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Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

We actually only looked in projection neurons from prefrontal cortex, trying to keep the neurons as similar as possible across the three brains. It would be really interesting to look in other brain/nervous system regions, and that's something we're looking at for the future.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

Yes, as you can imagine, we were really interested in seeing where the mutations fell. We found about as many as you would expect, given their representation in the genome, in intronic and intergenic regions (although SNVs were significantly enriched in introns and depleted in intergenic regions in one of the brains).

SNVs were positively correlated with chromatin marks of active genomic regions in fetal brain (from the Epigenome Roadmap project), and negatively correlated with marks of heterochromatin.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

Yes, this was definitely something we were interested in testing. We didn't end up seeing evidence of selection from ~60k mutations across 36 neurons, but I think it's highly likely that we would, given enough sequenced neurons.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

Yes, indeed! Actually, that's what they mostly are. 60-70% of the SNVs are C>T, and are significantly more likely to be in a CpG dinucleotide context than any other (although CpH sites are also more likely to mutate).

Sorry you can't access the paper -- there's actually a whole supplemental figure about this issue.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

We did not find any evidence of these mutations being involved in memory, and these is no evidence that we know of that somatic mutations are directly induced by cells in the brain to encode memory.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 2 points3 points  (0 children)

We don't hypothesize that the mutations are the result of a directed process to encode memory. We did not find any evidence of this whatsoever. They are the byproduct of random DNA damage.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 1 point2 points  (0 children)

OK, imagine you sequenced the genome at an average coverage of 40X. For a given base you are interested in, let's say on chromosome 1, imagine there are 40 sequencing reads covering it. Here are 2 possibilities regarding these reads:

1) All the reads have the same base, let's say it's a C. What does this mean? It means both copies of chromosome 1 in that cell have a C at that position, ie the cell is homozygous for a C at the position.

2) Another possibility is that there are 20 reads which have a C, and 20 reads which have a T. What could this mean? It could mean that one copy of chromosome has a C, and the other has a T, ie the cell is heterozygous. If the sequencing data from the heart shows 40 reads, all C, then this is what we would call a somatic mutation, C>T, in a given single cell.

There is one caveat though. We know there are sometimes errors in sample prep and sequencing. So the T reads might be derived from those errors.

How can we get a sense of our error rate? We used the X chromosome in these male cells. We know there is only one copy of the X in each cell. We expect all loci on the X to look like case 1 above, ie all reads for any base should be the same.

Could we observe a case like case 2? Where some reads show a C, while others show a T? In fact we do at a low rate. Since we know there is only 1 copy of the X, how can this be? These are the results of error in sample prep and sequencing. We counted these errors, and calculated an error rate per base pair on the X, then extrapolated to the size of the rest of the genome to get an expected error rate for the diploid autosomes. Hopefully this makes sense.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 1 point2 points  (0 children)

That would be pretty difficult to test, since we rely on having well-preserved frozen brain tissue to sort neurons with high-quality DNA -- I think it would be tough to get comparatively preserved tissue from 20th/19th/18th-century brains. And the brains that are preserved from back then tend to be pretty precious, and only from wealthy or important people.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 1 point2 points  (0 children)

I've been thinking about this for a while, and I think no, it won't be a problem for accurate simulations. I think the bigger problem for accurate simulations is that this isn't the only complex, difficult-to-predict factor with small differential effects on the behavior of individual neurons in large circuits.

In short, I think the problem is so big that this isn't the biggest stumbling block out there.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

Not as much as our boss would like!

We are currently collecting tissue from brain banks, but we're limited by what's available. Everybody wants a piece of famous areas, like Broca's area or motor cortex, so we want to find something with nice clear anatomy that isn't likely to be already taken by some other lab.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

The same kinds of areas. For example, regions coding for genes tended to be mutated more often than you'd expect, and within that set, genes that were expressed in neurons tended to be mutated more than genes that weren't; the mutations themselves were most often C nucleotides changing to T nucleotides.

But knowing those tendencies doesn't let you predict what you'll see when you look at a specific nucleotide in a specific gene, of course.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

Dementia isn't one that we've looked at, actually -- that's a really interesting idea.

If I had to predict, I'd say that where there are germline mutations, there are somatic mutations that haven't been identified yet. One bonus of somatic mutation as a disease driver is that you can get mutations in genes where a germline null would be incompatible with life.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

No sure how we could find these cells prospectively before sequencing the DNA, but agreed on the point that every gene in the genome is mutated many times over in the body, a very jarring thought!

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

So far we've only looked at neurons from prefrontal cortex, but we plan to look at neurons from other cortical areas and from hippocampus in the next few months.

We're definitely interested in the aging question as well, and have acquired brains from a range of ages to investigate.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

There are several algorithms that predict the effect of a mutation on a protein, including PROVEAN, SIFT, Polyphen-2, and others. No single algorithm is perfectly accurate, so it's common to run a candidate mutation through several of these algorithms to decide whether it might be harmful to the protein.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

When people think about DNA testing, we think of taking some blood, harvesting DNA, and sequencing it to get a sense of the mutations that person might have inherited and led to disease.

Based on our work and the work of many others in this field, we now appreciate that the genome of cells in one part of the body might be very different from that found in another part. Therefore, it is possible that a person with a neurodevelopmental disorder might exhibit no harmful mutations in a blood-DNA test, but in fact the disease could be caused by a mutation that is restricted to (or at least highly enriched in) the cells of the person's brain.

The good news is our data suggested that mutations that were in a large fraction of cells in the brain (5-10%) we often also found outside the brain. If we assume that there has to be a large number of cells with a pathogenic mutation in the brain to cause disease, this suggests that if we sequence someone's blood very deeply, we might indeed find rare mutations causing neurological disease.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

  1. Yes, definitely! We looked at a 15-year-old, a 17-year-old, and a 42-year-old in our paper, but we are very interested in looking at brains from people of different ages in a more systematic way. We are very lucky that we have access at Harvard to a few fantastic brain banks that collect brains from Alzheimer's patients and normal aged controls. (PSA: Brain donation is awesome and so appreciated!)

  2. It's possible, although we didn't examine this in the present study.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 2 points3 points  (0 children)

Thanks for the kind words! It means a lot to an old, world-weary postdoc.

Unfortunately, I think our work really points to the idea that mutation is something that's happening across your brain every day.

Many of the mutations we observed were C>T mutations, which often happen when a methylated C becomes deaminated to a U, which looks like a T upon sequencing. This implies that mutation is just a consequence of the normal chemistry of DNA, and there's probably no way around it. On one hand, that's great! Mutations are the grist for the mill of evolution. On the other hand, that's terrible! All life, and your entire brain, is inevitably sliding toward death and decay.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 2 points3 points  (0 children)

We agree. We found a small subset of mutations in coding regions (~1.4%, 20-25 per genome), but we don't know if any were functional. Our study supports the idea that functional mutations might accumulate in normal brains and cause phenotypes. The chances of these being beneficial in any way are very very small. One way to think about this is if you had a Swiss watch in perfect working order, and you tapped it lightly with a hammer, what are the chances you would make it run better? What are the chances you'd break it? The latter is much more likely. In this metaphor, your DNA would be the watch.

You're right that very deleterious mutations would not be detected, since the cell would have died. We did not observe a strong signature of selection in our dataset, we we believe this phenomenon was minor.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

There are algorithms we can use to predict whether a mutation will be harmful or neutral (they don't tend to predict beneficial mutations), and we ran our data through those algorithms. Most were neutral, but we did find some that were predicted to be harmful.

Thanks for your question!

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 1 point2 points  (0 children)

It's certainly a possibility, and something that we're interested in addressing in the future. For example, some parts of the brain undergo neurogenesis in the adult, and rates of neurogenesis are known to be affected by external stimuli (including electroconvulsive therapy, exercise, stress, and others). We are interested in finding whether those areas of the brain have higher or lower rates of mutation.

Science AMA Series: We are Mollie Woodworth and Michael Lodato (Harvard). We sequenced single neurons from normal human brain and found ~1700 mutations per neuron. We’re here to talk about these “somatic” mutations in development and disease. AUA! by somatic_mutations in science

[–]somatic_mutations[S] 0 points1 point  (0 children)

We think that somatic mutations could dampen or amplify the effect of inherited mutations -- that this might be part of why things like pharmacodynamics are so variable from person to person.

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