Thoughts on the latest schizophrenia genetics report

The news and social media were headlining a report last week that presented some genetic findings, and even aspects of a possible causal mechanism, related to schizophrenia.  As habitually skeptical readers of these daily stories, we wondered how substantial this claim is.

The report in question was a Nature paper by Sekar et al. that identifies variation in the very complex MHC genome region that, based on the authors' analysis, is statistically associated with schizophrenics relative to unaffected controls. These are variants in the number of copies of particular genes in the C4 'Complement' system.  The authors show that gene copy number is correlated with gene expression level and, in turn, with some changes in brain tissue that may be related to functional effects in schizophrenia patients.

Comparing genotypes and disease status, in ~30,000 cases and controls of European ancestry, in 40 cohorts from 22 countries, the authors find that genotypes with higher C4 gene copy numbers are more frequent in schizophrenics, and there is a quantitative relationship between copy number and expression level in postmortem-tested neural tissue.  The relevant potential mechanism involved may have to do with the pruning of synapses among neurons in the brain.

The authors estimate that the relative risk of the highest-copy number genotype is 1.27 times that of the lowest. The lowest risk genotype is rare in the population, comprising only about 7% of the sample population, meaning that almost everyone has a middling relative-risk genotype.  That is comparable, say, to most of us having middling height or blood pressure. But the net population absolute risk of schizophrenia is about 1%, so that the absolute risks associated with these various genotypes are small and not even very different from each other.  The careful work done by the authors has many different components that together consistently seem to show that these copy number differences do have real effects, even if the absolute risks are small.

How that effect or association arises is not clear, and the findings are certainly not the same as explaining schizophrenia as a C4 disease per se.  As the authors note, around 100 or so other chromosome locations have been associated with the disease in genome-wide mapping studies that have been done.  That means that if their results stand up to scrutiny, C4 variation is one component of what is basically a polygenic disorder.  The association for each C4 genotype category is the effect averaged over all other contributing causes in those people. The absolute risk in individuals with a given copy number is still very small, and may depend on other genetic or environmental factors.

Schizophrenia is not a single disorder and has a spectrum of onset age, sex, symptoms, and severity of occurrence.  Many authors have been warning against using a single term for this variety of traits. Whether that is relevant here or not remains to be seen, but at least as presented in the their paper, some of the current authors' results seem not to vary with age.  This study doesn't address whether there is a smallish subset of individuals in each C4 category who are at much higher risk than the average for the category.  However, the familial clustering of schizophrenia suggests this may be so, because family members share environments and also genomic backgrounds.  One might expect that C4 genotypes are interacting with, or if not, being supplemented by, many other risk factors.

Even if average risk is not very high in absolute terms, this paper received the attention it did because it may be the first providing a seemingly strong case for a potentially relevant cellular mechanism to study, even if the specific effect on risk turns out to be quite small.  It could provide a break in understanding the basic biology of schizophrenia, given the dearth of plausible mechanisms know so far.

Because the statistically riskier genotypes are found in a high percentage of Europeans, one would expect them to be found, if at varying frequencies, in other populations than Europeans. Whether their associated risks will be similar probably depends on how similarly the other risk factors are in other populations.  C4 copy number variation must be evolutionarily old because there is so much of it, clearly not purged by natural selection--another indicator of a weak effect, especially because onset is often in the reproductive years and would seem to be potentially 'visible' to natural selection. So why is the C4 variation so frequent?  Perhaps C4 provides some important neural function, and most variation causes little net harm, since schizophrenia is relatively rare at roughly 1% average risk.  Or, copy number changes must happen regularly in this general MHC genome region, and can't effectively be purged, but is generally harmless.  But there is another interesting aspect to this story.

The Complement system is within a large, cluster of genes generally involved in helping destroy invading pathogens that have been recognized.  It is part of what is called the 'innate' immune system. Innate here means it does not vary adaptively in response to foreign bodies, like bacterial or viruses, that get into the blood stream.  The adaptive immune system does that, and is highly variable for that reason; but once a foreigner is identified, the complement system takes part in destroying it.  So it is curious that it would be involved in neural degeneration, unless it is responding to some foreign substance in the brain, or is an autoimmune reaction. But if the latter, how did it become so common?  Or is the use of C4 genes in this neural context a pleiotropy--a 'borrowed' use of existing genes that arose for immunity-related functions but then came also to be used for a different function?  Or is neural synapse regulation a kind of 'immune' function that hasn't been thought of in that way?  Whatever it's doing, in modern society it contributes to problems about 1% of the time, for reasons for which this paper clearly will stimulate investigation.

Why does this system 'misfire' only about 1% of the time?  One possible answer is that the C4 activity prunes synapse connections away normally in a random kind of way, but occasionally, by chance, prunes too much, leading to schizophrenia.  The disease would in that sense be purely due to random bad luck, rather than interacting with other mechanisms or factors. The higher the copy number the more likely the bad luck but too weakly for selection to 'care'.  However, that reason for the disease seems unlikely, for several reasons.  First, mapping has identified about 100 or so genome regions statistically associate with schizophrenia risk, suggesting that the disease is not just bad luck. Secondly, schizophrenia is familial: close relatives seem to be at elevated risk, 10-fold in very close relatives and almost 50-fold in identical twins.  This should not happen if the pathogenetic process is purely random, even though since haplotypes are inherited in close family members there could be a slight correlation in risk.  Also, the authors cite several incidental facts that suggest that C4 plays some sort of systematic relevant functional role.  But thirdly, since the absolute risk is so small, about 1%, one has to assume that C4 is not acting alone, but is directly interacting with, or is complemented by (so to speak) many other factors to which the unlucky victims have been exposed.

Something to test?
This might be a good situation in which to test a variant of an approach that British epidemiologist George Davey Smith has suggested as 'Mendelian randomization'.  His idea is basically that, when there is a known candidate environmental risk factor and a known gene through which that environmental factor operates, one can compare people with a genetic variant exposed to an environmental risk factor to people with that genetic risk factor but not exposed to test whether the environmental factor really does affect risk.

Here, we could have a variant of that situation.  We have the candidate gene system first, and could sort individuals having, say, the highest 'risk' genotypes, compared to the lowest, and see if any environmental or other systematic genomic differences are found that differentiates the two groups.

Interesting lead but not 'the' cause
Investigating even weakly causal factors could lead the way to discovering major pathogenic mechanisms or genetic or environmental contributors not yet known that interact with the identified gene region. There will be a flood of follow-up studies, one can be sure, but hopefully they will largely be focused investigations rather than repeat performances of association studies.

Given the absolute risks, which are small for given individuals, there may or may not be any reason to think that intervening on the C4 system itself would be a viable strategy even if it could be done. This still seems to be a polygenic--many-factorial--set of diseases, for which some other preventive strategy would be needed.  Time will tell.

In any case, circumspection is in order.  Remember traits like Alzheimer's disease, for which apoE, presenilins, beta-amyloid, and tau-protein associations were found years--or is it decades?--ago and still mystify to a great extent.  Or the critical region of chromosome 21 in in Down syndrome that has, as far as we know, eluded intensive study for similarly long times. And there are many other similar stories related to what are essentially polygenic disorders with major environmental components.  This one is, at least, an interesting one.

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.

You are what you're infected with?

Rats infected with the parasite Toxoplasma gondii do crazy things.  They find the scent of cat urine sexy and attractive, they don't run from the actual beasts; they are more active in running wheels, which might indicate that the parasite induces increased activity which may more readily attract a cat's attention. When an infected rat is eaten by a cat, the T. gondii is passed on in the cat's feces to infect again.  T. gondii can only reproduce inside the cat.  Great survival strategy on the part of the parasite, this trick of making the rat no longer fear cats -- now that's really building a better mouse-trap! Did this strategy evolve by adaptive selection, or is it just something that happened?

Czech biologist, Jaroslav Flegr, thinks T. gondii infections do much the same to humans -- his story is told in the March 2012 Atlantic Monthly.  Toxoplasmosis, the infection caused by T. gondii, infects a significant segment of the world's population -- perhaps 20% of Americans, but 55% of French people are infected, probably because the French diet includes more rare or raw meat than the American diet.  The usual mode of transmission is from a member of the cat family to another warm-blooded animal via ingestion of feces from an infected cat, but raw or rare meat can be another source.  It can also be transmitted from mother to fetus, and can result in serious complications in an infected fetus, including stillbirth.  This is why pregnant women are told to avoid litter boxes.

Infection has long been supposed to cause mild flu-like symptoms in otherwise healthy individuals, but then it was assumed that the parasite lay dormant in cysts sequestered away inside brain cells.  People with weakened immunity were at greater risk, however; in the days before antiretroviral drugs for treating HIV, toxoplasmosis infections are thought to have caused much of the dementia in patients with end-stage AIDS.

But maybe the parasite actually does more damage than has been thought.

...if Flegr is right, the “latent” parasite may be quietly tweaking the connections between our neurons, changing our response to frightening situations, our trust in others, how outgoing we are, and even our preference for certain scents. And that’s not all. He also believes that the organism contributes to car crashes, suicides, and mental disorders such as schizophrenia. When you add up all the different ways it can harm us, says Flegr, “Toxoplasma might even kill as many people as malaria, or at least a million people a year.” 

Flegr's hypothesis comes directly from his own experience.  He wondered for years why he was willing to take risks that others wouldn't, like crossing a street in the middle of traffic, or speaking out against communism in Communist Czechoslovakia.  Entirely by fluke, he was tested for T. gondii by someone in his institution looking for infected people to study a diagnostic kit they were developing, and he was discovered to be positive. To him, this explained his bizarre risk-taking behavior.

He reasons that T. gondii is not the only parasite that affects behavior.  The rabies virus incites fury in infected animals, ensuring that they bite others, and thus pass on the infection.  Ants infected with parasitic Cordyceps fungi do all kinds of bizarre, self-destructive things, including climbing onto a blade of grass and then clamping on with their mandibles. Soon the fungus consumes the ant's brain, and fungal fruiting bodies sprout from the ant's head (as in the video) and burst, releasing spores into the air, to settle and find a home in another unsuspecting, soon to be robotic ant. Apparently the Cordyceps fungi release chemicals that change an ant's pheromone reception, which alters their sense of navigation. Is this coincidence, not specific enough to have evolved per se?  Or is it a specific adaptation?



Another example of zombie ants involves infection by the lancet liver fluke, Dicrocoelium dendriticum.  When infected, the ant again climbs onto a blade of grass where it clamps on, there to be eaten by a grazing sheep or cow.  The ant does this only in the evening, when the air cools, and if it survives the night uneaten, it climbs down and behaves normally again until the following evening, when the fluke regains control. Again, this is remarkable, but it is it specific enough and frequent enough to be a Darwinian adaptation?  And what's in it for the poor manipulated ant? 

Things that seem (to human observers) so bizarre probably would be expected to have a balance, or else the victim species would have evolved resistance.  So many questions are raised by these examples.  And there are many more like them. 

But in any case, parasite-induced behavior changes are not unprecedented.  Could T. gondii really do the same?

In the Soviet-stunted economy, animal studies were way beyond Flegr’s research budget. But fortunately for him, 30 to 40 percent of Czechs had the latent form of the disease, so plenty of students were available “to serve as very cheap experimental animals.” He began by giving them and their parasite-free peers standardized personality tests—an inexpensive, if somewhat crude, method of measuring differences between the groups. In addition, he used a computer-based test to assess the reaction times of participants, who were instructed to press a button as soon as a white square popped up anywhere against the dark background of the monitor.

The subjects who tested positive for the parasite had significantly delayed reaction times. Flegr was especially surprised to learn, though, that the protozoan appeared to cause many sex-specific changes in personality. Compared with uninfected men, males who had the parasite were more introverted, suspicious, oblivious to other people’s opinions of them, and inclined to disregard rules. Infected women, on the other hand, presented in exactly the opposite way: they were more outgoing, trusting, image-conscious, and rule-abiding than uninfected women.

Flegr confirmed these surprising findings with further research, finding that infected men were suspicious, sloppy dressers, and introverted, while infected women were well-dressed and gregarious.  Reaction times of infected people were considerably slower than uninfected, and he found that they were 2 1/2 times more likely to be in traffic accidents -- this statistic has been replicated in other countries.  Flegr says that the personality changes are generally subtle, only detectable on a statistical basis.  But, it turns out that a fairly substantial percentage of people diagnosed with schizophrenia are T. gondii positive. 



What's the mechanism? 

Many schizophrenia patients show shrinkage in parts of their cerebral cortex, and Flegr thinks the protozoan may be to blame for that. He hands me a recently published paper on the topic that he co-authored with colleagues at Charles University, including a psychiatrist named Jiri Horacek. Twelve of 44 schizophrenia patients who underwent MRI scans, the team found, had reduced gray matter in the brain—and the decrease occurred almost exclusively in those who tested positive for T. gondii.

That's not clearly a mechanism, however, as the shrinkage could be entirely unrelated to schizophrenia.  Indeed, since only 1/4 of the patients tested showed reduced gray matter.  Anything more convincing?

Apparently, sequencing of the T. gondii genome suggests that it has 2 genes that can make the infected animal increase production of dopamine, and elevated dopamine levels are a mark of schizophrenia. Infection also, apparently, increases the infected animal's gregariousness, and in humans, increases sociability -- even infection with the influenza virus.  Infection can, apparently, even increase a person's (or a rat's) sex drive, and because many of these infections can be transmitted sexually, this improves their chances of being passed on.  This relates to any kind of infection that has been tested, not just T. gondii.

As it turns out, schizophrenia has been associated with a number of infections ("maternal rubella (German measles), influenza, Varicella zoster (chicken pox), Herpes (HSV-2), common cold infection with fever, or poliovirus infection while in childhood or adulthood, coxsackie virus infection (in neonates) or Lyme disease (vectored by the Ixodes tick and Borrelia Burgdorferri) or Toxoplasmosis" -- from a 2011 paper by C.J. Carter), and in fact, while genomewide association studies haven't found genes with major effects, or reliably replicated what they have found, for schizophrenia, itself, they have found 600 genes with small effect, many associated with inflammatory response, others implicated in the life cycle of the associated pathogens.  The same paper suggests that:

Schizophrenia may thus be a “pathogenetic” autoimmune disorder, caused by pathogens, genes, and the immune system acting together, and perhaps preventable by pathogen elimination, or curable by the removal of culpable antibodies and antigens.

That is, the authors suggest that the susceptibility genes code for proteins that are homologous to the pathogen's proteins, and that the latter might be intermingling or replacing endogenous proteins, and they are different enough to disrupt normal function, and lead to disease.

Pathogens' proteins may act as dummy ligands, decoy receptors, or via interactome interference. Many such proteins are immunogenic suggesting that antibody mediated knockdown of multiple schizophrenia gene products could contribute to the disease, explaining the immune activation in the brain and lymphocytes in schizophrenia, and the preponderance of immune-related gene variants in the schizophrenia genome. 

Further,

All of the pathogens implicated in schizophrenia express proteins with homology to multiple schizophrenia susceptibility gene products. The profile of each individual pathogen is again specific for different types of gene product, but all target key members of the schizophrenia network including dopamine, serotonin and glutamate receptors as well as neuregulin and growth-related or DISC1 related pathways.

So, the idea is that our genomes, our particular DNA variants, determine which human/viral matches we carry, and thus which pathogens we're susceptible to damage from.  So, in that sense, Carter, and others, suggest, schizophrenia and other behavioral disorders may be 'genetic', but environmental exposures, our vaccination history and so on determine the pathogens we might be infected with, and our immune system determines how we respond.

To be sure, these are statistical findings and there are so many genes associated with schizophrenia -- or perhaps more accurately so many genes not clearly but weakly, possibly, maybe, but not replicably associated, that it is possible one could almost always find some potential association with these pathways.  That makes it hard to evaluate the infectious scenario.

One clear point, though, is that even when what we are is genetic, the genes need not be those we were born with.  Bacteria, and hence their genes are vital to our survival and that appears just to be for starters.  When parasites affect our gene expression or function, their genomes become part of ours.  And from a biological point of view, our genetic battle for persistence -- for staying alive -- may have more to do with microbial challenges than with wearing out, which is basically what many GWAS targets are about (cancer, diabetes, etc.)

Even more important, perhaps, and a hint that we need to pay more attention to, is that many GWA kinds of studies are finding genes in immune-related systems, or those related to 'inflammation' for what appeared to be totally non-infectious and non-behavioral diseases, even including diabetes, intestinal disorders, retinal disorders of the eye, and many others.  These would be genetic in the sense that genetic susceptibility is involved, but not in the sense of intrinsically harmful genetic variants.

Is this behavioral parasite work definitive?  Do we now know that schizophrenia, and other disorders, are infectious in origin?  No.  Many questions have yet to be answered.  Maternal or early childhood exposure seem to be associated with risk, but why does schizophrenia have such a relatively late age of onset, given early age of exposure?  And why so stereotypically in late adolescence?  And so on.

But, it's intriguing that many GWAS have found an albeit small proportion of risk of many diseases explained by immune genes.

The Zen of GWAS: the sound of one hand clapping

So we've come to this: Nature is applauding the latest genomewide association study (GWAS) on schizophrenia as "welcome news" because it is "zeroing in on a gene" to explain this devastating disease whose etiology has been frustratingly elusive for so long (Hugh Piggins, "Zooming in on a Gene").  Many authors have found 'hits' by mapping, but most of them, if not perhaps all, have not been replicable.  The largest recent study we know of, prominently published (Nature, 2009), estimated that hundreds of genes contribute to schizophrenia.  Piggins does acknowledge that GWAS have been much criticized for explaining so little, but, he says, this one's different (well, at the very least, it'll sell more copies of Nature).

Speaking of copies, rare copy number variants (CNVs) have been found to be associated with schizophrenia and other neurodevelopmental disorders including autism. The operative word here being 'rare'. Copy number variants are generally large (1000 basepair or greater) genomic insertions or deletions, that, by definition, vary widely among individuals.  They're either inherited from a parent who carries the CNV, or arise anew. When CNVs were first recognized, it was thought that they would be found to be associated with many diseases, but the most common CNVs seem to not be disease-related at all.  After all, genomes evolve largely by segment duplication.
 
So given how Nature touts this result, we thought we must have misread, surely.  But, no, the paper confirms:

Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample.

That's 0.35%, as in 3 schizophrenics per thousand.  That's a signal so weak that even a smoke alarm couldn't detect  it.  So, what's the real import of this finding?  Nothing new at all -- schizophrenia  is a complex disorder, or suite of disorders, that is multigenic, and/or has multiple different causes.  Like most other complex diseases, as has been shown over and over.

But the authors go on to discuss the gene (VIPR2) at the identified chromosomal locus that they think might be causative, and conclude, in what may be the Oversell of the Century to date:

The link between VIPR2 duplications and schizophrenia may have significant implications for the development of molecular diagnostics and treatments for this disorder. Genetic testing for duplications of the 7q36 region could enable the early detection of a subtype of patients characterized by overexpression of VIPR2. Significant potential also exists for the development of therapeutics targeting this receptor. For instance, a selective antagonist of the VPAC2 receptor could have therapeutic potential in patients who carry duplications of the VIPR2 region. Peptide derivatives and small molecules have been identified that are selective VPAC2 inhibitors, and these pharmacological studies offer potential leads in the development of new drugs. Although duplications of VIPR2 account for a small percentage of patients, the rapidly growing list of rare CNVs that are implicated in schizophrenia indicates that this psychiatric disorder is, in part, a constellation of multiple rare diseases. This knowledge, along with a growing interest in the development of drugs targeting rare disorders, provides an avenue for the development of new treatments for schizophrenia.

You may not have heard of this infamous gene, so for your edification, it's name is Vasoactive intestinal peptide receptor 2 (hence VIPR2).  The ultra high plausibility of this Major Gene for--what was it?  schizophrenia--is made clear by the sites in which it is expressed: the uterus, prostate, smooth muscle of the GI tract, seminal vescicles, blood vessels, and thymus.  Wiki adds as an afterthought that VIPR2 is also expressed in the cerebellum (whew!  A narrow escape chance for relevance?).

In fact, here's a section from GenePaint showing VIPR2 expression in a 14.5 day mouse embryo.  The gene is expressed where you see the darker blue -- the snout, the vertebrae, the ribs and lungs....  Not in the brain, but then this is only one stage in development, so it's relevance to brain function can't be ruled out.

But from the evidence, targeting this for therapy might ease digestion and calm the nerves.....including those in the genitals.  (So maybe schizophrenia is a sex problem, after all.)
 
We thought and thought what would be the right way to characterize this stunning discovery.  A supernova of genetics?  Darwin redux?  The sting of the VIPR2?  No, those images are too pedestrian.  We needed to go much deeper, to something with much more profound imagery, to capture what has just been announced.

Of course, it's possible that we've missed something in the story that is far more important than our impression has been.  It's always possible since we're no less fallible than the next person.

Nonetheless, based on our understanding of the story, we thought, well, Zen Buddhism is about as profound as it gets, in human thought and experience.  So we decided that the clamour of this new finding, the glory of GWAS, was the roaring sound of one hand clapping.  Listen very, very (very) carefully, and you, too, may be able to hear it!

The complexity of schizophrenia and how to understand it

An excellent, measured and thoughtful paper about the causes of schizophrenia appears in this week's Nature, a special issue on current knowledge about the disease.  Much recent research into this devastating disease has been gene-based, including of course genomewide association studies, but, as with all other complex traits, no simple genetic basis has been identified.  In this paper ("The environment and schizophrenia"), Jim van Os et al discuss reasons for this, and suggest ways to move the research forward.

GWAS have identified hundreds of genes for schizophrenia, or more, but currently only accounting for a small percent of the variation in disease presence or test-scores.  Depending on some assumptions and on what data one considers, estimates are that hundreds or even thousands of genes (including regulatory and other functional regions) contribute.  There are few that seem to make strong individual contributions, and one region that has been found to do that is in the HLA part of the immune system, a strange kind of finding.  Even with optimistic assumptions, predictive power will vary from sample to sample and population to population.  These will not, as currently designed, assess epigenetic changes, due to DNA modification.  And then there's the little trivial thing called the 'environment.'

Briefly, in this new paper van Os et al. argue that schizophrenia, and other 'psychotic syndromes', are the result of the interaction of the developing brain with environmental triggers during developmental sensitive periods in those with what is presumably a genetic susceptibility to disruptions in normal functioning of the brain.  We condense a long argument into one inadequate sentence here, but even so please note that it's a paean to complexity, flexibility as a response to the environment during development, and that genetic susceptibility is only one part of the picture.  

These researchers believe, based on a body of prior evidence, that the development of normal neuronal connections in the brain requires interaction with a variable environment, as they portray in the figure to the left.   Based on epidemiological data for associations of psychosis with environmental risk factors, they define risk as the stressors of urban environments, belonging to a minority group, developmental trauma, and/or cannabis use.  However, these factors are extremely common, while what they call the 'psychotic syndrome' is not.  This is where the genetic susceptibility comes in.

This suggests that beneath the relatively small marginal risks linking the environment to psychotic syndrome at the population level, vulnerable subgroups exist that are more sensitive to a particular environmental risk factor at a much larger effect size. Thus, the validity of observed associations with urban environment, developmental trauma, cannabis use and minority group position hinges on evidence of vulnerable subgroups. Genetically sensitive studies indicate that differential sensitivity to the psychosis-inducing effects of environmental factors may be mediated by genetic factors. For example, in siblings of patients with a psychotic disorder, who are at increased genetic risk to develop psychotic disorder, the psychotomimetic effect of cannabis is much greater than in controls, as is the risk to develop psychotic disorder when growing up in an urban environment

It's one thing to state this, and another thing to suggest ways to test this complex interaction of risk factors.  They do just this, however, describing a number of animal studies that can now be done to look at the effect of environmental factors on the developing brain of susceptible and non-susceptible animals.  The authors recognize that this won't be easy -- sensitive periods during development, risk factors, at-risk animals, and evidence of psychosis all must be well-defined and observed.

And then any potentially disastrous effects of methodological shortcomings, such as bias or confounding -- including genetic confounding -- must be ruled out before results can be considered credible.  E.g., are at-risk adolescents self-medicating with cannabis, making it appear that cannabis is the trigger when it's something intrinsic instead?  Does genetic susceptibility predispose to the use of cannabis, again making it look as though cannabis is the risk factor when it's genes?  They in fact include a box devoted to issues related to weighing the evidence.

Whether van Os et al. are correct in the details of which factors are most important to account for, or in the timing of the sensitive periods for specific aspects of normal growth we certainly can't say, but even so this is a beautifully nuanced and well-reasoned argument for accepting complexity, with suggestions for how to move forward from there, all based on the mountains of data that have come before.  And, the authors don't over claim, or say it will be easy. 

Indeed, the existence of many 'sensitive stages' during development means any number of pathways could be affected at any time to lead to disease.  Schizophrenia, like any trait, of course has genetic underpinnings, but given the 4-dimensional complexity of the developmental pathways in the brain,  this means that there are many ways that things could go awry, which only increases the difficulty with which they can be found, or, if found, be useful for prediction.

The authors conclude:

The human brain has evolved as a highly context-sensitive system, enabling behavioural flexibility in the face of constantly changing environmental challenges. There is evidence that genetic liability for psychotic syndrome is mediated in part by differential sensitivity to environments of victimization, experience of social exclusion and substances affecting brain functioning, having an impact during development. Given the complexity of the phenotype and evidence of dynamic developmental trajectories, with environmentally sensitive periods, longitudinal research on gene–environment interplay driving variation in behavioural expression of liability, that subsequently may give rise to more severe and more ‘co-morbid’ expressions of psychopathology and need for care, is required to identify the causes and trajectories of the psychotic syndrome. Examination of differential sensitivity to the environment requires technology to assess directly situated phenotypes indexing dynamic, within-person environmental reactivity as substrate for molecular genetic studies; parallel multidisciplinary translational research, using novel paradigms, may help identify underlying mechanisms and point the way to possible interventions.

So, this is an unusually sober and realistic treatment of a complex disease.  Is there anything surprising here?  Only that Nature is giving a number of pages to a nuanced treatment of a subject that is so often treated simply.

GWAS: Carry on regardless....(?)

From 1958-78,29 very funny British 'Carry on' movies, with titles like 'Carry on nurse,' were produced. It's a British phrase that a boss would say to an employee, but in this case, no matter how bollixed up the situation was, the idea was that people just 'carry on' regardless. The movies parodied British institutions and their behavior.

Well, though it's neither satirical nor funny, the same is often the case in science, where the thing-to-do is what's done regardless of whether it's the best thing or is working very well. We cling to our flotsam if it's all we know or seems the safest.

On July 14, we wrote about a Nature paper describing the results of a genome-wide association study (GWAS) of the genetics of schizophrenia, published online. This and two accompanying papers appear in the journal this week, reinforcing the story of schizophrenia as a polygenic trait, with many genes involved, each with very small effect. The papers suggest that the immune system may somehow be involved, as genes in the HLA system are found to have a significant, if limited effect, as well as some aspects of brain development, cognition and memory.

This is potentially very interesting because if GWAS are finding anything it may be that immune or inflammatory system genes are involved in a wide array of traits, perhaps not always previously suspected as such. Could infectious or autoimmune causes be more widespread than we have suspected? If so, it may say a lot. Partly it could be the things that go wrong over a life that's decades long, in terms of exposures and/or mutations that attack self.

But this and a host of other GWA studies have had minimal findings--hyped to death, perhaps, but usually accounting for only a minor fraction of all causation, even the known genetic component as revealed by the family cluster of disease. Each study finds one or a few genes that contribute detectable amounts to the trait. Some of these have been replicated and begin to be believable for that reason (though many if not most have no a priori plausibility as causes of the mapped disease).

This should be providing geneticists with plenty of targets for real genetics, real in the sense of figuring out what the genes do and how to attack them therapeutically. Modest they may be, but they're the strongest candidates we have.

So why, then are new GWAS still being done all over the place on the same diseases? Even strong proponents of this method have acknowledged that they are finding many genes with small effect. The same traits are being studied over and over again, often finding different genes, but which almost invariably explain very little risk.

It's time to stop paying for ever more of this, and to demand proof of principle. The principle is that (1) we can show how, why, and when these candidates (and their many mutations and regulatory sequence variants) are involved in disease, and then (2) that we can do something about them. Yet, because many investigators are set up for mapping, which is after all a rather mechanical button-pushing kind of enterprise (and very grant-able), one often hears investigators saying that 'mapping is what I do' .

That's a very poor excuse, even if in a careerist society that depends on the grant system and on intellectual inertia. What we need now is some accountability: to show that the fruits of GWAS labor to date are worth it and do, after all, have important biomedical use.

That being done, we'll know better whether we should continue down the reductionist mapping road, or whether better, more effective approaches to causation, even genetic causation, are the proper course.

In the 'Carry on' movies, things muddled along despite all the confusion, and in science we'll muddle along, too. Nobody can say that persistence with GWAS or other tactics is useless, even if it's inefficient and we know better what would be better. But biomedical science can do better, and we think that it should.

The mind boggles

Schizophrenia is one of those important human traits that has eluded understanding despite heavy research investment. It is elusively variable and hence challenging to diagnose as a single entity or to decide how to split it up into causally distinct subsets. It seems highly familial in terms of its increased risk among family members, and hence seems clearly to have a genetic component. But the specific genes have been elusive--they must be there in the genome, but where are they?

A recent paper in Nature ("Common polygenic variation contributes to risk of schizophrenia and bipolar disorder", The International Schizophrenia Consortium, published online 1 July 2009) looked at large amounts of data on schizophrenia from several study populations. The authors did an extensive amount of genotyping and then various kinds of analysis (they looked, for example, at about a million variable sites (SNPs) in the genome, to identify regions where a particular variant marker was found more often in some 3322 cases than 3587 controls--pretty large studies for this kind of trait.

No really strong signal, that is that explained a high fraction of the disorder, was found. But through a series of analytic approaches, including computer simulations to test a range of possible genetic causal models to see which fit best, the authors (and this is one of those papers with a huge list of authors) concluded that many thousands of genes (classically they'd be known as 'polygenes') contribute to the trait. Most of the contributing variants are rare, but more importantly, they have individually very small effects.

Regardless of the details of the study, which could include all sorts of artifacts or be affected by the methods and assumptions of the authors, the study seems convincing that schizophrenia is like many other traits of a polygenic nature. The authors confirmed current ideas that bipolar disorder may involve many of the same genes, as well.

There are good evolutionary and biological reasons why this makes sense. In a nutshell, it's because so many processes are involved in brain development and function, each of them subject to mutational variation, that there are many ways to end up with the same trait. Natural selection only prunes those who can't reproduce as successfully, but the effect is distributed across these many parts of the genome, and hence acts only very weakly against any one of them. The result is an accumulation of variation that, at each individual region is essentially undetectably abnormal. The frequency of the individual variants changes over generations (and over geographic space in our species) mainly by chance (genetic drift).

The individual components have to work together--the 'cooperation' that is at the core of life as we outline in our book The Mermaid's Tale, but there is plenty of tolerance for variation, what we refer to as functional 'slippage'. It all makes sense biologically, evolutionarily, and causally.

In addition to its consistency with evolutionary expectations, this flies in the face of current predominant thinking about the prospects for what is being called 'personalized medicine', that is, medicine based on each individual person's genotype. If genotypes are poorly predictive, as in this case they seem to be, then they are of no real use to a clinician. In fact, as with so many similar studies, the total identified effect was small: based on various assumptions, the polygenic component identified by this geomewide search accounted for only 3 to 20% of the total disease risk, which itself is only 1%! Schizophrenia is an important problem (1% of the population is a lot of people), but clearly the predictive power of these gene-sets is modest, and this assumes that environmental effects will retain their current overall nature and impact (many of the genes probably have effects that vary depending on environment).

Many researchers will try to develop synthesizing methods to make individual sense of polygenotypes, so that treatment might be varied accordingly. How well they succeed only time will tell. But this is another case in which extensive study of a trait based on modern high-intensity technology has documented the nature of complex traits.