Leber's hereditary optic neuropathy (LHON) is a blinding disorder recognized as the most frequent mitochondrial disease. LHON is associated with mtDNA point mutations in complex I subunit genes, but remains characterized by poorly understood features such as tissue specificity, incomplete penetrance and male prevalence. Environmental factors (tobacco and alcohol) and specific mtDNA backgrounds (haplogroup J) are confirmed to play a role in modulating penetrance. We have also demonstrated that mitochondrial biogenesis and mtDNA copy number are major determinants on penetrance and gender difference in LHON. We are currently pursuing a multilayered “tour de force” to identify genetic modifiers by combining traditional tools, such as linkage analysis, with high-throughput techniques, such as MitoExome sequencing, functional and tag SNPs genotyping and microarray expression studies. Our results indicate that penetrance in LHON is modulated by polymorphisms in different genes rather than by a single mutation. Co-variates (age, sex, smoke and mtDNA copy number) have been instrumental to generating a list of candidate genes for which validation is currently ongoing.