Amplification Refractory Mutation System PCR Research Articles

Comparison of diagnostic methods for detection of BRAFV600E mutation in ameloblastoma.

Ameloblastoma is an aggressively growing, highly recurrent odontogenic jaw tumor. Its association with BRAFV600E mutation is an indication for BRAFV00E-inhibitor therapy The study objective was to identify a sensitive low-cost test for BRAFV600E-positive ameloblastoma. We hypothesized that immunohistochemical staining of formalin-fixed paraffin-embedded tissues for BRAFV600E mutation is a low-cost surrogate for BRAFV600E gene sequencing when laboratory resources are inadequate for molecular testing. Tissues from 40 ameloblastoma samples were retrieved from either formalin-fixed paraffin-embedded blocks, RNAlater™ stabilization solution or samples inadvertently pre-fixed in formalin before transfer to RNAlater™. BRAFV600E mutation was assessed by Direct Sanger sequencing, Amplification Refractory Mutation System-PCR and immunohistochemistry (IHC). BRAFV600E mutation was detected by IHC, Amplification Refractory Mutation System-PCR and Direct Sanger sequencing in 93.33%, 52.5% and 30% of samples respectively. Considering Direct Sanger sequencing as standard BRAFV600E detection method, there was significant difference between the three detection methods (𝜒2 (2) = 31.34, p < 0.0001). Sensitivity and specificity of IHC were 0.8 (95% CI: 0.64-0.90) and 0.9 (95% CI: 0.75-0.99) respectively, while positive predictive value and negative predictive value (NPV) were 0.9 and 0.8 (Fischer's test, p < 0.0001) respectively. Sensitivity and specificity of Amplification Refractory Mutation System-PCR detection method were 0.7 (95% CI: 0.53-0.80) and 0.9 (95% CI = 0.67-0.98) respectively, while PPV and NPV were 0.9 and 0.6 respectively (Fischer's test, p < 0.0001). Low-cost and less vulnerability of IHC to tissue quality make it a viable surrogate test for BRAFV600E detection in ameloblastoma. Sequential dual IHC and molecular testing for BRAFV600E will reduce equivocal results that could exclude some patients from BRAFV600E-inhibitor therapies.

Methylenetetrahydrofolate reductase gene polymorphisms, lipid profiles, and basic renal functional markers as risk for myocardial infarction: A case-control study and haplotype analysis

Myocardial infarction (MI) is a serious cardiovascular disease and the primary cause of mortality, with a complex etiopathology. Identifying the genetic basis of myocardial infarction (MI) is essential for developing personalized medical treatments. This study examined the possible association between polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene and MI. In the study, 120 patients with MI and 120 age-and-sex-matched controls were genotyped for C677T and A1298C MTHFR polymorphisms by the allele-specific or amplification refractory mutation systempolymerase chain reaction (ARMS-PCR). In the case of the C677T polymorphism, the T/T and C/T genotypes were associated with a significantly increased risk of MI under the dominant genetic model (odds ratio (OR)=2.060; P=0.006). Although there was no significant association between the A1298C variant and MI, this polymorphism was linked to a higher level of creatinine in MI patients (P&lt;0.002). A similar association was observed for the C677T polymorphism (P=0.003). An A-T haplotype represented an increased risk for MI (OR=1.630; P=0.014), whereas the A-C haplotype had a protective role (R=0.517; P=0.002). These findings indicate that C677T MTHFR polymorphism is strongly associated with and increased risk of MI, making it a potential genetic risk factor and a possible predictor of MI.

Analysis of EGFR Gene Mutations in Lung Adenocarcinoma in Karamay, Xinjiang

Abstract Objective To investigate the mutation types and mutation rate of the epidermal growth factor receptor (EGFR) gene in patients with lung adenocarcinoma and the clinical features of lung adenocarcinoma with EGFR gene mutations in Karamay, Xinjiang, China. Methods Paraffin-embedded tissue samples of adenocarcinoma patients were collected in the Karamay Central Hospital from March 2016 to June 2019, and mutations in exon 18–21 of the EGFR gene were detected by the allele-specific amplification polymerase chain reaction (Amplification Refractory Mutation System–PCR) method. The relationships between the mutation types, mutation incidence, and clinical features were analyzed. Results Of the 170 patients with lung adenocarcinoma, 83 had EGFR mutations. The total mutation rate of EGFR in patients with lung adenocarcinoma was 48.8%, which included mutations in exons 18 (1.2% [2/170]), 19 (19.4% [33/170]), 20 (2.4% [4/170]), and 21 (20.6% [35/170]). Intriguingly, there was a case with 9 mutations in exons 20 and 21. The mutations in exon 19 of EGFR resulted in the deletion of codons 746 to 750. The main mutation in exon 21 was L858R (91.4% [32/35]). There was no significant difference in exons 19 and 21 mutation rates (P &gt; 0.05). The mutation rate of EGFR in female patients was significantly higher than that in male patients (P &lt; 0.05) but had no correlation with the age, smoking status, and clinical stage of patients with non–small cell lung cancer (P &gt; 0.05). The EGFR mutation rate may be related to the degree of tumor differentiation. Conclusion Among patients with lung adenocarcinoma in Kelamayi (city in Xinjiang), EGFR mutations were more frequently detected in female patients, and the main sites of mutations were exons 19 and 21.

A novel tetra-primer ARMS-PCR for genotyping of the OPRM1 gene rs1799971 variant associated with opioid use disorders

ObjectivesA SNV is a single nucleotide change that can occur at any point in the genome. SNVs are the most common genetic variants that occur in the human genome, and a number of SNVs have been found to be associated with human traits and disease. Researchers genotype SNVs using TaqMan technology, DNA microarray, MALDI-TOF mass spectrometry, and automated sequencing, which are expensive and time-consuming. The OPRM1 gene rs1799971 (A118G) has been identified for its association with Opioid use disorder (OUD). The present study focused on developing a single step identification test using Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) to detect the presence of SNV OPRM1 rs1799971 (A118G). This study was performed to optimize the protocol for the designed four primers and validate it using a total of 52 buccal samples from volunteers who are currently under rehabilitation for the drug abuse disorder.ResultsUtilizing 52 DNA samples, a novel T-ARMS-PCR assay was successfully developed, tested, and validated. The products of the T-ARMS PCR for rs1799971 contained 395 bp as the control band, 186 bp as G allele (variant) and 257 bp as A allele (wild type), which were observed in the gel image. The genotype frequencies for the OPRM1 gene rs1799971 (A118G) were 44% (22/52) of homozygous variant type (GG), 28.9% (15/52) of homozygous wild type (AA) and 28.9% (15/22) of heterozygous (AG). The G allele frequency was 56.7% and A allele frequency was 43.3%.

One-step amplification refractory mutation system-PCR/high-resolution melting curve assay for carrier detection of red blood cell membranopathy caused by common SPTB mutations.

Hereditary pyropoikilocytosis (HPP) is the most common cause of non-thalassemic severe inherited hemolytic anemia in Thai population. Up to 90% of affected patients harbor biallelic mutations of SPTB Providence (SPTB c.6055T>C), SPTB Buffalo (SPTB c.6074T>G), and SPTB Chiang Mai (SPTB c.6224A>G). This study aimed to develop a simple assay for mass screening of the three common SPTB mutations and to study their carrier frequencies in a healthy Thai population. We combined multiplex amplification refractory mutation system-PCR (ARMS-PCR) and high-resolution melting (HRM) curve analysis to create a one-step single-tube assay. The primers were designed to generate products with different melting temperatures in the presence of 6055C, 6074G, and 6224G. Internal control primers were added for quality control. Residual samples from blood donors and healthy adolescents were collected and tested for the three common SPTB mutations using the newly developed assay. Optimized multiplex ARMS-PCR/HRM curve assay yielded well-separated melt curves to detect the three SPTB mutations with 4-h turnaround time. The assay was validated in screening of 2261 non-repetitive blood donors and 89 adolescents, in which 10 (0.43%), 2 (0.09%), and 3 (0.13%) individuals were identified as carriers of SPTB Providence, SPTB Buffalo, and SPTB Chiang Mai, respectively. All mutated SPTB and 20 random wild-type samples were confirmed using Sanger sequencing with 100% accuracy. The novel ARMS-PCR/HRM curve assay is simple, accurate, and time-effective for mass screening of the common SPTB mutations. This can be employed to prevent HPP birth in a Thai population.

Association between TNFα-308 G/A and IFN-γ+874A/T Polymorphisms with Oral Lichen Planus

Background: Oral lichen planus (OLP) is a chronic disease that presents with inflammation and has a global prevalence of 0.1-4%. Lesions of the disease occur in the oral mucosa, gums, and rarely in the palate. This study aimed to investigate the relationship between this disease and TNFα-308G/A and IFN-γ+874A/T polymorphisms. Methods: In this case-control study, oral mucosal samples were collected from 50 healthy subjects, and 50 OLP patients presented to the Kerman Faculty of Dentistry were enrolled using a simple sampling method. Subsequently, we used the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) technique followed by sequencing to determine the presence of TNFα-308G/A and IFN-γ+874A/T polymorphisms in cases and controls. Results: Compared to the control group, the prevalence of A and GA alleles of the TNF gene was higher than the prevalence of G and GG alleles in OLP patients, while the AA genotype of the gene was not found in OLP patients. Regarding IFN gene polymorphism, the relationship between the T allele and the risk of disease was discovered, but it was not statistically significant (P value: 0.068). Conclusion: Although there is a strong relation between the A allele of TNF-α (-308G/A) polymorphism and the risk of OLP, this association between IFN-γ+874A/T genotype and the disease was not strong enough to predict the possibility of developing OLP.

An optimized method for PCR-based genotyping to detect human APOE polymorphisms

BackgroundApolipoprotein E (APOE) is one of the most polymorphic genes at two single nucleotides (rs429358 and rs7412). The various isoforms of APOE have been associated with a variety of diseases, including neurodegenerative, type 2 diabetes, etc. Hence, predicting the APOE genotyping is critical for disease risk evaluation. The purpose of this study was to optimize the tetra amplification refractory mutation system (Tetra-ARMS) PCR method for the detection of APOE mutations. Material and methodsHere, in our optimized Tetra-ARMS PCR method, different factors like cycle conditions, using HiFidelity enzyme instead of Taq polymerase and setting its best concentration, and the lack of using dimethylsulfoxide (DMSO) for amplifying the GC-regions were set up for all primer pairs. The sensitivity and accuracy were tested. For validation of the assay, the results were compared with known genotypes for the APOE gene that were previously obtained by two independent methods, RFLP and Chip-typing. ResultsSuccessful Tetra-ARMS PCR and genotyping are influenced by multiple factors. Our developed method enabled us to amplify the DNA fragment by 25 cycles without adding any hazardous reagent, like DMSO. Our findings showed 100 % accuracy and sensitivity of the optimized Tetra-ARMS PCR while both criteria were 95 % for RFLP and 100 % for the chip-typing method. In addition, our results showed 91 % and 100 % consistency with RFLP and chip typing methods, respectively. ConclusionsOur current method is a simple and accurate approach for detecting APOE polymorphisms within a large sample size in a short time and can be performed even in low-tech laboratories.

Analysis of Correlation Between Coronary Heart Disease and Genetic Polymorphism Detected by Gold Magnetic Nanoparticles Chromatography.

It aimed to explore the correlation of Glu504Lys locus mutation of aldehyde dehydrogenase-2 (ALDH2) with coronary heart disease (CHD) based on gold magnetic nanoparticles (GMNPs) chromatography and amplification refractory mutation system-PCR (ARMS-PCR). 120 CHD patients admitted to the cardiovascular Department of Wenling First People's Hospital affiliated to Wenzhou Medical University from December 2020 to December 2021 were selected as Case group and 80 non-CHD patients admitted during the same period were selected as Ctrl group. The venous blood and indexes of Total Cholesterol (TC), Triglyceride (TG), Low Density Lipoprotein Cholesterol (LDL-C), High Density Lipoprotein Cholesterol (HDL-C), and Fasting Blood Glucose (FBS) were collected. The ARMS-PCR GMNPs chromatography based on ARMS-PCR and immunochromatography assay was adopted to detect gene polymorphism of ALDH2. Correlation between ALDH2 gene polymorphism and risk factors of CHD was analyzed via logistic regression. In contrast to Ctrl group, the genotypes of GG, GA, and AA in Case group were evidently different (P < 0.05), and the frequency of A allelic gene was obviously increased (P < 0.05). Under the dominant model, frequency of GA + AA genotype in Case group was remarkably higher in contrast to Ctrl group (P < 0.05). Under the recessive model, there was no obvious difference in genotype frequency between two groups. In contrast to Ctrl group, TC, LDL-C, and FBS in Case group were notably increased (P < 0.05), while HDL-C was notably decreased (P < 0.05). The distribution frequency of abnormal LDL-C, HDL-C, and FBS in Case group was notably higher in contrast to Ctrl group (P < 0.05). LDL-C and FBS had no obvious effect on the genotypes and frequency distribution of alleles in CHD patients. However, the frequency distribution of genotypes of GA and AA and A allelic gene in patients with abnormal HDL-C was notably lower in contrast to those with normal HDL-C (P < 0.05). Logistic regression analysis showed that abnormal HDC-C with A allelic gene were independent risk factors for CHD (P = 0.001, OR = 1.934). The gene polymorphism of Glu504Lys locus of ALDH2 was closely related to the pathogenesis of CHD, A allelic gene may be a susceptibility gene for CHD, and patients with abnormal HDC-C and carried A allelic gene had relatively higher incidence of CHD.

FRI031 A Novel Strategy To Detect Leptin Receptor (Lepr, fa) Mutation In Obesity And Diabetic Animal Model (Zucker Rats)

Abstract Disclosure: X. Xu: None. X. Hu: None. G. Ma: None. T. Wang: None. J. Wang: None. G. Chen: None. L. Zhao: None. J. Chen: None. Globally, diabetes is among the leading causes of premature morbidity and mortality. Early intervention is crucial to achieve optimal outcomes and minimize the burden of diabetes. Animal models are indispensable tools to investigate etiology and explore novel strategies for diabetes treatment and prevention. Zucker strain and its derivatives, Zucker Fatty (ZF) and Zucker Diabetic Fatty (ZDF) rats, are one of the most used rodent models to study diabetes and diabetes-associated metabolic disorders. Homozygous Zucker rats with leptin receptor gene mutation (Lepr, fa-/-) spontaneously develop obesity, hypercholesterolemia, and hyperlipidemia. The wildtype (fa+/+) and heterozygous (fa+/-) littermates phenotypically are indistinguishable. Furthermore, early stages of diabetes are asymptomatic, and the obese phenotype is not appreciable in homogenous (fa-/-) Zucker rats until week 4 of age. Therefore, a rapid, cost-effective, and reliable genotyping method is essential to differentiate homozygous (fa-/-) rats from either wildtype (fa+/+) or heterozygotes (fa+/-) in early interventional studies. We reported a tetra-primer Amplification-Refractory Mutation System (ARMS)—PCR approach to detect leptin receptor fa mutation using genome DNA collected from ear and tail tissues of Zucker rats. Three different types of amplicon patterns are easily identified by agarose gel electrophoresis. Wildtype (fa+/+) genotype displays a 401 bp and a 201 bp amplicon band; heterozygous (fa+/-) consists of a 401 bp, a 263 bp, and a 201 bp amplicon band on the gel; homozygous (fa-/-) comes up with two bands of 401 bp and 263 bp. The genotypes identified by our tetra-primer ARMS—PCR method are further confirmed by sequence analysis. This method requires neither DNA purification nor restriction enzyme digestion, which is suitable for research exploring gene-environment interaction, as well as implementing appropriate intervention approaches in the research areas of diabetes and metabolic diseases. Presentation: Friday, June 16, 2023

An Association Between GAS5 rs145204276, NEAT1 rs512715, and MEG3 rs4081134 Gene Polymorphisms and Papillary Thyroid Carcinoma.

This study explores the association between growth arrest-specific 5 (GAS5) rs145204276, nuclear paraspeckle assembly transcript 1 (NEAT1) rs512715, and Maternally Expressed 3 (MEG3) rs4081134 polymorphisms and their impact on susceptibility to papillary thyroid carcinoma (PTC), considering differential expression of long noncoding RNAs (lncRNAs) in PTC. A case-control study involving 125 papillary thyroid carcinoma (PTC) patients and 125 controls was conducted. Genotyping of polymorphisms was performed using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods. No significant association was found between the two groups regarding genotypes and allelic frequencies of GAS-5 145204276 and MEG3 rs4081134 polymorphisms. Genetic models also showed the same results. Regarding NEAT1 rs512715, The PTC group had more GC genotypes and over-dominant models of NEAT1 rs512715 than controls, while controls showed a higher frequency of recessive models. GAS5 rs145204276 and MEG3 rs4081134 polymorphisms showed no significant association with papillary thyroid carcinoma (PTC) risk. In contrast, NEAT1 rs512715 exhibited a significant impact on PTC development.

Non-Small Cell Lung Cancer: Targetable Variants in Concurrent Tissue and Liquid Biopsy Testing in a North Indian Cohort.

Testing for EGFR, ALK, ROS1 and MET alterations in paired tissue and plasma samples of treatment-naïve patients of NSCLC and correlating their status with overall survival. One hundred treatment-naïve patients were recruited after obtaining informed consent. Ten ml of blood was collected within a period of two weeks from histological diagnosis, prior to the start of any treatment. DNA & RNA extraction was done from formalin-fixed paraffin embedded (FFPE) tissue and total cell-free nucleic acid extraction was done from plasma samples. EGFR mutation, ALK, ROS1 and MET rearrangements were tested by ARMS (Amplification Refractory Mutation System) PCR. All statistical analyses were conducted in R version 4.1.1. A total of 61 cases showed molecular alterations in tissue samples which included EGFR mutations (47), ALK rearrangements (12), ROS1 fusion (2). MET alteration was not detected. Forty-three cases showed EGFR mutations in plasma, 26 of which were concurrently positive in tissue. Concordance observed was 62%. ALK-EML4 rearrangement, ROS1 fusion and MET were not detected in plasma samples. Sensitivity and specificity for detection of EGFR mutation in plasma were 55.3% and 67.9% respectively. Univariate Cox regression analysis showed a positive association between EGFR mutation in tissue and overall survival (HR = 0.4; 95% CI: 0.2-0.7; p = 0.003) and improved overall survival in those who received targeted therapy (HR = 0.29; 95% CI: 0.1-0.8; p = 0.02). Concurrent testing in tissue and liquid biopsy in NSCLC increased the detection of EGFR mutations (47% to 64%). This has substantial implications in deciding treatment and administration targeted therapy and the consequent overall survival.

Analysis of Clinicopathological Features on Spread Through Air Spaces of Lung Adenocarcinoma

The biological and molecular characteristics of spread through air spaces (STAS), a newly recognized invasive mode of lung cancer, remain controversial. The aim of this study was to investigate the clinicopathological features and molecular characteristics of STAS in patients with pulmonary adenocarcinoma. A total of 694 resected invasive non-mucinous lung adenocarcinomas diagnosed by clinicopathology from July 2019 to March 2021 in the First Affiliated Hospital of Guangzhou Medical University were collected, and the relationship between STAS and clinicopathological factors was analyzed. The state of protein expression of anaplastic lymphoma kinase (ALK) was detected by immunohistochemical method. Epidermal growth factor receptor (EGFR) was detected by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). ROS proto-oncogene 1-receptor (ROS1) was detected by reverse transcription-PCR (RT-PCR). A total of 344 STAS positive cases and 350 STAS negative cases were collected. By univariate analysis, STAS positivity was statistically associated with tumor maximum diameter (P<0.001), pleural invasion (P<0.001), lymphovascular invasion (P<0.001), nerve invasion (P=0.013), lymph node metastasis (P<0.001), clinical stage (P<0.001) and histological type (P<0.001). There was a statistical correlation between STAS and ALK protein expression (P=0.001). Multivariate analysis showed that STAS positive was correlated with pleural invasion (P=0.001), vascular invasion (P<0.001), lymph node metastasis (P=0.005)and ALK protein expression (P=0.032). STAS is associated with highly aggressive biological behavior of lung adenocarcinoma, suggesting a poor prognosis.

CYP2C19*17 association with higher plasma 4-hydroxy tamoxifen in Pakistani (estrogen-positive) breast cancer patients.

Breast cancer (BC) continues to be the most common cancer in the women worldwide. Since estrogen receptor (ER)-positive BC accounts for the majority of newly diagnosed cases, endocrine therapy is advised to utilize either tamoxifen (Tam) or aromatase inhibitors. The use of Tam as a monotherapy or in conjunction with an aromatase inhibitor following two or three years of endocrine therapy has long been recommended. When used adjuvantly, Tam medication reduces BC mortality and relapses, while it extends survival times in metastatic BC when used in conjunction with other treatments. Unfortunately, the efficiency of Tam varies considerably. This study was conducted to explore the influence of genetic polymorphisms in CYP2C19 gene on Tam's pharmacogenetics and pharmacokinetics in estrogen-positive BC patients. Data from healthy, unrelated individuals (n = 410; control group) and ER-positive BC patients (n = 430) receiving 20 mg of Tam per day were recruited. Steady-state plasma concentrations of Tam and its three metabolites were quantified using the high-performance liquid chromatography in the patients. The CYP2C19 polymorphisms were genotyped using an Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) approach. More than 65% of healthy individuals were extensive metabolizers (*1/*1) for CYP2C19, whereas more than 70% of ER-positive BC patients were rapid and ultrarapid metabolizers (*1/17*, *17/17*). The polymorphism CYP2C19*17 is significantly associated with higher 4-hydroxytamoxifen (4-OH-Tam). Patients with the *17/*17 genotype exhibited 1- to 1.5-fold higher 4-OH-Tam, which was also high in patients with the *1/*2 and *2/*2 genotypes.

Detecting fa leptin receptor mutation in Zucker rats with tetra-primer amplification-refractory mutation system (ARMS)-PCR

Due to the genetic mutation (fa) in the gene encoding for leptin receptor, homozygous Zucker rats (fa−/−) develop excessive adiposity and become an experimental animal model in obesity and metabolic-related diseases research. Based on tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), we developed a method to quickly genotype Zucker rats with a mutated fa allele from their wildtype littermates. The three genotypes are clearly discriminated on 2.0% agarose gel. Our method can be used as a reliable tool to set up and maintain the breeding colony in animal facilities as well as assign animals to control and treatment groups based on their genotypes for animal studies.

Genetic and epigenetic alterations in MGMT gene and correlation with concomitant chemoradiotherapy (CRT) in cervical cancer.

The MGMT (O6-methylguanine-DNA methyltransferase) gene plays a crucial role in repairing DNA damage caused by alkylating agents, including those used in chemotherapy. Genetic and epigenetic alterations can influence the regulation of MGMT gene, which in turn may impact the response to concomitant chemoradiotherapy (CRT) in cervical cancer. The present study was undertaken to evaluate the correlation of such variations in MGMT gene with the treatment outcome of concomitant chemoradiotherapy (CRT) in cervical cancer. A total of 460 study subjects (240 controls and 220 patients) were subjected to genotypic analysis of MGMT gene variants rs12917(T/C) and rs2308327(A/G) by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). Out of them, 48 each of controls and patients were analyzed for promoter methylation and expression by methylation-specific PCR and real-time PCR, respectively. Patients (n = 48) were followed up and evaluated for treatment (CRT) outcome. Statistical analyses were done using GraphPad (9.0) and SPSS version 18.0. Individuals with GG genotype, G allele of rs2308327, and haplotype 'TA' of both variants showed a significant increase in the development of cervical cancer (P ≤ 0.05). In epigenetic regulation, there was a significant hypermethylation of MGMT gene and down-regulation of their expression in patients compared to control individuals. In treatment outcome of CRT, GG genotype of rs2308327(A/G) gene variant showed better response and GG + AG was significantly associated with vital status (alive). Unmethylated MGMT gene showed better median overall survival up to 25months significant in comparison to methylated MGMT promoter. Gene variant rs2308327(A/G) and promoter hypermethylation regulated MGMT gene can be a good prognostic for treatment response in cervical cancer patients.

Comprehensive Molecular Evaluation of HNF-1 Alpha, miR-27a, and miR-146 Gene Variants and Their Link with Predisposition and Progression in Type 2 Diabetes Patients.

Type 2 diabetes (T2D) is a metabolic condition induced by insulin resistance and pancreatic beta cell dysfunction. MicroRNAs (miRNAs) have biological significance because they regulate processes such as the molecular signaling pathways involved in the pathophysiology of diabetes mellitus. The hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a transcription factor found in hepatocytes and the pancreas. Mutations in the HNF-1 alpha gene were reportedly associated with maturity-onset diabetes of the young (MODY). The objective of the present study was to examine the associations between MiR-27a, MiR-146, and HNF-1 alpha single-nucleotide variations (SNVs) with T2D risk in the Saudi population. We evaluated the association of SNVs of miR-27a rs895819 A>G, 146a-rs2910164 C>G, and HNF-1 alpha rs1169288 G>T (I27L) with the risk of T2D in Saudi patients with the Amplification Refractory Mutation System PCR (ARMS-PCR). For the miR-27a SNVs, we used 115 cases (82 males, 33 females) and 117 matched healthy controls (HCs); for the Mir-146 SNVs, we used 103 cases (70 males, 33 females) and 108 matched HCs; and for the HNF-1 alpha, we employed 110 patients (80 males, 30 females) and 110 HCs. The blood biochemistry of the participants was essayed using commercial kits, and the methods of statistical analysis used were the Chi-square test, the Fisher exact test, and a multivariate analysis based on logistic regression, like the odds ratio (OD) and risk ratio (RR), with 95% confidence intervals (CIs). The MiR-27a rs895819 AG genotype was linked to increased T2D susceptibility, with OR = 2.01 and p-value = 0.011, and the miR-146 rs2910164 CG genotype and C allele were linked to an elevated risk of T2D, with OR = 2.75, p-value < 0.0016, OR = 1.77, and p-value = 0.004. The results also showed that the GT genotype and T allele of the HNF-1 alpha (rs1169288) G>T is linked to T2D, with OR = 2.18, p-value = 0.0061, and 1.77, p-value = 0.0059. The SNVs in miR-27a, miR-146, and HNF-1 alpha can be potential loci for T2D risk. The limitations of this study include the relatively small sample size and the fact that it was a cross-sectional study. To our knowledge, this is the first study to highlight the association between miR-27a, miR-146, and HNF-1 alpha SNVs and the risk of T2D in the Saudi population. Future large-scale case-control studies, as well as studies on the functions of the proteins and protein interaction studies for HNF-1 alpha, are required to verify our findings. Furthermore, these findings can be used for the identification and stratification of at-risk populations via genetic testing for T2D-prevention strategies.

Genetic analysis of TMPRSS6 catalytic domain variants in Mexican patients with iron treatment refractoriness.

To identify the TMPRSS6 gene variants in Mexican patients with iron treatment refractoriness, to describe hematological and iron profile parameters, and to use bioinformatic prediction and protein modeling tools to assess a possible biological impact for the detected missense variants. Nineteen patients referred with iron treatment refractoriness were studied. Peripheral blood was collected to determine hematic cytometry, iron profile, hemoglobin electrophoresis, and quantification. Molecular screening was carried out for exons 15 through 18 of the TMPRSS6 gene by Sanger sequencing and for frequent thalassemia variants by amplification-refractory mutation system-polymerase chain reaction (PCR) and gap-PCR. The biological impact of the detected missense variants was assessed using bioinformatic prediction and protein modeling tools. We found 5 genetic variants in the matriptase-2 catalytic domain: 1 at intron-15/exon-16 junction (rs60484081) and 4 exonic, 3 missense (rs377054987, p.Gly626Asp; rs1384127820, p.Ser672Thr; rs855791, p.Val727Ala) and 1 synonymous (rs2235321, p.Tyr730=), with frequencies ranging from 0.18 to 0.53. No significant differences were observed in the hematological parameters or iron profile, considering type and number of variants. Bioinformatic predictions suggested a possible biological impact only for rs377054987. The TMPRSS6 variants observed in Mexican patients with oral iron treatment refractoriness have high frequencies; nevertheless, their relationship with hematological and iron profile parameters needs further research. The possible biological impact for rs377054987 is due to size and amino acid hydrophobicity changes and hydrogen bond modifications.

Investigating pathogenic SNP of PKCι in HCV-induced hepatocellular carcinoma

Hepatocellular carcinoma is a leading cause of cancer-related deaths due to its complexity in diagnosis, chemo-resistance, and aggressive nature. Identifying pathogenic single nucleotide polymorphism (SNP) in protein kinase C iota (PKCι) can be a potential biomarker in the prognosis and treatment of HCC. This study investigated the association between a SNP in PRKCI and the Pakistani population's hepatocellular carcinoma (HCC) risk. Obtained samples were first evaluated for ALT measurements and viral load quantification through reverse transcriptase-PCR. The PKCι nsSNP rs1199520604 was evaluated computationally by multiple consensus bioinformatics tools for predicting its potential deleterious effects. Its association with hepatitis C virus- (HCV) mediated HCC was then investigated through ARMS-PCR (Amplification Refractory Mutation System Polymerase Chain Reaction). SNP analysis of rs1199520604 was performed in 100 cases and 100 controls. Variant rs1199520604’s homozygous T genotype is a risk factor allele for the HCV-induced HCC (odds ratio: 4.13, relative risk: 2.01, P-value < 0.0001). The heterozygous genotype is determined to protect HCV patients from HCC development (P < 0.001). The study highlighted the disease association of variant rs1199520604 with HCV-induced HCC in the Pakistani populations. This variant, after further validation through high-throughput investigation on a larger cohort, has the potential to be translated at the clinical level.

The importance of SOCS1 - 1478 CA/del polymorphism and expression in breast cancer: a case-control study in the north of Iran.

This project aimed to evaluate the relationship between the suppressor of cytokine signaling-1 (SOCS1) - 1478 CA > del genetic variation and breast cancer susceptibility. Moreover, we investigated the SOCS1 mRNA expression level in cancerous tissues. A total of 100 patients with breast cancer and 120 healthy individuals were selected. Genomic DNA was extracted from blood. SOCS1 genotyping and relative gene expression were performed using ARMS-PCR (Amplification-Refractory Mutation System-Polymerase Chain Reaction) and real-time PCR, respectively. In breast cancer patients, the prevalence of genotype frequencies of SOCS1 (- 1478 CA > del) CA/CA, CA/del, and del/del was 52, 31, and 17%, respectively. Among controls, the distribution of CA/CA, CA/del, and del/del was 63, 15, and 22%, respectively. The chi-square test reported that a significant difference was observed in the genotypic distribution of SOCS1 (- 1478 CA > del) polymorphism between cases and controls (χ2 = 8.08, P = 0.01). In addition, the presence of the CA/del genotype was associated with an elevated risk of breast cancer (in the codominant model: OR 2.51; 95% CI 1.27-4.96, P = 0.007 and in the over dominant model: OR 2.54; 95% CI 1.32-4.90, P = 0.005). However, there was no significant difference in allelic distributions between the groups (P > 0.05). There was no significant difference in the breast cancer risk associated with the dominant and recessive genetic models when the reference was CA/CA and CA/CA + CA/del genotype, respectively (P = 0.09 and P = 0.38). Moreover, the expression of SOCS1 decreased in cancerous tissues as compared to the adjacent non-cancerous tissues (P < 0.0001). In conclusion, a functional SOCS1 promoter polymorphism (- 1478 CA > del) may affect breast cancer susceptibility.

Frequency of secondary modifiers in Beta Thalassemia intermedia in patients from Northern Punjab.

The aim of this study was to determine frequency of secondary modifiers in non-transfusion dependent thalassemia. This descriptive cross sectional study was done at Fauji Foundation Hospital Islamabad. Seventy diagnosed patients of thalassemia intermedia were included. Deoxyribonucleic acid (DNA) was extracted using Chelex method. The Xmn-1 and BCL11A polymorphisms were analyzed by Amplification Refractory Mutation System (ARMS) and Restriction Fragment Length Polymorphism (RFLP) PCR. The PCR amplified products were run on Polyacrylamide Gel Electrophoresis (PAGE). The Xmn-l polymorphism was seen in 26/70 (37.1%) and BCL11A polymorphism was seem in 50/70 (71.4%) of the patients. BCL11A and Xmn-l polymorphisms are important secondary modifiers in patients with thalassaemia intermedia in Northern Punjab.