Allele-specific Polymerase Chain Reaction Method Research Articles

Association between COMMD1 gene polymorphism rs11125908 and rheumatoid arthritis in the Cuban population.

To evaluate the association of the rs11125908 polymorphism in the COMMD1 gene in the Cuban population with rheumatoid arthritis (RA). In this case-control study, 161 RA patients and 150 control subjects were genotyped for rs11125908 by the allele-specific polymerase chain reaction method. DNA sequencing was used to verify the assignation of the polymorphism. The odds ratios (OR) and their 95% confidence interval were calculated by logistic regression to determine the associations between genotypes and RA using the SNPStats software. An association of the single nucleotide polymorphism with the disease was found in the overdominant model (p=0.025; OR=1.91) for the AG genotype. Our analyses revealed an association between rs11125908 and the subgroup of patients with swollen joints < median under the codominant model for AG (p=0.034; OR=2.30) and GG genotype (p=0.034; OR=0.82) and with the overdominant model (p=0.01; OR=2.38). The subgroup of patients with an age of onset lower than the mean and AG genotype showed an association in the overdominant model (p=0.027; OR=2.27). Disease activity score 28 with erythrocyte sedimentation rate and disease duration variables were not associated with the rs11125908 polymorphism. rs11125908 was associated with RA and with the number of swollen joints and age of onset subgroup analyses. We provide concepts for treatments for RA, based on pharmacological management of COMMD1 expression.

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.

Study the Effect of RAGE and HMGB1 Genes Polymorphism on Breast Cancer Susceptibility in Iraqi Female

Background: The most frequent malignancy among women globally is breast cancer (BC). Breast lobules or ducts are the genesis of breast cancer, which is an uncontrolled development of epithelial cells. High mobility group protein box 1 (HMGB1) possesses both pro- and anti-tumorigenic bioactivities, which contribute to its complicated function in carcinogenesis. Damage associated Molecular Pattern molecules (DAMPs) generated following tissue injury include HMGB1, many S100 proteins, and others. RAGE is thought to be a receptor for these moleculAim of the study: The study was aimed to find the High mobility group protein box 1 (HMGB1) HMGB1 (rs1412125) and Receptor for Advanced Glycation End Products RAGE (rs1800624) genotypic and allelic frequency. Using Allele Specefic polymerase chain reaction AC-PCR and Polymerase Chain Reaction Restriction Fragment Length Polymorphism RFLP-PCR in BC patients and healthy controls, as well as their correlation with breast cancer susceptibility.Material and methods :. Samples were taken at Marjan Cancer Hospital, Babylon. HMGB1 rs1412125 and RAGE rs18400624 polymorphisms were evaluated for patients and controls using the PCR-RFLP and allele-specific polymerase chain reaction (AS-PCR) method. SPSS software was used to perform the statistical analysis.Results : There is non-significant difference between patients and control in TT, AA, and TA genotyping of (rs1800624) in RAGE gene (p=0.22), non-significant difference between patients and control in TT, TC, and CC genotyping of (rs1412125) in HMGB1 gene (p=0.09).Conclusion : Our results showed no association between HMGB1 polymorphisms and RAGE and breast cancer risk.

Highlighting allelic variations at the interleukin-19 locus in term of preeclampsia predisposing factors and access to an accurate diagnostic/screening option

BackgroundPreeclampsia is the main cause of preterm parturition and maternal–fetal complications. T helper 1 and T helper 2 cytokines balance is a requirement in normal pregnancy and aberrant in this immunologic balance, play an important role in the pathology of preeclampsia. In previous studies single nucleotide polymorphisms have been associated with the alteration of serum cytokine levels.ObjectiveThis study was aimed to discover association between interleukin-13 (rs20541, and rs56035208) and interleukin-19 (rs1028181 (T/C) and rs2243191(T/C)) polymorphisms with susceptibility to preeclampsia.MethodsIn this case-control study 300 women with and without preeclampsia (n = 150/each) who referred to Zeynabieh Hospital- Shiraz, Iran, from February 2021 to April 2022 were enrolled. For genotyping the interleukin-13 and interleukin-19 polymorphisms, the Allele-specific polymerase chain reaction and direct sequencing method was carried out.ResultsOur statistical results revealed no significant differences in allele and genotype frequencies for interleukin-13 polymorphisms compared to controls. We found that the interleukin‐13 polymorphisms are significantly associated with vulnerability to edema at rs20541 position and maternal drinking at rs56035208 position. But it was interesting to note that the differences of both the allele and genotype frequencies of interleukin-19 polymorphisms and their contribution to the risk of preeclampsia susceptibility were significant.ConclusionsNo risk of preeclampsia was found in all comparisons for interleukin-13 polymorphisms. However, the interleukin-19 polymorphisms were found to confer the risk of preeclampsia in our population.

Determining the Genotype of the RH Blood Group System in Maltese Blood Donors

Aims: The Rh blood group system is a complex protein based system having the D, C, c, E and e as the most immunogenic antigens. This blood group system has a vital role in transfusion medicine thus by DNA typing, important medical issues in transfusion practice can be resolved when serological techniques fail. The aim of the study was to determine the frequency of the RH genotype in the Maltese blood donors using molecular techniques.&#x0D; Methodology: 797 blood donor samples were enrolled in this study. An allele-specific polymerase chain reaction (AS-PCR) method was used to determine the presence of RHD, RHCE*E and RHCE*e, while multiplex PCR was used to test for RHCE*C/c. &#x0D; Results: Out of 797 samples studied, the most common allele was RHCE*e with a percentage of 98. The most common genotype in RhD positive samples was DCcee and in RhD negative samples, the most common was dccee. The most frequent haplotype was DCe.&#x0D; Conclusion: Like in previous studies, this research also concludes that the distribution of the RH genotype varies in different geographical areas. Further studies working with larger sample size and better techniques would share better light on the molecular function of these loci.

Simple confirmation methods for rare but impaired variants of human flavin-containing monooxygenase 3 (FMO3) found in an updated genome resource databank

Forty-seven new nonsense or missense human flavin-containing monooxygenase 3 (FMO3) variants were recently identified in an updated Japanese population reference panel. Of these, 20 rare single-nucleotide substitutions resulted in moderately or severely impaired FMO3 activity. To easily identify these 20 FMO3 variants (2 stop codon mutations, 2 frameshifts, and 16 amino-acid substitutions) in the clinical setting, simple confirmation methods for impaired FMO3 variants are proposed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) or allele-specific PCR methods. Using PCR-RFLP, FMO3 variants p.Arg51Gly, p.Met66Lys, p.Asn80Lys, p.Val151Glu, p.Val187fsTer25, p.Gly193Arg, p.Val283Ala, p.Asp286His, p.Val382Ala, and p.Phe451Leu were digested by the designated restriction enzymes and confirmed using reference cDNAs. In contrast, the FMO3 variants p.Gly39Val, p.Arg238Ter, p.Arg387Cys, p.Arg387His, p.Leu457Trp, and p.Met497Arg were not digested, whereas the wild type was digested. FMO3 variants p.Gly11Asp, p.Lys416fsTer72, p.Gln427Ter, and p.Thr453Pro were confirmed using allele-specific PCR systems. The previously identified FMO3 p.Arg500Ter variant has a relatively high frequency and was differentiated from p.Arg500Gln in two steps, i.e., enzyme restriction followed by allele-specific PCR, similar to the method for p.Arg387Cys and p.Arg387His. These systems should facilitate easy detection in the clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearance possibly caused by impaired FMO3 function.

Detection of Knockdown-Resistance Homozygous Mutant C1534C using Allele-Specific Polymerase Chain Reaction in Aedes Albopictus and Aedes Aegypti

Highlights: This research analyzed a previously understudied subject in Surabaya, Indonesia, and discovered knockdown resistance (kdr) mutations in Aedes albopictus. The findings of this study can prompt further research, including bioassay testing and the development of more potent insecticides. Abstarct : Dengue hemorrhagic fever (DHF) has been endemic in Indonesia for decades. Insecticides are necessary to manage the transmission of the dengue virus. However, prolonged use of insecticides can lead to insecticide resistance. This study aimed to examine the genotype of mosquitoes using the allele-specific polymerase chain reaction (ASPCR) method. The ASPCR method was chosen for genotype detection due to its high sensitivity, affordability, and ease of design. Five mosquitoes were collected from human habitation in four different areas of Surabaya, Indonesia, namely Kranggan, Ulul Azmi Mosque, Ploso, and Kalijudan. Among them, three samples were identified as Aedes albopictus (A1, A2, and A5) and two samples were identified as Aedes aegypti (A3 and A4). The frequency of resistant alleles was analyzed using the Hardy-Weinberg package in RStudio version 2023.03.1. This study revealed that two mosquitoes carried homozygous mutant alleles with a band of 113 bp and three mosquitoes carried homozygous wild-type alleles with a band of 93 bp. Cysteine-to-cysteine (C/C) mutations and phenylalanine-to-phenylalanine (F/F) mutations at codon 1534 were observed in Aedes aegypti and Aedes albopictus mosquitoes. The homozygous mutant alleles were found in Kranggan, Surabaya, Indonesia. Further research is required to assess insecticide resistance and knockdown resistance (kdr)-like mutation by collecting more representative samples from larger areas within the region of Surabaya. Nevertheless, this study can be used as a reference for vector control and early prevention of dengue fever.

Relationship between SHP2 gene polymorphisms and systemic lupus erythematosus risk.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder. SHP2, a non-transmembrane member of the protein tyrosine phosphatase (PTP) family, can be involved in multiple signaling pathways in inflammatory response. To date, it remains to be investigated whether polymorphisms in the SHP2 gene are correlated with SLE in the Chinese Han population. A study comprising 320 SLE patients and 400 healthy individuals was performed. Three single nucleotide polymorphisms (rs4767860, rs7132778, rs7953150) of the SHP2 gene were genotyped using the Kompetitive Allele-Specific Polymerase Chain Reaction method. Genotypes of rs4767860 (AA, AG + AA) and rs7132778 (AA, AC + AA), and alleles of rs4767860 (A) and rs7132778 (A) were associated with SLE risk. Genotype AA of rs7132778 and allele A of rs7132778 and rs7953150 were associated with oral ulcers in SLE patients. Allele C of rs7132778 and genotype AA and allele A of rs7953150 were associated with pyuria. Patients who carried AA genotype and allele A of rs7953150 are more likely to develop hypocomplementemia. AA and AG genotype frequencies are more raised in patients with SLE with alopecia than in those without alopecia. Patients who carried AA and AG genotypes of rs4767860 had elevated C-reactive protein levels. Gene polymorphisms of SHP2 (rs4767860, rs7132778) are relevant to SLE susceptibility.

Single Nucleotide Polymorphism at rs7903146 of Transcription Factor 7-like 2 gene Among Subjects with Type 2 Diabetes Mellitus in Myanmar.

To investigate the association between the single nucleotide polymorphism (SNP) rs7903146 in the transcription factor 7-like 2 (TCF7L2) gene and type 2 diabetes mellitus (T2DM) and to examine the impact of this variant on pancreatic beta-cell function in the Myanmar population. A case-control study was undertaken in 100 subjects with T2DM and 113 controls. The SNP rs7903146 was genotyped using the allele-specific polymerase chain reaction method. Plasma glucose and serum insulin levels were determined using the enzymatic colorimetric method and ELISA respectively. Beta-cell function was calculated by the HOMA-β formula. The frequencies of carrier genotypes (CT and TT) were higher in subjects with T2DM than in controls. The minor T alleles of rs7903146 were found to statistically increase type 2 diabetes risk than the C allele with an allelic odds ratio of 2.07 (95% CI 1.39-3.09, p=0.0004). The mean HOMA-β level of the group with non-carrier genotype (CC) was significantly higher than that of the groups with carrier genotypes (CT and TT) in subjects with T2DM and controls with a p-value of 0.0003 and less than 0.0001, respectively. The rs7903146 variant of the TCF7L2 gene was found to be associated with T2DM and low β-cell function among Myanmar subjects.

Identification of Self Incompatibility (S) Alleles in Turkish Apple Gene Sources using Allele-specific PCR

Self-incompatibility (SI) is a genetic mechanism in many flowering plants by which generative reproduction is prevented. The self-incompatibility caused by the genetic functions of the cell is controlled by genes called S genes or self-incompatibility genes. Self-incompatibility results in decreased pollination and ultimately yield loss. In apple (Malus domestica L.), self-incompatibility is controlled by multi-allelic S-locus. Approaches in the S-glycoprotein profiles and allele-specific PCR methods using the gene profiles and S-glycoprotein profiles for determination of the incompatibility levels are of great importance. In current study, the self-incompatibility status of 192 apple genotypes (such as, Amasya, Hüryemez, Şah elması, Tokat, Demir elması etc.) obtained from the National Collection of Atatürk Horticultural Central Research Institute, Yalova, Turkey, has been determined. For this purpose, genotype-specific allele status and compatibility levels were screened via PCR (Polymerase Chain Reaction) using 4 different S-alleles (Sd, Sf, S26 and S9). 181 genotypes containing at least 1 S-allele were identified as ‘Partially Incompatible’ and 12 genotypes involving 4 S-alleles were assigned ‘Totally Incompatible’. No S-alleles were observed in 2 genotypes (Pancarlık and Hüryemez) which exhibited ‘Compatibility’ status.

Detection of homozygous wildtype V1016V using allele-specific polymerase chain reaction in Aedes albopictus

Abstract. Setiawan AR, Fadila SZ, Sucipto TH, Fauziyah S, Madaniyah S, Dewi EC, Naw SW, Tukiran, Cahyaningrum SE. 2023. Detection of homozygous wildtype V1016V using allele-specific polymerase chain reaction in Aedes albopictus. Biodiversitas 24: 62-67. Aedes sp. is a carrier of several viruses that can infect humans and cause diseases such as zika, yellow fever, chikungunya, and dengue fever. Symptoms of dengue infection vary, consisting of classic dengue fever (DD), dengue hemorrhagic fever (DHF), and dengue shock syndrome. Insecticide spray can be used to manage Aedes mosquitoes chemically. Insecticide substances target nervous system proteins. Voltage-gated sodium channels (VGSC) are rendered inactive by pyrethroid binding. Knockdown is a signal indicating an insect has been knocked down in response to a specific insecticide. However, using insecticides for a long time can cause mosquitoes to become resistant. The pesticide resistance of mosquitoes is known as knockdown resistance (kdr). This study aims to detect kdr mutations (V1016G) in two male Aedes albopictusmosquitos named A1 and A2 collected from settlements in Kranggan, Sawahan, Surabaya, East Java, Indonesia, using allele-specific polymerase chain reaction (AS-PCR) assay. RNA was extracted from the two mosquito samples using an RNA extraction kit. After that, the extracted RNA was tested for kdr mutations using the AS-PCR method. After assaying, both samples are homozygous wildtype (V1016V) because the results showed bands appearing from samples A1 and A2 at 60 bp. On the other hand, this study has the potential to serve as preliminary monitoring for the program controlling vectors.

A high-throughput KASP assay provides insights into the evolution of multiple resistant mutations in populations of the two-spotted spider mite Tetranychus urticae across China.

The two-spotted spider mite (TSSM), Tetranychus urticae (Acari: Tetranychidae), is a cosmopolitan phytophagous pest in agriculture and horticulture. It has developed resistance to many acaricides by target-site mutations. Understanding the status and evolution of resistant mutations in the field is essential for resistance management. Here, we applied a high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) method for detecting six mutations conferring resistance to four acaricides of the TSSM. We genotyped 3274 female adults of TSSM from 43 populations collected across China in 2017, 2020, and 2021. The KASP genotyping of 24 testing individuals showed 99% agreement with Sanger sequencing results. KASP assays showed that most populations had a high frequency of mutations conferring avermectin (G314D and G326E) and pyridaben (H92R) resistance. The frequency of mutation conferring bifenazate (A269V and G126S) and etoxazole (I1017F) resistance was relatively low. Multiple mutations were common in the TSSM, with 70.2% and 24.6% of individuals having 2-6 and 7-10 of 10 possible resistant alleles, respectively. No loci were linked in most populations among the six mutations, indicating the development of multiple resistance is mainly by independent selection. However, G314D and I1017F on the nuclear genome deviated from Hardy-Weinberg equilibrium in most populations, indicating significant selective pressure on TSSM populations by acaricides or fitness cost of the mutations in the absence of acaricide selection. Our study revealed that the high frequency of TSSMs evolved multiple resistant mutations in population and individual levels by independent selection across China, alarming for managing multiple-acaricides resistance. © 2023 Society of Chemical Industry.

Association of TCF7L2 Variants in Type 2 Diabetes Mellitus with Hypertriglyceridemia - A Case-Control Study.

Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic condition involving various genetic and environmental factors leading to impaired insulin secretion, resulting in hyperglycemia. The transcription factor 7-like 2 (TCF7L2) gene is an element of the Wnt signaling pathway that plays an important role in glucose and lipid metabolism. The aim of this study is to evaluate the association of TCF7L2 rs7903146 and rs12255372 polymorphisms in T2DM with hypertriglyceridemia. We investigated the effect of rs7903146 and rs12255372 on T2DM with high triglyceride (TG) levels in 60 patients and 20 controls. The anthropometric measurements and biochemical tests were assessed. Peripheral blood samples were collected, and genomic DNA was extracted. The genotyping of TCF7L2 polymorphisms was carried out using polymerase chain reaction (PCR)-based direct sequencing and allele-specific PCR methods. The T2DM patients and controls were compared by means of the t-test, Chi-square test, odds ratio (OR), and 95% confidence interval (CI) using Epi Info v7. The HbA1c was found to be 9.7 ± 2.1 and 5.4 ± 0.5% in patients and controls, respectively. The average TG levels (P < 0.005) in patients were 205.2 ± 145.7 and 106.4 ± 27.4mg/dl in controls. Significant evidence of association was found in T2DM patients having high TG levels with rs7903146 CT/TT (OR: 4.89; P = 0.0105) and rs12255372 GT/TT (OR: 5.23; P = 0.0101) genotypes when compared to controls. The results of this study show that TCF7L2 rs7903146 CT/TT and rs12255372 GT/TT genotypes are significantly associated with the risk of hypertriglyceridemia in individuals with T2DM among the studied population.

Association of Human Leukocyte Antigen Alleles with Carbamazepine- or Lamotrigine-Induced Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis in an Iranian Population: A Case-control Study.

Genetic diversity in human leukocyte antigen (HLA) alleles across populations is a significant risk factor for drug-induced severe cutaneous adverse reactions (SCARs), e.g., carbamazepine (CBZ)- and lamotrigine (LTG)-induced Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). The present study aimed to investigate the frequency of different HLA alleles in Iranian patients with CBZ- and LTG-induced SJS/TEN. A case-control study was conducted from 2011 to 2018 at various hospitals affiliated with Shiraz University of Medical Sciences (Shiraz, Iran). A total of 31 patients receiving anticonvulsant drugs (CZB or LTG) were recruited and divided into two groups. The drug-induced group (n=14) included hospitalized patients due to CBZ- or LTG-induced SJS/TEN. The drug-tolerant group (n=17) included individuals receiving CBZ or LTG for at least three months with no adverse effects. In addition, 46 healthy individuals (control group) were recruited. The frequency of HLA-A, -B, and -DRB1 alleles in patients with CZB- or LTG-induced SJS/TEN was investigated. HLA typing was performed using the allele-specific polymerase chain reaction method. The Chi square test and Fisher's exact test were used to determine a potential association between SJS/TEN and HLA alleles. P<0.05 was considered statistically significant. CBZ- or LTG-induced SJS/TEN was not significantly associated with HLA alleles. However, HLA-DRB1*01 showed a significantly higher frequency in patients with CBZ-induced SJS/TEN than the CBZ-tolerant patients (30% vs. 9%, P=0.07). Overall, no significant association was found between CBZ- or LTG-induced SJS/TEN and HLA alleles. Further large-scale studies are required to substantiate our findings.

Prevalence of CYP2C9 and CYP2C19 variants and the impact on clopidogrel efficacy in patients having CYPC19*2 variant.

Human cytochrome p450 enzymes play an important role in the metabolism of various substances. The CYP2C subfamily consists of various important drug-metabolizing enzymes such as CYP2C9 and CYP2C19. The objectives of the study include the determination of the frequency of genetic variants (CYP2C9*2, CYP2C9*3, and CYP2C19*2) of selected enzymes using allele-specific polymerase chain reaction (ASPCR) and its comparison with Indian as well as global past frequencies. We also aimed to study the impact of genetic mutation on clopidogrel efficacy and compare the efficacies between patients with and without CYP2C19*2 genetic variant. In this study, the prevalence of variants CYP2C19*2, CYP2C9*2, and CYP2C9*3, the most popular variants of the respective enzymes, was determined using the ASPCR method. The correlation between the CYP2C19*2 variant and the antiplatelet activity of clopidogrel was studied using platelet aggregation assay (PAA). The determined frequencies of CYP2C19*2, CYP2C9*2, and CYP2C9*3 are 46%, 9%, and 12%. These frequencies are indicative of homozygous as well as heterozygous mutations. Reduced clopidogrel efficacy was observed in patients with a heterozygous mutation of CYP2C19*2 variant. The observed frequencies are not significantly different from that observed in earlier reported studies conducted across India and the world. Antiplatelet activity, as measured using the PAA method, was significantly lesser in patients having the CYP2C19*2 variant. The therapy failure in these patients can lead to serious cardiovascular consequences, and we propose determining the presence of the CYP2C19*2 variant before initiation of clopidogrel therapy.

Fast and more affordable CYP2C19 LOF testing to personalized of clopidogrel therapy

Abstract Background Clopidogrel is an antiplatelet drug used to treat coronary artery disease (CAD) to reduce blood clotting by inhibiting platelet activity. However, many patients experience ischemic events due to changes in clopidogrel metabolism attributed to various genetic and non-genetic factors. CYP2C19 is one of the important genes responsible for the metabolism of clopidogrel, but this gene has polymorphisms in cytochrome P450 (CYP), and this CYP2C19 Loss-of-function (LoF) polymorphisms impair clopidogrel metabolism between individuals and races. Therefore, genetic testing is an effective strategy to personalized clopidogrel and reduce the incidence of ischemic events in CAD patients. Purpose We sought to develop a nested allele-specific multiplex polymerase chain reaction (PCR) method for the detection of CYP2C9 LOF alleles. Method Genomic DNA was extracted from blood taken from 7 healthy volunteers using a commercial DNA extraction kit. A two-step PCR method was developed. First, DNA was subjected to a first PCR used to amplify exons 4 and 5 simultaneously in a single reaction tube. Second, The products of 1st PCR were then used as a template in the second PCR to detect CYP2C9 polymorphisms alleles (CYP2C19*2 and *3) using allele-specific primers. The test results were validated via sequencing. Result We successfully detected clinically important CYP2C19 LOF polymorphisms alleles by the nested allele-specific multiplex PCR method (Figure 1). This method is fast and doesn't require high-quality and expensive laboratory equipment. It was reproducible and specific and can reliably detect CYP2C19 variants. Direct sequencing of target variants has been used to validate this strategy further, and the amplified sequences were 100% identical to the CYP2C9 sequence. Conclusion The developed methods were specific to identify polymorphisms in CYP2C19 (rs4244285 and rs4986893). However, to understand ethnic differences and their potential implications for clopidogrel therapy, we highly recommend identifying relevant genetic polymorphisms before clinical manifestations arise. Funding Acknowledgement Type of funding sources: None.

Molecular characterization and A1/A2 genotyping of casein beta gene in zebu and crossbred cattle of Bangladesh

Casein beta (CSN2) is the most explored gene in cattle due to its potential impact on human health. Here, we investigated the entire coding sequence of CSN2 gene except the last two codons for detection of polymorphisms in different cattle populations of Bangladesh as well as genotyping of A1 and A2 allelic variants using a newly developed allele specific polymerase chain reaction (AS-PCR) based the genotyping protocol. Five primer pairs were used to amplify the coding sequences of CSN2 gene. This study included a total of 258 DNA samples from six Bangladeshi zebu populations and one Holstein Friesian (HF) derived crossbred cattle population. Sequence analysis detected five nonsynonymous mutations in the coding sequence of CSN2 gene that defined five allelic variants as A1, A2, B, F and I. In addition, substitution of GTA (Val) by GCA (Ala) at position 197 resulted in an undefined allele in the zebu cattle population of Bangladesh that has not yet been reported elsewhere. Like other Bos indicus cattle populations, A2 allele is predominant in the studied zebu populations. The mean frequencies of A1A1, A1A2 and A2A2 genotypes were 0.02, 0.16 and 0.82, respectively, in zebu cattle populations while the corresponding allele frequencies were A1 (0.10) and A2 (0.90). In opposite, the aforesaid genotype frequencies were 0.14 (A1A1), 0.50 (A1A2) and 0.36 (A2A2) in the HF crossbred population with allele frequencies of 0.39 (A1) and 0.61 (A2). The adopted AS-PCR method was found cost-effective, rapid and had high specificity for genotyping of A1 and A2 allelic variants. Altogether, this study provides information for the selection of desired zebu and crossbred individuals in order to produce premium quality milk as well as to design a breeding plan in the crossbreeding program.

Phenotypic Expression of Known and Novel Hemoglobin A2-Variants, Hemoglobin A2-Mae Phrik [Delta 52(D3) Asp > Gly, HBD:c.158A > G], Associated with Hemoglobin E [Beta 26(B8) Glu > Lys, HBB:c.79G > A] in Thailand

The interactions of δ-globin variants with α- and β-thalassemia or other hemoglobinopathies cause complex thalassemic syndromes and potential diagnostic problems. Understanding the molecular basis and phenotypic expression is crucial. Four unrelated Thai subjects with second hemoglobin (Hb) A2 fractions were studied. A standard automated cell counter was used to acquire initial hematological data. Hb analysis was carried out by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assays. Globin gene mutations and haplotype were identified by appropriate DNA analysis. An allele-specific polymerase chain reaction method was developed to provide a simple molecular diagnostic test. Hb analysis revealed a Hb A2 variant in all cases. DNA analysis of the δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu > Lys] variant in combination with Hb E in three cases. Analysis of the remaining case identified a novel δ-Hb variant, namely Hb A2-Mae Phrik [δ52(D3)GAT > GGT; Asp > Gly], found in association with Hb E and α+-thalassemia, indicative of the as yet undescribed combination of triple heterozygosity of globin gene defects. An allele-specific PCR-based assay was successfully developed to identify this variant. The β-haplotype of the Hb A2 Mae-Phrik allele was strongly associated with haplotype [+ − − − − ± +]. This study advanced our understanding of the phenotypic expression of known and novel δ-Hb variants coinherited with other globin gene defects, routinely causing problems with diagnosis. Therefore, knowledge and recognition of this Hb variant and molecular assessments are crucial to improving diagnosis.

Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA.

Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.

Association of -675 4G/5G PAI-1 and -2518A/G MCP-1 genetic polymorphisms with polycystic ovary syndrome in Kashmiri women: A case control study.

Polycystic ovarian syndrome (PCOS) is a highly prevalent endocrine disorder among females of fertile age. It has been speculated to be associated with low-grade chronic inflammation like other inflammatory response-driven multifactorial illnesses such as diabetes mellitus (DM) and cancer. Monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) are biomarkers of inflammation and endothelial dysfunction, respectively. These have been found to be elevated in PCOS patients. The current research reveals that single nucleotide polymorphisms (SNPs) in their genes are strongly associated with the elevation of these biomarkers. The goal of this study was to see if there was a link between PAI-1 -675 4G/5G and MCP-1 -2518 A/G polymorphisms with the occurrence of PCOS. This study included 220 PCOS participants and 220 healthy controls. The allele-specific polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods were used to investigate PAI-1-675 4G/5G and MCP-1 -2518A/G SNPs, respectively. The -675 4G/5G SNP in the PAI-1 gene was strongly linked to PCOS. The odds ratio (OR) for the 4G/4G genotype was (OR = 3.2; P = 0.001), whereas the OR for the 4G/5G genotype was (OR = 2.39; P = 0.001). The carriers with the 4G/4G and 4G/5G genotypes showed significantly increasing trends in the triglyceride levels (P < 0.05). The genotypic frequency of the -2518 A/G MCP-1 SNP differed significantly between the PCOS patients and healthy controls; the GG genotype remained a strong predictor of PCOS (OR = 8.7; P = 0.01) and the AG genotype (OR = 2.40; P = 0.01), indicating an elevated risk of predisposing women to PCOS. There was a significant variation in the glucose 2-h levels between -2518A/G MCP-1 genotypes with AG heterozygous and GG mutant genotype showing increasing trends of glucose 2-h levels (P < 0.05). Both PAI-1 -675 4G/5G and MCP-1 -2518A/G polymorphisms are associated with predisposition to PCOS and its complications in Kashmiri women.