Ligase Detection Reaction Assay Research Articles

Association of gene polymorphisms of MyD88 and TICAM1 and their interactions with community-acquired pneumonia in children

To investigate the association of single nucleotide polymorphisms (SNPs) of myeloid differentiation factor 88 (MyD88) and Toll-like receptor adaptor molecule 1 (TICAM1) and their interactions with community-acquired pneumonia (CAP) in children. Improved multiple ligase detection reaction assay was used for detecting the polymorphisms of nine tagging SNPs of the MyD88 and TICAM1 genes in 375 children with CAP who attended the Department of Pediatrics of the Second Affiliated Hospital of Yan'an University Medical School from August 2015 to September 2017 and 306 healthy children who underwent physical examination. A logistic regression analysis was used to evaluate the association between the distribution of genotypes and their interactions with CAP in children. The polymorphism of the TICAM1 gene at rs11466711T/C locus was closely associated with the susceptibility to CAP in children (P<0.05). The AA genotype of rs35747610G/A locus significantly reduced risk of sepsis in children with CAP (P<0.05). The AA genotype of rs6510826G/A locus was significantly associated with the increase in C-reactive protein level in children with CAP (P<0.05). The GG genotype of the MyD88 gene at rs7744A/G locus significantly increased the risk of respiratory failure and circulatory failure (P<0.05). The multiplicative interactions between MyD88 gene rs7744A/G and TICAM1 gene rs11466711T/C, rs2292151G/A, rs35299700C/T, and rs35747610G/A loci were significantly associated with the susceptibility to CAP, the severity of CAP, and the risk of sepsis in children (P<0.05). The gene polymorphisms of MyD88 and TICAM1 and their interactions are closely associated with CAP in children, with a synergistic effect on the development and progression of CAP in children.

Long Noncoding RNA HULC Promoter Polymorphism rs1041279 Is Associated with an Increased Risk of Cervical Squamous Cell Carcinoma.

Hepatocellular carcinoma upregulated long noncoding RNA (HULC), identified as an oncogene in cervical cancer, is involved in not only the clinical stage, lymph node metastasis, and depth of cervical invasion but also outcome. In this study, we aimed to investigate the association between 3 polymorphisms (i.e., rs1041279, rs3005167, and rs7770772) in the promoter of HULC and the risk of cervical squamous cell carcinoma (CSCC). The polymorphisms were genotyped using the multiplex ligase detection reaction assay. The promoter activity was measured using the dual-luciferase reporter assay kit. The rs1041279 GG genotype and G allele revealed a significantly higher risk of CSCC compared with the rs1041279 CC genotype and C allele (GG vs. CC, adjusted OR = 1.79, 95% CI, 1.17-2.73, P = 0.007; G vs. C, adjusted OR = 1.36, 95% CI, 1.09-1.69, P = 0.006). Haplotype analysis revealed that the rs3005167C-rs7770772G-rs1041279C or rs3005167C-rs7770772G-rs1041279G haplotype had a significantly higher risk of CSCC compared to the rs3005167G-rs7770772G-rs1041279C haplotype (CGC vs. GGC, OR = 2.38, 95% CI, 1.53-3.75, P < 0.001; CGG vs. GGC, OR = 3.76, 95% CI, 2.12-6.68, P < 0.001). Dual-luciferase reporter assay showed that the rs1041279 G promoter resulted in higher transcriptional activity compared with the rs1041279 C (P < 0.01). Additionally, the rs1041279 GG genotype carriers had an increased level of HULC expression (P = 0.03). These findings suggest that the HULC rs1041279 may be a useful marker for the etiology of CSCC.

Association of GWAS-supported noncoding area loci rs404860, rs3117098, and rs7775228 with asthma in Chinese Zhuang population.

BackgroundAsthma is a complicated and polygenic inheritance disease, and its prevalence increases worldwide. Recent genome‐wide association studies (GWASs) identified a significant association of single nucleotide polymorphism with asthma in the Japanese population. This study aimed to examine the association of GWAS‐supported noncoding area loci, namely rs404860, rs3117098, and rs7775228, with asthma in Chinese Zhuang population.MethodsA case‐control study involving 223 individuals, comprising 123 patients with asthma and 100 healthy controls, was conducted. Genotypes were determined by polymerase chain reaction (PCR)/ligase detection reaction assay. The association between gene polymorphisms and asthma risk was calculated by logistic regression analysis using different genetic models through comparisons of alleles (A vs a), homozygote genotypes (AA vs aa), heterozygote genotypes (Aa vs aa), dominant models (AA+Aa vs aa), and recessive models (AA vs. Aa+aa).ResultsThe distribution of the genotype frequency of rs3117098 was statistically different between the case and control groups. For rs3117098, significant associations were observed through comparisons of alleles (OR: 1.832, 95% CI: 1.048‐3.204, P = .034) and dominant models (OR: 2.065, 95% CI: 1.001‐4.260, P = .050). The statistical analysis showed no significant difference for loci rs404860 and rs7775228 between patients with asthma and controls.Conclusionrs3117098 may be the risk factor for asthma in Chinese Zhuang population.

Epigenetic modifications but not genetic polymorphisms regulate KEAP1 expression in colorectal cancer.

Kelch-like ECH-associated protein 1 (KEAP1), as a negative regulator of nuclear factor erythroid 2 like 2 ( NRF2), plays a pivotal role in NRF2 signaling pathway and involves in tumorigenesis. Polymorphisms and methylation in gene promoter region may influence its expression and be related to cancer susceptibility. In this study, we examined the effect of the KEAP1-NRF2 interaction on the risk of colorectal cancer (CRC). The polymorphisms of NRF2 and KEAP1 were genotyped using the improved multiplex ligase detection reaction assay. KEAP1 promoter methylation and histone modification were analyzed using bisulfite genome sequencing and chromatin immunoprecipitation (ChIP) assay, respectively. The KEAP1 rs1048290 CC genotype and C allele were associated with increased risks of CRC (CC vs GG: odds ratio [OR] = 1.39; 95% confidence interval [CI], 1.08-1.78; CC vs GG/GC: OR = 1.29; 95% CI, 1.05-1.58; C vs G: OR = 1.18; 95% CI, 1.04-1.34). The rs1048290-rs11545829 GT haplotype was associated with a reduced risk of CRC. KEAP1-NRF2 interaction analysis revealed that the rs6721961, rs35652124, rs1048290, and rs11545829 conferred the susceptibility to CRC. The hypermethylation of KEAP1 promoter resulted in lower levels of KEAP1 messenger RNA (mRNA). After treatment with 5-aza-2'-deoxycytidine/trichostatin A, KEAP1 promoter methylation was decreased and KEAP1 mRNA levels were increased. ChIP-quantitative polymerase chain reaction results showed an enhanced enrichment of H3K4Me3 and H3K27Ac to the promoter of KEAP1. In vitro methylation analysis showed that the methylated plasmid decreased the transcriptional activity by 70%-84%. These findings suggest that the KEAP1- NRF2 pathway could potentially impact CRC risk and the downregulation of KEAP1 could be explained in part by epigenetic modifications.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Associations SELE gene haplotype variant and hypertension in Mongolian and Han populations.

Genetic variation is thought to contribute to the etiology of hypertension, and E-selectin is a candidate essential hypertension-associated gene. In this study, we attempted to test the hypothesis that subtle haplotype variants of SELE genes may be sources of essential hypertension in Mongolian and Han populations. A total of 429 unrelated Mongolian herdsmen and 416 Han farmers were enrolled, including 212 Mongolian essential hypertension (EH) patients, 217 Mongolian normotensives (controls), 200 Han EH patients and 216 Han normotensives (controls). All nine tag single-nucleotide polymorphisms (SNPs) within the SELE gene were retrieved from HapMap and the genotyping was performed using a polymerase chain reaction (PCR)/ligase detection reaction assay. Results The distributions of the A-allele frequency of rs3917458 and the C-allele frequency of rs2179172 differed significantly between the hypertensive subjects and controls in the Han population. The frequency of haplotype GGC was significantly higher in the EH group than in the controls in the Mongolian population. In the Han population, a significant difference was observed in the haplotype frequency of TCC between the patients and controls, whereas haplotype ACA was detected significantly less often in the EH subjects than in the controls. Meanwhile, the haplotype TCC in the Han hypertensive patients and the haplotype GGC in the Mongolian patients had independent effects in increasing the risk for EH and maybe used as risk factors for predicting high blood pressure. However, the haplotype ACA had an independent effect in decreasing the risk of hypertension and may be protective in normotensive subjects in the Han population. Therefore, multiple SNPs in combination in SELE may confer a risk of hypertension.

SLC29A1 single nucleotide polymorphisms as independent prognostic predictors for survival of patients with acute myeloid leukemia: an in vitro study

The mechanism behind poor survival of acute myeloid leukemia (AML) patients with 1-barabinofuranosylcytosine (Ara-C) based treatment remains unclear. This study aimed to assess the pharmacogenomic effects of Ara-C metabolic pathway in patients with AML. The genotypes of 19 single nucleotide polymorphisms (SNPs) of DCK, CDA and SLC29A1from 100 AML patients treated with Ara-C were examined. All the SNPs were screened with ligase detection reaction assay. The transcription analysis of genes was examined by quantitative real time polymerase chain reaction. The association between clinical outcome and gene variants was evaluated by Kaplan-Meier method. Genotypes of rs9394992 and rs324148 for SLC29A1 in remission patients were significantly different from those in relapsed ones. Post-induction overall survival (OS) significantly decreased in patients with the CC genotype of rs324148 compared with CT and TT genotypes (hazard ratio [HR] = 2.997 [95% confidence interval (CI): 1.71-5.27]). As compared with CT and TT genotype, patients with the CC genotype of rs9394992 had longer survival time (HR = 0.25 [95% CI: 0.075-0.81]; HR = 0.43 [95% CI: 0.24-0.78]) and longer disease-free survival (DFS) (HR = 0.52 [95% CI: 0.29-0.93]; HR = 0.15 [95% CI: 0.05-0.47]) as well As compared with CT and TT genotype, patients with the CC genotype of rs324148 had shorter DFS (HR = 3.18 [95% CI: 1.76-5.76]). Additionally, patients with adverse karyotypes had shorter DFS (HR = 0.17 [95% CI: 0.05-0.54]) and OS (HR = 0.18 [95% CI: 0.05-0.68]). AML patients with low activity of SLC29A1 genotype have shorter DFS and OS in Ara-C based therapy. Genotypes of rs9394992 and rs324148 may be independent prognostic predictors for the survival of AML patients.

Enhanced discrimination of single nucleotide polymorphisms using 3′ nucleotide differences in ligase detection reaction probes

The ligase detection reaction (LDR) is a highly specific genotyping method for single nucleotide variations. Although LDR typically discriminates single nucleotide polymorphism (SNP) alleles at the 3′ end of so-called LDR discriminating probes, we designed probes in which the position of nucleotide differences for discrimination was shifted to the second and third nucleotides from the 3′ end. Using the 3′-modified probes, we targeted SNPs of the human ABO group and investigated the specificity and efficiency of ligation by a universal LDR assay. We demonstrated that one or two nucleotide shifts of differences in discriminating probes improve the allele balance in detecting both base substitutions and short deletions. In regard to short deletions, moreover, the shifts of nucleotide differences in discriminating probes form the perfect-machted or multiple-mismatched structures (the bulge structures) in the discriminating probe-target DNA duplex and may contribute to enhance ligation efficiency.

Detection of the rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil using a ligase detection reaction assay

Background Adefovir dipivoxil (ADV) is effective for treatment of chronic hepatitis B virus (HBV) infection, but long-time ADV therapy leads to drug resistance because of HBV reverse transcriptase mutations. We developed a sensitive and specific method for detecting the rtA181V/T and rtN236T mutations associated with ADV resistance in chronic hepatitis B patients, based on a ligase detection reaction (LDR). Methods HBV templates were amplified by polymerase chain reaction (PCR), followed by LDR and electrophoresis on a sequencer. The assay was evaluated using 165 serum samples, and plasmid controls. Results In a mixture of wild-type and mutant plasmids, the assay could detect mutant plasmid at 1%. Complete concordance between the PCR–LDR assay and sequencing analysis was observed for 141 of 148 samples (95.3%) at codon 181, and 143 of 148 samples (96.6%) at codon 236. Discordant results were confirmed to be consistent with the PCR–LDR assay by subclone sequencing. Seventeen samples could not be detected by both of the methods due to low HBV DNA levels. Conclusions The PCR–LDR assay can sensitively and specifically detect the rtA181V/T and rtN236T mutations, and may be used for monitoring ADV resistance in patients infected with HBV.

ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design.

16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences.