講演情報
[P9-4]患者血液由来全長cDNAを用いた新規オンデマンド遺伝子検査法の確立とその評価
○研 澄仁1,2, 浦 大樹1,2, 新井田 要1,2 (1.金沢医科大学 総合医学研究所 先端医療研究領域 ゲノム疾患研究分野, 2.金沢医科大学病院 ゲノム医療センター)
We have developed a new genetic test method (combined long amplicon sequencing, CoLAS) that can detect splicing mutations and intragenic structural abnormalities, by combined targeted full-length mRNA (cDNA) and genomic DNA (including deep intron) sequencing using patient-derived peripheral mononuclear cells, by long-range PCR based next generation sequencing.
To evaluate this new method, we have tested 48 genes by CoLAS, and identified 25 splicing mutations in 15 genes, and intragenic large deletion in 2 genes. Especially, 7 of 25 aberrant splicing events were caused by deep intronic mutations. Furthermore, we demonstrated that CoLAS can be applied to the phase test by analysing full-length cDNA.
On the other hand, it has been well known that a limited number of genes are expressed in blood, which makes difficult to analyse mRNA in genetic test using blood. In this study, we confirmed that 44 of the 48 genes could be analyzed by CoLAS, including the genes with extremely low expression level (transcripts per million reads, TPM ＜0.1) in peripheral mononuclear cells. Another 4 genes were not expressed at all with 0 TPM, and failed to amplify the full-length cDNA. This result indicated that the most of disease-related genes will be covered by CoLAS in genetic test using peripheral blood.
We concluded CoLAS offers an inexpensive, faster and useful on-demand genetic testing that can detect wider mutation spectrum compared to conventional panel test.
To evaluate this new method, we have tested 48 genes by CoLAS, and identified 25 splicing mutations in 15 genes, and intragenic large deletion in 2 genes. Especially, 7 of 25 aberrant splicing events were caused by deep intronic mutations. Furthermore, we demonstrated that CoLAS can be applied to the phase test by analysing full-length cDNA.
On the other hand, it has been well known that a limited number of genes are expressed in blood, which makes difficult to analyse mRNA in genetic test using blood. In this study, we confirmed that 44 of the 48 genes could be analyzed by CoLAS, including the genes with extremely low expression level (transcripts per million reads, TPM ＜0.1) in peripheral mononuclear cells. Another 4 genes were not expressed at all with 0 TPM, and failed to amplify the full-length cDNA. This result indicated that the most of disease-related genes will be covered by CoLAS in genetic test using peripheral blood.
We concluded CoLAS offers an inexpensive, faster and useful on-demand genetic testing that can detect wider mutation spectrum compared to conventional panel test.