ELSEVIER Biochimica et Biophysica Acta 1226 (1994) 49-55 Biochi f ic~a etBiophysica A~ta Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy Massoud Houshmand a, Nils-G6ran Larsson a,d,,, Elisabeth Holme a, Anders Oldfors b, Mfir H. Tulinius d Oluf Andersen c a Department of Clinical Chemistry, Gothenburg University, Sahlgren's Hospital, Gothenburg, Sweden, Department of Pathology, Gothenburg University, Sahlgren's Hospital, S-41345 Gothenburg, Sweden, c Department of Neurology, Gothenburg University, Sahlgren's Hospital, S-41345 Gothenburg, Sweden, d Department of Pediatrics, Gothenburg University, East Hospital, S-41685 Gothenburg, Sweden (Received 1 September 1993) Abstract We have investigated nine children with infantile onset of mitochondrial myopathy and two adults with myoclonus epilepsy and ragged-red fibers (MERRF) and chronic progressive external ophthalmopl.egia (CPEO), respectively. These patients lacked any of the previously known pathogenic tRNA mutations. Southern blot analysis of muscle mtDNA revealed no deletions. The tRNA genes of muscle mtDNA were sequenced. Restriction enzyme analysis of PCR fragments was performed to verify the presence of the mutations identified by automatic sequencing. Several tRNA mutations were found, but they were all homoplasmic. Furthermore, the mutations were either present in controls or did not change nucleotides conserved between species. This strongly suggests that none of the tRNA mutations identified in the 11 patients with mitochondrial encephalomy- opathy was pathogenic. It can thus be concluded that mitochondrial tRNA mutations and mtDNA deletions probably are an infrequent cause of mitochondrial disorders in infants. Patients with MERRF and CPEO may lack both pathogenic point mutations of tRNA genes and deletions of mtDNA. Key words: Mitochondria; mtDNA; tRNA; Myopathy; encephalomyopathy I. Introduction Human mtDNA contains the genes for 13 proteins (that all are subunits of the respiratory chain enzyme complexes), 22 tRNAs and 2 rRNAs [1]. The majority of the respiratory chain subunits and the proteins that are needed for replication and transcription of mtDNA and mitochondrial translation are encoded in the nu- cleus and imported into the mitochondria [1]. Muta- tions of mtDNA are a frequent cause of mitochondrial encephalomyopathies [2,3]. Single large deletions of mtDNA [4] are found in children with multisystem disorders [5,6] and in adults with chronic progressive ophthalmoplegia (CPEO) or Kearns-Sayre syndrome [7,8]. The deletions affect several protein and tRNA genes and there is always heteroplasmy with a mixture * Corresponding author. Fax: (+46) 31 82 76 10. 0925-4439/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0925-4439(93)E0136-Y of normal and deleted mtDNA. In vitro studies have demonstrated that deletions of mtDNA impair mito- chondrial protein synthesis, most likely due to lack of tRNAs encoded in the deleted region [9]. Several het- eroplasmic point mutations of tRNA genes are associ- ated with different mitochondrial encephalomyopathic syndromes, e.g., myoclonus epilepsy and ragged-red fibers (MERRF) [10-12], mitochondrial encephalomy- opathy, lactic acidosis and stroke-like episodes (MELAS) [13-15] and other syndromes [16-18]. Exper- imental studies have demonstrated that the point mu- tations at nucleotide (nt) 8344 of the tRNA TM gene and nt 3243 of the tRNA L~u(uuR) gene (associated with MERRF and MELAS, respectively) impair mitochon- drial protein synthesis [19,20]. Patients with single mtDNA deletions or tRNA point mutations often have biochemical and morphological evidence of respiratory chain dysfunction in muscle with reduced activity of complex I and IV and cytochrome c oxidase (COX) deficient and ragged red fibers (RRF) [7,8,21]. There is