And in a final flourish, one of my favourite analyses, using the DOGMA output, Sonia was able to establish that the heteroplasmy threshold for a transcriptional phenotype of our example rRNA hotspot was somewhere ~20-30%.

Using the powerful DOGMA-seq method from Leif Ludwig (no Bsky) and @caleblareau.bsky.social, we were able to tease apart heteroplasmic dosage of m.1227G>A on cellular transcriptional phenotype, showing a clear effect on mitochondrial transcripts, but also (gasp) non-mitochondrial ribosomes...

This was coupled to a clearly perturbed mitoribosome assembly/translational profile, again from a low heteroplasmy (38%). If you're not seeing it immediately don't sweat - it took @mitogene.bsky.social ~5 years to teach me how to interpret these things

Interestingly, even after all that screening we couldn't get the bulk heteroplasmy of this recurrent mutation, m.1227G>A, above ~40%. And even more interesting, we saw massive effects of this mutation on metabolism, mitochondrial function and protein levels starting from heteroplasmy of ~10%.

Employing fantastic mitochondrial base editing tools developed in David Liu's lab @broadinstitute.org institute.org, we engineered in vitro models to bear recurrent rRNA mutations, which we then profiled extensively to see what was happening.

However, the heteroplasmy of these genetically recurrent and structurally clustered rRNA mutations was pretty low - rarely rising over 30% in the bulk sequencing from tumor genomes (probably an underestimate, given how much of a tumor isn’t cancer cells, but still rather low).

With beautiful structures from @amunts.bsky.social nts.bsky.social lab as our roadmap, we were able to pin the most recurrent of these onto the structure - where they also appear to cluster in two regions where they are likely to impact ribosome function - but maybe not totally catastrophically.

We were intrigued by this, as recurrence suggests positive selection pressure. Going deeper, we found that these hotspot mutations were often in positions that do not tolerate variation in the germline, and were in regions likely to impact RNA folding (WC basepairs).

In this new superpowered dataset we found again that there are quite a lot of single nucleotide variant mutations in mitochondrial rRNA of tumors, but these are not randomly distributed - they fall into clearly recurrent hotspots, affecting 4% of all tumors.

Back in the mists of time (2021) we noticed that a decent proportion of cancers (~10%) had mutations in the ribosomal RNA genes required for mitochondrial ribosome assembly and function. But we weren't powered to say much more about it then.

www.nature.com/articles/s42...

Of mtDNA mutations found in cancer, there is some overlap with those in mito disease, but the majority are unique to cancer. In the past we tried to predict how damaging these mutations are, and while it seems higher pathogenicity is tolerated in cancer, ultimately this is a limited approach.

mtDNA mutations in the germline cause primary mitochondrial disease, a group of rare metabolic disorders. Because mtDNA is present in 100's-1000's of copies per cell, an amount of these mtDNA molecules have to bear the mutated allele before symptoms arise; this is known as the Threshold Effect.