TaqMan Assay Research Articles

Polymorphisms and dental age in non-syndromic cleft lip and palate: a cross-sectional study.

Children with non-syndromic cleft lip with or without palate (CL ± P) may present alterations in dental development. The purpose of this cross-sectional study was to compare the dental age (DA) between children with and without CL ± P, and whether single nucleotide polymorphisms (SNPs) in genes encoding growth factors are associated with DA variations. Children aged between 5 and 14 years with and without CL ± P were recruited to participate in this study. DA was evaluated by calibrated examiners (kappa > 0.80) using the method proposed by Demirjian et al. (1973). Genomic DNA was extracted from buccal cells, and SNPs in Epidermal Growth Factor (EGF) - rs4444903 and rs2237051, Epidermal Growth Factor Receptor (EGFR) - rs2227983 -, Transforming Growth Factor Beta 1 (TGFB1) - rs1800470 and rs4803455 -, and Transforming Growth Factor Beta Receptor 2 (TGFBR2) - rs3087465 - were genotyped by real-time polymerase chain reactions using the TaqMan assay. The Student T-test was used to compare the variations in DA between the phenotypes "with CL ± P" and "without CL ± P", and the ANOVA two-way test was performed to compare the variations in DA among the genotypes (α = 0.05). A post-hoc analysis was performed using Bonferroni correction. Two hundred and nine (n = 209) children (100 with CL ± P and 109 without CL ± P) with a mean chronological age of 8.66 years - standard deviation (SD) = 1.92 - were included. The group with CL ± P demonstrated a significantly delayed DA (mean=-0.23; SD = 0.71) compared to the group without CL ± P (mean=-0.01; SD = 0.88) (p = 0.049). Genotype distributions were in Hardy-Weinberg equilibrium. The SNP rs4803455 in TGFB1 was significantly associated with DA variations in children without CL ± P (p < 0.01). In the group with CL ± P, no significant differences in DA were observed among the genotypes. Children with CL ± P presented delayed DA compared with children without CL ± P. The SNP rs4803455 in TGFB1 is associated with variations in DA in children without CL ± P.

The Relationship Between Differential Expression of Non-coding RNAs (TP53TG1, LINC00342, MALAT1, DNM3OS, miR-126-3p, miR-200a-3p, miR-18a-5p) and Protein-Coding Genes (PTEN, FOXO3) and Risk of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive interstitial lung disease of unknown pathogenesis with no effective treatment currently available. Given the regulatory roles of lncRNAs (TP53TG1, LINC00342, H19, MALAT1, DNM3OS, MEG3), miRNAs (miR-218-5p, miR-126-3p, miR-200a-3p, miR-18a-5p, miR-29a-3p), and their target protein-coding genes (PTEN, TGFB2, FOXO3, KEAP1) in the TGF-β/SMAD3, Wnt/β-catenin, focal adhesion, and PI3K/AKTsignaling pathways, we investigated the expression levels of selected genes in peripheral blood mononuclear cells (PBMCs) and lung tissue from patients with IPF. Lung tissue and blood samples were collected from 33 newly diagnosed, treatment-naive patients and 70 healthy controls. Gene expression levels were analyzed by RT-qPCR. TaqMan assays and TaqMan MicroRNA assay were employed to quantify the expression of target lncRNAs, mRNAs, and miRNAs. Our study identified significant differential expression in PBMCs from IPF patients compared to healthy controls, including lncRNAs MALAT1 (Fold Change = 3.809, P = 0.0001), TP53TG1 (Fold Change = 0.4261, P = 0.0021), and LINC00342 (Fold Change = 1.837, P = 0.0448); miRNAs miR-126-3p (Fold Change = 0.102, P = 0.0028), miR-200a-3p (Fold Change = 0.442, P = 0.0055), and miR-18a-5p (Fold Change = 0.154, P = 0.0034); and mRNAs FOXO3 (Fold Change = 4.604, P = 0.0032) and PTEN (Fold Change = 2.22, P = 0.0011). In lung tissue from IPF patients, significant expression changes were observed in TP53TG1 (Fold Change = 0.2091, P = 0.0305) and DNM3OS (Fold Change = 4.759, P = 0.05). Combined analysis of PBMCs expression levels for TP53TG1, MALAT1, miRNA miR-126-3p, and PTEN distinguished IPF patients from healthy controls with an AUC = 0.971, sensitivity = 0.80, and specificity = 0.955 (P = 6 × 10-8). These findings suggest a potential involvement of the identified ncRNAs and mRNAs in IPF pathogenesis. However, additional functional validation studies are needed to elucidate the precise molecular mechanisms by which these lncRNAs, miRNAs, and their targets contribute to PF.

Association of Genetic Polymorphisms in SLC45A2, TYR, HERC2, and SLC24A in African Women with Melasma: A Pilot Study

Melasma is a chronic skin disorder characterized by hyperpigmentation, predominantly affecting women with darker skin types, including those of African descent. This study investigates the association between genetic variants in SLC45A2, TYR, HERC2, and SLC24A5 genes and the severity of melasma in women of reproductive age. Forty participants were divided into two groups: twenty with facial melasma and twenty without. Deoxyribonucleic acid (DNA) was extracted from blood samples and genotyped using TaqMan assays to identify allele frequencies and genotype distributions. Significant associations were observed for the TYR gene (rs1042602), HERC2 gene (rs1129038), and SLC24A5 gene (rs1426654) polymorphisms, highlighting their potential roles in melasma susceptibility. For example, the rs1042602 Single Nucleotide Polymorphisms (SNP) in the TYR gene showed a strong association with melasma, with the AA genotype conferring a markedly increased risk. Similarly, the rs1129038 SNP in the HERC2 gene and the rs1426654 SNP in the SLC24A5 gene revealed significant genetic variations between groups in women of African descent. These findings underscore the influence of genetic polymorphisms on melasma’s pathogenesis, emphasizing the need for personalized approaches to its treatment, particularly for women with darker skin types.

Prevalence of Mutations Associated with QoI, QiI, QioSI and CAA Fungicide Resistance Within Plasmopara viticola in North America and a Tool to Detect CAA Resistant Isolates.

Grape downy mildew, caused by Plasmopara viticola poses a threat to grape cultivation globally. Early detection of fungicide resistance is critical for effective management. This study aimed to assess the prevalence and distribution of mutations associated with resistance to Quinone oxide inhibitors (QoI, FRAC 11), Quinone inside inhibitors (QiIs, FRAC 21, cyazofamid), Carboxylic acid amides (CAA, FRAC 41), and Quinone inside and outside inhibitor, stigmatellin binding mode (QioSI, FRAC 45, ametoctradin) in P. viticola populations in the eastern United States and Canada; and evaluate whether these mutations are linked to fungicide resistance correlate with specific P. viticola clades. A total of 658 P. viticola samples were collected from commercial vineyards across different states and years in the eastern United States and Canada and sequenced for the PvCesA3 and cytb genes and the ITS1 region. Results showed P. viticola clades aestivalis, vinifera, and riparia were prevalent in the eastern United States and Canada. QoI resistance was widespread, with the A-143 resistant genotype prevalent in P. viticola clades aestivalis and vinifera. CAA resistance, associated with the G1105S mutation, was mainly identified in P. viticola clade aestivalis from Georgia, New York, and Ontario. A TaqMan-probe based assay was developed to detect G1105S mutation in P. viticola conferring CAA fungicide resistance. The TaqMan assay demonstrated sensitivity at even low DNA concentrations and specificity in distinguishing between sensitive and resistant genotypes. This study provides insights into geographic distribution of fungicide resistance in P. viticola populations and presents a reliable method for detecting CAA resistance in P. viticola.

P1202 Metabolic-dysfunction associated liver disease in patient with Inflammatory Bowel Disease: Is obesity the major driver of disease progression?

Abstract Background Prevalence of MASLD in patients with Inflammatory Bowel Disease (IBD) has been reported 24.4% in a recent metaanalysis and 21.6% in a previous cross-sectional study from our center. However, data on the progression of MASLD in IBD patients are scanty. We aimed to investigate the prevalence and the progression of MASLD, diagnosed by non-invasive tools, in a homogeneous cohort of patients with IBD followed in our referral center. Methods Consecutive IBD patients without known chronic liver disease were retrospectively enrolled and prospectively followed between 2020 and 2024. MASLD was defined as Controlled Attenuation Parameter (CAP) ≥275 dB/m according to EASL criteria. Significant fibrosis was defined as Liver Stiffness (LSM) ≥8 kPa by FibroScanÒ. Demographic, metabolic and biochemical data were collected at baseline and at the last follow-up visit. All patients were genotyped for PNPLA3 rs738409 C&amp;gt;G by Taqman assay. Univariate logistic regression analysis was performed to identify risk factors associated with the prevalence of MASLD and its progression. Results 230 patients (mean age was 46.1±13.4 years, 49.2% were males, 50.8% had Crohn’s disease, mean disease duration was 9.4±7.5 years) were recruited. 14.8% of them were obese (BMI &amp;gt; 30 kg/m2), 9.8% diabetic. 49.2% were PNPLA3 rs738409 CG/GG. 62 out of 230 patients had complete metabolic and biochemical assessment at follow-up. Mean follow-up time was 4 years. At baseline, according to EASL criteria, 31.1% (19/62) had MASLD. 19.1% (8/42) of patients without steatosis at baseline, developed steatosis at follow-up; while 42.1% of those having steatosis at baseline, did not show steatosis at follow-up. Two patients without fibrosis assessed by LSM at baseline developed significant fibrosis, while two patients with fibrosis at baseline experienced fibrosis regression. 13.5% developed obesity. Obesity was significantly associated with the prevalence of MASLD both at baseline (p=0.01) and on follow-up (p=0.01) and was a predictor of steatosis progression (p=0.01); no association was found in relation to sex, PNPLA3 polymorphism, disease characteristics and the use of biologics. Diabetes was not significantly associated with MASLD in our cohort, but the number of patients was low. Conclusion One third of IBD patients had MASLD and obesity was the main predictor of its prevalence and progression, highlighting the relevance of active surveillance of MASLD and appropriate nutritional and lifestyle counselling especially in obese IBD patients. The use of biologics to control inflammation seems to have no influence on MASLD progression but data need further confirm in larger cohorts.

Dynamic changes in RNA integrity, gene expression, and tissue pathomorphology of experimental mice in the postmortem period

Aim. To examine the pattern of morphological changes, RNA quality number, and gene expression in mouse tissues sampled at autopsy under controlled experimental conditions.Materials and methods. Balb/c mice were euthanized and subsequently subjected to necropsy at 0, 3, 12, 24, 48, and 72 hours of the postmortem period. During the first three hours following euthanasia, the mice were maintained at room temperature, after which they were transferred to a refrigerator (4 º С). Total RNA was extracted from tissue samples taken from the kidney, liver, and brain; the integrity of the RNA samples was assessed by capillary electrophoresis, and the RNA quality number (RQN) was calculated. The expression levels of Actb, Epas1, and Rps18 housekeeping genes were evaluated by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) with original primers and probes using the TaqMan assay. The histologic examination was performed according to standard techniques.Results. Degradation of RNA extracted from mouse kidney tissues appeared to be greater than that of RNA taken from the liver. In the meantime, a negative linear correlation was observed between RQN and the duration of the postmortem interval for liver and kidney samples. In contrast, no significant changes in the RQN score were observed for brain RNA samples at any of the time points. The expression of the Epas1 and Rps18 genes was significantly decreased in mouse kidney and liver tissues. However, the level of Epas1 and Rps18 gene expression in the brain remained stable at all time points and did not exhibit a significant decrease at 72 hours after euthanasia. No obvious morphological changes were detected by the histologic examination, which does not exclude the presence of ultrastructural pathological changes.Conclusion. RQN in autopsy tissues serves as a crucial predictor of sample quality for molecular biology studies, including gene expression analysis.

NOS3 Gene Polymorphisms (rs2070744 and rs1799983) and Differentiated Thyroid Cancer: Investigating Associations with Clinical Outcomes.

Differentiated thyroid cancer (DTC) is the most common endocrine malignancy, with genetic factors playing an important role in its development and progression. This study investigated the association between nitric oxide synthase 3 (NOS3) gene polymorphisms (-786T>C or rs2070744 and Glu298Asp or c.894T>G or rs1799983) and the clinical characteristics and outcomes of DTC, aiming to evaluate their potential as biomarkers for prognosis. A case-control study was conducted, enrolling 172 individuals from the Endocrinology Clinics of Târgu Mureș and Iași, Romania, between 2021 and 2023. This study included 88 patients with DTC and 84 healthy controls, matched for age and sex. DNA was extracted from blood samples, and the NOS3 polymorphisms were genotyped using TaqMan assays. Statistical analysis included chi-square tests with a significance level set at p < 0.05. The distribution of the rs2070744 and rs1799983 polymorphisms showed no significant differences between the patients with DTC and healthy controls (p = 0.387 and p = 0.329, respectively). Furthermore, no significant associations were found between these polymorphisms and key clinical outcomes such as biochemical control, structural control, or loco-regional metastases. Our findings indicate that NOS3 rs2070744 and rs1799983 gene polymorphisms do not significantly influence the clinical outcomes of DTC, suggesting their limited utility as biomarkers for DTC prognosis.

A family with gallstone disease: defining inherited risk in the era of clinical genetic testing.

Gallstones are among the most frequent hepatobiliary conditions. Although in most cases, they remain asymptomatic, they can cause complications and, in such cases, invasive treatments like endoscopic retrograde cholangiography (ERC) or cholecystectomy are required. Here, we present the results of genetic testing of a single family with a high incidence of symptomatic gallstones and cholestatic liver phenotypes. Gallstone disease was detected among seven family members spanning three generations, and DNA samples were available from five of them. Genotyping was performed using TaqMan assays for known, selected genetic risk factors for gallstones and cholestasis, as well as next generation sequencing (NGS) of three genes involved in hepatobiliary transport. In all genotyped patients, we detected at least one copy of the gallstone-predisposing p.D19H variant in the hepatobiliary sterol transporter ABCG5/8, and in three cases, this variant was found in the rare homozygous state. In addition, the patients were all homozygous carriers of two intronic variants (c.2211+16C >T and c.3508-16T>C) and two common polymorphisms (c.504C>T and c.711A>T) in the ABCB4 gene, as well as the ATP8B1 gene variant c.696T>C. All genotyped patients also carried the predisposing variants c.1331C>T and c.3084A>G of the hepatobiliary bile salt export pump ABCB11 in either heterozygous or homozygous form. Hence, we propose that these variants taken together may have contributed to the high frequency of gallstone disease in this family, although functional studies for some variants are still lacking. In this report, we present these findings and discuss the challenges associated with interpreting sequencing data.

Click chemistry-based dual nanosystem for microRNA-122 detection with single-base specificity from tumour cells

MicroRNAs (miRNAs) have been recognised as potential biomarkers due to their specific expression patterns in different biological tissues and their changes in expression under pathological conditions. MicroRNA-122 (miR-122) is a vertebrate-specific miRNA that is predominantly expressed in the liver and plays an important role in liver metabolism and development. Dysregulation of miR-122 expression is associated with several liver-related diseases, including hepatocellular carcinoma and drug-induced liver injury (DILI). Given the potential of miR-122 as a biomarker, its effective detection is important for accurate diagnosis. However, miRNA detection methods still face challenges, particularly in terms of accurately identifying miRNA isoforms that may differ by only a single base. Here, with the aim of advancing accessible methods for the detection of miRNAs with single-base specificity, we have developed a robust dual nanosystem that leverages the simplicity of click chemistry reactions. Using the dual nanosystem, we successfully detected miR-122 at single-base resolution using flow cytometry and analysed its expression in various tumour cell lines with high specificity and strong correlation with TaqMan assay results. We also detected miR-122 in serum and identified four single nucleotide variations in its sequence. The chemistry employed in this dual nanosystem is highly versatile and offers a promising opportunity to develop nanoparticle-based strategies that incorporate click chemistry and bioorthogonal chemistry for the detection of miRNAs and their isoforms.Graphical

Association Between Common Variants in the LAG3/CD4 Genes and Risk for Essential Tremor.

Many clinical, neuroimaging, neuropathological, epidemiological, and genetic data suggest a relationship between essential tremor (ET) and Parkinson's disease (PD). Several hypothesis-based gene association studies attempted to find a genetic association between these diseases. Recent case-control association studies in Chinese and Spanish populations showed a marginal association between the CD4 rs1922452 and CD4 rs951818 single nucleotide variants (SNVs) and the risk of PD. The proteins encoded by the CD4 and LAG3 genes have an important role in modulating inflammatory responses, and some recent data associated inflammatory markers to ET. This study investigates a possible association between the most common SNVs in the LAG3/CD4 genes and the risk of ET in the Spanish Caucasian population. We genotyped 267 patients diagnosed with familial ET and 270 age- and sex-matched controls using specific TaqMan assays for CD4 rs1922452, CD4 rs951818, and LAG3 rs870849 variants. We found a decreased risk for ET in carriers of the LAG3 rs870849 C/C genotype and the LAG3 rs870849C allelic variant exclusively in men. The mean age of onset of ET was not related to any of the variants studied. These data suggest no association of the gene variants studied with the overall risk for ET, except for a slight decrease in risk in male ET patients carrying the variant LAG3 rs870849C. However, such an association lost significance after correcting for multiple comparisons.

Direct TaqMan assay for the detection and genotyping of bovine viral diarrhea virus types 1 and 2

Bovine viral diarrhea (BVD), caused by bovine viral diarrhea virus (BVDV), has a significant economic impact on affected farms worldwide. For effective disease control, it is crucial to select an appropriate vaccine based on the specific genotype of BVDV. Therefore, developing a rapid and reliable assay to detect and genotype BVDV is imperative for controlling the spread of disease. In this study, we developed a TaqMan assay to detect and genotype BVDV types 1 and 2 directly in bovine serum without extraction of RNA. The direct BVDV TaqMan assay effectively detected both BVDV1 and BVDV2 with confirmed specificity and showed no cross-reactivity with any of the other viruses tested, including bovine respiratory syncytial virus, bovine coronavirus, Akabane virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine immunodeficiency virus, and bovine leukemia virus. The assay could detect the virus in serum samples with a titer as low as 102 TCID50/mL in two out of three trials for BVDV1 and all three trials for BVDV2, indicating that its sensitivity is equivalent to that of virus isolation. Our findings represent a significant advancement in BVDV detection and typing directly from bovine serum.

Evaluation of the antibacterial activity of some plant essential oils against Ralstonia solanacearum and the molecular diagnosis of the bacterium

Background Evaluation of the antibacterial activity of some essential oils for controlling potato bacterial wilt. Objective In vitro and planta trials, five essential oils, lemongrass, eucalyptus, thyme, ginger, and peppermint, were evaluated for their aptitude to impede the progress of Ralstonia solanacearum. Materials and methods Five essential oils were assessed for their activity to inhibit R. solanacearum growth using the disc diffusion method. Certain volumes of each tested plant essential oil were added on filter paper discs using tetrazolium chloride agar medium, which contained the tested bacterium to obtain the proposed concentrations used to evaluate the most efficient oil for controlling potato bacterial wilt. Serological and molecular identification was also conducted. Results and conclusion R. solanacearum was characterized using cultural characteristics on the selective medium South Africa, “which is the most effective in suppressing the growth of contaminating microorganisms, and the highest recovery rate of the tested bacterium,” immunofluorescence antibody staining tomato bioassay test and molecular identification via real-time PCR (Taq-man) assay. In-vitro studies showed that all tested plant essential oils (lemongrass, eucalyptus, thyme, ginger, and peppermint) significantly reduced R. solanacearum progress. Eucalyptus (Eucalyptus globulus) and peppermint (Mentha piperita) essential oils were the most efficient in suppressing R. solanacearum growth, where it showed the highest mean inhibition zone, followed by thyme oil (Thymus vulgaris), lemongrass oil (Comybogen citratus), and ginger oil (Zingiber officinale), respectively. Phenotypic variances in bacterial cell construction have been noticed via a transmission electron microscope. Eucalyptus oil led to cell wall lysis, cell laceration, bubble-like structures, and degraded cellular components in bacterial cells.

Association Between NK Cell Genetic Variants and the Development of Long COVID Associated- and Prepandemic Small Fiber Neuropathy.

Long coronavirus disease 2019 (COVID) (LC) symptoms including pain and autonomic dysfunction are in some patients associated with small-fiber neuropathy (SFN). The pathomechanisms underlying SFN are mostly unclear. Natural killer (NK) cells play a crucial role in immune regulation, viral clearance and nerve metabolism. The aim of this study was to identify associations between development of small-fiber dysfunction dependent and independent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and human genetic markers associated with specific NK cell functions. The genetic markers assessed in all cohorts included: FCGR3A, IGHG1, HLA-E, NKG2C, and rs9916629. Genotyping was performed using TaqMan assays, Sanger sequencing and touchdown polymerase chain reaction. We assessed human cytomegalovirus (HCMV) IgG serostatus in all participants, and screened for anti-neuronal, anti-glial and anti-ganglioside autoantibodies in both patient cohorts. We included 50 LC patients with newly-emerged symptoms of small-fiber dysfunction after SARS-CoV-2 infection, 27 prepandemic SFN patients and 320 control persons. Markers associated with low NKG2C response, that is, deletion of the NKG2C gene and lack of prior HCMV infection (IgG seronegativity), occurred significantly more frequently in prepandemic SFN patients compared to LC patients and controls (p = 0.0109 and 0.0005, respectively). In conclusion, markers of impaired NKG2C pathways are associated with prepandemic SFN, but not with Long COVID-associated small-fiber dysfunction.

Interaction of Clock gene variants and behavioral parameters influences adiposity-related traits

ABSTRACT Obesity, a major health concern, is influenced by an individual’s genetic makeup and lifestyle factors (eating, sedentary lifestyle, and sleep disruptions) that affect circadian clock and metabolism. This study investigates the impact of Clock gene variants rs6843722 and rs4864548 on obesity measures in the Pakistani population. Genetic-behavior interaction studies have focused on Western populations, overlooking South Asians. We included 306 overweight/obese and 306 normal-weight controls, matched for age and gender. Anthropometric measures (BMI, waist circumference, waist-to-height ratio, and body fat percentage) were taken using standard procedures while behavioral data (dietary and sleep-related behaviors, shiftwork, and physical activity) was collected by questionnaire. Genotyping was performed by Taqman assays. Data was analyzed using SPSS 19.0. Rank-based inverse normal transformation was executed for quantitative non-normal variables. The linear regression revealed that both Clock gene variants interacted significantly with dietary and sleep-related behaviors and low physical activity, impacting adiposity measures regardless of age and gender. Shiftwork interacted specifically with rs6843722, increasing body fat percentage. This study indicates that Clock gene variants, when interacting with lifestyle factors, play a substantial role in increasing obesity risk highlighting the link between lifestyle choices and disturbances in circadian rhythms controlled by Clock gene variations, ultimately leading to obesity.

Validation and TaqMan Conversion of a Molecular Chronotype Assessment Approach.

The present study aimed to develop a TaqMan genotyping card for molecular chronotype assessment based on a predictive panel of 35 previously identified genetic variants. A reliable TaqMan assay was successfully developed for 33 out of the 35 chronotype-predictive variants. The resulting TaqMan genotyping card was utilized to genetically characterize 196 new individuals (in addition to the previously studied 96) and the Morningness-Eveningness Questionnaire was utilized for their phenotypical chronotype assessment. The predictive panel performance was validated on (a) a group of morning and evening individuals (logistic regression model), (b) a representative sample of the original study population also including intermediate chronotypes (linear regression model) and, (c) 25,986 individuals from the Estonian Biobank, for whom Munich Chronotype Questionnaire scores were available. The validation of the morningness-eveningness logistic regression model on 25 morning and 21 evening types resulted in a predictive value of 72%, confirming the reliability of the predictive panel and the success of its conversion into a TaqMan genotyping card. By contrast, the inclusion of intermediate individuals in the model led to a significant decrease in predictive performance (45% on 100 individuals [25 morning, 54 intermediate, and 21 evening]), with intermediate types being the most affected. No significant associations were observed between the genotype panel and chronotype in the Estonian Biobank sample. In conclusion, our genotyping card might represent a promising molecular chronotyping tool for the Italian population. Its performance in other populations is worthy of further study.

YTHDF3 rs7464 A > G polymorphism increases Chinese neuroblastoma risk: A multiple-center case-control study.

Neuroblastoma (NB), a rare childhood cancer originating in nerve tissue. YTHDF3, a member of the YTH domain protein family, is involved in RNA m6A modification and cancer progression. Polymorphisms in YTHDF3 may influence its expression and biological function. Herein, this study estimated the association between YTHDF3 polymorphisms (rs2241753, rs2241754, and rs7464) and NB susceptibility in a multicenter study comprising 898 cases and 1734 controls. We genotyped YTHDF3 candidate polymorphisms by the TaqMan assay. Logistic regression analysis was applied to indicate the possible relationships between these polymorphisms and NB susceptibility, using odds ratios (ORs) and 95% confidence intervals (CIs). Logistic regression analysis revealed that the rs2241753 GA versus GG decreased NB risk (Adjusted OR = 0.84, 95% CI = 0.71-0.997, p = .047), while the rs7464 GG versus AA enhanced NB risk (Adjusted OR = 1.62, 95% CI = 1.20-2.18, p = .002). Additionally, rs7464 GG versus AA/AG showed a higher risk (Adjusted OR = 1.66, 95% CI = 1.24-2.22, p = .0006). Combination analysis showed that having 1-3 risk genotypes versus 0 was associated with increased NB risk (Adjusted OR = 1.28, 95%CI = 1.09-1.51, p = .003). The significance of rs7464 and the risk genotypes combination persisted across multiple subgroups, whereas rs2241754 was significant only in mediastinal NB. False-positive report probability (FPRP) confirmed the reliability of results. Notably, the interaction between rs7464 and rs2241754 may increase NB risk dramatically. Our study demonstrated that YTHDF3 rs7464 A > G significantly affected NB susceptibility, warranting validation in larger sample sizes.

MIR27A rs895819 CC Genotype Severely Reduces miR-27a Plasma Expression Levels.

Background/Objectives:MIR27A rs895819 polymorphism has emerged as a potential additional pharmacogenomic marker of fluoropyrimidine response. Current evidence on its potential effect on miR-27a expression, which represses DPD activity, leading to DPD deficiency and increased fluoropyrimidine-associated toxicity risk, is scarce and inconsistent. We have analyzed the effect of MIR27A rs895819 polymorphism on miR-27a-3p plasma expression levels under different models of inheritance to contribute further evidence on its plausible biological role in miR-27a expression. Methods: A total of 59 individuals with no medical history of cancer were included in this study. MIR27A rs895819 genotyping and miR-27a-3p expression were analyzed by using predesigned TaqMan assays. Results: The frequency of TT, TC, and CC genotypes was present at a prevalence of 50.8%, 44.1%, and 5.1%, respectively. Individuals carrying the CC genotype presented with decreased miR-27a-3p expression (0.422 fold-change versus TT, p = 0.041; 0.461 fold-change versus TC, p = 0.064), whereas no differences were present between TT and TC individuals (1.092 fold-change, p = 0.718). miR-27a-3p expression was decreased in CC individuals under a recessive model of inheritance (0.440 fold-change, p = 0.047). No differences were found in dominant (TT vs. TC+CC, 0.845 fold-change, p = 0.471) or over dominant (TT+CC vs. TC, 0.990 fold-change, p = 0.996) models of inheritance. Conclusions:MIR27A rs895819CC genotype leads to severely reduced miR-27a-3p expression in plasma. Further study of this association is warranted in cancer patients to apply MIR27A genotyping in therapeutics to identify fluoropyrimidine-treated patients who are at a decreased risk of experiencing fluoropyrimidine-induced severe toxicity.

Biomarkers improving genetic and metastatic disease prediction in paraganglioma: insights from a prospective study.

Identifying the risk of malignancy and genetic status in primary paraganglioma or pheochromocytoma (PPGL) is a key challenge. The aim was to assess the diagnostic accuracy of genomic, metabolomic and histopathological biomarkers for predicting metastatic and genetic status. COMETE-TACTIC is a prospective study (NCT02672020) conducted from November 2015 to March 2019 across 16 referral centers. Tumor samples and liquid biopsies from 231 consecutive patients with PPGL were collected. Germline and somatic genetic status were determined by NGS. SDHB, SDHA and CA9 immunohistochemistries were performed on tumor tissues. TERT promoter methylation was assessed by pyrosequencing. Metabolomic profile and circulating miRNAs were measured in liquid biopsies by gas chromatography MS/MS and TaqMan assay quantified by droplet digital PCR, respectively. Tumor analysis outperformed germline analysis for determining genetic status. Positive SDHA and SDHB staining combined with negative CA9 labeling indicated the absence of SDHx and VHL variants. Plasma succinate levels above 4.94µM identified SDHx mutation carriers with 65% sensitivity and 92% specificity (AUC-ROC 0.82, 95%CI 0.70-0.93). Among circulating miRNAs, miR-483-5p was the best classifier of metastatic status (AUC-ROC 0.64, 95%CI 0.52-0.77). A sum of dinucleotide methylation rate of TERT promoter CpGs above 42% predicted metastatic status (AUC-ROC 0.75, 95%CI 0.65-0.85). Multivariate analyses showed that biomarker combinations significantly predicted SDHx status (AUC-ROC 0.99, 95%CI 0.98-1.00) and metastatic potential (AUC-ROC 0.93, 95%CI 0.84-1). Circulating miR-483-5p, plasma succinate, TERT promoter methylation, and SDHB immunostaining are valuable for PPGL risk stratification. Combining biomarkers with clinical data provides excellent diagnostic accuracy for metastatic patients (AUC-ROC 0.97, 95%CI 0.93-1).

Molecular methods for the detection and identification of parasitoids within larval wheat midges

Three species of cecidomyiid midges (Diptera: Cecidomyiidae) cause significant yield losses on wheat in Europe: Sitodiplosis mosellana (Géhin), Contarinia tritici (Kirby) and Haplodiplosis marginata (von Roser). Eggs and young larvae may be parasitised by a complex of hymenopteran parasitoids belonging to the Pteromalidae and Platygastridae families which contributes to natural pest control. We have developed molecular tools for detecting and identifying seven parasitoid species previously encountered in Belgium inside individual wheat midge larvae. Barcode DNA sequences from COI, 18S and 28S genes were obtained from the midges and parasitoid species. Each of the three genes allowed all the species to be distinguished although 18S was the only one displaying a barcoding gap, both between parasitoids and midges, and at the species level. Based on the 18S gene, we developed a TaqMan assay to assess parasitism in midge larvae, regardless of the midge and parasitoid species. Next, two group-specific PCR primer pairs were generated, allowing the separate amplification of midge DNA or parasitoid DNA in parasitised individuals and subsequent identification by Sanger sequencing. Finally, species-specific primers were designed to identify six parasitoid species by simple PCR amplification. These tools were successfully applied to assess the parasitism rate of S. mosellana larvae in seven Belgian fields.

Association Between the rs13306703 and rs8192288 Variants of the SOD3 Gene and Breast Cancer and an In Silico Analysis of the Variants' Impact.

Background/Objectives: This study investigated the association between the rs13306703 and rs8192288 variants of the superoxide dismutase 3 (SOD3) gene and breast cancer (BC) in the Mexican population, conducting both genetic and in silico analyses. Methods: 357 healthy women and 386 BC patients were studied using TaqMan assays, qPCR, and RFLP-PCR. Results: The TT genotype and a recessive pattern of these variants were risk factors for BC (p < 0.05). Specifically, the TT genotype of rs13306703 was associated with metastatic lymph nodes, tumor progression (III-IV), luminal A, nonresponse to chemotherapy, and ki-67 ≥ 20% with diabetes mellitus (DM). Meanwhile, the GT genotype of rs8192288 was associated with menopause, luminal A, tumor progression (III-IV), ki-67 ≥ 20%, and a positive estrogen receptor with nonresponse to chemotherapy. Additionally, the TT genotype combined with DM was identified as a BC risk factor (p < 0.05). The TT haplotype was also found to be a risk factor for BC. In silico analysis suggested that these variants might influence SOD3 regulation by affecting transcription factors and active enhancer sites. Conclusions: The rs13306703 and rs8192288 variants of the SOD3 gene were associated with an increased risk of BC and may alter SOD3 regulation through effects on transcription factors, active enhancers, and transcription start sites, with modified motifs in breast epithelium cells.