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Single-Cell Sequencing Pinpoints Genetic Mechanisms Underlying Brain Lesions

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Cerebral cavernous malformations (CCMs) are a blood vessel abnormality that can ultimately lead to brain hemorrhages. CCMs are often found in the proximity of developmental venous anomalies (DVAs), irregular arrangements of the brain vasculature that are usually harmless. In a new paper, Duke University Professor Douglas Marchuk and colleagues examined CCM samples taken from three patients using single-cell sequencing. They identified that mutations in the genes MAP3K3 and PIK3CA, commonly linked to CCMs, co-occurred in the same cells.


Their analysis revealed a course of disease progression for CCMs. They showed that PIK3CA mutations are the first to occur in the benign DVA. Progression to CCMs, the team found, only occur when an additional MAP3K3 mutation happens in the DVA. The researchers also identified circulating mRNAs related to the pathway in patients’ plasma, suggesting that tracking of these mutations in DVAs could one day become a viable clinical approach for predicting CCM risk.

We spoke to Marchuk to find out more about CCMs and the advantages and potential of single-cell sequencing.


Ruairi Mackenzie (RM): What are the challenges of assessing genetic mechanisms of disease with bulk sequencing?

Douglas Marchuk (DM): Bulk sequencing is great for identifying mutations that contribute to disease, but when you have a complex mosaic lesion with multiple mutations that are interacting in unknown ways, it becomes important to have sequence information for single cells so you can determine which mutations occur in which cells, as well as the temporal organization of those mutations.

RM: Do your findings suggest that sporadic and familial CCMs operate via distinct genetic mechanisms?

DM: Yes, though there is some nuance. Familial and sporadic CCMs appear to develop from the same mutations, but the order in which those mutations occur seems to be different. We showed that sporadic CCMs may result from an initial mutation in PIK3CA leading to a DVA and a subsequent mutation in one of several genes to form the CCM. Conversely, familial CCMs are not known to be associated with DVA and therefore the order of mutations is likely the reverse of what we see in sporadic CCMs (though this is still speculative at this time).

Molly Campbell (MC): The study utilized samples from three patients. Can you discuss how these findings could be translated, and whether you plan to conduct further studies using larger cohorts?

DM: This study was very limited in sample size because DVA are generally not removed surgically (they themselves are benign). We were fortunate to have a few cases where it was both safe and practical to sample from the DVA, however it will be challenging to scale this up to a larger cohort. However, what we can do is non-invasively assay blood to look for biomarkers of PIK3CA activation which may help address this question in a larger (though less direct) study.

MC: How can the information garnered from the single-cell analysis be utilized to guide diagnostics or personalized treatment approaches?

DM: At this time, our use of single-cell sequencing is to answer fundamental mechanistic questions about CCM pathogenesis and at this time does not have direct utility for CCM diagnostics. However, single-cell sequencing has helped us learn about CCM pathology in a way that is clinically useful (e.g., it may be appropriate to monitor known DVA for CCM formation in the future).

MC: In your opinion, what role do you see single-cell DNA sequencing playing in the future of medicine?

DM: I think it will (and partially has already) revolutionize medical research similar to the impact of next-generation sequencing compared to traditional Sanger sequencing.

MC: Do you plan on applying single-cell DNA sequencing in the study of any other neurological diseases?

DM: Yes, we have ongoing studies applying single-cell sequencing to brain arteriovenous malformations present in individuals with hereditary hemorrhagic telangiectasia. 


Dr. Douglas Marchuk was speaking to Ruairi J Mackenzie and Molly Campbell, Senior Science Writers for Technology Networks.