Abstract
Purpose :
To determine the prevalence of mitochondrial DNA (mtDNA) mutations in a cohort of patients with primary open-angle glaucoma (POAG) using massively-parallel sequencing and correlate genotypic findings with mitochondrial functional assays performed on human Tenon fibroblasts (TFs) in a subset of the cohort.
Methods :
Caucasian patients with POAG (high tension glaucoma: HTG and normal tension glaucoma: NTG) and matched disease-negative controls were recruited following phenotyping from the Royal Liverpool Hospital, UK. DNA was extracted from peripheral blood leucocytes and cultured Tenon’s fibroblasts harvested at the time of glaucoma and/or cataract surgery. Long-range PCR was used to amplify the mtDNA in two overlapping fragments followed by Nextera library preparation and sequencing on the Illumina platform followed by bioinformatics analysis using Partek Genomics Suite. Analysis of the TFs included (a) 8-hydroxy-2’deoxyguanosine (8-OH dG) level using ELISA; (b) qPCR of mtDNA copy number; (c) citrate synthase activity and (d) mitochondrial physiology using a Seahorse XF24 analyser.
Results :
Lymphocyte DNA was collected from 96 HTG, 25 NTG and 96 controls. From this cohort cultured TFs were obtained from 15 of the HTG patients, 5 NTG patients and 15 controls. Massively-parallel sequencing on the Illumina platform and bioinformatics analysis using Partek Genomics Suite identified the mtDNA mutational load in this extensive cohort. Genotypic data was correlated with mitochondrial functions assays: (a) There was no significant difference in the levels of 8-OHdG in TFs between the three phenotypic groups (n=15): HTG= 28.9 (±13.3)ng, NTG =22.5 (±5.2) ng, and controls= 29.6 (±13.7) ng; (b) citrate synthase activity was higher in HTG TFs: 3.15 (±1.35)x10-2 nmole/min/ml (n=6) compared to controls: 2.47 (±1.22) x10-2 nmole/min/ml (n=6) although not significantly (p=0.3845); (c). there was no significant difference in mtDNA copy number in 15 HTG and 15 controls and (d) Seahorse mitochondrial physiological findings are reported.
Conclusions :
We describe an efficient method to perform massively- parallel sequencing of mtDNA in a larger cohort of POAG patients and controls. Combining genotypic data with functional correlates adds further insights and can identify potential therapeutic manipulations to treat glaucoma
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.