Next-generation sequencing

Studies focusing on mitochondrial genomics are particularly important since mitochondria have their own copy of DNA (mtDNA)

Mitochondrial research is a major component of the BRC which has its own cross-cutting theme looking at mitochondria and Neuromuscular Disease and its impact in common age related diseases. Studies focusing on mitochondrial genomics are particularly important since mitochondria have their own copy of DNA (mtDNA). Somatic mtDNA mutations (i.e. mutations that are acquired and not inherited) accumulate over our lifespan, and are a feature of the ageing process.

Mitochondria provide energy for normal cellular processes and are more numerous in the cells of the body that have higher energy demands such as muscle, brain and heart, the same tissues affected by age related diseases. Each cell has multiple copies of mitochondria, which in turn have multiple copies of mtDNA. Current opinion speculates that a somatic mutation in a copy of mtDNA replicates, giving rise to a mixture of mutated and normal mtDNA in each cell, this is termed heteroplasmy. At a certain point the number of mutated copies tips the balance within the cell impeding its normal function. Alternatively it could be that there are multiple copies of mtDNA with varying mutations i.e. low-level heteroplasmy.

A recent study carried out by the mitochondrial research group based at the institute of genetic medicine used next-generation sequencing technology (NGS) to observe low level heteroplasmy at an unprecedented level of detail. The researchers designed an experiment to assess the long term effects of nucleoside analog anti-retroviral drugs (NRTIs) on the mtDNA mutation rate among HIV patients. NRTIs had been reported to have an inhibitor effect on the mtDNA polymerase, pol γ (a molecule involved in copying mtDNA), and patients taking the drug showed signs of prematurely ageing. It wasn’t known if this was occurring through irreversible damage to mtDNA.

Polymerase chain reaction (PCR) was used to select and artificially copy two different regions of mtDNA from muscle tissue i) HVS2, a region with many genetic variants, and ii) CO3 a region associated with minimal variants. Using NGS technology (GS-FLX Roche) the genetic code of these regions was read for a group of HIV patients who had taken NRTIs long term, never taken NRTIs, POLG patients who had an inherited defect in pol γ and healthy cases of the same age. The researchers were able to look at thousands of copies of each fragment in parallel at a sensitivity that allowed them to distinguish variants at the 0.2% level, giving a rare insight into low level heteroplasmy. The outcome of this work helped show that the premature ageing of NTRI treated HIV patients arose through the rapid clonal expansion of somatic mtDNA mutations.

The field remains unclear as to whether mutations in mtDNA are a cause or consequence of the ageing process, however innovation in genomic methods as described above are allowing us to overcome the technical challenges associated with observing the expansion of rare somatic mtDNA mutations, thus giving us an important tool to observe the human ageing process. Furthermore, this particular method has been adopted by another two studies assessing low level heteroplasmy in human ageing in the 12 months since publication.