Journal of diabetes research · 2026
Clinical Validation of Digital PCR for Precise m.3243A>G Heteroplasmy Quantification in Early-Onset Diabetes.
СтатьяВалидационное исследованиеНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Kong Xiaomu, Bei Lei, Gao Peng, Lu Lixia, Zhu Haoyan, Jiang Yongwei, Zhao Meimei, Liu Yi, Wang Guozhen, Li Mo, Feng Ziyi, Deng Guoxiong, Cao Yongtong, Ma Liang
Аннотация
BACKGROUND: Mitochondrial DNA m.3243A>G variant causes maternally inherited diabetes with deafness. We developed a droplet digital PCR (dPCR) assay to quantify m.3243A>G heteroplasmy and investigated its prevalence in Chinese Han early-onset diabetes patients.
METHODS: The dPCR method was validated using plasmid mixtures (104 copies) for sensitivity, specificity, dynamic range (10-105 copies/μL), accuracy, and precision. We screened 1506 unselected early-onset diabetes patients, analyzing blood and urinary sediment DNA. This population comprised 1474 diagnosed type 2 diabetes patients (97.88%), 31 diagnosed type 1 diabetes patients (2.06%), and one patient with mitochondrial diabetes (0.07%). The dPCR results were compared with Sanger sequencing and next generation sequencing (NGS) to evaluate the accuracy of heteroplasmy rate quantification.
RESULTS: The dynamic range spans from 10 to 105 copies/μL. The assay detected heteroplasmy as low as 0.05% with 100% specificity. The method can reliably quantify the heteroplasmy rate at 0% to 100% for an input copy number of 104. Using the dPCR method, we identified 0.80% (n = 12) had >5% m.3243A>G heteroplasmy in peripheral blood and/or urinary sediment, and 0.40% (n = 6) had 1%-5% among the early-onset diabetes patients. The dPCR approach was more sensitive than Sanger sequencing. When compared with NGS results, the dPCR method accurately genotyped m.3243A>G and quantified its heteroplasmy rate in clinical samples. Furthermore, it demonstrated that urine is a reliable noninvasive sample source for determining the m.3243A>G heteroplasmy rate.
CONCLUSION: The developed method can provide an accurate, reliable, rapid, simple, and low-cost evaluation of the heteroplasmy rate of m.3243A>G for both peripheral blood and urinary sediment. Thus, it is highly promising as a genetic screening method for early-onset diabetes related to m.3243A>G.
METHODS: The dPCR method was validated using plasmid mixtures (104 copies) for sensitivity, specificity, dynamic range (10-105 copies/μL), accuracy, and precision. We screened 1506 unselected early-onset diabetes patients, analyzing blood and urinary sediment DNA. This population comprised 1474 diagnosed type 2 diabetes patients (97.88%), 31 diagnosed type 1 diabetes patients (2.06%), and one patient with mitochondrial diabetes (0.07%). The dPCR results were compared with Sanger sequencing and next generation sequencing (NGS) to evaluate the accuracy of heteroplasmy rate quantification.
RESULTS: The dynamic range spans from 10 to 105 copies/μL. The assay detected heteroplasmy as low as 0.05% with 100% specificity. The method can reliably quantify the heteroplasmy rate at 0% to 100% for an input copy number of 104. Using the dPCR method, we identified 0.80% (n = 12) had >5% m.3243A>G heteroplasmy in peripheral blood and/or urinary sediment, and 0.40% (n = 6) had 1%-5% among the early-onset diabetes patients. The dPCR approach was more sensitive than Sanger sequencing. When compared with NGS results, the dPCR method accurately genotyped m.3243A>G and quantified its heteroplasmy rate in clinical samples. Furthermore, it demonstrated that urine is a reliable noninvasive sample source for determining the m.3243A>G heteroplasmy rate.
CONCLUSION: The developed method can provide an accurate, reliable, rapid, simple, and low-cost evaluation of the heteroplasmy rate of m.3243A>G for both peripheral blood and urinary sediment. Thus, it is highly promising as a genetic screening method for early-onset diabetes related to m.3243A>G.