Long-read sequencing across the DM2 repeat expansion reveals unique insight of repeat expansion structure

2022 Fiscal Year Final Research Report

• Project/Area Number: 20K07740
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 51030:Pathophysiologic neuroscience-related
• Research Institution: Jichi Medical University
• Principal Investigator: Matsuura Tohru 自治医科大学, 医学部, 教授 (90402560)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: 筋強直性ジストロフィー2型 (DM2) / 非翻訳領域リピート病 / 次世代シーケンサー / ハプロタイプ解析 / 伸長リピート内シーケンス / DNA メチル化

Outline of Final Research Achievements

The aim of this study is to characterize the origin and features of expanded CCTG repeats in Japanese DM2 patients. Over the last 15 years, we have identified 7 DM2 patients from 5 unrelated families in Japan. We used Nanopore long-read sequencing (LRS) to investigate the following measures: 1) the phase of SNPs flanking the repeat, 2) repeat length of the expanded repeat, 3) sequence of the repeat tract, and 4) adjacent DNA modifications. LRS revealed that Japanese DM2 patients share a common haplotype, which differs from European DM2 patients. Simultaneous detection of the CNBP repeat region revealed that each read has different number of repeats, suggesting somatic mosaicism of the expanded repeat allele. The sequence within the repeat showed interruptions of the CAGA repeat. We also detected the CpG modifications in the native DNA molecules, which were not substantially altered in normal and expanded alleles.

Free Research Field

神経遺伝学、神経内科学

Academic Significance and Societal Importance of the Research Achievements

ナノポア・ロングリードシーケンサーを用いて、従来解析困難であった非翻訳領域リピート伸長の一つであるDM2 CCTGリピート伸長変異を解析した。ナノポア・ロングリードシーケンシングは、単一実験でリピート長・リピート内部配列・ハプロタイプ解析・CpGメチル化の同時解析が可能である。本研究は、同手法が伸長リピート変異解析に極めて有用なツールであることを実証した。