Clinical Validation of Digital PCR for Precise m.3243A>G Heteroplasmy Quantification in Early‐Onset Diabetes

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 (10 4 copies) for sensitivity, specificity, dynamic range (10–10 5 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 10 5 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 10 4 . 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.