• Genomics & Informatics
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• v.17 no.4
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• pp.42.1-42.6
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• 2019

Robust identification of genetic alterations is important for the diagnosis and subsequent treatment of tumors. Screening for genetic alterations using tumor tissue samples may lead to biased interpretations because of the heterogeneous nature of the tumor mass. Liquid biopsy has been suggested as an attractive tool for the non-invasive follow-up of cancer treatment outcomes. In this study, we aimed to verify whether the mutations identified in primary tumor tissue samples could be consistently detected in plasma cell-free DNA (cfDNA) by digital polymerase chain reaction (dPCR). We first examined the genetic alteration profiles of three colorectal cancer (CRC) tissue samples by targeted next-generation sequencing (NGS) and identified 11 non-silent amino acid changes across six cancer-related genes (APC, KRAS, TP53, TERT, ARIDIA, and BRCA1). All three samples had KRAS mutations (G12V, G12C, and G13D), which were well-known driver events. Therefore, we examined the KRAS mutations by dPCR. When we examined the three KRAS mutations by dPCR using tumor tissue samples, all of them were consistently detected and the variant allele frequencies (VAFs) of the mutations were almost identical between targeted NGS and dPCR. When we examined the KRAS mutations using the plasma cfDNA of the three CRC patients by dPCR, all three mutations were consistently identified. However, the VAFs were lower (range, 0.166% to 2.638%) than those obtained using the CRC tissue samples. In conclusion, we confirmed that the KRAS mutations identified from CRC tumor tissue samples were consistently detected in the plasma cfDNA of the three CRC patients by dPCR.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.8
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• pp.1084-1089
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• 2015

Chemerin, highly expressed in adipose and liver tissues, regulates glucose and lipid metabolism and immunity in these tissues in ruminants and mice. Our previous reports showed that chemerin is involved in adipogenesis and lipid metabolism in adipose tissue as an adipokine. The aim of the present study was to identify single nucleotide polymorphisms (SNPs) in the coding region of the chemerin gene and to analyze their effects on carcass traits and intramuscular fatty acid compositions in Japanese Black cattle. The SNPs in the bovine chemerin gene were detected in 232 Japanese Black steers (n = 161) and heifers (n = 71) using DNA sequencing. The results revealed five novel silent mutations: NM_001046020: c.12A>G (4aa), c.165GT (92aa), c.321 A>G (107aa), and c.396C>T (132aa). There was no association between 4 of the SNPs (c.12A>G [4aa], c.165GG [107aa], and c.396C>T) and carcass traits or intramuscular fatty acid compositions. Regarding the remaining SNP, c.276C>T, we found that cattle with genotype CC had a higher beef marbling score than that of cattle with genotype CT, whereas cattle with genotype CT had a higher body condition score (p<0.10). Further, cattle with genotype CC had significantly higher C18:0 content in their intramuscular fat tissue than that of cattle with genotype CT (p<0.05). On the other hand, cattle with genotype CT had significantly higher C14:0 and C16:0 content in their intramuscular fat tissue (p<0.05). Moreover, the number of individuals carrying the minor allele of c.276C>T SNP is small. It is suggested that the c.276C>T SNP of the chemerin gene has potential in cattle breeding using modern methods, such as marker assisted selection. So, further functional and physiological research elucidating the impact of the chemerin gene on bovine lipid metabolism including fatty acid synthesis will help in understanding these results.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.1
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• pp.25-31
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• 2010

Objective: X inactivation is the silencing one of the two X chromosomes in female mammals for gene dosage on the X-chromosome between female and male. X inactivation is controlled by X inactive-specific transcript (XIST) gene, untranslated RNA. XIST is expressed only from the inactive X (Xi), not expressed from the active X (Xa). The Xist promoter is methylated on the silent Xist allele on the Xa in somatic cells, and less methylated on the Xist-expressing Xi. We investigated the difference of XIST methylation pattern of the promoter and 5'-region of XIST from male (XY) and female (XX) subjects. Methods: The direct quantification of XIST methylation is required for clinical application of normal XX and XY blood. Methylation percentage of eight CpG sites (-1696, -1679, -1475, -1473, -1469, +947, +956, +971) of XIST gene were diagnosed by pyrosequencing. Results: We directly quantitated the methylation percentage of the promoter and 5'-end of XIST by pyrosequencing. The average methylation percentages at CpG6-8 sites (+947, +956, +971) were 45.2% at CpG6, 49.9% at CpG7, and 44.2% at CpG8 from normal female and normal male were 90.6%, 96.7%, 87.8%, respectively. Nether CpG 1-5sites (-1696, -1679, -1475, -1473, -1469) had any effect on XX and XY. Conclusion: This method is sensitive for quantifying the small percentage change in the methylation status of XIST, and may be used for diagnosis.