|Budget Amount *help
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
1)In order to know the steady-state levels of RNA 19 in normal human tissues and the actual levels of it in MELAS patients, we analyzed the total RNA from various human tissues from 8 normal individuals, and biopsied muscles from 4 MELAS patients having MELAS-3243 point mutation. Normal tissues include skeletal muscle, brain, heart, kidney, liver, uterus, and spleen. We also analyzed the percentage of point mutation in RNA 19 fraction in RNAs from muscles, using rTth reverse transcriptase PCR-RFLP methods. We observed significantly increased levels of RNA 19 in muscles from all 4 MELAS patients (5 - 8 times more than that of the control) and from one patient having glycogen storage disease (3 times more than that of the control). The level of RNA 19 in the patient muscles ranged from 20 to 35% of total ND 1 signal is significantly higher than that of the control muscle (less than 4% of total ND I signal), which is well harmonized with the percentage of mutation analysed in their mitoch
ondrial DNA from muscles. The RNA 19 is also recognized in various human tissues including muscle, heart, brain, kidney, liver, spleen and uterus, ranging 3.5%, 11.6%, 13 6%, 8.4%, 4.2%, 1.7% and 6.5% of their total ND 1 signal, respectively. This accumulation of RNA 19 observed in MELAS-muscle seems to be specific for the tRNALeu(UUR) mutation and related to the pathogenetic mechanism of MELAS.
2)Five unrelated patients harboring the A3243G mutation in the mitochondrial DNA (mtDNA) but presenting with different clinical phenotype were studied for their percentage of mutation at the single muscle fiber levels. One patient had a clinically and pathologically defined Leigh syndrome (LS), two showed mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS), another showed progressive external ophthalmoplegia (PEO), and the other showed mitochondrial diabetes mellitus (MDM). The mutation load was greater in the muscle from the patient with LS (92%), who showed more than 80% even in the non-ragged red fibers (RRF) and also presented the highest proportion of RRF.The patients with MELAS had lower mutation levels as well as lower proportion of RRF, and these two parameters were even lower in the PEO and MDM patients. These results confirm the concept that differences in the mutation load and in the spatial distribution of the mutation among different cells and tissues are responsible for the differences in phenotypical expression of the disease. Less