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ID 117206
Title Alternative
バイサルファイト変換とARMS PCRを用いた膵β細胞傷害の新規定量法
Detecting pancreatic β-cell cfDNA
Author
Okada, Asami Tokushima University
Yamada-Yamashita, Misuzu Tokushima University
Tominaga, Yukari Tokushima University
Jo, Kyoka Tokushima University
Suzuki, Reiko Tokushima University
Akehi, Yuko Tokushima University
Takashi, Yuichi Tokushima University
Koga, Daisuke Otsuka Pharmaceutical Co., Ltd.
Shimokita, Eisuke Tokushima University
Mitsui, Yukari Tokushima University
Masuda, Shiho Tokushima University
Ferreri, Kevin City of Hope
Fujitani, Yoshio Gunma University
Keywords
cell free DNA
DNA methylation
Quantitative RT-PCR
amplification-refractry mutation system PCR
Type 1 diabetes
Content Type
Thesis or Dissertation
Description
Aims/Introduction: Several research groups have reported methods for quantifying pancreatic beta cell (β-cell) injury by measuring β-cell-specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next-generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment.
Materials and Methods: We established a novel method for detecting four CpG unmethylations of the insulin gene using two-step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults.
Results: The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose β-cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA-positive type 1 diabetes.
Conclusions: We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of β-cells in human disease such as type 1 diabetes.
Journal Title
Journal of Diabetes Investigation
ISSN
20401124
NCID
AA12488319
Publisher
Asian Association for the Study of Diabetes|Wiley
Volume
13
Issue
7
Start Page
1140
End Page
1148
Published Date
2022-04-09
Remark
内容要旨・審査要旨・論文本文の公開
本論文は,著者Asami Okadaの学位論文として提出され,学位審査・授与の対象となっている。
Rights
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/ ), which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
FullText File
language
eng
TextVersion
ETD
MEXT report number
甲第3641号
Diploma Number
甲医第1535号
Granted Date
2022-06-23
Degree Name
Doctor of Medical Science
Grantor
Tokushima University
departments
Institute of Advanced Medical Sciences
Medical Sciences
Bioscience and Bioindustry
University Hospital