Project Abstract |
Mitochondria are the principle site of oxidative energy metabolism in eukaryotic cells. Mitchondrial respiratory chain deficiencies in humans result in a wide range of clinical presentations. Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). However, mtDNA has been shown to accumulate 10 to 15 times more mutations compared to the nuclear genome. Since each cell contains hundreds of mitochondria and each mitochondrion may contain multiple copies of the genome, these mutations are frequently heteroplasmic with both mutated and normal mtDNA within the same cell. The heteroplasmy level, or percentage of mtDNA that is mutated, can differ between individuals as well as between tissues within an individual; and has been shown to correlate to the severity of symptoms. The Surveyor Nuclease has been used to identify mutations in the mitochondrial genome. The proband in our study, experienced a relatively mild progressive encephalopathy and was found to have a novel 15952C>T mutation in a highly conserved stem region of tRNAThr gene. Variable degrees of heteroplasmy were found in all patient tissues tested and a high percentage of mutant mtDNA was observed in muscle. Mutations found in the tRNA encoding regions of mtDNA have been linked to mitochondrial dysfunction, and bases in the stem regions of the tRNA molecules are more tightly constrained against substitutions. To determine if the proband 15952C>T mtDNA adversely affected mitochondrial enzyme activities, lymphoblastoid cells from the proband were chemically enucleated and fused to mtDNA-deficient 143B(TK-) ρo cells. Three independent patient transmitochondrial cybrids were generated with different mutant loads. These cells are currently being tested at the biochemical level to demonstrate a mitochondrial enzyme defect due to the mtDNA mutation using spectrophotometry and polarography. A correlation between the mutant load and severity of the biochemical defect will demonstrate the pathological nature of the novel mtDNA mutation. |
