AGE-RELATED HUMAN MTDNA DELETIONS - A HETEROGENEOUS SET OF DELETIONS ARISING AT A SINGLE PAIR OF DIRECTLY REPEATED SEQUENCES

Deletions in mtDNA accumulate during the human aging process, arising from either intramolecular illegitimate recombination or strand slippa ge during replication, which results in subgenomic mtDNA molecules. We identify here two classes of mtDNA deletions-class A deletions, which are homogeneous at the breakpoints, with all subgenomic molecules the refore being identical in size, and class B deletions, which arise fro m a less stringent process that gives rise to heterogeneity at the bre akpoints, with the subgenomic molecules being of slightly different si zes. A novel approach is described that offers a global overview of th e populations of different deletions in individual tissues. It is base d on PCR cycle-sequencing reactions that are carried out directly on m tDNA segments, amplified by PCR from total cellular DNA. The results s how a clear size homogeneity of the subgenomic mtDNA molecules represe ntative of class A, which carry a commonly detected 4,977-bp deletion occurring at a pair of 13-bp directly repeated sequences. In this case , precisely one copy of the repeat is retained in the subgenomic molec ules. We then describe a class B situation comprising a family of at l east nine closely related 8.04-kb deletions involving the same pair of 5-bp direct repeats. In this situation, the breakpoints differ at the base-pair level (8,037-8,048-bp deletions); the subgenomic molecules retain >1 copy, 1 copy, or <1 copy of the 5-bp repeat. In different ti ssues from either the same individual or among different individuals, there is a widely variable occurrence of particular deletions in the s ubgenomic mtDNA population within this class B set. Class B deletions offer a new approach for studying the accumulation of mtDNA deletions, thereby providing insight into the independent somatic origin of muta ted mtDNA molecules, both in aging and in mitochondrial diseases. We a lso report a convenient method for ascertaining whether a given PCR pr oduct results from the amplification of a subgenomic mtDNA template, o n the basis of the selective degradation of full-length mtDNA molecule s prior to PCR.