Commercial TaqMan Research Articles

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR.

The declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance. Using 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples. The study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes. The employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method's robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method's independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves.

Genetic risk score for gestational weight gain

Gestational weight gain (GWG) involves health consequences for both mother and offspring. Genetic factors seem to play a role in the GWG trait. For small effect sizes of a single genetic polymorphism (SNP), a genetic risk score (GRS) summarizing risk-associated variation from multiple SNPs can serve as an effective approach to genetic association analysis.The aim of the study was to analyze the association between genetic risk score (GRS) and gestational weight gain (GWG).GWG was calculated for a total of 342 healthy Polish women of Caucasian origin, aged 19 to 45 years. The SNPs rs9939609 (FTO), rs6548238 (TMEM18), rs17782313 (MC4R), rs10938397 (GNPDA2), rs10913469 (SEC16B), rs1137101 (LEPR), rs7799039 (LEP), and rs5443 (GNB3) were genotyped using commercial TaqMan SNP assays. A simple genetic risk score was calculated into two ways: GRS1 based on the sum of risk alleles from each of the SNPs, while GRS2 based on the sum of risk alleles of FTO, LEPR, LEP, and GNB3.Positive association between GRS2 and GWG (β = 0.12, p = 0.029) was observed. Genetic risk variants of TMEM18 (p = 0.006, OR = 2.6) and GNB3 (p < 0.001, OR = 3.3) are more frequent in women with increased GWG, but a risk variant of GNPDA2 (p < 0.001, OR = 2.7) is more frequent in women with adequate GWG, and a risk variant of LEPR (p = 0.011, OR = 3.1) in women with decreased GWG.GRS2 and genetic variants of TMEM18, GNB3, GNPDA2, and LEPR are associated with weight gain during pregnancy.

Impact of the mare colostral immunoglobulins on the expression of TLR3, TLR4, and TLR7 in foals.

Colostrum contains substances such as hormones, enzymes, polyamides, nucleic acid derivatives, and amino acid derivatives and also includes immunomodulating substances, proline-rich polypeptides, immunoglobulins (Ig) as well as bacteriostatic compounds. The aim of the study was to evaluate the impact of colostral Ig on the level of expression of selected Toll-like receptor genes (TLR3, TLR4, and TLR7). The experiments were conducted on 25 Polish Pony foals. Blood samples were collected according to the following scheme: before first suckling, at the 1st, 3rd, 5th, 10th, 20th, and 30th, day of age. Colostrum was obtained before the first suckling, 24 hr, and 3 days after the delivery. Gene expression analyses were performed on Illumina Eco using a commercial kit TaqMan®MGB probes. The quality of colostrum was assessed via refractometer and BRIX value. The total Ig level was calculated from the spectrophotometric method. IgG levels were measured using ELISA assay. The quality of colostrum significantly correlated with TLR4 and TLR7 expression. Foals that ingested colostrum with low Ig content displayed a higher level of TLR4 gene expression, while in the case of TLR7 the opposite trend was shown. This result indicates that colostrum may play an important role in shaping the mechanisms of building the immune system's response to bacterial pathogens.

Optimization and Application of Quantitative In-House ELISA for Diagnosis of HBsAg and its Correlation with Commercial ELISA and Molecular Kits

Background: Several studies have indicated the role of quantitative hepatitis B surface antigen (HBsAg) evaluation in managing and prognosis of hepatitis B virus (HBV) infection. Thus, quantitative evaluation of HBsAg using cost-effective assays can be an important approach to managing HBV patients. Objectives: This study aimed to set up and apply an in-house quantitative enzyme-linked immunosorbent assay (ELISA) to evaluate HBsAg in research and diagnosis. Methods: New Zealand white rabbits were immunized with HBsAg. Sera were collected 28 days after immunization, and polyclonal HBsAb (antibody to hepatitis B surface antigen) were purified and evaluated. Then, the in-house quantitative ELISA was optimized. Finally, the functional characteristics of the assay were evaluated using 200 plasma samples compared to commercial ELISA and quantitative TaqMan real-time PCR. Results: The assay has a limit of detection (LOD) of 0.5 ng/mL with a specificity and sensitivity of 94% and 97%, respectively. The assay's highest coefficient of variation (CV) values for intra- and inter-assays were 7.23% and 8.59%, respectively. The correlation of the developed assay with commercial ELISA was 0.987 (P-value = 0.024). The correlations of the developed assay and commercial ELISA with quantitative TaqMan real-time PCR were 0.739 and 0.658, respectively (P-value = 0.017). Conclusions: The developed assay has a suitable sensitivity and specificity. It is also reproducible and well-correlated with commercial assays. Most importantly, it is cost-effective and, thus, can be used for detecting and quantifying HBsAg in research and diagnosis.

Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas.

The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is beginning to be tested. We evaluated the efficiency of the automated extraction of viral RNA for studies of pediatric dengue and zika. Clinical samples of children with dengue that were well characterized through RNA isolation by silica columns and serotype-specific nested RT-PCR (DENV-1 n = 7, DENV-2 n = 5, and negatives n = 8) in addition to 40 pediatric plasma samples spiked with ZIKV (strain PRVA BC59) and 209 from negative pre-epidemic children were analyzed. RNA from patients was extracted by two automated standard and high-throughput protocols on the KingFisher™ Flex instrument. The isolated RNA was evaluated for concentration and purity by spectrophotometry, for structural and functional integrity by electrophoresis and expression of the RNase P gene, and usefulness in serotype-specific DENV detection by conventional and real-time RT-PCR. For the evaluation of ZIKV RNA, the commercial TaqMan Triplex® assay was used, along with a well-tested in-house RT-qPCR assay. The concentration of RNA (5.2 vs. 7.5 ng/μL, P=0.03) and the number of integral bands (9 vs. 11) were higher with the high-throughput protocol. However, the number of specimens serotyped for DENV by RT-qPCR was comparable for both protocols. The cycle thresholds of the TaqMan Triplex® commercial kit and the in-house assay for the detection of plasma ZIKV RNA isolated with the standard protocol showed a strong association (r = 0.93, P < 0.0001) and a Cohen Kappa index of 0.98 when all 249 samples were analyzed. These preliminary results suggest that automated instruments could be used in studies of cocirculating flaviviruses that have represented a public health problem in recent decades in Colombia. They boast advantages such as efficiency, precision, time savings, and lower risk of cross-contamination.

Expression of microRNA-590 in patients with chronic coronary heart disease, atrial fibrillation, and their combination

Background. The role of microRNAs in the processes of remodeling, proliferation, and fibrogenesis of myocardial cells is not clear enough.&#x0D; Aim. Analysis of the microRNA-590 expression level in patients with chronic coronary heart disease, atrial fibrillation, as well as their combination to assess the potential prognostic significance of the expression level.&#x0D; Material and methods. The study included 94 patients divided into three clinical groups: the first with non-valvular atrial fibrillation without coronary heart disease (39 people); the second with non-valvular atrial fibrillation and coronary heart disease (22 patients); the third with ischemic heart disease without atrial fibrillation (23 patients). The comparison group consisted of 10 people without atrial fibrillation and coronary heart disease. Venous blood was taken from all subjects, from the plasma of which microRNA was isolated. The relative level of microRNA expression was estimated based on real-time polymerase chain reaction data obtained during the reaction on a cycler using commercial TaqMan probes and primers. The statistical significance of differences between groups was determined using one-way analysis of variance, followed by post-hoc analysis using Tukey's contrasts, differences were considered statistically significant at p 0.05. The ShapiroWilk test was used to assess the normality of the distribution of residuals.&#x0D; Results. A statistically significant decrease in the expression level of microRNA-590 was registered in the third (p=0.0104) and in the second (p=0.0046) groups compared with the control group, as well as in patients with atrial fibrillation and left atrial dilatation (p=0.0313), with recurrent arrhythmias after radiofrequency ablation (p=0.0083) and with permanent atrial fibrillation (p=0.0242).&#x0D; Conclusion. Ischemic heart disease, including when combined with atrial fibrillation, and aggravating factors, such as left atrial dilatation, permanent atrial fibrillation, recurrence of arrhythmia after radiofrequency ablation, lead to a decrease in the expression level of microRNA-590.

Rapid and accurate quantification of isomiRs by RT-qPCR

Currently, microRNAs (miRs) are annotated as a single defined sequence (canonical), even though high-throughput small RNA sequencing has identified miR isoforms (isomiRs) that differ from their canonical counterparts in length, sequence, or both. Here we describe a simple reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)-based assay for quantification of the miR-100-5p_iso_3p:−2 variant. We chose miR-100-5p because the canonical sequence was underrepresented in our evaluation of human plasma. The quantification of miR-100-5p_iso_3 p:−2 from 57 plasma samples demonstrated high concordance between high-throughput RNA sequencing and RT-qPCR results (r = 0.55, p < 0.0001). Of note, we could not detect or quantify miR-100-5p in our plasma samples using a commercial TaqMan canonical miR-100-5p RT-qPCR kit. With these 57 samples, we also adapted this assay to specifically quantify the canonical sequences of miR-122-5p and miR-192-5p. Similar to the results obtained with miR-100-5p_iso_3p:−2, RT-qPCR results for miR-122-5p and miR-192-5p highly correlated with high-throughput RNA sequencing data (miR-122-5p: r = 0.44, p = 0.0005; miR-192-5p: r = 0.72, p < 0.0001). The assay described here can be easily adapted to many different identified isomiRs. Because of the high specificity of isomiRs, their reliable RT-qPCR-based quantification could provide greater resolution and higher accuracy than using canonical sequences.

Evaluation of Pharmacogenetics of Drug-Metabolizing Enzymes and Drug Efflux Transporter in Renal Transplants Receiving Immunosuppressants.

Cytochrome P450 (CYP) enzymes, such as CYP3A4, and CYP3A5, P450 oxidoreductase (POR), peroxisome proliferator activated receptor alpha (PPAR-alpha), and drug transporter (ABCB1) were observed to influence concentrations of immunosuppressants (cyclosporine, everolimus, sirolimus, and tacrolimus) and outcomes in renal transplants. We carried out the present study to evaluate the prevalence and impact of these single nucleotide polymorphisms (SNPs) in adult renal transplants. SNPs were evaluated using commercial TaqMan® assays. Serum drug concentrations were estimated using immunoassays. One hundred and forty-six patients were recruited. SNPs in CYP3A5*3 were significantly associated with greater dose-adjusted cyclosporine and tacrolimus concentrations. SNPs in POR*28 were observed with significantly lower dose-adjusted concentrations, particularly with cyclosporine and tacrolimus. ABCB1 homozygous polymorphisms were observed with significantly lower time spent in the therapeutic range with cyclosporine and everolimus/sirolimus. Cyclosporine was observed in a significantly greater proportion of patients with elevated GGT, and SNPs in PPAR-alpha were significantly associated with an increased risk of this adverse event. Hypertriglyceridemia with everolimus was significantly associated with POR*28 polymorphisms. There is a need to validate the influence of these SNPs in a prospective study and develop an algorithm predicting the achievement of target concentrations.

Frequency of ADIPOQ 276 and ADIPOQ 45 Polymorphisms in Obese and Eutrophic Adolescents with and without Asthma and their Relationship with Serum Adiponectin Levels

Background: Asthma is a chronic allergic disease characterized by variable airflow limitation; Obesity is a chronic disease that has reached epidemic proportions globally. Both are diseases with a significant inflammatory component, and their relationship suggests being weight dependent. Adiponectin (ADIPOQ) is the main adipokine secreted by white adipose tissue, it is an insulin synthesizer and regulator of energy homeostasis, and its plasma levels are inversely correlated with obesity and asthma. The effect of genetic factors in both diseases has been investigated, and haplotypes of the ADIPOQ 45 T/G (rs2241766) and ADIPOQ 276 G/T (rs1501299) polymorphisms have been related. Aims: To know de polymorphisms frequency of ADIPOQ 45 and ADIPO 276 in obese and eutrophic adolescents with and without asthma, likewise, link the adiponectin levels with the presence of such polymorphisms. Methods: An observational, analytical, and cross-sectional study in which 169 Mexican adolescents were recruited. Thirty mL of blood was taken from each individual; genomic DNA was extracted using the saline expulsion technique and quantified by spectrophotometry; two polymorphisms located in the promoter region were studied: ADIPOQ 45 and ADIPOQ 276; the determination of the different polymorphisms was carried out using TaqMan probes using real-time PCR (qPCR) using the commercial kit TaqMan One Step RT-PCR mastermix, the RNA extraction was carried out with Trizol Ls (Invitrogen), and the fluorescence was quantified employing the 7900HT ABI PRISM real-time computer SDS software. Results: There were no statistically significant differences between ADIPOQ 276 and ADIPOQ 45 polymorphisms in asthmatic and obese patients. Compared to the control group, a negative correlation was observed between adiponectin plasmatic levels in obese and asthmatic individuals. Conclusion: The ADIPOQ 276 and ADIPOQ 45 polymorphisms do not seem to be associated with asthma and obesity in the Mexican population. It is necessary to continue studying these polymorphisms and consider larger populations.

Analiza polimorfizma rs34637584 u genu LRRK2 kod bolesnika sa Parkinsonovom bolešću

Introduction: Parkinson's disease (PD) belongs to neurodegenerative diseases, and since the prevalence is 1% to 2% in people older than 65 years and over 4% in people older than 85 years, it is the second most common disease in this group. The cause of PD is the death of dopaminergic neurons in the CNS, primarily in the basal ganglia and the substantia nigra. The LRRK2/PARK8 gene is located on the short arm of chromosome 12. There are many variants in this gene associated with PD, and the most common of which is mutation c.6055G&gt;A (p.Gly2019Ser), also referred to as rs34637584. Aim: The aim of this study was to determine the frequency of the c.6055G&gt;A (rs34637584) mutation in the LRRK2 gene in a group of patients with PD from Serbia. Material and methods: The study included a group of 127 patients with PD from tertiary healthcare institutions in Serbia, as well as an appropriate control group without neurological diseases. Molecular genetic analysis was performed by real-time PCR, using a commercial TaqMan assay. Results: Mutation c.6055G&gt;A ie. rs34637584 variant was detected in 1 of 127 examinees with PD (0.7%). That patient was without a previous family history of PD. This mutation was not found in the control group. Conclusion: The results obtained align with findings of previous studies for the European, especially the southern European population. If these results would be confirmed in a larger patient sample, testing of LRRK2 mutation c.6055G&gt;A (rs34637584) should become part of the PD test protocol.

EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.

Comparison of Seven Commercial TaqMan Master Mixes and Two Real-Time PCR Platforms Regarding the Rapid Detection of Porcine DNA

A pig-specific real-time PCR assay based on the mitochondrial ND5 gene was developed to detect porcine material in food and other products. To optimize the performance of assay, seven commercial TaqMan master mixes and two real-time PCR platforms (Applied Biosystems StepOnePlus and Bio-rad CFX Connect) were used to evaluate the limit of detection (LOD) as well as the PCR efficiency and specificity. The LODs and PCR efficiencies for the seven master mixes on two platforms were 0.5–5 pg/reaction and 84.96%–108.80%, respectively. Additionally, non-specific amplifications of DNA from other animal samples (human, dog, cow, and chicken) were observed for four master mixes. These results imply that the sensitivity and specificity of a real-time PCR assay may vary depending on master mix and platform used. The best combination of master mix and real-time PCR platform can accurately detect 0.5 pg porcine DNA, with a PCR efficiency of 100.49%.

DNA Variants Involved in Regulation of Fetal Hemoglobin in Mexican Patients with Sickle Cell Disease. a Preliminary Analysis

HbS (HBB:c.20A&amp;gt;T, beta 6 Glu&amp;gt;Val) is a frequent Hb variant in Mexico, in particular in some regions close to the Pacific and Atlantic Oceans, with carrier frequencies from 0.5 to 12.8%, therefore Sickle Cell Disease patients are often seen in those regions. It is well known that increased levels of Fetal Hemoglobin (HbF) ameliorates the clinical complications of sickle cell disease. Several genetic studies have identifiedBCL11A,HBS1L-MYB(intergenic region),HBG2andHBBP1genes, among others, to be involved in HbF regulation; DNA variants in theselociare associated to elevated HbF. The aim of present study was to analyze 15 variants in HbF regulatorylociin Mexican patients with Sickle Cell Disease. About 10 mL of peripheral blood was collected in EDTA for hematological and molecular testing from 24 sickle cell anemia patients (S/S) and 15 sickle cell trait carriers (S/A) from Southern Mexico, 13 were from the state of Guerrero and 26 from Chiapas. All subjects voluntarily agreed to participate in this study and gave signed informed consent; underaged patients' consent was obtained from their parents; all procedures were performed according to the ethical principles of the Declaration of Helsinki. Hemoglobin S genotype was determined by Sanger sequencing; DNA variants genotyping was performed by qPCR using commercial Taqman probes for the following variants: inBCL11Ars11886868, rs4671393, rs7557939, rs1427407, rs766432, rs6706648, rs7599488; inHBS1L-MYBrs7776054, rs28384513, rs9399137, rs4895441, rs9402686, rs1320963, inHBG2rs7482144 and inHBBP1rs10128556. Hematological and clinical data were analyzed by IBM SPSS v24®. A total of 39 sickle cell patients were studied, twenty-one patients were male and 18 were female (53.8% and 46.2% respectively). Thirty-two were pediatric age patients and 7 adults. Anemia was observed in all S/S patients, eight had severe anemia (&amp;lt;8 g/dL); on the other hand, only 5/15 S/A patients had anemia (10 - 12 g/dL); HbF over 5% was observed in 23/24 S/S patients and in 13/15 S/A had HbF from 2% - 5%. Nineteen S/S patients were treated with hydroxyurea and presented less severe phenotype and more elevated HbF, however, the statistical analysis showed no significant differences (12.9%vs8.9%p=0.248) (Table 1). Genotype and allele frequencies are displayed in Table 2. All minor alleles were observed in frequencies over 0.05, the mo2st commonly observed minor allele wasBCL11Ars1427407 (0.69), and the less frequently observed wasHBBP1rs101028556 (0.08). The allele frequencies of four HbF regulating variants were significant different from those reported for Mexican ancestry population (MXL) in 1000 Genome database (rs11886868 C&amp;gt;T, rs4671393 A&amp;gt;G, rs7599488 C&amp;gt;T and rs10128556 C&amp;gt;T), however, the analyzed sample in this study is undersized and is not representative of the global Mexican population. No differences were observed when comparing allele frequencies from GuerrerovsChiapas patients, nor S/SvsS/A. The correlation analysis of minor allele count (MAC) and HbF demonstrated no association when comparing it to HbF levels. Likewise, the analysis was performed onBCL11AandHBS1L-MYBvariants and showed no significant correlations. There was also no correlation between MAC and the other hematological parameters (RBC, Hb, PCV, MCV and MCH). We report the first study of HbF regulating DNA variants in Mexican sickle cell disease patients. The preliminary analysis of HbFvsminor allele count showed no relation of total MAC and increased HbF, neither with the variants ofBCL11AorHBS1L-MYB. Disclosures No relevant conflicts of interest to declare.

Влияние злоупотребления алкоголем на развитие острого небилиарного панкреатита у носителей полиморфного варианта rs7674870 гена SLC7A11 A&gt;G

The aim was to determine the contribution of the rs7674870 single nucleotide polymorphism of the SLC7A11 A&gt;G gene and alcohol abuse to the risk of acute pancreatitis. Materials and methods. The material for the study was DNA samples obtained from 469 unrelated patients with acute non-biliary pancreatitis and 572 unrelated individuals without gastrointestinal diseases. The average age of the patients was 48.9 ± 13.1, healthy individuals - 47.8 ± 12.1. The diagnosis and severity of ANP were based on clinical symptoms, laboratory and instrumental methods of investigation. The study participants were divided into two groups depending on the amount of alcohol consumed per week: (1) - less than 200 g per week and (2) - more than 200 g per week; according to frequency: (1) - 1 to 2 days a month or less and (2) - 1 or more days a week; and duration: (1) - up to 10 years and (2) - for 10 or more years. Genomic DNA was purified from the thawed blood samples by phenol chloroform extraction method. Genotyping was performed by real-time PCR by discriminating alleles using TaqMan probes on a CFX96 Bio-Rad Laboratories amplifier (USA) using commercial TaqMan SNP Genotyping Assays reagent kits from Applied Biosystems (USA). Associations of alleles and genotypes with a risk of ANP were evaluated by the odds ratio (OR). Statistical analysis was performed with Statistica 10 (StatSoft, USA), SNPstats. Results. We did not find any association of the rs7674870 single nucleotide polymorphism of the SLC7A11 A/G gene with the risk of ANP. However, the genotype-environment analysis revealed an association between the G/G SLC7A11 A/G rs7674870 genotype with a reduced risk of ANP in the absence of alcohol abuse in terms of frequency (OR=0.54, 95% CI=0.31-0.96, Р=0.02), duration (OR=0.66, 95% CI=0.44-0.99, Р=0.03), and amount of alcohol consumed per week (OR=0.63, 95% CI=0.41-0.97, Р=0.01). Conclusion. The results obtained may indicate that, in carriers of the G/G SLC7A11 A/G rs7674870 genotype, moderate consumption of alcohol compensates for pathological changes that can lead to the development of ANP.

SUN-711 Association of Single Nucleotide Polymorphisms of CYP11B2, CYP11B1 and CYP17A1 with Primary Aldosteronism in a Multi-Ethnic Malaysian Cohort

Primary aldosteronism (PA), also known as Conn’s syndrome, is a common curable cause of hypertension. Family studies of essential hypertensive patients suggest that heritable genetic factors play a role in blood pressure regulation1. Interestingly, single nucleotide polymorphisms (SNP) in genes encoding enzymes involved with adrenal steroidogenesis, CYP11B2, CYP11B1 and CYP17A1, associate with increased risk of hypertension2. Therefore, we analysed whether selected SNPs in these genes are associated with PA. We performed an association study using genotype imputation for selected SNPs of the steroidogenic enzyme genes CYP11B2 (rs4546, rs1799998, rs13268025), CYP11B1 (rs6410, rs149845727), and CYP17A1 (rs1004467, rs138009835, rs2150927) from a pilot genome wide association study of Malaysian PA patients and healthy controls which was merged with the Singapore Genome Variation Project (SGVP) population dataset3. Genotype imputation for minor and major alleles was validated using PCR sequencing (n>10 for each genotype). Further, one SNP from each steroidogenic enzyme (CYP11B2:rs1799998, CYP11B1:rs6410 and CYP17A1:rs1004467) was validated using commercial TaqMan genotyping assays on the ABI 7000 Sequence Detection System which was performed on 149 PA patients and 78 non-hypertensive healthy individuals. Case-control genetic association analysis was performed at http://www.oege.org/software/orcalc.html and the association between genotypes and phenotypes was done using the independent-samples Kruskal-Wallis test on SPSS (version 25). The Minor Allele Frequencies (MAFs) for rs1004467, rs6410 and rs1799998 were similar to East Asian populations but differed significantly different from European, African, American and South Asian populations (rs1004467 MAF: C=0.258/298, rs6410 MAF: A=0.265/298, rs1799998 MAF: C=0.225/298). In Chinese patients matched by gender, heterozygotes for rs6410 had significantly increased risk of PA compared to common homozygotes (OR: 3.15, 95% CI: 1.01–9.8, p=0.04). Across patients of different ethnicity, the distribution of aldosterone levels was significantly different (p=0.039). In summary, only SNP rs6410 in Chinese patients matched by gender showed association with PA in our South East Asian cohort. More functional experiments need to be done to find out whether this is causal for PA or whether the SNP is in linkage disequilibrium with the actual functional causative SNPs. Once the functional SNP is known, identification of these germline variants in asymptomatic family members would allow early screening of PA to be offered and potentially provide novel drug targets to treat the disease.

The Role of Phosphatidylethanolamine-N-methyltransferase (PEMT) Gene rs12449964 Polymorphism in the Development of Acute Pancreatitis and its Complications

Background. Acute pancreatitis is considered to be an important issue in modern medicine. The phosphatidylethanolamine-N-methyltransferase enzyme plays a significant role in the regulation of lipid metabolism by catalyzing the process of methylation of phosphatidylethanolamine to phosphatidylcholine. These lipids are key components of mitochondrial and cell membranes, providing their fluid and plastic properties and participating in the transport of fats, fatty acids and cholesterol. Along with its function in the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine promotes the turnover of S-adenosylmethionine for the synthesis of cysteine and glutathione through transulphurisation. PEMT is a gene encoding the phosphatidylethanolamine-N-methyltransferase enzyme.Aim. To determine the role of PEMT C/T rs12449964 polymorphism in the risk of developing acute pancreatitis and its complications among Russian residents in Central Russia.Materials and methods. Whole blood samples were collected from 502 unrelated patients with acute non-biliary pancreatitis (97 women and 405 men) of Russian nationality who had been admitted to the surgical departments of the city of Kursk from 2015 to 2018, as well as from 513 unrelated individuals of Russian nationality without gastrointestinal diseases (101 women and 412 men). The average age of patients and healthy individuals was 48.9 ± 13.1 and 47.89 ± 12.1 years, respectively. Genomic DNA was isolated by a standard phenol-chloroform extraction method. Genotyping of rs12449964 polymorphism was performed using real-time PCR by allelic discrimination using a CFX96 Bio-Rad Laboratories amplifier (USA) with TaqMan probes and commercial TaqMan SNP Genotyping Assays reagents purchased from Applied Biosystems (USA).Results. The study has shown that the frequency of the C allele and the C/C PEMT C/T rs12449964 genotype was higher in the group of patients with acute pancreatitis, while the C/T genotype was predominant in the control group. C/T — T/T genotypes demonstrated a protective effect on the development of infected pancreatic necrosis, purulent necrotic peripancreatitis and severe acute pancreatitis.Conclusions. The disruption of phosphatidylethanolamine methylation processes increases the sensitivity of cells to oxidative stress, which can lead to the development of acute pancreatitis.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/μL of AMDV plasmid or 3 pg/μL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

Association of NTCP polymorphisms with clinical outcome of hepatitis B infection in Thai individuals

BackgroundSingle nucleotide polymorphisms (SNPs) in the sodium taurocholate co-transporting polypeptide (NTCP) have been showed to be associated with natural history of hepatitis B virus (HBV) infection. However, it is unclear whether the SNPs are related to the clinical outcome of HBV infection in Thai individuals.MethodsThe rs2296651 and rs4646287 polymorphisms of NTCP were determined by allelic discrimination using commercial TaqMan probes in blood samples of 1021 Thai individuals. These subjects included 610 patients with chronic HBV infection [CHB, 305 with hepatocellular carcinoma (HCC) and 305 without HCC], 206 subjects with spontaneous HBV clearance and 205 healthy controls who were age and gender-matched.ResultsThe frequencies of rs2296651 A minor allele in the CHB group, the HBV clearance group and healthy controls were 7.8, 7.3 and 13.9%, respectively. For rs4646287, the frequencies of T minor allele of the corresponding groups were 10.4, 8.0 and 9.5%, respectively. Compared with healthy controls, the frequencies of rs2296651 GA + AA genotypes were significantly lower in the CHB group (P < 0.001) and in the HBV clearance group (P = 0.001). There was no difference in their distribution between the HBV clearance and CHB groups. Among the CHB group, the distribution of GA + AA genotypes in patients with HCC were significantly lower than in patients without HCC (P = 0.014). The frequencies of HBeAg positivity in patients harboring GG and GA + AA genotypes were 39.8 and 23.5%, respectively (P = 0.004). Among patients with HCC, the mean HBV DNA of the corresponding genotypes were 4.9 ± 1.3 vs. 2.7 ± 1.0 log10 IU/mL, respectively (P < 0.001). There was no difference in genotype and allele frequencies of rs4646287 polymorphism among all studied groups.ConclusionsOur results showed that rs2296651 polymorphism was associated with a decreased risk of susceptibility to HBV infection and the development of HCC. These data suggest that the NTCP polymorphism might have an influence on natural history of HBV infection in Thai individuals.This abstract was partly presented at the American Association for the Study of Liver Diseases (AASLD) Meeting 2018, November 9–13, 2018, in San Francisco, CA, USA and was published in Hepatology 2018; 68:1237A-1238A.

Cytokine Gene Polymorphism Profiles in Kidney Transplant Patients - Association of +1188A/C RS3212227 SNP in the IL12B Gene Prevents Delayed Graft Function

Transplantation is the best treatment option for end stage kidney disease. The most common early complications in post-transplant period are acute rejection (AR) of the graft and delayed graft function (DGF). The underlying mechanisms in these events are heterogeneous and at least in part involve cytokine genes which regulate immune response to allograft. We have investigated whether functional single nucleotide polymorphisms (SNP) in the genes encoding IFN-γ (IFNG), TNF (TNFA), IL-10 (IL10) and p40 subunit of IL-12/IL-23 (IL12B) could predict risk of AR and DGF in kidney allograft recipients. Our study involved 152 kidney transplant recipients on standard immunosuppressive regimen which included calcineurin inhibitors, mycophenolic acid derivatives and corticosteroids. Genotyping of IFNG, TNFA, IL10 and IL12B was performed using commercial TaqMan assays. We found association between the carriers of AA genotype of IL12B+1188A/C polymorphism (rs3212227) and a lower rate of DGF (p=0.037, OR=0.45, 95% CI=0.21-0.96), implying protective role of A allele in the pathogenesis of DGF in kidney transplant recipients, whereas no such association was observed with AR. None of the analyzed SNPs in TNFA (-308G/A), IFNG (+874T/A), IL10 (-1082G/A, -819T/C, -592C/A) were associated with AR or DGF in our patients. Our study shows a preliminary evidence that the AA genotype of rs3212227 SNP in the IL12B gene might be associated with a lower risk for DGF after kidney transplantation. In the future, additional well-designed large studies are required for the validation of our results.

Evaluation of the miRNA-146a and miRNA-155 Expression Levels in Patients with Oral Lichen Planus.

Oral Lichen Planus (OLP) is a chronic autoimmune disease that could be considered as a potential premalignant status. To evaluate the miRNA-146a and miRNA-155 expression levels in patients with oral Lichen planus lesions compared to healthy subjects with normal oral mucosa. Forty patients with oral lichen planus and 18 healthy age and gender-matched controls were recruited in this case-control study. Oral lichen planus was diagnosed clinically and pathologically. The expression levels of two miRNAs in peripheral blood samples were determined using commercial TaqMan MicroRNA Assays. Relative quantification of gene expression was calculated by the 2-ΔΔct method. The expression levels of miRNA-146a and miRNA-155 in patients with oral Lichen planus were significantly higher than those of healthy controls. Also, a direct but insignificant correlation was found between miRNA-155 and miRNA-146a expression levels among the patient group. Our findings indicate that miRNA-146a and miRNA-155 could be potential biomarkers for the immunopathogenesis of oral lichen planus.