We propose using mitochondria as a model system to evaluate methods for finding human disease genes and to understand the complexity of the genetics that underlies the group of mitochondrial disorders. We will pilot this approach on Leber's Hereditary Optic Neuropathy (LHON), which is characterized by the selective degeneration of retinal ganglion cells leading to optic nerve atrophy and blindness. We propose two specific aims.
Aim 1 (Capture and sequencing the human exome by hybridization capture and long padlock probes, LPPs) will combine two DNA sequence capture technologies and next-generation sequencing platforms for the targeted resequencing of human exons at high accuracy and completeness. This involves optimizing capture and hybridization protocols and developing molecular barcode tagging for multiplexed sequencing of exome libraries from multiple individuals as well as extending LPP-based capture to all 1,200 nuclear- encoded mitochondrial genes. LPPs will enable capture of exons that are missed by the exome library (e.g. hard to target sequences) and exons with insuficient read coverage. To assess capturing eficiency and accuracy of the basecalling algorithms, such as to estimate and control the false discovery rate (FDR) for novel variants, we will utilize sample sequences with known genotype content.
Aim 2 (Characterization of LHON candidate genes by functional complementation, silencing and subcellular localization studies) will determine the contribution of the identified candidate genes, which is performed in two ways: i) by transduction of a patient cell line harboring the putative causative allele with a wild-type cDNA to complement the candidate gene defect, and ii.) by transduction of a wild-type control cell line with a lentiviral vector to produce stable RNAi-mediated silencing of a candidate gene. The assayed molecular phenotypes include mitochondrial respiration and redox balance, while subcellular localization of candidate genes is performed to identify new mitochondrial proteins and expand proteome annotations. The candidate genes for these studies are prioritized through bioinformatics filters based on DNA variant function including variant effects on coding regions with a primary focus on detecting rare (<1% MAF) loss-of-function variants. The identified candidate genes are mapped to mitochondrial networks of genes and diseases to gain insight into specific pathways and the LHON neurodegenerative process with implications for clinically similar disorders.
Mitochondrial dysfunction has been recognized in the last decade to play a primary role in several common diseases, including diabetes, aging, cancer, and neurodegeneration. Most mitochondria- related diseases are caused by defects in nuclear-encoded genes. The identification of new mitochondrial disease genes with our cost-effective and high quality clinical resequencing approach has important implications to better understand the role of mitochondria in human health.
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