The detection and partial localisation of heteroplasmic mutations in the mitochondrial genome of patients with diabetic retinopathy

Diabetic retinopathy (DR) is a common complication of diabetes and a major cause of acquired blindness in adults. Mitochondria are cellular organelles involved in energy production which contain mitochondrial DNA (mtDNA). We previously showed that levels of circulating mtDNA were dysregulated in DR patients, and there was some evidence of mtDNA damage. In the current project, our aim was to confirm if mtDNA damage can be detected in DR patients. Total DNA was isolated from blood samples (n=59) from diabetes patients with and without DR. Regions of mtDNA were amplified as overlapping amplicons and analysed using mismatch-specific Surveyor Nuclease to detect heteroplasmic mtDNA mutations. An initial screen of the entire mtDNA genome of 6 DR patients detected a higher prevalence of mutations in amplicon P, covering nucleotides 14,443 to 1,066 and spanning the control region. Further analysis of 42 subjects showed the presence of putative mutations in amplicon P in 36% (14/39) DR subjects and in 10% (2/20) non-DR subjects. The prevalence of mutations in DR was not related to the severity of the disease. The detection of a high prevalence of putative mtDNA mutations within a specific region of the mitochondrial genome, supports the view that mtDNA damage contributes to DR. Future work will involve next generation sequencing to better characterize these mutations in terms of location, heteroplasmy and potential functional impact.