Clinical and analytical validation of an 82-gene comprehensive genome-profiling panel for identifying and interpreting variants responsible for inherited retinal dystrophies.

Inherited retinal dystrophies comprise a clinically complex and heterogenous group of diseases characterized by visual impairment due to pathogenic variants of over 300 different genes. Accurately identifying the causative gene and associated variant is crucial for the definitive diagnosis and subsequent selection of precise treatments. Consequently, well-validated genetic tests are required in the clinical practice. Here, we report the analytical and clinical validation of a next-generation sequencing targeted gene panel, the PrismGuide IRD Panel System. This system enables comprehensive genome profiling of 82 genes related to inherited retinal dystrophies. The PrismGuide IRD Panel System demonstrated 100% (n = 43) concordance with Sanger sequencing in detecting single-nucleotide variants, small insertions, and small deletions in the target genes and also in assessing their zygosity. It also identified copy-number loss in four out of five cases. When assessing precision, we evaluated the reproducibility of variant detection with 2,160 variants in 144 replicates and found 100% agreement in terms of single-nucleotide variants (n = 1,584) and small insertions and deletions (n = 576). Furthermore, the PrismGuide IRD Panel System generated sufficient read depth for variant calls across the purine-rich and highly repetitive open-reading frame 15 region of RPGR and detected all five variants tested. These results show that the PrismGuide IRD Panel System can accurately and consistently detect single-nucleotide variants and small insertions and deletions. Thus, the PrismGuide IRD Panel System could serve as useful tool that is applicable in clinical practice for identifying the causative genes based on the detection and interpretation of variants in patients with inherited retinal dystrophies and can contribute to a precise molecular diagnosis and targeted treatments.

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death.

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 µM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.