DETECTION AND CHARACTERIZATION OF MITOCHONDRIAL-DNA REARRANGEMENTS INPEARSON AND KEARNS-SAYRE SYNDROMES BY LONG PCR

We used a strategy based on long PCR (polymerase chain reaction) for d etection and characterization of mitochondrial DNA (mtDNA) rearrangeme nts in two patients with clinical signs suggesting Pearson syndrome an d Kearns-Sayre syndrome (KSS), respectively, and one patient with myop athic symptoms of unidentified origin. Mitochondrial DNA rearrangement s were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantit ative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a co mmon 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues t ested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that w as present in the form of deletions as well as duplications. In the pa tient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearr anged mtDNA populations in skeletal muscle, a previously described 7-k b deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be t he cause of the myopathic symptoms as they were present at low levels (10-40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA r earrangements in three patients.