1. When did man get his somatic mutations in mitochondrial DNA? Konstantin Khrapko Harvard Medical School, Boston, USA

2. The fraction of many somatic mtDNA mutations increases as we age Age (years) % of homoplasmic point mutations

3. Since the fraction of mutations increases as we age, the natural answer to our question: “ When does man get his mtDNA mutations?” Seems to be simply: We get them as we age. This may or may not be true.

4. Each cell contains hundreds or thousands of mtDNA subject to turnover with a half-life of about a few weeks What does it mean “ to get ” (a mtDNA mutation) in a population of mtDNA molecules?

5. In a dynamic population of mtDNA molecules the fraction of mutant molecules can conceivably be increased by: 1. Clonal expansion of pre-existing mutant molecules a. preferential replication, b. slower demise (or both) or 2. de novo mutation of a non-mutant molecule a. inaccurate replication, b. sequence rearrangement, … “to get a mutation” should, of course, mean “to acquire a de novo mutation”.

6. The re-formulated title of the talk is: At what developmental stage are most mutant mtDNA molecules (or their ancestors) acquired de novo? two antithetic answers: Late in the development. acute de novo mutations account for the increasing mutant fraction (makes sense: high oxidative stress should ensure high de novo mutagenesis) Early in development. expansion of early de novo mutations accounts for the increasing mutant fraction (also makes sense: there is enough time for the mutants to expand if early enough, mutants can be widely seeded and then expanded throughout the tissue) Which answer (or both) is correct?

7. Single-cell level : “When did cell get its mtDNA mutations?” mtDNA mutations are clonally expanded within cells.

8. WT mtDNA 16.6 kb  mtDNA 6.3 kb 6.1 kb 16.4 kb Long-distance PCR Detection of mtDNA with large deletions in single cells by long-distance PCR ori primers Single human cardiomyocyte Single human neuron Tissue before dissection Tissue after dissection primers

9. 16.4 kb, wt mtDNA 8 kb 4 kb Long-distance DNA amplification from single myocytes reveals cells bearing clonally expanded mitochondrial genomes with deletions Cells containing deleted mtDNAA typical normal cell PCR from a normal cell (overloaded) PCR from:

10. Replicate PCR, sequencing Single-cell analysis of point mutations 251 G > C PCR, Sequencing Cells collected individually DNA isolation,

11. Since mtDNA mutations are clonal in cells, the ancestor mutants should have been created some time ago (expansion takes time depending on expansion rate). Many clonal expansions are incomplete (<100%), so the rate of expansion is probably not too fast. (otherwise mutations would instantly progress through intermediate stages and we would not observe partially mutant cells) But the estimate of the rate is too uncertain to infer the age of the ancestors of intracellular mutant clones. What about tissue level?

12. Mutational spectra: a signature of developmental history? Early mutations Tissue growth Late mutations Tissue growth Mutations are redundant. (One ancestral mutation gives rise to a large proportion of all mutants) Mutations are non-redundant. (There are many different mutations it’s unlikely they will repeat themselves.) CellmtDNA Mutant mtDNA

13. 10% in tissue (Fayet et al. 2002 Neuromusc. Disord. 12:484-493) Total: 14 people 69+y.o., 109(+)&109(-) fibers COX(-)<0.5% Single-fiber analysis of human skeletal muscle Essentially all mutations are redundant. Are they all early mutations?

14. More examples of redundant mutations: Human brain. Single molecules. delG(66-71): 19/100; 189A>G: 10/100 (Jazin (1996) PNAS 98:12382-12387) Rat liver. Single cells 16007G>A: 4/33 (Khaidakov (2003) Mutation Research 526:1) Human substantia nigra. Single molecules. Del(6320-14956): 4/18 (unpublished) Human fibroblasts 414T>G Michikawa (1999) Science 286:774 Human muscle 189A>G, 408A>T Calloway (2000) AmJHG 66:1384, Wang (2001) PNAS 98:4022, Del Bo (2002) J.Neurol.Sci 202:85

15. Problem with redundancy interpretation: Redundancy of a mutation can also be caused by high mutational rate (mutational “hotspot”). This is particularly plausible with commonly abundant mutations like 189A>G. ( Non-common mutations are unlikely candidates for hotspots.)

16. Multiple mutations: “linkage analysis” in mtDNA. Are mtDNA mutations linked (i.e. located on the came DNA molecule)? Early mutations Tissue growth Mutations redundant, unlinked (or all linked) Late hotspot mutations Tissue growth Mutations redundant, some are linked

17. Single cells Single molecules Cell#8, Linkage analysis in mtDNA (contd.) mtDNA of a person with two proximal mtDNA mutations (one of them 189A>G) shows no linkage between the mutations in individual cells, nor in single molecules from a mixed mutant cell: Tentative conclusion: apparently, both mutations were introduced on separate DNA molecules in early development. 185G/189A 185A/189A 185A/189G There is no 185G/185G

18. Alternative explanations : 1. “Repulsion” between mutations (a molecule with both mutations is selected against). 2. Different types of mtDNA mutations are generated and or selected on different nuclear backgrounds.

19. What does it have to do with oxidative stress and aging? 1. Oxidative stress is considered a mitochondrial mutagen. It may be modulating mutational rate by affecting mitochondrial turnover rate rather than by direct mutation. 2. A similar idea of early origin of nuclear DNA mutations has been proposed recently (Frank (2003) Nature 422:494) 3. Pertinent to the HIDL (High Initial Damage Load) hypothesis. (L.Gavrilov, Monday at 14:45) 4. Implications to measuring mtDNA mutations. Example: the use of common deletion as a measure of the total load of deletions maybe misleading.

20. Conclusions : 1. It seems plausible that a significant fraction of mtDNA mutations originate in the early development. 2. The hypothesis successfully accounts for redundancy and “linkage disequilibrium” of mtDNA mutations. 3. The conclusion is preliminary though as alternative explanations are possible and more data are needed. 4. Single cell/molecule analysis offer powerful tools in studying of the various aspects of mtDNA behavior.

21. Acknowledgements: Katya Nekhaeva Genya Kraytsberg Wolfram Kunz (University Bonn) Aubrey de Grey