2DM. The interaction of genotypes between studied variants in the P2XR4 gene increased the risk of T2DM. Haplotype analysis showed that Ars1169727/Grs25644 haplotype caused an increase in the risk of T2DM. Conclusions The findings suggest that rs25644A > G plays a role in our population’s increased risk of T2DM.

Association of TNFα and IL1β genotypes with the risk of Staphylococcus aureus causing recurrent tonsillitis in a sample of Iraqi patients

Background and aim: Tonsillitis is an inflammation of the tonsils, usually caused by an infection by viruses or bacteria; recurrent tonsillitis occurs when tonsillitis occurs several times a year. The current study aimed to investigate the association of TNFα and IL1β genotypes with the increased risk of recurrent Staphylococcus aureus tonsillitis in a sample of Iraqi patients. Methods: A total of 50 blood samples were collected from patients with recurrent tonsillitis of S. auerus from three hospitals in Najaf city, and 50 samples were collected from apparently healthy volunteers with a free medical history of recurrent tonsillitis as control group. Genomic DNA was extracted from peripheral blood and genotyped for TNF-α 308G/A and IL1β C/T SNP using Nested tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR). Results: The results of the distribution IL β –rs1143627 C/T alleles in S. aureus tonsillitis patients showed CC wild type genotype was in 11 patients (22%), CT genotype was in 30 patients (60%) and the TT genotype was in 9 patients (18 %) also C allele frequency was (0.42), whilst T allele frequency was (0.58) with Odds Ratio (1.507) at significant difference (P≤ 0.01), while prevalent of TNF-α –rs1800629 G/A alleles genotype in S. aureus tonsillitis patients revealed that GG wild type genotype was 37 patients (74%), GA genotype was 13 patients (26%) and AA genotype was 0(0%) also, G allele frequency was (0.87) and A allele frequency was (0.13) with Odds Ratio (1.507) at no significant differences in both the patient and control.

Multiplex fluorescent amplification-refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay.

The common deleterious genetic defects in Holstein cattle include haplotypes 1-6 (HH1-HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single-tube multiplex fluorescent amplification-refractory mutation system (mf-ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf-ARMS PCR method introduced 10 sets of tri-primers optimized with additional mismatches in the 3' end of wild and mutant-specific primers, size differentiation between wild and mutant-specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf-ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf-ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects.