The application of droplet digital PCR technology to measure heteroplasmy levels of the mitochondrial DNA mutation m.3243A>G associated with maternally inherited diabetes and deafness

Abstract

The pathogenic mitochondrial DNA mutation m.3243A>G causes two syndromes; maternally inherited diabetes and deafness (MIDD) and Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS). There is considerable clinical variation in these syndromes, and although clinically distinct patients can have overlapping features. m.3243A>G is highly heteroplasmic and mutation load varies significantly between different tissues and between individuals. Heteroplasmy levels have been shown to positively correlate with disease burden in MELAS, and very low levels of heteroplasmy (1%) have been considered diagnostic. However the effect of low level heteroplasmy on diagnostic test sensitivity is unknown and the association between heteroplasmy and clinical traits has rarely been studied in patients with MIDD. We developed a quantitative droplet digital PCR assay to measure m.3243A>G heteroplasmy to 0.01% in blood. We then tested 190 patients from suspected MIDD families previously tested by a TaqMan genotyping assay capable of detecting ≥5% heteroplasmy. The aim was to determine if cases had been missed by TaqMan due to low heteroplasmy, and to look for an association between heteroplasmy and clinical features in MIDD patients. We confirmed all previous positive TaqMan results and did not identify any additional low heteroplasmy cases in the negative patients. The mean heteroplasmy level was 24.9% ±13.9%. All positive patients had heteroplasmy >2% and all negative patients G. There may be a role for ddPCR in a research setting, although recent reconfigurations to NHS genomic testing services will likely result in the replacement of most single variant genotyping assays with next generation sequencing over time.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	William Newman (Supervisor) & Cynthia Morton (Supervisor)