Somatic mutation and neurological traits. Part II. Relevant Somatic Mutation discovered?

The first installment of this brief series discussed some possible manifestations of somatic mutation in helping to account for the biology and epidemiology of epilepsies.  From a cellular point of view, epilepsies seem to be functionally close to gene action, but mostly have eluded gene mapping in families, GWAS, or mouse models.  Yesterday, I referred to a paper that I published in 2005 which mused about the possibility that some epilepsies and other behavioral traits might be due to genetic effects that arose through somatic mutation rather than being inherited in the parents' germline, and I tried to suggest reasons why that idea seems plausible  But finding direct evidence for relevant somatic mutation in relevant neural (brain) tissue, just a subset of cells, would have been technically very challenging.  That was then, however, and technology marches on.

A new paper by Wei et al. in the February 2016 issue of Cell ("Long Neural Genes Harbor Recurrent DNA Break Clusters in Neural Stem/Progenitor Cells") may suggest that we're getting to a point where it might be possible to address my speculation directly--and perhaps go far beyond that to understand epilepsies much better but, even more, to address variation in normal behavior and abnormal psychological function in general.

Wei et  al. report using a clever, sophisticated, if very technical method to make a systematic search for a class of somatic mutations in neural cells.  The experiments are done in mouse cell culture and, basically, Wei et al looked genomewide for chromosomal changes or rearrangements that arise out of double-strand chromosomal breaks.  The detail is far beyond what I could competently describe here, and would be out of place in a blog.  However, the upshot of their work is that the authors expanded on what had been known, that there are very large mutational rearrangements detectable in repeatable locations in the genomes of early neural precursor cells.

As shown in the figure below, the rearranged locations are found genomewide, and on most chromosomes. The changes varied among cells tested.  I am not qualified either to defend or critique the method itself, but have no reason to doubt the findings, and if anything it must be fair to assert that because the method only finds one sort of mutation, the finding implies that other methods would find expanded evidence for somatic mutation of various sorts in these cells, as are clearly known to happen in other cell types. The authors used methods that have passed muster before, they cite ample other literature on somatic rearrangements generally.  Experiments like this are artificial in various ways, and theirs doesn't prove that the mutations they detected do in fact have phenotypic effects in vivo, but the case for that is certainly and strongly plausible.

Neural and other genes undergoing SoMu in (mouse) brain. Circle numbers are chromosomes  From Wei et al., Cell, 2016

The importance of this finding, and what justifies its being published in Cell in my view, is that it shows not just tolerance of somatic mutation, but perhaps even active genomic modification in the early mouse brain. Moreover, the authors find that a major fraction if perhaps even preponderance, of the genes altered in this process, whatever other roles they may serve, are involved in neural cell adhesion and synapse formation and/or function.  One can be properly skeptical about assigning function to genes (gene ontologies are often quite shaky and genes typical serve many functions), but even if there is a sort of after-the-fact bias in interpretation, the involvement in the modified genes in synapse function seems well established.

For a partial summary of the approach, the original authors as well as a short, cogent and somewhat more digestible accompanying commentary by Weissman and Gage ("A Mechanism for Somatic Brain Mosaicism"), explain the potential this may have for the determination of individuality. This is speculation in detail, naturally, but the proto-neurons develop into relevant areas of the adult brain, and the authors note that the affected cells are, or reasonably may be, involved in a variety of higher functions.  They mention learning and mental functions such as autism, bipolar depressive disorders, schizophrenia, and intellectual disability.  They also speculate that the somatic changes could be relevant to various forms of cancers of brain cells.  And, perhaps an artifact of the way science is done these days, I saw no mention of the obvious likely fact, that if these changes can be involved in disease they can also be involved in any other psychological traits.

The authors also did not mention epilepsies, nor whether genes known or suspected of being involved in some epilepsies were detected in any of these rearranged regions. I'm not any sort of expert on that, but on a very cursory check, I did not spot any specific known epilepsy related genes in the above figure, but one can hardly take that as in any way definitive. Since epilepsy is itself relatively rare, only rare somatic mutational changes need be involved, for epilepsy to be an occasional consequence and to help account for the variation in the types and parts of the brain affected, the seriousness, age of onset, and so on.  We'll see.

Phenotype amplification
In the previous post, I referred to to means of phenotypic amplification, a process by which mutations in a single or small subset of cells could reach organismal (whole-person) detectability or effect.  One means would be if a somatic mutation happens early enough in development for a large enough set of descendant cells to be affected, almost as if the mutation were inherited in the germ line.  Given the n numbers of at-risk embryonic cells and the number of people produced each year, and the surely large number of genes that could have an effect on epilepsy (or any other psychological trait), this simply must occur!  Depending on when in development the somatic mutation occurred, its descendant set could be general and bilateral, or could be restricted to a small part of the brain on just one side, depending on the way these tissues differentiate during development.  That is, the types of epilepsy that are focal, affecting only a restricted, identifiable part of the brain, might be susceptible to genomic analysis to identify the mutations (illustrated in the figure in yesterday's post).  That is, the rest of the person's neural cells would not be mutated.

The other method of phenotype amplification I mentioned yesterday could occur when one or just a few mutant, misfiring neurons misfires, and that then induces a cascade of firing in the many other neurons to which it synapses.  This kind of phenotype amplification might not be easily detectable, because the single or few mutant culpable neurons might be invisible in sequences of the entire affected brain region, because most of the misfiring cells would, themselves, be genetically normal. That is, their misbehavior would be induced by the abnormal neuron(s) to which they were synapsed.

The variable and usually highly restricted, focal nature of epilepsies (i.e., suggested in yesterday's figure) cries out for explanation that involves the unique features of these small areas.  The methods in the Cell paper are very complex, technical, and certainly nothing I ever did in my lab, so I must acknowledge that my thoughts could be off the speculative mark.  Still, the paper and others it cites show that the ideas have more than totally circumstantial support.  There are lots of mutations in neural cells, as indeed, there are in any set of somatic cells.  They affect the individual in which they occur but are not transmitted in the germ line and hence not discoverable by GWAS and family studies.

No matter what one may think of the idea, a point worth making is that there is now at least one major study systematically documenting the pervasive frequency of somatic mutation of particular types (rearrangements),  that is active, common, and genomewide in developing neural precursor cells (and the authors cite other relevant results).  How or even whether a role of somatic mutation in epilepsy can be shown in real mice, much less humans, and not just cell culture or sequencing of the brains of deceased patients, is of course an open question.

However, unlike 2005 when I wrote my speculations, high throughput and even single-cell sequencing are now at least beginning to be practicable in mouse models and deceased humans who had epilepsies of known focality and expression.  If the ideas are cogent, investigators may find them compelling subjects to study.

Searching for somatic mutational causes of traits that seem to have specific tissue locations, seems now at least more possible, inroads might be possible and could, in principle at least, lead to substantial advances in understanding the mechanisms involved, as well as the epidemiology and phenotypes of epilepsy.

Somatic mutation and neurological traits. Part I. Background and Epilepsy

Epilepsies are disorders of cascades of uncontrolled neural firings.  Local or large parts of the brain can be involved in seizures, and the affected person's functioning is seriously impaired until the wave or storm of 'undisciplined' synapse firing has passed....until the next episode.  Epilepsy can be triggered by different stimuli in different people, and can affect specific parts of the brain, either quite localized or on just one side, or more general and bilateral. Epilepsies also  have variable onset triggers and ages in different people.

Waves of unconstrained neural firing seem to be related to signal transfers between connections between neurons (synapses) in the brain.  Since signal molecules are basically genetic (that is, not dietary or due to exogenous chemicals), one would expect epilepsy to be basically 'genetic' in its basic causal physiology.  Indeed, some epilepsies have long been known to result from mutations in single genes, occur in Mendelian fashion in families, involve neural signaling molecules, and some of these genes are known to cause epileptic symptoms when mutated in laboratory mice.  But most epilepsies are sporadic, not obviously familial, and have resisted attempts to map the responsible genes.

This was the general picture in around 2004, when I was asked to give a presentation to a meeting at NIH, of people working on epilepsy, to discuss what I saw as the general genetic causal landscape at the time.  This was early in the age of GWAS and other genomewide approaches.

I don't remember the details, but in my presentation I gave a general picture and issues in genetic mapping, and relevant evolutionary genetics.  But I also wanted to make a contribution beyond just a review, and I had thought about the genetics, physiology, and epidemiological patterns of the epilepsies.  I suggested what I thought would be relevant, based on previous work I had done over the years on the genetics of cancer and aging.  Basically, I hypothesized that many epilepsies might, like many cancers, be due not to inherited mutations but to somatic mutations--that is, mutations in relevant genes that occurred in neural cells during embryonic development or later in life, rather than being inherited from parents.

I found considerable interest in this idea, which I thought (and still think) could be a positive, potentially innovative way to understand the biology and epidemiology of epilepsies.  Some interest was shown, and a leading epilepsy neurobiologist offered to work on a paper about this idea and how one might test for it.  But for whatever reason, he lost interest before any paper could be published.  Partly perhaps, that was because of the difficulty of testing the idea, which would have required identifying the specific misfiring neurons in epilepsy victims and, when they died, sequencing those areas looking for mutations not found in neighboring normal neurons.

After my potential collaborator had more important things to do (or, probably, more important people to please) and dropped the idea of doing a collaborative review paper, I published my ideas in Trends in Genetics in 2005 (paywalled; if you're interested, email me for a pdf).  With some helpful discussion with a then-junior colleague, Dan Burgess (now working at Roche, I believe), we thought about ways in which, depending on embryonic or postnatal age, somatic mutation in neural cells or their precursors, could generate epileptic effects.  The idea is shown in this figure, drawn in collaboration with Dan, and taken from the paper.

Possible Epileptogenic somatic mutation scenarios.  From Weiss, TiGs, 2005; drawn with Dan Burgess

There are at least two main ways this might happen.  First, a somatic mutation arising early in embryonic development in a cell that was the precursor of neurons, could set up that descendant lineage  or clade of cells (and their respective part of the brain) to be vulnerable to uncontrolled firing, that is, to epilepsy.

Alternatively, a single later-occurring mutation might make the affected neuron too ready to fire under some conditions, and that could entrain firing in the often thousands of other neurons with which it synapses, and their neurons in turn  In a sense, such an episode could 'burn in' the synaptic connection in this set of neurons, all genetically normal except for the mutant triggering cell, making the set vulnerable to later episodes triggered by the original offending cell.  The location in the brain of the 'parent' aberrant cell(s) and its or their synaptic network would determine the side and part(s) of the brain that were affected by the seizures.

For somatic mutation to have detectable effect at the organismal level, there must be some sort of phenotype amplification, such that even a single or few aberrantly firing cells, that might on their own not be particularly noticeable would entrain enough other cells to cause a seizure. Cancers are now extensively studied for somatic mutation, because of their exuberant growth and histological characteristics.  One transformed cell leads to a large number of descendant cells--a tumor.

As noted above, aberrantly behaved neurons even if not individually detectable could, in principle at least, entrain so many other otherwise normal cells in a synaptic network, as to amplify the effect to make it noticeable to the person as a whole and to clinicians.  Thus in a variant of the precedent of cancer, one mutant transformed neural cell early in the embryo may be unnoticeable in itself, but after millions of cell divisions it can lead to a clone of misfiring cells.

With this sort of thing in mind, one can naturally also ask about a potential role for somatic mutation, arising in developing neural cells after fertilization in the embryo's later life, in other aspects of brain function--even including traits like intelligence, behavior, learning ability, memory, and personality, or other pathologies such as schizophrenia?  Behaviors and especially behavior genetics are highly contentious areas.  Some would like these traits and abilities to be due entirely to environments, while others are fervid in their belief that you are what you inherited in your genome--what you are is inborn.  That was the view, of course, of the eugenics movement and it's still around today in the belief system of many.

We've posted on this general topic before.  The argument for inherency has always been assumed to be about germ-line genotypes, that is, inheritance from parent to offspring. But could similar effects arise by somatic mutation--or, rather, if they can be inherited how could they not in some instances be due to somatic mutation?

Testing for somatic mutation in regard to brain function, even specific traits like epilepsy, has been prohibitively difficult, because specific somatic mutations would need to be identified systematically in specific brain areas or subsets of neurons.  Detecting and characterizing somatic mutation in traits like epilepsy will be challenging and may not quickly lead to therapy, but it could at least illuminate etiology and mechanisms.

At least, the idea that I laid out (and others may have as well, though not that I know of), hasn't been tested.  But that may be changing, as we'll discuss tomorrow.

[Since first being posted, this has been edited, twice, for clarity and to correct inapt phrasing]

Causal genes hiding in the "p"-patch!

We've posted many times about the problems we face today in dealing with multifactorial causation. In metaphoric terms, we wand to find causes that satisfy a statistical criteron of 'significance', by using some test, often some probability, p, of unusualness of the result that points to causation, that we can symbolically refer to as a p-value.

This applies to human genetics and the fashionable 'omics' approach, and to much else in biology.  One thing we talked about before and recently is the hypothesis that rare variants cause human trait variation in the sense of the difference between cases and controls. Some investigators have been arguing that rare variants with strong effect, rather than common variants, account for a substantial fraction of disease (combinations of variants, some of them rare, each with small effects, is another version of the rare-variant arguments).

But rare variants present a problem, which is that you don't see them often enough for statistical significance to be achieved. Yet they may be causal.  We recently noted that finding the same rare variant in affected family members is one possible way to identify them where significance is less of an overwhelming requirement.  Our last couple of posts deal with this subject.

Two back-to-back papers in the August 10 American Journal of Human Genetics are of interest here, because of what they confirm about this problem.  These are two reports from David Goldstein's lab, both large-scale searches for genetic causation, one of idiopathic generalized epilepsy and the other of schizophrenia (both open access).  Goldstein has argued for some time that genomewide association studies (GWAS) aren't finding genes with large effects because most complex diseases are caused by rare variants, with small effects. They don't reach significance, though they're real causes (one thinks): we're caught in the p-patch!

Idiopathic generalized epilepsy
Idiopathic generalized epilepsy (IGE) is a complex disease that, like many such diseases, is highly heritable but its genetic architecture has been difficult to parse ('Idiopathic' means cause not known).  According to the paper, rare copy number variants have been found to explain the disorder in only 3% of affected individuals.  So Goldstein's purpose was to test whether rare variants with moderate effect could be found to explain IGE.

The group compared the exomes -- all the exons, DNA coding regions --  of 118 people with IGE with those of 242 controls, and found no variants significantly associated with the disorder.  They then looked at almost 4000 variants that they considered to be candidates for epilepsy susceptibility and genotyped 878 cases and 1830 controls for these variants, with no statistically significant finding.

They report that close to 1/2 of these variants were only in cases, which suggested to them that at least some of these must be genetic risk factors.  However, the high heterogeneity of epilepsy disorders means that any single variant will be difficult to find, and/or that single-nucleotide variants have small effects.  E.g., they estimate that the variant they observed most frequently here accounts for 0.6% of the cases of IGE in this study, if it is indeed turns out to be causal, and this is the ballpark figure for causal variants they've identified for other complex diseases.  And, a recent study of epilepsy published in Cell by a group at Baylor compared cases to controls looking at all exons, and found potentially pathologic variants statistically as often in controls as in cases.

The current paper concludes that "moderately rare variants with intermediate effects ("goldilocks alleles") do not play a major role in the risk of IGE."  Current methods are not adequate for detecting variants with very small effects, even when they exist. The epilepsies are considered to be channelopathies, disorders in which an ion channel disruption plays a major part.  Thus, it has been assumed that mutations in ion channel genes would be found to be causal, but the list of candidate genes identified by these authors is not enriched for such genes, suggesting that "the pathophysiology governing epilepsy might be far more complex than simply a disorder of disrupted ion channels..."

Finally, the authors conclude that results from small studies must be treated with caution as they can't provide comprehensive lists of candidate variants.  But, studies large enough to detect variants that are at a frequency of, say, 0.06%, as some of the variants in this study, are essentially impossible.  Such variants, they say, "will probably only be securely implicated through gene-based association analyses in large sample sizes and, where available, cosegregation analyses within multiplex families."

Schizophrenia
Schizophrenia is another complex trait with high heritability, high phenotypic heterogeneity, and a low success rate with respect to identifying genetic risk factors.  As with most traits, GWAS have identified some genes with very low effect, but not always replicably.  Again, the question is whether the causal variants are moderately rare but identifiable in large studies, or so heterogeneous and rare as to remain hidden with current large-population based methods. 

In the study reported in the AJHG, Goldstein's group followed the same 2-step analysis as described above for IGE, ultimately assessing selected variants in 2,617 cases and 1800 controls.  No single variant was statistically significant, though, again, they identified case-specific variants, some of which may actually be causal.  They conclude that risk of schizophrenia is unlikely to be due to moderately rare variants with moderate effect, and that "multiple rarer genetic variants must contribute substantially to the predisposition to schizophrenia, suggesting that both very large sample sizes and gene-based association tests will be required for securely identifying genetic risk factors."

In essence, this is either polygenic control in which each case is due to some combination of large numbers of individually weak, mainly rare, contributing variants, or that individual strong-variants exist but are so rare that we may struggle to get enough samples.  Follow-up or family studies that find many different variants in the same gene, and where the gene's function seems plausible for the trait, could help.  But it could be that there aren't enough humans on earth to achieve significance in the statistical sense....and that in important ways means the variant or gene isn't 'significant' in the public health or clinical setting either: approaches to aggregate causation may be needed. A way to escape from the p-patch.  We think so, at least, as we've said many times here before.