Amplification Refractory Mutation System-polymerase Chain Research Articles

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

Abstract Objectives 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. Methods 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 96 months for the prognostic impact of the mutation. Results 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: 96 months) was 81.8 % in patients harbouring T315I mutation. Patients in the blastic phase had significant odds of harbouring T315I mutation. Conclusions 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.

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.

Genetic evidence for predisposition to acute leukemias due to a missense mutation (p.Ser518Arg) in ZAP70 kinase: a case-control study

BackgroundThe apparent lack of additional missense mutations data on mixed-phenotype leukemia is noteworthy. Single amino acid substitution by these non-synonymous single nucleotide variations can be related to many pathological conditions and may influence susceptibility to disease. This case-control study aimed to unravel whether the ZAP70 missense variant (rs104893674 (C > A)) underpinning mixed-phenotype leukemia.MethodsThe rs104893674 was genotyped in clients who were mixed-phenotype acute leukemia-, acute lymphoblastic leukemia- and acute myeloid leukemia-positive and matched healthy controls, which have been referred to all major urban hospitals from multiple provinces of country- wide, IRAN, from February 11’ 2019 to June 10’ 2023, by amplification refractory mutation system-polymerase chain reaction method. Direct sequencing for rs104893674 of the ZAP70 gene was performed in a 3130 Genetic Analyzer.ResultsWe found that the AC genotype of individuals with A allele at this polymorphic site (heterozygous variant-type) contribute to the genetic susceptibility to acute leukemia of both forms, acute myeloid leukemia and acute lymphoblastic leukemia as well as with a mixed phenotype. In other words, the ZAP70 missense variant (rs104893674 (C > A)) increases susceptibility of distinct cell populations of different (myeloid and lymphoid) lineages to exhibiting cancer phenotype. The results were all consistent with genotype data obtained using a direct DNA sequencing technique.ConclusionOf special interest are pathogenic missense mutations, since they generate variants that cause specific molecular phenotypes through protein destabilization. Overall, we discovered that the rs104893674 (C > A) variant chance in causing mixed-phenotype leukemia is relatively high.

The Association of Vitamin D Receptor Gene Polymorphism with Polycystic Ovary Syndrome in a Southeast Iranian Population

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women of reproductive age, characterized by hyperandrogenism, amenorrhea, and polycystic ovaries. Metabolic disorders, including insulin resistance (IR), are common comorbidities of PCOS, with vitamin D receptors playing a crucial role. Objectives: We investigated whether the rs2228570T > C variant of the vitamin D receptor (VDR) gene is associated with the risk of PCOS. Methods: In the current study, 112 women with PCOS and 150 healthy women participated. Genomic DNA was extracted using the standard salting-out method. Genotyping of rs2228570T > C was conducted via amplification-refractory mutation system polymerase chain reaction. Additionally, VD3 levels were measured using the ELISA system. Statistical analysis of genotyping data was performed using the SPSS V.22 package. Results: The statistical analysis showed that the T-allele of rs2228570C/T significantly decreased the risk of PCOS. Moreover, the T-allele in the codominant model significantly eliminated the risk of PCOS in our population. Our results also showed a statistically significant difference in VD3 levels between the CC and TT genotypes. Conclusions: Based on our result, rs228570C/T had a protective role for PCOS risk in our population.

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.

Mannose-Binding Lectin Gene Variants as Disease Susceptibility Biomarkers in Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease characterized by progressive destruction of peripheral joints. About 1% of the human population worldwide is suffering from this disease. The pathophysiology of RA is largely being influenced by immune dysregulation. Mannose-binding lectin (MBL), an acute-phase protein, has been reported to play an important role in pathogenesis of RA by the activation of complement pathway. Various studies documented the established the role of MBL in pathogenesis of various autoimmune diseases, including RA. MBL protein is encoded by gene MBL2, mapped on chromosome 10q11.2-q21. Objective: Both MBL serum levels and activity are mainly determined genetically by its variants. So considering the putative clinical role of MBL2, this case-control association study was designed to assess its six functional variants in a northwestern Indian cohort. Methods: Genetic typing of six MBL2 variants was done by amplification refractory mutation system-polymerase chain reaction. Data were analyzed using suitable statistical tools. Results: Significant difference has been observed in genotypic and allelic distribution between cases and controls for rs11003125. Comparison of allelic distribution for rs1800450 showed significantly high prevalence of A allele in cases than controls. Conclusion: These results indicate that MBL2 variants may act as plausible marker for susceptibility toward RA. Keeping this in view, it is pertinent to screen these variants in other population groups of India.

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.

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.

Hematological Indices and Genetic Variants of Premature Ovarian Insufficiency: Machine Learning Approaches.

Premature Ovarian Insufficiency (POI) is associated with infertility. Little is known about the potential circulating biomarkers that could be used to predict POI. We have investigated the possible association between white and red blood cells, platelet indices, and eight established single nucleotide polymorphisms (SNPs) associated with POI risk. 117 women with premature menopause (PM) and 183 healthy women without a history of menopause before age 40 were recruited for this study. The tetra-primer amplification refractory mutation system-polymerase chain reaction (Tetra ARMS PCR) and allele-specific oligonucleotides-polymerase chain reaction (ASO-PCR) were carried out for genotyping for eight SNPs reported to be associated with POI. Decision tree analysis was applied to test the diagnostic value of hematological parameters to identify the risk of POI. Women with POI had lower neutrophil (NEUT) and white blood cell (WBC), whereas red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean cell hemoglobin (MCH) were higher. Platelet (PLT) count was also lower in affected women. Our data also indicated that HGB and HCT count were significantly associated with rs16991615 and rs244715. Mean Platelet volume (MPV) and platelet distribution width (PDW) were associated with rs244715, rs1046089, rs4806660, and rs2303369. The rs16991615 was also associated with RBC count, and rs451417 was associated with NEUTs. The decision tree (DT) model reveals that women with the NEUT count at a cut-off value of less than 2.8 and HCT equal to or more than 38.7% could be identified as high-risk cases for POI. Overall, we found the DT approach had a sensitivity = 85%, specificity = 72%, and accuracy = 74%. The genetic variants involved in POI are associated with changes in reproductive hormone levels and with changes in hematological indices.

Evaluation of interleukin-4 and tumor necrosis factor-alpha in patients with breast cancer

Abstract Background: Breast cancer is a complicated, multifaceted condition that affects a wide range of entities and exhibits significant heterogeneity in its clinical, morphological, and molecular characteristics. Objective: This study intended to determine whether there is a correlation between the serum level of IL-4 expression and the single nucleotide polymorphism in tumor necrosis factor-alpha (TNF-α) in the development of breast cancer. Materials and Methods: IL-4 serum levels in 70 patients (35–65 years old) and 70 control groups (30–50 years old) were determined using the enzyme-linked immunosorbent assay (ELISA) technique. Genomic DNA was obtained from blood samples for molecular analysis to investigate the TNF-α-308 G→A gene polymorphism in patients and the control groups. Genotyping done by using tetra amplification refractory mutation system-polymerase chain reaction technique. Results: Patients with breast cancer had significantly higher serum IL-4 levels than the control group (P &lt; 0.001), 133.27 (66.00) vs. 38.66 (38.00), respectively. The frequency distribution of the TNF-α genotype in the patients with breast cancer and control groups was studied. In the patient group, there were 56 out of 70 heterozygous AG genotypes compared to 6 out of 70 in the control group (P &lt; 0.001). With an odds ratio of 37.33 (95% confidence interval: 7.99–174.51) and an etiologic fraction (EF) of 0.88, the AG genotype existed indeed a risk factor. Conclusion: In the current investigation, the heterozygous AG genotype was shown to be substantially more linked with IL-4 serum and TNF-α genotype in breast cancer patient groups. Also, the homozygous GG genotype was significantly higher in correlation between IL-4 serum and TNF-α genotype in breast cancer patient group.

Genetic association of Interleukin-17A polymorphism in Bangladeshi patients with breast and cervical cancer: a case-control study with functional analysis

BackgroundBreast and cervical cancer are the two leading cancers in terms of incidence and mortality. Previous studies reported different interleukins, including interleukin-17A (IL-17A) to be responsible for the development and progression of these malignancies. Therefore, we speculated that the variants in this gene might be associated with these cancer developments in Bangladeshi population. For evaluating the hypothesis, we investigated the association of IL-17A rs3748067 polymorphism with the susceptibility of both breast and cervical cancer. MethodsThis case-control study was performed on 156 breast cancer patients, 156 cervical cancer patients, and 156 controls using the tetra-primer amplification refractory mutation system-polymerase chain reaction. The statistical software package SPSS (version 25.0) was applied for analyses. The genetic association was measured by the odds ratio (OR) and 95% confidence intervals (CIs). A statistically significant association was considered when p-value ≤ 0.05. Functional analysis was performed using GEPIA and UALCAN databases.ResultsFrom the calculation of the association of IL-17A rs3748067 with breast cancer, it is found that no genotype or allele showed a statistically significant association (p>0.05). On the other hand, the analysis of IL-17A rs3748067 with cervical cancer demonstrated that CT genotype showed a significant association (CT vs. CC: OR=1.79, p=0.021). In the overdominant model, CT genotype also revealed a statistically significant association with cervical cancer, which is found to be statistically significant (OR=1.84, p=0.015).ConclusionOur study summarizes that rs3748067 polymorphism in the IL-17A gene may be associated with cervical cancer but not breast cancer in Bangladeshi patients. However, we suggest studies in the future with a larger sample size.

Association between Interleukin-6 Gene Polymorphism (rs1800795 and rs1800796) and Type 2 Diabetes Mellitus in a Ghanaian Population: A Case-Control Study in the Ho Municipality.

There is no conclusive evidence on the association between interleukin- (IL-) 6 gene polymorphism and type 2 diabetes mellitus (type 2 DM). Thus, this study is aimed at evaluating the role of rs1800795 and rs1800796 polymorphisms in the pathogenesis of type 2 DM among Ghanaians in the Ho Municipality. We recruited into this hospital-based case-control study 174 patients with type 2 DM (75 DM alone and 99 with DM+HTN) and 149 healthy individuals between 2018 and 2020. Demographic, lifestyle, clinical, anthropometric, and haemodynamic variables were obtained. Fasting blood samples were collected for haematological, biochemical, and molecular analyses. Genomic DNA was extracted, amplified using Tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) technique, and genotyped for IL-6 gene polymorphism. Logistic regression analyses were performed to assess the association between IL-6 gene polymorphism and type 2 DM. The minor allele frequency (MAF) of the rs1800795 and rs1800796 polymorphisms was higher in DM alone (57.5%, 62.0%) and DM with HTN groups (58.3%, 65.3%) than controls (33.1%, 20.0%). Carriers of the rs1800795GC genotype (aOR = 2.35, 95% CI: 1.13-4.90, p = 0.022) and mutant C allele (aOR = 2.41, 95% CI: 1.16-5.00, p = 0.019) as well as those who carried the rs1800796GC (aOR = 8.67, 95% CI: 4.00-18.90, p < 0.001) and mutant C allele (aOR = 8.84, 95% CI: 4.06-19.26, p = 0.001) had increased odds of type 2 DM. For both polymorphisms, carriers of the GC genotype had comparable levels of insulin, HOMA-IR, and fasting blood glucose (FBG) with those who carried the GG genotype. IL-6 levels were higher among carriers of the rs1800796GC variant compared to carriers of the rs1800796GG variant (p = 0.023). The rs1800796 polymorphism, dietary sugar intake, and exercise status, respectively, explained approximately 3% (p = 0.046), 3.2% (p = 0.038, coefficient = 1.456), and 6.2% (p = 0.004, coefficient = -2.754) of the variability in IL-6 levels, suggesting weak effect sizes. The GC genotype and mutant C allele are risk genetic variants associated with type 2 DM in the Ghanaian population. The rs1800796 GC variant, dietary sugar intake, and exercise status appear to contribute significantly to the variations in circulating IL-6 levels but with weak effect sizes.

Association of Fibroblast Growth Factor-1 Promoter Polymorphism and its Serum Concentrations with Repeated Implantation Failure after In vitro Fertilisation: A Cross-sectional Study.

Fibroblast growth factors (FGFs) play a key role in embryo implantation and support endometrial trophoblastic interaction. The aim of the study was to evaluate the association between FGF-1 (rs34011) gene variety and its serum concentration with repeated implantation failure (RIF). The design of the study was a cross-sectional study. Four hundred infertile women with a history of RIF and 400 healthy women undergoing the first in vitro fertilisation-embryo transfer attempt with successful delivery (controls) were enrolled in the study. Genomic DNA was extracted from peripheral blood leucocytes and genotyped by Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction. Serum FGF-1 concentration was evaluated with enzyme-linked immunosorbent assay. The ANOVA test was used to analyse the difference between the means of the groups. In RIF group, the genotype frequencies of the GG, GA and AA were 59%, 33.5% and 7.5%, respectively, whereas in controls were 72.5%, 24% and 3.5%, respectively. The G and A allele frequencies in the RIF group were 75.75% and 24.25%, while in controls were 84.5% and 15.5%, respectively (P < 0.0001). We have also shown that serum FGF-1 concentration in RIF and control groups was 17 ± 3.55 and 23.62 ± 4.91 pg/mL, respectively (P = 0.008). We have also shown that AA genotype is significantly associated with decreased serum FGF-1 concentration in RIF (AA, GA and GG serum levels were 9.55 ± 2.65, 14 ± 3.35 and 22.55 ± 7.26 pg/mL, and in controls were 12.22 ± 2.27, 18.44 ± 5.98 and 26.66 ± 8.29 pg/mL, respectively). The current study suggests that a significant association between FGF-1 (rs34011) promoter polymorphism and its serum concentration with RIF. The study also suggests that AA genotype is linked to lower FGF-1 serum levels and may play a risk factor for RIF.

Angiotensin-Converting Enzyme Genetic Polymorphism rs4343 as Risk of Diabetic Nephropathy in Jambi-Malay Population

Background: Diabetic nephropathy (DN) is one of the frequent complications of type II diabetes mellitus (T2DM) in Jambi province. Controlling blood glucose and blood pressure does not guarantee DN prevention, since genetic factors may also contribute to this disease. Multi-ethnic studies showed that one of the strongest genetic factors associated with DN was single nucleotide polymorphism rs4343 of angiotensin-converting enzyme (ACE) gene. Study regarding phenotype-genotype association of ACE rs4343 and DN has not yet been performed in Jambi Province, which is dominated by Malay ethnicity. This study was conducted to reveal the association between ACE rs4343 and the risk of DN in the Jambi-Malay population.Materials and methods: This was a cross-sectional study involving 75 subjects (44 with DN and 31 without DN) who suffered from T2DM and hypertension. DN was defined as albumin to creatinine ratio (ACR) ≥30 mg/g. Genotyping was performed with one-step tetra amplification refractory mutation system-polymerase chain reaction (PCR) using specific primer for ACE rs4343. Bivariate and multivariate analyses were performed to analyze the genetic risk for DN.Results: The bivariate analysis showed the proportion of DN subjects was higher than non-DN within the AG genotype (11:1) than within the AA (33:30) genotype. This difference was statistically significant (p=0.012; OR (95% CI): 10.00 (1.22-82.15)). Multivariate analysis showed that AG genotype (p=0.047; OR (95% CI): 10.04 (1.03-97.31)) and uncontrolled blood pressure (p=0.001; OR (95% CI): 6.72 (2.08-21.71)) were the risk factors of DN in the Jambi-Malay population.Conclusion: Polymorphism of ACE rs4343 is a risk factor of DN in the Jambi-Malay Population.Keywords: rs4343, angiotensin-converting enzyme gene, diabetic nephropathy, Malay, Jambi

The impact of MIR196A2 and MIR423 genes polymorphisms on the development of type 2 diabetes mellitus in a sample of the Iraqi population

PurposeMicro-RNAs, play a significant role in regulating essential physiological processes by controlling post-transcriptional gene expression. Several disorders have been linked to variations in the miRNA genes that generate sequences of mature micro-RNA. The current research aims to determine if the genetic variations rs6505162; C > A in the MIR423 gene and rs11614913; T > C in the MIR196A2 gene are associated with type 2 diabetes mellitus (T2DM). MethodThe tetra-primers amplification refractory mutation system-polymerase chain reaction technique was used to identify these two single nucleotide polymorphisms (SNPs). ResultsThe allele frequency of MIR423- rs6505162; C > A SNP was 0.47 for the wild allele (A) and 0.53 for the mutant allele (C) in the control group, while in the patients group the frequency was 0.54 and 0.46 for the A and C alleles respectively. No association was discovered between this SNP and T2DM (O.R = 0.75, P = 0.47 for AC genotype, and O.R = 0.53, P = 0.15 for CC genotype). The wild type of MIR196A2 rs11614913; C > T SNP showed a 100% prevalence in the study subjects, thus this SNP has no association with T2DM as well. ConclusionThe current investigation demonstrates that the incidence of T2DM is not associated with the MIR196A2-rs11614913; C > T, and MIR423-rs6505162; C > A SNPs.

Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F Mutation in Fluconazole Resistant Candida parapsilosis.

There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.

Common mutation analysis of beta-thalassemia carriers by amplification refractory mutation system-polymerase chain reaction in cases diagnosed by capillary zone electrophoresis at National Public Health Laboratory, Nepal

Abstract BACKGROUND: Beta (β)-thalassemia is a globally prevalent genetic disorder with over 350 known mutations in the β-globin gene. Intervening-sequence (IVS) 1–5 (G→C), 619 bp deletion, IVS 1–1 (G →T), C-15 (G→A), frameshift (FS) 41/42 (−CTTT), FS 8/9 (+G), codon 19, A-G, and codon 17 (A→T) are some common mutations in β-thalassemia. This study aimed to identify and analyze the common mutation by amplification refractory mutation system polymerase chain reaction in β-thalassemia carriers diagnosed by capillary zone electrophoresis (CZE) at National Public Health Laboratory. MATERIALS AND METHODS: On the basis of archived data, the CZE performed on 781 samples between June 2019 and July 2022 was analyzed. Without regard to age, gender, race, or address, 121 samples were ultimately selected for mutation analysis during this time because they were identified as positive for the β-thalassemia traits based on high HbA2 levels of more than 3.5%. RESULTS: IVS 1–5 (G→C) was the most common mutation followed by 619 bp deletion. Five mutations namely IVS 1–5 (G→C), 619 bp deletion, FS 41/42 (−TTCT), FS 8/9 (+G), and codon 15 (G→A) accounted for 84.3% of cases. A single mutation was seen in 87 cases (71.9%), more than one mutation in 15 cases (12.4%) whereas 19 cases (15.7%) did not show any of the mutations tested. CONCLUSION: IVS 1–5 (G-C) is the most prevalent mutation in Southeast Asian nations, with the 619 bp deletion predominant in the Tharu community. Further research is needed to understand diverse ethnic groups and regions, enabling prenatal diagnostics and genetic counseling for β-thalassemia prevention.

The Genetic Polymorphisms of CYP3A4*1G and CYP3A5*3 in Javanese Indonesian Population

Polymorphisms of CYP3A4*1G and CYP3A5*3 affect the pharmacokinetic profile of various drugs, e.g., fentanyl, tacrolimus, diltiazem, simvastatin. Tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) is a simple and economical method for SNP determination. The polymorphisms in the CYP3A4*1G and CYP3A5*3 genes have not yet been examined using this method in Javanese Indonesian. Our aim was to determine the frequency of polymorphisms in the CYP3A4*1G and CYP3A5*3 genes in Indonesian Javanese using the ARMS-PCR method. Eighty-six patients at the Kalasan Community Health Centre in Yogyakarta, Indonesia, were chosen based on the inclusion criteria, which is Javanese ancestry. They gave their informed consent to blood collection by completing a form. Genetic variants were detected using Tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR). The chi-square test was used to determine genotype deviations from Hardy-Weinberg equilibrium, with a significant threshold of 0.05. For homozygous wild types, CYP3A4 *1/*1 dominated overall among study participants (73.35%), whereas for CYP3A5*3/*3, homozygous mutants were more prevalent (83.72%). Hardy-Weinberg equilibrium is consistent with genotype frequencies (p &gt; 0.005). One participant carried a homozygous mutation for both CYP3A4*1G and CYP3A5*3, while the other 49 subjects were heterozygous for CYP3A4*1G and homozygous mutant for CYP3A5*3, which is the highest number of SNP combinations. The findings of the current investigation demonstrate that the population has the highest proportion of homozygous CYP3A4*1G wild-types (CYP3A4*1/*1) and homozygous mutants for CYP3A5*3 (CYP3A5*3/*3)

The Exploring of Growth Differentiation Factor-15 and H63D Gene Polymorphisms in β-thalassemia Major: Implications for Cardiovascular Risk and Iron Overload

BACKGROUND: β-thalassemia major (βTM) is a genetic disorder characterized by a deficiency in hemoglobin production, ineffective erythropoiesis, chronic hemolysis, lifelong blood transfusions, iron overload, and increased risk of cardiac complications. OBJECTIVE: The study aimed to evaluate the growth differentiation factor-15 (GDF-15) concentration in βTM patients and its correlation with cardiac complications. H63D refers to a specific mutation in the HFE gene, which is associated with hereditary hemochromatosis (HH), a genetic disorder characterized by excessive accumulation of iron in the body. This mutation involves a change of histidine (H) to aspartic acid (D) at position 63 in the HFE protein. This mutation is often only written abbreviated as (H63D). MATERIALS AND METHODS: This case–control study was done on 120 subjects. A total of 60 patient samples were randomly collected from the Genetic Hematology Center at the Babylon Hospital, with an age range of 10–26 years. In addition, 60 samples were collected from healthy children in the same age range as the control group; patients and controls were subdivided into (10–18) and (18–26) year groups. GDF-15 was measured by enzyme-linked immunosorbent assay, and the genotyping of mutation was done by amplification refractory mutation system-polymerase chain reaction technique. RESULTS: The study revealed a significant increase in ferritin (FER) and GDF-15 levels in the patients compared to controls (P &lt; 0.001). GDF-15 showed a direct correlation with age (r = 0.244, P = 0.02) and FER (r = 0.215, P = 0.04). There was a significant difference in H63D mutations between controls and patients (P = 0.044), with a higher proportion of the C-G (heterozygous for the mutant allele) genotype observed in βTM patients (31.67%). Additionally, a notable effect of the H63D mutation on serum ferritin (higher) levels within the βTM group was observed. CONCLUSION: Elevations of the GDF-15 in βTM patients indicate a high risk of cardiovascular complications in patients with βTM. The H63D mutation of the hemostatic iron regulator (HFE) gene is frequently found in βTM. Although a significant effect of the mutation was obtained on serum FER levels, it did not act as a risk factor in βTM patients. However, the frequent presence of the H63D mutation in patients indicated a possible association between single-nucleotide polymorphism and the iron regulation pathway.

Correlation Between DCAMKL-1 Protein Expression and K-ras Gene Mutation in Colorectal Cancer.

To investigate the correlation between doublecortin and CaM kinase-like-1 (DCAMKL-1) protein expression, K-ras gene mutation, and their impact on patient prognosis in colorectal cancer (CRC). Immunohistochemistry was used to detect the expression of DCAMKL-1 protein in 60 cases of colorectal adenoma, 82 cases of CRC (including 65 cases of lymph node metastasis) and paraffin-embedded paracancerous intestinal mucosal tissue. K-ras gene mutations in primary CRC lesions were detected using an amplification-refractory mutation system and fluorescent polymerase chain reaction. The relationship between DCAMKL-1 protein expression and K-ras gene mutations with the clinicopathological characteristics of patients with CRC was analyzed. Univariate Kaplan‒Meier survival analysis and multivariate Cox regression analysis were performed using follow-up data. The mutation rate of the K-ras gene in 82 cases of CRC was 48.8% (40/82). The positivity rate for the presence of DCAMKL-1 protein in CRC was 70.7% (58/82), significantly higher than that for colorectal adenomas (53.3%; 32/60) and paracancerous intestinal mucosa (0%; 0/82) (P<0.05). The positive expression rate for the presence of DCAMKL-1 protein in 65 patients with lymph node metastasis was higher in the primary lesions (69.2%; 45/65) than in the lymph node metastases (52.3%; 34/65) (χ2=12.087, P=0.001). The K-ras gene mutation status was positively correlated with DCAMKL-1 protein expression (r=0.252, P=0.022). In this study, a potential positive correlation between K-ras gene mutation and DCAMKL-1 protein expression was identified in CRC tissues. The assessment of K-ras gene mutation status and DCAMKL-1 protein expression holds promise for augmenting early diagnosis and prognosis evaluation in CRC. This approach may improve the overall prognosis and survival outcomes for CRC patients.