MLC-Seq: de novo Sequencing of Full-Length tRNAs and Quantitative Mapping of Multiple RNA Modifications

Despite the extensive use of next-generation sequencing of RNA, simultaneous sequencing and quantitative mapping of multiple RNA modifications remain challenging. Herein, we develop MLC-Seq, a mass spectrometry-based direct sequencing method allowing for simultaneously unravelling the RNA sequences and quantitatively mapping different tRNA nucleotide modifications site-specifically. Importantly, MLC-Seq reveals the stoichiometric changes of tRNA modifications upon treatment with the dealkylating enzyme AlkB, and led to the discovery of new nucleotide modifications.

Study of UGT1A1*28 genetic polymorphism related to irinotecan response in Kinh Vietnamese

Irinotecan is a medicine commonly used to treat cancer. Carboxylesterase converts irinotecan into SN-38, a substance with 100 times more cytotoxicity than the original compound. SN-38 is inactivated by glucuronidation in the liver to the inactive form of SN-38 glucuronidation. UGT1A1 is the main enzyme responsible for the glucuronidation SN-38 secretion. Neutropenia and diarrhea are dose-limiting toxicity of the drug. UG1A1*28 variant is believed to increase the risk of neutropenia and is closely related to the risk of severe diarrhea. In this study, we used the direct sequencing method of the promoter UGT1A1 gene to determine the genotype and allele frequency of UGT1A1*28 in 95 individuals of healthy Kinh Vietnamese . The results showed that UGT1A1*28 variant is present in homozygousand heterozygous genotypes with frequencies of 14.74% and 1.05%, respectively. The allele frequency of the UGT1A1*28 variant was 8.421% which was small compared with the wild type allele *1 (91.579%). The data obtained from the study contribute to an effective cancer treatment solution using the drug irinotecan.

The genetic association between TLR-1, -2, -4, and -6 gene polymorphisms and rheumatoid arthritis (RA) susceptibility in a Chinese Han population

Background: The toll-like receptor (TLR) genes were shown to be involved in the pathogenesis of RA. We aimed to investigate the genetic associations between the TLR-1, -2, -4, and -6 genes polymorphisms and RA susceptibility in a Chinese Han population. Methods : Six polymorphisms (TLR-1 (rs5743610, rs5743618), -2 (rs5743708), -4 (rs4986790, rs4986791), and -6 (rs5743810)) in TLRs genes were genotyped in 360 patients with RA and 560 matched healthy controls by using direct sequencing method. The ORs and 95% CIs were evaluated using a standard logistic regression analysis. Results : No significant association between the allelic, dominant and recessive models of TLR-1 rs5743618, TLR-2 rs5743708, TLR-4 rs4986790 and rs4986791, and TLR-6 rs5743810 polymorphisms and RA risk was observed (p>0.05). However, significant associations were detected between the allelic, dominant and recessive models of TLR-1 rs5743618 and RA risk (allelic: OR[95%CI]= 2.21 [1.73, 2.81], p<0.0001; dominant: OR[95%CI]= 2.33 [1.75, 3.09], p<0.0001; recessive models: OR[95%CI]= 3.70 [1.85, 7.41], p=0.0002), In addition, the TLR6 rs5743810 was found to be associated with the RF - and anti-CCP - antibody in RA group (RF: OR[95%CI]= 2.29 [1.42, 3.69], p=0.0007; anti-CCP: OR[95%CI]= 2.33 [1.39, 3.89], p=0.001). Conclusions : The allelic, dominant, and recessive models of TLR1 rs5743618 might be associated with RA susceptibility. And the TLR6 rs5743810 might be associated with RF and anti-CCP antibody in RA in Chinese Han population.

A Functional Polymorphism in the Promoter Region of Interleukin-12B Increases the Risk of Colorectal Cancer

Objective. To investigate whether the polymorphisms of interleukin-12B (IL-12B) were associated with the risk of developing colorectal cancer (CRC). Patients and Methods. Genotypes of rs17860508 and rs3212227 were determined by polymerase chain reaction with a direct sequencing method in 329 CRC patients and 342 matched healthy control subjects. The expression of IL-12B mRNA was determined by RT-qPCR in 50 pairs of CRC tissues and their adjacent normal tissues. Results. Compared with TTAGAG/TTAGAG genotype of rs17860508, the GC/GC and TTAGAG/GC genotypes may significantly increase the risk of CRC (OR = 1.81, 95% CI = 1.18–2.78; OR = 1.46, 95% CI = 1.01–2.12, respectively). Furthermore, the mRNA levels of IL-12B were significantly higher in the CRC tissues from patients with the rs17860508 GC/GC genotype than those with the TTAGAG/GC (P=0.009) and TTAGAG/TTAGAG (P=0.001) genotypes. Conclusion. These data suggested that the rs17860508 GC/GC genotype might upregulate IL-12B expression at the transcriptional level and thus increase the risk of CRC.

POST-OPERATIVE PATIENTS;

Objective: To explore the effect of genetic polymorphism -100_-102AAG deletion in the 5-hydroxytryptamine type 3B (5-HT3B) gene on the incidence of post-operative vomiting (POV). Study Design: A prospective, clinical trial. Place and Duration of study: Clinical data collection and blood sampling was carried out at Combined Military Hospital, Rawalpindi. Genetic analysis was carried out at Institute of Biomedical and Genetic Engineering, Islamabad from 01 Aug 2012 to 22 Sep 2013. Methods: This study included two hundred and sixty patients planned for elective laparoscopic cholecystectomy. 4 mg ondansetron was administered intravenously thirty minutes before the end of surgery. A total of 140 patients with the complaints of vomiting and 120 patients without vomiting were analyzed for -100_-102delAAG deletion polymorphism with the help of direct sequencing method. Results: A significant association was found between the incidence of vomiting and the -100_-102AAG insertion/deletion polymorphism of the 5-HT3B gene at 2 hours after surgery. Conclusion: The -100_-102AAG deletion variant of the 5-HT3B gene may affect POV and predict the responsiveness to ondansetron.

Variants of Interleukin-22 Gene Confer Predisposition to Autoimmune Thyroid Disease

As there are no previous studies on the interleukin-22 (IL-22) variants in autoimmune thyroid disease (AITD), the present study aimed to explore the association between polymorphisms of IL-22 and the predisposition to AITD. The study had 975 AITD patients, including 639 Graves’ disease (GD) and 336 Hashimoto’s thyroiditis (HT) individuals and 851 healthy cohorts. Ligase detection reaction (LDR) and direct sequencing method were used for genotyping the IL-22 gene polymorphisms at rs2046068, rs2227478, rs2227485, rs11611206, and rs1179251. In comparison to female controls, genotype CC of rs1179251 was increased in the female AITD patients. Alleles C at rs2046068, C at rs2227478, and C at rs1179251 linked to the susceptibility of HT males. Genotype CC in rs1179251 was higher in male HT. Variants at rs2046068, rs2227478, and rs1179251 were associated with the AITD teenagers. Besides, genotype GG in rs11611206 was correlated with thyroid-associated ophthalmopathy (TAO). Moreover, allele G at rs11611206 was associated with decreased risk for TAO by 28.9%. Similarly, genotype CC of rs1179251 and genotype GG of rs11611206 were associated with Graves’ ophthalmopathy (GO). Allele G in rs11611206 increased people with HT towards the predisposition of hypothyroidism. In conclusion, genetic variants of IL-22 are associated with the occurrence of AITD.

Detected EGFR mutation in cerebrospinal fluid of lung adenocarcinoma patients with meningeal metastasis

ObjectiveTo discuss the application of ARMS method to detect EGFR gene mutation in cerebrospinal fluid of lung adenocarcinoma patients with meningeal metastasis.Methods5 cases of lung adenocarcinoma were identified with meningeal metastasis that were cleared EGFR gene mutation by gene sequencing method. From each patient 5ml cerebrospinal fluid was obtained by lumbar puncture. ARMS method was used to detect EGFR mutations in cerebrospinal fluid.Results5 samples of cerebrospinal fluid were successfully detected by ARMS method, 3 samples found that EGFR gene mutations, the mutations in line with direct sequencing method.ConclusionARMS method can be used to detect EGFR gene mutations of cerebrospinal fluid samples in lung adenocarcinoma with meningeal metastasis. But cerebrospinal fluid specimens from histological specimens, blood samples need to be confirmed by further comparative study whether there is advantage.

The application of molecular methods in the diagnostics of infection of swine caused by Porcine circovirus 2

Porcine circovirus 2 (PCV2) belongs to the family Circoviridae, genus Circovirus. Infection of swine caused by this virus is manifested in several different clinical forms, leading to significant economic losses in swine production worldwide. For this reason, prompt and precise diagnostics of this swine infection is of great importance. For this purpose today there are used molecular methods of virological diagnostics such as polymerase chain reaction (PCR), and real-time method PCR, that is direct sequencing method by Sanger.

Investigation of Ser315 Substitutions withinkatGGene in Isoniazid-Resistant Clinical Isolates ofMycobacterium tuberculosisfrom South India

Mutation at codon 315 ofkatGgene is the major cause for isoniazid (INH) resistance inMycobacterium tuberculosis(M. tuberculosis). Substitution at codon 315 ofkatGgene was analyzed in 85 phenotypically resistant isolates collected from various parts of southern India by direct sequencing method. The obtained results were interpreted in the context of minimum inhibitory concentration (MIC) of INH. Of the 85 phenotypically resistant isolates, 56 (66%) were also correlated by the presence of resistance mutations in thekatGgene; 47 of these isolates had ACC, 6 had AAC, 2 had ATC, and one had CGC codon. The frequency of Ser315 substitution inkatGgene was found to be higher (70%) amongst multidrug-resistant (MDR) strains than among non-MDR (61%) INH-resistant isolates. Further, the frequency of mutations was found to be greater (74%) in isolates with higher MIC values in contrast to those isolates with low MIC values (58%). Therefore, the study identified high prevalence of Ser315Thr substitution inkatGgene of INH-resistant isolates from south India. Also, isolates harboring this substitution were found to be associated with multidrug and high level INH resistance.