Safety tests and clinical research on buccal and nasal microneedle swabs for genomic analysis.

Conventional swabs have been used as a non-invasive method to obtain samples for DNA analysis from the buccal and the nasal mucosa. However, swabs may not always collect pure enough genetic material. In this study, buccal and nasal microneedle swab is developed to improve the accuracy and reliability of genomic analysis. A cytotoxicity test, a skin sensitivity test, and a skin irritation test are conducted with microneedle swabs. Polymer microneedle swabs meet the safety requirements for clinical research and commercial use. When buccal and nasal microneedle swabs are used, the amount of genetic material obtained is greater than that from commercially available swabs, and DNA purity is also high. The comparatively short microneedle swab (250 µm long) cause almost no pain to all 25 participants. All participants also report that the microneedle swabs are very easy to use. When genotypes are compared at five SNP loci from blood of a participant and from that person's buccal or nasal microneedle swab, the buccal and nasal microneedle swabs show 100% concordance for all five SNP genotypes. Microneedle swabs can be effectively used for genomic analysis and prevention through genomic analysis, so the utilization of microneedle swabs is expected to be high.

Association of PD-L1 Expression with Tumor-Infiltrating Immune Cells and Mutation Burden in High-Grade Neuroendocrine Carcinoma of the Lung.

INTRODUCTION: The immune microenvironment of high-grade neuroendocrine carcinoma of the lung, including programmed death ligand 1 (PD-L1) expression, has not been well characterized. METHODS: On the basis of immunohistochemistry (IHC) results, PD-L1 expression on tumor cells (TCs) and tumor-infiltrating immune cells (ICs) was scored as follows: TC0 and IC0 were defined as PD-L1 expression less than 1%, TC1 and IC1 as at least 1% but less than 10%, TC2 and IC2 as 10% or more but less than 50%, and TC3 and IC3 as 50% or more. Phosphatase and tensin homolog (PTEN) IHC was scored as either lost or retained expression. The Ion AmpliSeq Comprehensive Cancer Panel (ThermoFisher Scientific, Waltham, MA) was used to identify mutations in all coding exons of 409 cancer-related genes. RESULTS: A total of 192 patients with large cell neuroendocrine carcinoma (LCNEC) (n = 72) and SCLC (n = 120) were studied. The prevalence of PD-L1 expression on TCs was 15.1% (29 of 192). IC infiltration and PD-L1 expression on ICs were observed in 34.4% of patients (66 of 192) and 31.3% of patients (60 of 192), respectively. The prevalence of IC infiltration and PD-L1 expression on IC were more strongly correlated with LCNEC than with SCLC (57.6% versus 23.3%, p < 0.01; 45.8% versus 22.5%, p < 0.01) and high nonsynonymous mutations (p = 0.05 and .04). PTEN loss was found in 9.5% of patients (18 of 189) and showed no correlation with PD-L1 expression. Progression-free survival was better in patients with IC infiltration than in those without IC infiltration (median 11.3 versus 6.8 months [p < 0.01]) and in patients with PD-L1 expression of IC1/2/3 than in those with expression of IC0 (median 11.3 versus 7.0 months [p = 0.03]). CONCLUSION: These findings suggest that the PD-1/PD-L1 pathway is activated in the microenvironment of pulmonary high-grade neuroendocrine carcinoma and correlated with a higher mutation burden.

HEpD: a database describing epigenetic differences between Thoroughbred and Jeju horses.

With the advent of next-generation sequencing technology, genome-wide maps of DNA methylation are now available. The Thoroughbred horse is bred for racing, while the Jeju horse is a traditional Korean horse bred for racing or food. The methylation profiles of equine organs may provide genomic clues underlying their athletic traits. We have developed a database to elucidate genome-wide DNA methylation patterns of the cerebrum, lung, heart, and skeletal muscle from Thoroughbred and Jeju horses. Using MeDIP-Seq, our database provides information regarding significantly enriched methylated regions beyond a threshold, methylation density of a specific region, and differentially methylated regions (DMRs) for tissues from two equine breeds. It provided methylation patterns at 784 gene regions in the equine genome. This database can potentially help researchers identify DMRs in the tissues of these horse species and investigate the differences between the Thoroughbred and Jeju horse breeds.

Genome-wide analysis of DNA methylation before-and after exercise in the thoroughbred horse with MeDIP-Seq.

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.

Genome-wide analysis of DNA methylation patterns in horse.

BACKGROUND: DNA methylation is an epigenetic regulatory mechanism that plays an essential role in mediating biological processes and determining phenotypic plasticity in organisms. Although the horse reference genome and whole transcriptome data are publically available the global DNA methylation data are yet to be known. RESULTS: We report the first genome-wide DNA methylation characteristics data from skeletal muscle, heart, lung, and cerebrum tissues of thoroughbred (TH) and Jeju (JH) horses, an indigenous Korea breed, respectively by methyl-DNA immunoprecipitation sequencing. The analysis of the DNA methylation patterns indicated that the average methylation density was the lowest in the promoter region, while the density in the coding DNA sequence region was the highest. Among repeat elements, a relatively high density of methylation was observed in long interspersed nuclear elements compared to short interspersed nuclear elements or long terminal repeat elements. We also successfully identified differential methylated regions through a comparative analysis of corresponding tissues from TH and JH, indicating that the gene body regions showed a high methylation density. CONCLUSIONS: We provide report the first DNA methylation landscape and differentially methylated genomic regions (DMRs) of thoroughbred and Jeju horses, providing comprehensive DMRs maps of the DNA methylome. These data are invaluable resource to better understanding of epigenetics in the horse providing information for the further biological function analyses.

Quantitative analysis of transcript variants of CHM gene containing LTR12C element in humans.

Choroideremia (CHM) is essential for the posttranslational activation of retina-specific Rab protein. Transcript variants (a and b) of the CHM gene were detected in human cancer cells and tissues. Sequence analysis of the both variants found that isoform b is caused by an LTR12C element offering an alternative splicing site within the CHM gene. We performed real-time RT-PCR analysis to study expression levels of the CHM transcript variants in tumor and adjacent normal tissue samples. Our results showed that CHM isoform b was highly expressed in tumor tissues but its expression levels were relatively low in those of adjacent normal tissues including colon, testis, and lung. In addition, high expression levels of CHM isoform b were detected in the cancer cell lines of colon and lung, and colon cancer patient tissues. Thus, we suggest that expression levels of alternative transcripts of the CHM gene could be used as a molecular marker system to identify human cancers.