TaqMan Technology Research Articles

Clinical and genetic characteristics of simple central serous chorioretinopathy according to age

ABSTRACT Purpose To investigate whether genetic and clinical characteristics differ depending on generations using 326 patients (male/female, 259/67; mean age, 55.4 ± 12.5 years) with simple CSC. Methods All patients were diagnosed with simple CSC, defined as a retinal pigment epithelium alteration area equal to or smaller than 2-disc areas based on multimodal imaging at the initial presentation. We cross-sectionally evaluated clinical characteristics at the initial visit and genotyped CFH rs800292 and rs1329428 for all patients using TaqMan technology. Results As generations decreased, the proportion of males, subfoveal choroidal thickness, and prevalence of fibrin significantly increased (p < 0.001, p < 0.001, and p = 0.012, respectively), and the best-corrected visual acuity improved (p < 0.001); in contrast, the prevalence of pachydrusen significantly decreased (p < 0.001). The younger presentation was significantly associated with male and risk variants (T allele) of CFH rs1329428 (p = 9.1 × 10−7 and p = 0.042, respectively), and patients were estimated to present 2 years younger per one T allele of CFH rs1329428 (p = 0.042, β = −1.95, stepwise regression analysis). Conclusion Clinical and genetic characteristics differed significantly among patients with simple CSC, depending on their generation.

MiRNA signature related to atherosclerotic lesion induced shear stress modifications in familial hypercholesterolemia patients with subclinical atherosclerosis: a bioinformatics systems biology study

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Institute of Health Carlos III (ISCIII) Background Patients with familial hypercholesterolemia (FH), even when appropriately treated, are at risk of premature clinical atherosclerotic cardiovascular disease (ASCVD) presentation. Although atherosclerosis (AT) is directly related to hemodynamic disturbances affecting the vascular geometry, there is scarce information on whether there is miRNA-mediated/flow-mediated modulation of atherosclerosis in FH. Purpose To identify a differential plasma miRNA signature related to fluid shear stress (FSS) in well controlled FH-patients with asymptomatic atherosclerosis. Methods FH-patients (N=49) with subclinical atherosclerosis, identified by computed tomographic angiography, and a reference cohort of non-FH subjects (N=32), both from a national FH-Registry, were included in the study. Coronary plaque volume and phenotype was analyzed by means of the QAngio CT imaging system. miRNAs associated to FSS were defined in silico by bioinformatics (KEGG, Cytoscape, GTEX portal, miRWalk, miRBase tools) and validated in plasma samples by RT-qPCR using TaqMan technology. Results By in silico analysis, 7 miRNAs targeting genes involved in features of functions (ROS production, oxidative stress, leukocyte transendothelial/migration-inflammation and vasodilation) of shear stress-induced cellular dysfunction and atherosclerosis (KEGG, hsa05418) were identified by miRWalk and miRBase databases. Of the 7 miRNAs, a 6-miRNA signature showed significantly lower plasma levels in FH-patients than in the non-FH group (P&amp;lt;0.01 for: miR-30e-5p, miR-378a-3p, let-7d-3p, and miR-550-5p; P=0.03 for miR-324-3p). In the FH-group, miR-324-3p, miR-30e-5p, miR-378a-3p and let-7d-3p significantly discriminated patients stratified by the plaque volume of non-calcified lesions (fibro-lipidic phenotype), using the median plaque volume as cut-off value. And even further, a high percentage (65-70%) of FH-subjects with low levels of the 4 miRNA had plaque volumes above the median. Interestingly, this miRNA-signature did not differentiate FH-patients stratified by the volume of calcified plaques. Conclusion FH-patients show a significantly lower content of an atherosclerosis/shear stress signature of 4 miRNAs targeting genes involved in subclinical atherosclerosis progression in FH.

Systematic assessment of the reliability of quantitative PCR assays targeting IS900 for the detection of Mycobacterium avium ssp. paratuberculosis presence in animal and environmental samples

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's disease (JD), which is endemic to dairy cattle and also implicated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. Johne's disease is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative PCR (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD. Mycobacterium avium ssp. paratuberculosis IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry, and 107 used TaqMan technology. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.

Associations of the variable site rs2010963 of the VEGF-A gene with multifocal atherosclerosis in aluminum industry workers with chronic occupational intoxication with fluoride compounds

Introduction. The aluminum industry occupies a leading position among non-ferrous metallurgy industries in revealing somatic pathologies in workers that occur against the background of chronic fluoride intoxication. Long-term exposure to fluorides on the body has a cardiovasotoxic effect, leads to structural and functional disorders of the myocardium and the development of comorbid cardiovascular pathology. This dictates the need for a risk-oriented approach to its diagnosis, including one based on molecular genetic analysis, for the timely identification of risk groups for the development of atherosclerosis and the elaboration of preventive measures aimed at preserving the health and maintaining working longevity of the studied occupational cohort. Materials and methods. There were examined ninety seven metallurgists with a previously proven diagnosis of chronic occupational intoxication with fluoride compounds, engaged in aluminum production. The comparison group consisted of 33 workers of this enterprise without a proven occupational disease. All respondents underwent ultrasound investigation of the main arteries. Genotyping of the rs2010963 polymorphic locus of the VEGF-A gene was carried out in a 96-well format using TaqMan technology (allele-specific real-time PCR). Results. The associative connection of the G/G VEGF-A genotype is determined in metallurgists with the risk of developing chronic occupational intoxication with fluoride compounds and heterozygous G/C genotype in subjects with multifocal atherosclerosis. The high level of predictive value of the polymorphism -634 G/C (rs2010963) of the VEGF-A gene allows considering it a marker of the risk for the developing atherosclerosis in workers exposed to long-term impact to fluoride compounds on the body. Limitations. The study was limited to the number of long-term work experienced subjects undergoing periodic medical examinations and undergoing inpatient treatment at the Research Institute for Complex Problems of Hygiene and Occupational Diseases. Conclusion. It is recommended to conduct a screening molecular and genetic analysis in a clinical hospital setting for timely assessment of the prognostic significance of risk markers for the development of atherosclerosis and its complications in workers of basic occupations engaged in aluminum production.

Genetic association of TIE2 with diabetic retinopathy and diabetic macular edema

PurposeTo evaluate the associations of the TIE2 gene with diabetic retinopathy (DR) and diabetic macular edema (DME). MethodsThis study included a Chinese cohort of 285 non-proliferative DR patients and 433 healthy controls. The DR patients were classified further into those with or without DME. Thirty haplotype-tagging single-nucleotide polymorphisms (SNPs) in TIE2 were genotyped using TaqMan technology. Associations of DR and subtypes were analyzed by logistic regression adjusted for age and sex. Stratification association analysis by sex was performed. ResultsTIE2 rs625767 showed a nominal but consistent association with DR [odds ratio (OR) = 0.71, P = 0.005] and subtypes (DR without DME: OR = 0.69, P = 0.016; DME: OR = 0.73, P = 0.045). SNP rs652010 was consistently associated with overall DR (OR = 0.74, P = 0.011) and DR without DME (OR = 0.70, P = 0.016), but not with DME. Moreover, SNPs rs669441, rs10967760, rs549099 and rs639225 showed associations with overall DR, whilst rs17761403, rs664461 and rs1413825 with DR without DME. In stratification analysis, three SNPs, rs625767 (OR = 0.62, P = 0.005), rs669441 (OR = 0.63, P = 0.006) and rs652010 (OR = 0.64, P = 0.007), were associated with DR in females, but not in males. Moreover, one haplotype T-T defined by rs625767 and rs669441 was significantly associated with DR in females only. ConclusionsThis study revealed TIE2 as a susceptibility gene for DR and DME in Chinese, with a sex-specific association in females. Further validation should be warranted.

The Genetic Markers of Knee Osteoarthritis in Women from Russia.

Osteoarthritis is a chronic progressive joint disease that clinically debuts at the stage of pronounced morphologic changes, which makes treatment difficult. In this regard, an important task is the study of genetic markers of the disease, which have not been definitively established, due to the clinical and ethnic heterogeneity of the studied populations. To find the genetic markers for the development of knee osteoarthritis (OA) in women from the Volga-Ural region of Russia, we conducted research in two stages using different genotyping methods, such as the restriction fragment length polymorphism (RFLP) measurement, TaqMan technology and competitive allele-specific PCR-KASPTM. In the first stage, we studied polymorphic variants of candidate genes (ACAN, ADAMTS5, CHST11, SOX9, COL1A1) for OA development. The association of the *27 allele of the VNTR locus of the ACAN gene was identified (OR = 1.6). In the second stage, we replicated the GWAS results (ASTN2, ALDH1A2, DVWA, CHST11, GNL3, NCOA3, FILIP/SENP1, MCF2L, GLT8D, DOT1L) for knee OA studies. The association of the *T allele of the rs7639618 locus of the DVWA gene was detected (OR = 1.54). Thus, the VNTR locus of ACAN and the rs7639618 locus of DVWA are risk factors for knee OA in women from the Volga-Ural region of Russia.

Genetic and Clinical Characteristics of Central Serous Chorioretinopathy With Steroid Use.

To compare the genetic and clinical characteristics of central serous chorioretinopathy (CSC) in patients with and without steroid use. A total of 407 consecutive patients with CSC were included. Demographic data and clinical factors, including subfoveal choroidal thickness, bilateral involvement, descending tracts, pachydrusen, fibrin, and dome-shaped pigment epithelial detachment, were obtained. Variants of complement factor H (CFH)I62V (rs800292) and rs1329428 were genotyped in all cases using TaqMan technology. Of the total patients, 48 (11.8%) were steroid users. The majority of males were non-steroid users (82.5%) than steroid users (58.3%) (p = 9.8 × 10-5). Demographic data and the prevalence of clinical factors were comparable between the two groups (all p-values > 0.10). Risk allele frequencies of CFH rs800292 and rs1329428 were also comparable between the two groups (p = 0.76, rs800292: steroid users = 52.1% vs. non-steroid users = 50.4%; p = 0.62, rs1329428: steroid users = 47.9% vs. non-steroid users = 45.3%). Except for the male/female ratio, there were no significant differences in the clinical presentation or genetic characteristics, including variants of the CFH gene, between the two groups.

MTNR 1B (rs10830963) Gene Polymorphism, but not MTNR 1A (rs2119882), Associated with Type 2 Diabetes Mellitus Risk in Saudi Arabia.

Disrupted circadian rhythm has been linked to the pathogenesis of type 2 diabetes mellitus (T2DM). Single nucleotide polymorphisms (SNPs) in melatonin receptors (MTNR), MTNR 1A rs2119882 (T>C) and MTNR 1B rs10830963 (C>G) may interfere with the normal function of melatonin and increase the risk of T2DM. This study investigated the prevalence of MTNR 1A rs2119882 (T>C) and MTNR 1B rs10830963 (C>G) SNPs and tested their association with T2DM in Saudi Arabian population. A total of 459 Saudi Arabian participants from Jazan Province, Saudi Arabia, were selected and included 227 T2DM patients and 232 control subjects. DNA was extracted from all participants and genotyped for rs2119882 and rs10830963 SNPs using TaqMan technology. Genotype frequencies were determined for both SNPs, and logistic regression was fitted to test the association with T2DM. No association was found between MTNR 1A rs2119882 (T>C) SNP and T2DM (odds ratio (OR) = 0.69; 95% confidence interval (CI) = 0.44 - 1.08; p-value = 0.111). However, the MTNR 1B rs10830963 (C>G) SNP was significantly associated with T2DM (OR = 1.73; 95% CI = 1.18 - 2.55; p-value = 0.0065). Co-inheritance of the MTNR 1B rs10830963 G allele and MTNR 1A rs2119882 T allele further increased the risk of T2DM (OR = 2.80; 95% CI = 1.71 - 4.57; p-value < 0.0001). The minor G allele of the MTNR 1B rs10830963 SNP was significantly associated with T2DM in our population. This association further intensified with the presence of the T allele in MTNR 1A rs2119882 locus. This study sheds light on the importance of melatonin receptor polymorphisms as genetic candidates for the development of T2DM in Saudi Arabia.

Genetic variants of ABCB1 and CES1 genes on dabigatran metabolism in the Kazakh population.

Allelic variants of genes encoding enzymes of the esterase system (CES1) and P-glycoprotein (ABCB1) can change the metabolism and pharmacokinetics of dabigatran. Therefore, they act as determining factors in the development of side effects, especially bleeding. We analyzed the genotype-phenotype relationship of ABCB1 (rs1045642, rs4148738, rs2032582, and rs1128503) and CES1 (rs8192935, rs71647871, and rs2244613) polymorphisms in patients with atrial fibrillation who had been treated with dabigatran. A total of 150 patients were recruited for this study. TaqMan technology was used for SNP genotyping. Patients with the rs2244613 GG genotype had a lower concentration (55.27 ± 34.22 ng/ml) compared to those with the TT genotype (63.33 ± 52.25 ng/ml) (additive model, P = 0.000). Individuals with the rs8192935 AA genotype had a lower concentration (52.72 ± 30.45 ng/ml) compared to those with the GG genotype (79.78 ± 57 ng/ml) (additive model, P = 0.001). The APTT values among the different genotypes of the ABCB1 SNPs, rs4148738 and rs1045642, were significantly different (P = 0.035 and P = 0.024, respectively). Our research demonstrates that the CES1 polymorphisms, rs8192935 and rs2244613, are associated with the pharmacodynamics and pharmacokinetics of dabigatran in the Kazakh subpopulation.

HEPATOCELLULAR CARCINOMA IN OCCULT HEPATITIS B VIRUS INFECTION: A CASE REPORT

Occult hepatitis B virus infection is defined as the presence of hepatitis B virus DNA in the hepatocytes or serum of individuals who had tested negative for serum HBV surface antigen. Controversies remain as to the role of the occult HBV infection in the development of hepatocellular carcinoma (HCC) in immunocompetent individuals. It has been shown that the persistence of very low viral replicative activity during occult hepatitis B virus infection may induce mild liver necro-inflammation continuing for life, and substantial clinical evidence indicates that occult hepatitis B virus infection can accelerate the progression of liver disease towards cirrhosis that is considered the most important risk factor for hepatocellular carcinoma development. The aim of this case is to describe the clinical symptoms and laboratory data of the first case we encountered of HCC occurring in an OBI patient. A 52-year-old man came to the Infectious Diseases Unit because of right upper quadrant pain. Blood test showed negative for HBsAg, positivity for both anti-HBs and anti-HBc total, the quantitative assay of HBV DNA based on TaqMan technology was 2012 copies/mL, elevated serum alpha-fetoprotein level, the findings in abdominal ultrasonography and computed tomography showed a tumor in the right liver lobe with 12cm in size. The patient was transferred to the Military Hospital 175 Ho Chi Minh City for diagnosis of intermediate stage HCC, treated with transarterial chemoembolization (TACE) and antiviral treatment with tenofovir disoproxil fumarate (TDF).

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

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

Leptin Rs7799039 polymorphism is associated with type 2 diabetes mellitus Egyptian patients

Background Leptin (LEP) is an anti-obesity hormone that regulates food intake, energy expenditure, and glucose metabolism. The genetic variants in LEP and the LEP receptor (LEPR) gene may play an important role in the pathogenesis of type 2 diabetes mellitus (T2DM) and obesity. The current study aimed to investigate the association of serum LEP levels, and LEP polymorphisms in LEP (rs7799039, 2548 G/A) with T2DM in Egyptian patients. Methods A total of 205 subjects were included in the present case-control study, consisting of 100 T2DM patients and 105 healthy controls. The anthropometric, psychometric, and biochemical measurements were taken from all the subjects. The genotyping of LEP gene variants was carried out by polymerase chain reaction TaqMan technology. Serum LEP levels were measured by the ELISA technique. Results T2DM patients had significantly elevated levels of glycated haemoglobin (HbA1c), fasting blood sugar (FBS), postprandial blood sugar (PPBS), international normalisation ratio (INR), creatinine, urea, cholesterol, triglyceride (TG), and low-density lipoproteins (LDL) and significantly decreased high-density lipoprotein (HDL) compared to healthy subjects. serum LEP levels were significantly decreased p (<0.001) as compared to the control group. LEP gene SNP rs7799039 was associated with an increased diabetic risk with A allele being more frequent in T2DM patients than control subjects. The distribution of the AA genotype and GA genotype of LEP SNP rs7799039 was higher in the diabetic group than control one. In addition, AA + GA genotype carriers had significantly elevated HbA1c, FBS, PPBS, TG, and LDL levels and on the contrary, decreased serum LEP levels compared to GG homozygotes. Conclusion The genetic polymorphism rs7799039 showed a highly significant correlation with blood LEP. The co-dominant and dominant models of the LEP genetic polymorphism (rs7799039, 2548 G/A) were shown to have a significant correlation with complicated and uncomplicated diabetes individuals, but we have found that serum LEP levels were inversely related with control and diabetes patients. A positive significant association was found between LEP genetic polymorphism (rs7799039, 2548 G/A) and serum LEP in patients and controls. LEP levels and its rs7799039 genetic variant may play a vital role in increasing T2DM susceptibility.

ВАРИАБЕЛЬНОСТЬ ГЕНОВ НАТРИЙУРЕТИЧЕСКИХ ПЕПТИДОВ И АНТИОКСИДАНТНОЙ ЗАЩИТЫ У ПАЦИЕНТОВ С ИНФАРКТОМ МИОКАРДА

Introduction. Cardiovascular diseases (CVD) continue to be the subject of scientific research in relation to molecular genetic predictors of their development. It has been proven that diseases of the cardiovascular continuum have a multifactorial nature with a significant genetic component, including the hereditary risk of myocardial infarction (MI). CVDs are characterized by a complex genetic structure with diverse combinations of single nucleotide polymorphic variants (SNVs). A promising task is to study the relationship of SNV adaptogenesis genes: inflammatory response, myocardial and endothelial dysfunction with developed MI in the Siberian population. Purpose: to reveal the relationship between SNV genes of natriuretic peptides and the antioxidant defense system with the development of myocardial infarction. Materials and methods. The material for the study was genomic DNA isolated from the peripheral blood of patients (n=146, 38 women and 108 men) admitted for treatment at the Kuzbass Clinical Cardiological Dispensary, Kemerovo, with a diagnosis of MI. The control group is represented by a population sample of residents of the city of Kemerovo (n=300, 190 women and 110 men). 20 polymorphic variants of 7 natriuritic peptide genes (NPPA, NPPA-AS1, NPPB, NPPC, NPR1, NPR2, NPR3) and 5 genes of the antioxidant defense system (SOD2, NCF4, CBR1, CBR3, CAT) were selected for the study. Genotyping of selected polymorphic variants was performed by real-time PCR using TaqMan technology. Statistical data analysis was carried out using GraphPad Prism 8 and SNPstats software. Results. Protective associations of allelic variants of the CBR1 rs9024 genes (OR=0.21 (95% CI 0.13-0.34); p=0.0001) and CBR3 (OR=0.44 (95% CI 0.28-0) .69); p=0.001) regardless of gender according to the dominant model of inheritance. When separating patients by gender, it was found that in men, the rs9024 CBR1 allele A (OR=0.20 (95% CI 0.11-0.36); p&lt;0.0001) and the rs1056892 CBR3 allele A (OR=0, 51 (95% CI 0.28-0.91); p=0.022) have a protective effect in the development of MI according to the dominant model of inheritance. Allele T rs2236289 (OR=1.93 (95% CI 1.04-3.58); p=0.035) and allele A rs7034957 (OR=1.88 (95% CI 1.03-3.45); p =0.038) of the NPR2 gene are associated with a risk effect on the development of myocardial infarction in men according to the dominant model of inheritance. In women, rare alleles of the polymorphic loci rs13288085 (OR=0.25 (95% CI 0.08-0.73); p=0.0034) and rs7034957 (OR=0.30 (95% CI 0.11-0, 79); p=0.007) of the NPR2 gene, rs9024 (OR=0.21 (95% CI 0.09-0.47); p=0.00001) of CBR1, as well as rs1056892 (OR=0.31 (95% CI 0.15-0.64); p=0.0014) CBR3 are characterized by a protective effect on the development of MI according to the dominant model of inheritance. Conclusion. It has been shown that polymorphism of genes for natriuretic peptides and antioxidant protection may have a risky and protective effect in relation to predisposition to the development of MI. The obtained preliminary results indicate the need for further studies of the identified SNV genes of adaptogenesis in relation to the severity of MI and the risk of recurrent cardiovascular events in the long-term period after MI.

РЕГУЛЯЦИЯ СИСТЕМНОГО ВОСПАЛИТЕЛЬНОГО ОТВЕТА У ПАЦИЕНТОВ С МУЛЬТИФОКАЛЬНЫМ АТЕРОСКЛЕРОЗОМ

Introduction. Data on the role of inflammation in the progression and manifestation of multifocal atherosclerosis (MFA) are contradictory. The systemic process of multiple lesions of different vascular beds in patients with an established diagnosis of coronary heart disease (CHD) is due to several parallel pathological mechanisms. In general, in the pathogenesis of both coronary atherosclerosis and atherosclerosis of peripheral arteries, one of the factors is the variability of immune response genes and cytokines, regulators of the inflammatory response. The aim of this work was to determine the role of variability in the genes of cytokines and innate immune response receptors in the structure of mechanisms associated with the development of MFA in patients with coronary artery atherosclerosis. Materials and methods. 260 patients with CAD (mean age 58 years) were examined. Of all those included in the study, at the time of the survey, MFA was found in 180 people (69.23%), while the incidence of MFA in men and women is comparable (70.33 vs. 64.71%, respectively). There is also no significant difference in the incidence of MFA in the two age groups (up to 60 years, 45% have lesions of two or more vascular beds, and in people older than 60 years, MFA was found in 55% of cases). All patients were informed about the course of the study and voluntarily signed an informed consent. Blood was collected from the cubital vein into Vacutainer tubes with K3EDTA. DNA isolation was carried out using the phenol-chloroform extraction method. Genotyping of 47 polymorphic sites with a single nucleotide change (SNV) of 19 genes proven to be associated with the regulation of the systemic inflammatory response (cytokines, innate immunity receptors) was performed in a 96-well format using TaqMan technology (LifeTechnologies, USA) and real-time result detection with using the ViiATM 7 RealTime PCR System (LifeTechnologies, USA) for polymerase chain reaction in accordance with the instructions. Associations were analyzed using Snpstats (https://www.snpstats.net/start.htm). Odds ratio (OR) and 95% CI for OR were calculated for risk assessment. Results. One association has been established that demonstrates a negative impact and increases the risk of developing MFA in young and middle age. Carriage of the G rs11685424 IL1RL1 allele increases the risks of earlier development of peripheral arterial lesions by 2 times (OR 2.11 (95% CI 1.09-4.06), p = 0.024) according to the recessive inheritance model. At the same time, variability in three sites (rs1974675; rs6758936; rs3755276) of the IL18 receptor gene (IL18R1) reduces the risk of MFA detection by three times (p=0.12; p=0.034: p=0.026, respectively) and this pattern is also observed in patients older age group. In the carriers of the A/A rs1974675, T/T rs3755276 and A/A rs6758936 genotypes in persons over 60 years of age, MFA was diagnosed less frequently (p=0.024; p=0.037; p=0.011, respectively). Conclusion. It has been shown that in patients with significant coronary atherosclerosis, the progression of peripheral vascular atherosclerosis is more associated with the variability of the genes responsible for the regulation of the systemic inflammatory response, rather than genes and their variants that determine the severity and strength of the immune response.

The NLRP3 inflammasome rs35829419 C>A polymorphism is associated with type 2 diabetes mellitus in Saudi Arabia.

To investigate the frequency of NLRP3 gene rs35829419C>A single-nucleotide polymorphism (SNP) in a Saudi Arabian population from Jazan (Southwest Saudi Arabia) and test its potential association with type 2 diabetes mellitus (T2DM). This case-control study included 546 volunteers (271 patients with T2DM and 275 healthy controls) recruited from outpatient clinics at Jazan University Hospital and King Fahad Central Hospital in Jazan, Saudi Arabia, between December 2021 and July 2022. Genomic DNA was extracted from all samples and genotyped for the NLRP3 rs35829419C>A SNP using TaqMan technology. The association between the NLRP3 rs35829419 polymorphism and T2DM was examined using logistic regression analysis. Overall genotype distributions were 90.5% (CC), 9.3% (CA), and 0.2% (AA). The heterozygous CA genotype was more frequent in T2DM group (12.2%) compared to the control group (6.5%) and logistic regression analysis showed a statically significant association with T2DM risk under codominant (CA versus CC; odds ratio [OR]=1.99; 95% confidence interval [CI]= [1.11-3.61]; p=0.0270), and dominant (CA+AA versus CC; OR=2.05; CI=[1.16-3.75]; p=0.019) models of inheritance. This study revealed the frequency of NLRP3 rs35829419C>A polymorphism in our population and showed a direct correlation between having the minor allele for A and having a higher risk of developing T2DM. This study highlights the significance of NLRP3 rs35829419C>A polymorphism in the pathophysiology of T2DM.

Pharmacogenetic Practice of Anticancer Drugs: Multiple Approaches for an Accurate and Comprehensive Genotyping.

The application of pharmacogenetics in oncology is part of the routine clinical practice. In particular, genotyping of dihydropyrimidine dehydrogenase (DPYD) and UDP-glucuronosyltransferase (UGT1A1) is crucial to manage the treatment of patients taking fluoropyrimidines and irinotecan. The unique approach of our laboratory to the pharmacogenetic diagnostic service in oncology is to combine two real-time PCR methods, LightSNiP assay (TIB MOLBIOL), and more recently FRET (Fluorescent Resonance Energy Transfer) probes technology (Nuclear Laser Medicine), plus TaqMan assay (Thermo Fisher) for the confirmation of the presence of variant alleles on DNA from a second extraction. We found that both the FRET and LightSNiP assays, where detection occurs by melting curve analysis, offer an advantage over the competing TaqMan technology. Whereas unexpected genetic variants may be missed using a mutation-specific TaqMan assay, the information thus obtained can be useful to adjust the therapy in case of unexpected post-treatment toxicity. The combination of TaqMan and FRET assays helped us to achieve more accurate genotyping and a correct result for the patient. The added value of the DPYD FRET assay is the possibility of detecting, with the same amplification profile of the polymorphisms detailed in the guidelines, also the c.2194G>A (*6 rs1801160), cited in the recommendations as a variant to be investigated in case of severe toxicity. Regarding the UGT1A1 (TA)n promoter polymorphism (rs3064744), the distinctive and positive feature of the FRET assay is to allow clearly identifying all those potential variant alleles, including the (TA)5 and (TA)8 alleles, that are frequent in African Americans. Our clinical practice emphasizes the importance of not only rapid and easy-to-use assays, such as the new FRET ones, but also of accurate and comprehensive genotyping for good pharmacogenetic diagnostic activity.

Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood.

PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA.

The association of genetic polymorphisms in protocadherin 15 with sudden sensorineural hearing loss in a Chinese population

Sudden sensorineural hearing loss (SSNHL) is a multifactorial disease, and its etiology is still unknown. SSNHL may be caused by environmental factors and genetic changes. PCDH15 is associated with susceptibility to hearing loss. The relationship between PCDH15 and SSNHL remains unknown. In this study, the potential association between PCDH15 polymorphism and SSNHL in Chinese population was evaluated. Two single nucleotide polymorphisms PCDH15-rs7095441 and rs11004085 in 195 SSNHL patients and 182 healthy controls were determined by TaqMan technology. In Chinese population, the TT genotype and T allele of rs7095441 are associated with increased susceptibility to SSNHL. The relationships between rs7095441 and the degree of hearing loss were analyzed, and TT genotype increased the risk of hearing loss. Among SSNHL patients, patients with TT genotype of rs7095441 have an increased risk of vertigo. This study found that the TT genotype of SNP rs7095441 can increase the risk of SSNHL in Chinese population.

Frequency of rs2228570 single nucleotide polymorphism of Vitamin-D Receptor (VDR) gene among the Kazakh ethnic group

The article presents the results of the study of vitamin D receptor (VDR) gene rs2228570 single nucleotide polymorphism (SNP) genotypes and individual alleles frequency among the Kazakh ethnic group representatives living in the Karaganda region. This SNP was determined by real-time polymerase chain reaction using TaqMan technology. The study relevance is due to the fact that genetic variations in rs2228570 affect the synthesis of the VDR protein and its activity as a transcription factor that regulates the expression of other genes. This mechanism determines the association of individual rs2228570 genotypes or alleles with susceptibility, course, and outcomes of various diseases. The polymorphism frequency may be depending on ethnicity. According to the study results, the most common genotypes of rs2228570 SNP among the Kazakhs were AG (32.8 %) and GG (25.2 %). The rarest are homozygotes TT (1.7 %) and CC (0.8 %). The frequency of all rs2228570 alleles was detected simultaneously in the present study for the first time. G became the predominant allele (51.3 %), less common was A (31.0 %), and the C and T alleles were the rarest (11.8 % and 5.9 %, respectively). The potential value of this SNP further study as a possible factor influencing the body’s susceptibility to various diseases, including COVID-19, is shown.

DISTINCT CHARACTERISTICS OF SIMPLE VERSUS COMPLEX CENTRAL SEROUS CHORIORETINOPATHY.

To compare the clinical and genetic characteristics of simple and complex central serous chorioretinopathy using central serous chorioretinopathy international group criteria. Patients with idiopathic central serous chorioretinopathy were included. Depending on the presence or absence of retinal pigment alterations greater than 2-disc areas in either eye, patients were classified into complex or simple types. Demographic factors and clinical findings were compared between groups. CFH variants, including rs800292 and rs1329428, were genotyped using TaqMan technology. A total of 319 consecutive patients were evaluated at the initial presentation. Of them, 53 (16.6%) had the complex type. The complex type was exclusively seen in men (100% vs. 79.0%, P = 2.0 × 10 -4 ) and demonstrated a significantly higher proportion of bilateral involvement (75.5% vs. 17.7%, P = 6.2 × 10 -18 ) and descending tract(s) (83.0% vs. 0%, P = 1.2 × 10 -57 ) than the simple type. Increased choroidal thickness (425 ± 131 vs. 382 ± 110, P = 0.02) and decreased central retinal thickness (274 ± 151 vs. 337 ± 136, P = 2.9 × 10 -4 ) were observed for the complex versus simple type. The risk allele frequencies of both variants were significantly higher in the complex versus simple type (rs800292: 61.3% vs. 48.7%, P = 0.018; rs1329428: 65.1% vs. 54.3%, P = 0.04). In this new classification system, the complex type has distinct genetic and clinical characteristics compared with the simple type.