Novel method utilizing bisulfite conversion with dual amplification-refractory mutation system polymerase chain reaction to detect circulating pancreatic ß-cell cfDNA.
J Diabetes Investig
; 13(7): 1140-1148, 2022 Jul.
Article
em En
| MEDLINE
| ID: mdl-35396829
ABSTRACT
AIMS/INTRODUCTION:
Several research groups have reported methods for quantifying pancreatic beta cell (ß-cell) injury by measuring ß-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 ANDMETHODS:
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 ß-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 ß-cells in human disease such as type 1 diabetes.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Diabetes Mellitus Tipo 1
/
Células Secretoras de Insulina
/
Ácidos Nucleicos Livres
Tipo de estudo:
Diagnostic_studies
Limite:
Adult
/
Humans
Idioma:
En
Revista:
J Diabetes Investig
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Japão