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The BRCA1 Gene: Trouble, Quantified

Add this to the (increasingly long) list of papers whose basic research plans would once have gotten a net dropped over your head. It’s looking at variations in the BRCA1 gene, the one that is famously associated with breast cancer risk. There is no doubt at all that there are mutations in this gene that raise such risk – but at the same time, there are a lot of mutations out there. And it’s well known that all genes carry mutations if you go around carefully sequencing large numbers of individuals – it’s just that most of these mutations appear to be silent. So which are the silent mutations in BRCA1 and which are the alarming ones? There are plenty of single-nucleotide variations seen in it, and unfortunately, most of them are (1) rare by themselves and (2) of unknown risk.

BRCA1 (the gene, italicized) codes for BRCA1, a protein (un-italicized) that’s crucial in homology-directed DNA repair (the most common route of which is homologous recombination). This new paper uses a cell line that’s particularly susceptible to impairment of that pathway as a readout for how the mutated genes/proteins would function. Using the CRISPR system, everyone’s gene-editing friend, they targeted 13 of the gene’s 22 coding exons for study. These are the ones involved in its RING and BRCT domains, and some of those are already known to be hot spots, particularly exons 11-13. All the known single-nucleotide variations that are associated with increased cancer risk are in these regions. These exons were taken through saturation genome editing (SGE), which is just what it says: you make every change in every nucleotide base, and see what happens. They took it one exon at a time, and assayed the cells for how badly their growth (or survival) was hit in each variant.

That’s the aspect that (not so many years ago) would have caused that net to come down on you, because that represents a lot of mutations (3,893 of them, about 96% of all the possible ones in the targeted regions). Overall, about 73% of the variations were functional, 21% were nonfunctional, and 6% were intermediate. Looking more closely, the intermediate ones were enriched in missense mutations. Mutations that disrupted the known splice sites in the sequence gave proteins that were about 90% nonfunctional, as you’d imagine, while mutations deep in the introns or in the 5′ untranslated regions were almost never nonfunctional.

The correlation of what they found with the clinical picture for such mutations (where we know it) is very good indeed. There are 169 single-nucleotide variations in these regions that have been annotated in clinical practice as “deleterious”, and only 2 of these scored in this work as “functional”. And there are 22 variants that have been determined as clinically benign – of those, 20 scored as functional, one as intermediate, and one as nonfunctional. So the correlation is not perfect, but this assay is clearly on to something. (In fact, on closer inspection, in three cases where the two data sets completely disagree there’s reason to think that the clinical database is mistaken). And given the rate at which new variants are piling up (as more patients get sequenced), this sort of thing is probably the only way to keep even with the data. Modern sequencing methods will swamp us otherwise!

The next step, clearly, is to extend this approach to more of the BRCA1 sequence, and to try applying it to other targets. The first one should be pretty straightforward, but the second will depend on careful choice of the cell assay. Extending this to other cancer targets that are strongly affected through defects homologous DNA repair would be the most likely thing to try, you’d think, since the current cell line worked so well.

7 comments on “The BRCA1 Gene: Trouble, Quantified”

  1. Imaging guy says:

    I think the next step should be correcting the mutations in vivo, first in mice and then in human. I think CRISPR would be like siRNA, good at knocking down proteins in cell lines but not effective in vivo. 20 years after its discovery and Nobel prize, we have one FDA approved siRNA (against transthyretin gene in liver called Onpattro).

    1. Chem2Bio says:

      The knockdown in vivo is still limited by delivery. It is very hard to get the editing machinery in and especially so in a uniform way. If you miss a couple cancer cell it will be a problem. This kind of thing can be modeled, but usually involves cell that were engineered prior to be added to the mouse or engineered mice, neither of which is what therapeutic gene editing for cancer looks like.

      For some of the rare disease where you are replacing or fixing defective/missing proteins, I think this is a lot more straight forward. You don’t have to hit every cell in most cases and even moderate boosts in correct protein make a big difference one a tissue/organism level.

  2. Nesprin says:

    What a shame they did this beautiful work in a leukemia cell line instead of non-malignant breast or ovarian cells. The latter are where you expect BRCA1 to be the most crucial.

  3. Anonymous says:

    Just to be clear about the paper (paywall; I’ve read the abstract and Derek’s summary, above): BRCA1 mutations are associated with only ~3-8% of all (diagnosed) breast cancers. “73% functional” means that those variants (relative to ???) retained their proper function as tumor suppressors and did not cause and were not associated with cancerous growth. Basically, ‘not all mutations are created equal’: many variants are benign and only 21% or so lead to cancerous growth. Before rushing off to get a radical treatment, you need to know WHICH mutation you have, not just that it is a mutation.

    In light of Derek’s recent topics on “Dark Side of Investments,” “Moral Imperative,” and so on, I throw in some history of BRCA1. Back in 1994, Myriad Genetics (wikipedia link in my name) isolated and then patented the BRCA1 gene. There were, as yet, no treatments, but that gave MG the sole right to sell assays for BRCA1 mutations. They did so at a very high price. When the 1995 patent filing was announced, MG’s NIH collaborators found out that they had been left off the patent. (Their inclusion would have no doubt diverted some of the profits to the government which had funded much of the BRCA1 research.) When the NIH threatened to file its own competing patent (the US was still “first to invent” in 1995), Myriad had a change of $, I mean heart, and did a deal with the Feds. There was a Moral Imperative to find a gene and help people; the MI to make some money; the MI to try to screw collaborators out of their share of the credit and profits; and so on.

    (Myriad was established out of the U of Utah in Salt Lake City where they had access to the extensive family histories of the Mormon community. It made searching lineages and linkage maps much easier and faster than searching the general population.)

  4. gippgig says:

    Has anyone looked for BRCA1 mutations that decrease the chance of breast cancer?

    1. loupgarous says:

      I guess that’d be possible and ought to be investigated, but I’m not sure how it’d work. Would such a mutation give a “super BRCA1” protein which would be even more efficient at repairing DNA than the normal one is?

      That seems like a worthwhile thing to aim for (CRISPR therapy-wise) as a method of repairing damaged cells in breast cancer patients, but two things make me nervous:
      – what are the unknown consequences of more efficient BRCA1 activity – would it up or down-regulate another cellular action deleteriously?
      – are we sure CRISPR to create that “super BRCA1” protein wouldn’t do something horrible to off-target DNA sequences?

  5. Dr Hfuhruhurr says:

    This is beautiful work, but it is important to note that the large scale editing was possible because the study was done in the rather special Hap1 cell line, which is largely haploid . To Nesprin’s comments above, gene editing on all alleles in a diploid or greater cell line (i.e. most cancer cell lines) would be difficult to implement at this scale. I suspect that most of these mutations would not read out when covered by wild-type BRCA1. One could consider performing this study in an engineered cell background where all but one of the copies of BRCA1 were removed by CRISPR, but again this would be challenging to implement.

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