Clues Emerge About the Maternal Infection Risk Factor for Schizophrenia

15 February 2011. Studies suggest that mothers who are infected with various pathogens during pregnancy pass an unfortunate legacy on to their children: an increased risk of schizophrenia. Most animal studies exploring this issue have modeled infection by exposing mouse dams to polyriboinosinic-polyribocytidylic acid, a viral mimic better known as poly I:C. Javier González-Maeso and colleagues at the Mount Sinai School of Medicine in New York City took a different approach: they infected mice in their first trimester of pregnancy with a mouse-adapted H1N1 virus. Their paper in the February 2 issue of the Journal of Neuroscience describes the changes they observed in the adult offspring of infected mice. Citing parallels in the schizophrenia literature, the researchers report upregulation of serotonin 5-HT_2A receptors and downregulation of metabotropic glutamate mGlu₂ receptors in the frontal cortex; they also found a heightened sensitivity to hallucinogens.

Many infections, same outcome
This research fits within a larger set of hypotheses about infectious and immune factors in schizophrenia etiology (see SRF related news story; SRF news story; SRF news story; SRF news story). It builds on longitudinal epidemiological studies that link schizophrenia to maternal exposure to infections of various kinds, including the flu, cytomegalovirus, herpes simplex virus type 2, bacteria, and the parasite Toxoplasma gondii (for reviews of this field, see Brown and Derkits, 2010). In an interview with SRF, Paul Patterson, California Institute of Technology in Pasadena, said that the variety of infections associated with schizophrenia suggests that “it’s the mother’s response that’s important, not the specific kind of infection.” That response seems to include the release of cytokines, signaling proteins secreted by cells of the immune system (for more about the role of cytokines, see Patterson, 2009).

To explore the mechanisms by which maternal infections might foster schizophrenia, researchers have turned to animal models in which they activate the mother’s immune system by exposing her to infectious agents or to other substances that trigger the same kind of cytokine release as a viral or bacterial infection would. Such substances include lipopolysaccharide (LPS), a bacterial endotoxin, and poly I:C. As for the latter, Meyer and Feldon (Meyer and Feldon, 2011) see the poly I:C approach as “a very powerful neurodevelopmental animal model of schizophrenia-relevant brain disease.”

A roundup of recent findings
Researchers have been using cytokine-releasing agents to examine schizophrenia-related abnormalities in the offspring of immune-activated mothers and to seek clues to how infections might promote schizophrenia. For example, in a study by Dickerson and others (Dickerson et al., 2010), rats whose mothers had received one injection of poly I:C while pregnant with them showed altered synchrony between cortical regions in adulthood, reminiscent of the changes seen in schizophrenia (see SRF Current Hypothesis by Woo and colleagues). Using the same model, Ito and colleagues (Ito et al., 2010) found that the adult offspring of mice challenged with poly I:C showed evidence of abnormal hippocampal processing at the synaptic and behavioral levels. The offspring performed poorly at processing information about objects, as opposed to locations. The researchers suspect that dopamine abnormalities play a role.

In Switzerland, Bitanihirwe and colleagues (Bitanihirwe et al., 2010) report alterations, some sex-specific and some not, in basal levels of the neurotransmitters dopamine, glutamate, γ-aminobutyric acid, and glycine in cortical areas in mice whose mothers were exposed to poly I:C. This echoes earlier findings last year from this group (Vuillermot et al., 2010; Bitanihirwe et al., 2010) and from the O’Donnell group, who used an LPS challenge (Feleder et al., 2010). Both groups concluded that infection during pregnancy affects the development and function of dopaminergic signaling in the offspring of immune-activated mothers.

Baharnoori and associates (Baharnoori et al., 2010) pushed the analysis earlier than most studies, which examined adult offspring. In rats born of LPS-exposed mothers, they noted subtle behavioral changes soon after birth and changes in serotonin receptor gene expression in the cortex at postnatal day 3. In a poly I:C study, De Miranda and colleagues (De Miranda et al., 2010) found that maternal immune activation causes neuronal stem cell abnormalities in the mouse embryo by way of Toll-like receptors, which govern the mother’s immune response.

Because mice cannot report hallucinations
In the wake of these studies, González-Maeso, first author José Moreno, and others explored the biochemical correlates of the abnormal behaviors seen in the offspring of virus-infected mothers. They focused on serotonin 5-HT_2A receptors and metabotropic glutamate mGlu₂ receptors. The serotonin receptors play a role in the effects of hallucinogenic drugs, which can induce behavioral changes that resemble schizophrenia and, with enough use, may even cause psychosis. Some researchers think that mGlu_2/3 receptor agonists show promise as a schizophrenia treatment (see SRF related news story; SRF news story).

In the new study, Moreno and colleagues challenged the immune systems of pregnant mice on day 9.5 of their pregnancy, which corresponds to the end of the first trimester in humans. Mice generally do not get the flu, but the researchers exposed them to a mouse-adapted H1N1 virus, causing a flu-like illness in the mice. A second group of pregnant mice received the vehicle only. After the mothers gave birth, the researchers waited until the pups reached adulthood to learn how maternal immune activation had affected them.

Patterson told SRF that Moreno and colleagues make the “very important” contribution of “looking at what I would call the rodent equivalent of hallucinations.” While other studies have assayed the so-called negative symptoms of schizophrenia, Moreno and colleagues looked at “activation of the sensory cortex in the absence of sensory input, mimicked by injecting a hallucinatory drug.”

The Mount Sinai team injected the offspring of viral- or mock-infected mice with either the hallucinogenic 5-HT_2A agonist DOI (1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane) or vehicle alone. They noted that the offspring of infected mothers showed increased sensitivity to the drug, as shown by a greater head-twitching response. “Notably, our results demonstrate that alterations in the head-twitch behavioral response are only observed after puberty in prenatally infected mice—a finding that parallels the adult onset of the disease in humans and supports our mouse schizophrenia model of perinatal insult,” the researchers write.

Maternal infection also amplified the response of cortical neurons to DOI, as shown by greater expression of the genes c-fos, egr-1, and egr-2, which serve as markers of neural activity.

The study also looked at noncompetitive NMDA glutamate receptor antagonists, including MK-801, which change animal behavior in ways that echo the positive symptoms, negative symptoms, and sensorimotor gating deficits seen in schizophrenia. MK-801 increased locomotion, as expected, but it did so similarly in the offspring of infected versus uninfected mothers. LY379268, an mGlu_2/3 agonist, reduced its effects, but only in mice born of uninfected mothers.

Delving deeper, Moreno and colleagues found that mice born of virus-infected mothers had increased density of 5-HT_2A receptors and a lower density of mGlu_2/3 receptors in the frontal cortex. They also noted unusually high expression of 5-HT_2A mRNA and low expression of mGlu₂ mRNA in that group, which showed normal expression of 5-HT_2C and mGlu₃ mRNA. These results dovetail with earlier Mount Sinai findings in untreated humans with schizophrenia that fingered similar serotonin and glutamate receptor changes in the disease (see SRF related news story).

The changes seen in the offspring of infected mothers did not seem to reflect infection of the fetus: only two of the 31 youngsters showed an antibody response to the virus. “Together with the absence of detectable virus in embryo samples, these data also suggest that the changes observed in the offspring are a consequence of maternal response to viral infection, and not of direct fetal infection by mouse-adapted influenza virus,” Moreno and colleagues wrote.

This conclusion receives support from another study that used the H1N1 virus. Fatemi and colleagues (Fatemi et al., 2011) found that infection at embryonic day 7 led to structural and gene expression changes in the placenta, as well as altered gene expression in the hippocampus and prefrontal cortex of exposed offspring. Neither the placentas nor the brains showed signs of active infection, again fingering the mother’s immune response rather than the infection itself.

In the future, Patterson would like to see studies that examine genetic risk in conjunction with environmental risk factors like maternal infection. Given the small effect sizes for most mutations, he wonders whether they have to combine with an environmental insult to cause schizophrenia. “The effect size of maternal infection is much greater than virtually all of the mutations that have been found, so I think the epidemiology deserves a lot more attention than it has been getting,” he said.—Victoria L. Wilcox and Hakon Heimer.

Reference:
Moreno JL, Kurita M, Holloway T, López J, Cadagan R, Martínez-Sobrido L, García-Sastre A, González-Maeso J. Maternal Influenza Viral Infection Causes Schizophrenia-Like Alterations of 5-HT2Aand mGlu2 Receptors in the Adult Offspring. J Neurosci. 2011 Feb 2;31(5):1863-72. Abstract

Q&A With Paul Patterson. Questions by Victoria L. Wilcox

Q: As you think about the research looking for biological mechanisms that could transduce maternal infection into risks for schizophrenia, what do you think are the major insights of the field so far?

A: In terms of the mechanism of how maternal infection alters fetal brain development, the first thing to say is that it's not caused by infection of the fetus, but rather it's caused by the activation of the mother's immune system. In their recent paper (Moreno et al., 2011), Moreno and colleagues provide some evidence in favor of that—that is, they don’t see evidence of the virus in the fetus, but that's been published previously by us (Shi et al., 2005). I think the most compelling evidence, though, is that you can get very similar, maybe the same, behavioral abnormalities in the offspring without using the virus at all; you can just activate the mother's immune system. And we do that with double-stranded RNA, poly I:C, without a pathogen. It should be noted that the behavioral abnormalities are consistent with those seen in schizophrenia, but many of them are also consistent with symptoms in autism. This is not surprising, because maternal infection is a risk factor for both schizophrenia and autism. There was an important study in 2010 that analyzed over 10,000 cases of autism in the Danish medical registry and found an association with viral infection in the first trimester (Atladóttir et al., 2010). This is similar to findings by Alan Brown and colleagues in schizophrenia, where they found the risk factor for infection was likely to be in the late first, early second trimester (Brown et al., 2004). In the schizophrenia epidemiology, the infections can be of almost any kind; there's evidence for viral, bacterial, and parasitic toxoplasma infections as increasing risk. This is also consistent with the idea that it's the mother's immune response that's important, not the particular kind of infection.

Q: What are some of the challenges to this field? Can they be overcome and, if so, how?

A: On the behavioral side, of course, you're dealing with mice or rats, which have only analogous behaviors to those in humans. In that sense, the behaviors that we and others look at are consistent with those in human schizophrenia and autism, but, of course, these are rodent behaviors. In that sense, Moreno et al. provide a new piece of data that is, I think, very important, which is they provide evidence for what I call the rodent equivalent of hallucinations. They see activation of the sensory cortex, in the absence of sensory input, which can also be evoked by injecting a hallucinatory drug. The surrogate marker for neuronal activation that they use is early gene activation. The hallucinogenic drug induces these genes in the cortex, but the key point is that there is a stronger induction in the offspring of mothers who were infected compared to controls. This is what is seen in schizophrenia: schizophrenics are more sensitive than controls to hallucinogens. Moreno further reports that the behavioral response to hallucinogens is also greater in the offspring of infected mothers. Our group has unpublished data very similar to these using the poly I:C maternal immune activation model. A key point about this rodent version of hallucinations is that it is a behavior that one can measure in rodents that is more specific to schizophrenia than other behaviors that we and others have measured. Hallucinations can also be found in bipolar disorder, but they are very prominent in schizophrenia. This is a so-called positive symptom, rather than a negative symptom, which is what everybody is assaying normally.

Q: I'm glad you mentioned poly I:C because that leads into my next question. The Moreno study uses “mousified” influenza virus, whereas a lot of the studies seem to use poly I:C to simulate the flu. What are the pros and cons of the different approaches?

A: The influenza model is more like the human situation, where one is studying an actual infection of the mother. The poly I:C approach has the advantage that one can control the timing of the mother’s immune response very precisely, whereas in the infection, the mother is sick for many days after infection—in other words, for a good part of embryonic development. In contrast, the poly I:C drives up the cytokines in the mother very transiently, so one is looking at a very narrow time window of the effect. Also, one can control the dose very closely with poly I:C, while it is harder to control the dose with infection of the virus, which is dependent on the breathing pattern of the animal. One can use different concentrations of the virus, but it is never clear what dose the animal is actually absorbing in the respiratory tract. We've done infection with different doses of influenza virus, and we do see a dose-response curve in the behavior of the offspring, but it's difficult to specify exactly what dose the mouse is getting. Nonetheless, the cytokine response to poly I:C is certainly not identical to influenza infection.

Q: What stands out to you about the results of the Moreno study?

A: I mentioned that, to my mind, the most interesting result is the evidence that the offspring of infected mothers are more sensitive to the hallucinogen. They also report that there are changes in the serotonin and glutamate receptors in the brains of the offspring, which is useful, and adds to the neuropathological findings of others in this model.

Q: What do the results imply about the role of glutamate and serotonin in the relationship between the infection in the mother and schizophrenia in her offspring, and is that important in terms of the neuropathology?

A: Moreno et al. make the point that they think the change in these receptors might be related to the behavioral changes that are observed in the offspring, which could be true. One would have to, of course, investigate that in much more detail to see if there's really a relationship between changes in, say, a glutamate receptor and the behaviors.

Q: Are there any limitations or caveats about the study that are important to keep in mind?

A: I don't think it's a terribly important part of the paper, but the negative result that they do not detect the virus is not as compelling as the positive result that similar behaviors can be observed in the offspring without using a virus; one can just stimulate the immune system in the mother.

Q: This is a mouse model, and some people are skeptical of them. Obviously, there are differences and similarities between mice and humans, so what, if anything, does this study based on a mouse model mean for humans with schizophrenia? What's needed to connect those dots?

A: A key advantage is that, in the rodents, one can study the mechanism of how maternal infection alters fetal brain development—this can't be done in humans.

Q: You can't just infect pregnant women with a virus, for instance.

A: What's known so far is that it's the mother's immune response that's important. Second, we found that the rise in the cytokine interleukin-6 that occurs during infection is critical for the changes in behavior of the offspring. If one blocks the interleukin-6 response, you don't see the abnormal behaviors in the offspring. Conversely, if one simply injects IL-6 alone in pregnant mice, the abnormal behaviors are found in the offspring, so you don't need poly I:C or the virus; transiently increasing this cytokine is sufficient. Another point about the mechanism is that the placenta is at least one of the sites of action of the maternal immune response (Hsiao and Patterson, 2010). Cytokines are upregulated in the placenta, there are endogenous immune cells in the placenta that are activated, and this results in endocrine changes in the placenta that undoubtedly affect the fetus. In addition, maternal poly I:C administration (or maternal LPS, to mimic bacterial infection) increases cytokine levels in the fetal brain, and these changes are prolonged until at least the early postnatal period (see Patterson, 2009). In other words, the first steps are being taken toward understanding the cellular and molecular pathways of how maternal infection alters fetal brain development, which ultimately changes behavior. That's one point: the mechanism. Another feature of animal work is the ease of testing potential therapeutics. For example, the Meyer group in Zurich and the Weiner group in Tel Aviv recently published important papers on one type of intervention, which is treating adolescent offspring of immune-activated mothers with antipsychotic drugs before they exhibit certain behavioral abnormalities and neuropathology (Meyer et al., 2010; Piontkewitz et al., 2010). This prevents the onset of at least some of the abnormal behaviors and neuropathology, in particular, the enlargement of the ventricles, which is a cardinal neuropathology in schizophrenia. This, I think, adds fuel to the fire concerning the question of prodromal treatment of teenagers at extremely high risk of schizophrenia. Many people argue that such treatment is ineffective or perhaps even unethical, but the rodent results, both in rats and mice, are very compelling that adolescent treatment can prevent abnormal behavior and pathology in the offspring. Such results also suggest that other novel treatments could be tested in these offspring because, clearly, not all the abnormal behaviors are permanently set during embryonic development. One might have predicted that since fetal brain development is altered, it's too late to do anything postnatally. These results indicate otherwise.

Q: Are there any other potential approaches to treatment or some kind of preventive intervention that could occur prior to the prodrome, such as vaccinating the mothers?

A: We're particularly interested in modulating the immune system of the offspring in the early postnatal period. That's another place that is potentially important, particularly in autism, because you would like to intervene in autism as early as possible. Since the offspring of infected or immune-challenged mothers display the cardinal symptoms of autism, which we haven't talked about yet, you could test early interventions. These symptoms are 1) repetitive or stereotyped behaviors; 2) deficits in communication, which in the animals is tested by recording ultrasonic vocalizations; and 3) deficits in social interaction. These behaviors can be readily assayed in the rodents and have parallels in human autism. There's also a neuropathology that's commonly found in autism, which is a localized deficit in Purkinje cells. Regarding maternal vaccination, one issue arises from the CDC recommendation that all pregnant women be vaccinated for the flu. The rodent results raise the question: Is this really safe? Vaccination activates the immune system, and that's what poly I:C does. In my mind, it raises the quantitative question: Is vaccination strong enough to activate the mother's immune system to the extent that it could alter fetal brain development as we see in the mice with poly I:C or IL-6 injection?

Q: That's a sobering thought.

A: And this hasn't been clarified. In fact, a review by the Canadian CDC flu experts (Skowronski and De Serres, 2009) concludes that it is not yet clear that maternal vaccination is both safe and effective. It seems that more studies need to be done. In fact, the studies that are quoted by the U.S. CDC regarding maternal vaccination did not look for schizophrenia in the offspring, because, of course, that would involve following people for 20 or more years.

Q: Interesting. Is there anything else you want to say regarding what are the biggest unanswered questions that remain in the field?

A: In terms of the mechanism of how maternal infection changes fetal brain development, we're only taking the very first small steps to understanding that. We don't know what the cytokines do in the fetal brain; we're not exactly sure where they're acting, or which are the key changes in brain development that lead to abnormal behaviors. There are many, many steps between changes in fetal brain development and actually seeing how that plays out in behavior. In fact, that will be very difficult to do because the behaviors that we're looking at are complicated. There is also a lot to be done in terms of testing potential new treatments for both the autism- and the schizophrenia-like behaviors and pathology.

Q: Do you think the findings from the Moreno study and other studies exploring this area could lead to a treatment?

A: There's nothing in the Moreno study that speaks to that question directly, but the results on the antipsychotic drugs show that you can test the drugs, and you can get effects, so the field is wide open for that.

Q: Is there anything I haven't asked you about that you would like to say about this topic, about research in this area, or about the Moreno study in particular?

A: Two points: First, I think everybody recognizes the importance of genes in schizophrenia and autism, but the animal studies that we're talking about so far don't really speak to the question of genetic risk. Therefore, another area of future work that's very important is to combine the susceptibility genes with the environmental risk factors, such as maternal infection. There have been a couple of studies on this published so far, but there's much more that can be done. People have looked at maternal immune activation combined with the DISC1 gene knockout (Abazyan et al., 2010). There's also a recent paper from Dan Ehninger and Alcino Silva on the tuberous sclerosis mouse model (Ehninger et al., 2010). Mutations in the tuberous sclerosis genes can enhance the chance of developing autism features. This paper shows a synergism between the poly I:C environmental factor and the tuberous sclerosis mutation.

Q: So there could be some gene-environment interactions involving maternal infection and perhaps some gene that makes humans or animals more vulnerable.

A: Right. This hasn't been studied in humans yet—that is to say, people who are doing genetic studies in humans haven't been looking at the combined effects with environmental risk factors. That may be part of the puzzle for why the effect sizes of most mutations are so small, because maybe the mutations have to occur in combination with an environmental risk factor.

Q: That makes sense to me.

A: That's point number one: the genetics of it. Point number two: it's frustrating that so much less attention is paid to environmental risk factors compared to genetic risk factors, because the effect size of maternal infection is so great. I think the epidemiology deserves a lot more attention than it has been getting.

Q: Do you think maternal infection could be related to some of the epidemiological factors that have been found to be related to schizophrenia, such as social density?

A: Yes, the point has been made that a number of other risk factors found by epidemiology, which are seemingly so disparate and unrelated, could in theory all be related to this maternal infection. That would include being born and raised in an urban environment and being born in winter-spring months, both of which would enhance the probability of maternal infection. Also, obstetric complications and maternal stress increase the risk for schizophrenia, and these involve increases in IL-6 levels, which the mouse studies showed to be critical for schizophrenia-like behaviors in the offspring.

Q: In addition, certain immigrant communities face a higher risk.

A: It's conceivable that when people are transplanted into a different environment, they could be more susceptible to infection by microbes they have not encountered before. Obviously, there are many other factors that are new to the immigrant as well, but it is interesting to think about unifying hypotheses.

Q: Thank you so much for taking the time to talk with me; I really appreciate it.

Over the past six years, Alan Brown and colleagues have published an impressive series of epidemiological findings on schizophrenia in the offspring of a large cohort of carefully studied pregnant women (reviewed by Brown, 2006). Their work has confirmed and greatly extended prior findings linking maternal infection in the second trimester with increased risk for schizophrenia in the offspring. Moreover, Brown et al. found an association between anti-influenza antibodies in maternal serum and increased risk for schizophrenia, as well as a similar association with elevated levels of a cytokine in maternal serum. In a new paper (Babulas et al., 2006), this group reports a fivefold increase in risk for schizophrenia spectrum disorders in the offspring of women who experienced a genital/reproductive infection during the periconception period. The infections considered were endometritis, cervicitis, pelvic inflammatory disease, vaginitis, syphilis, condylomata, “venereal disease,” and gonorrhea. Strengths of the study include physician documentation of the infections and face-to-face assessments of schizophrenia. Although sample size was modest, these results extend a prior finding that elevated maternal anti-herpes simplex type 2 antibodies are associated with increased risk of psychotic disorders, including schizophrenia (Buka et al., 2001).

The mechanism of how maternal infection increases risk for schizophrenia could involve pathogens invading the fetus. Although this is certainly possible in the case of some of the infections studied by Babulas et al., in the case of a respiratory virus such as influenza, this explanation appears unlikely. A more parsimonious mechanism would involve activation of the maternal immune system, and action of soluble mediators such as cytokines at the level of the placenta or the fetus. Support for this hypothesis comes from animal studies. An antiviral immune response can be evoked in the absence of the pathogen by injection of synthetic double-stranded RNA (polyI:C). When this is done in pregnant rats or mice, the adult offspring display a number of behavioral abnormalities reminiscent of those observed in schizophrenia. These include deficits in prepulse inhibition, latent inhibition, and social interaction, as well as enhanced amphetamine-induced locomotion and anxiety under mildly stressful conditions (Shi et al., 2003; Zuckerman et al., 2003; Ozawa et al., 2005). Moreover, some of these deficits are ameliorated by treatment with antipsychotic drugs and exacerbated by psychotomimetics (Shi et al., 2003; Ozawa et al., 2005), and the offspring also exhibit dopaminergic hyperfunction (Zuckerman et al., 2003; Ozawa et al., 2005). Some of these abnormalities are also seen in the offspring of influenza-infected mothers or mothers injected with the bacterial cell wall component, LPS (Borrell et al., 2002; Fatemi et al., 2002; Shi et al., 2003).

The most recent advance in this growing cottage industry is the finding that there are critical periods of maternal immune activation that determine the type of adult behavioral dysfunction and neuropathology found in the offspring (Meyer et al., 2006). Injection of polyI:C during stages of mouse gestation corresponding to first-to-second versus second-to-third trimesters of human pregnancy yields different deficits in exploratory and perseverative behavior, postnatal reelin expression, and hippocampal apoptosis. Moreover, these two different stages of injection evoke diverse cytokine responses in the fetal brain. It would further be interesting to know which of these abnormalities is specific to the period corresponding to the human second trimester, as this is the key time of vulnerability for risk of schizophrenia associated with maternal infection.

Other fascinating questions for this increasingly popular model are, what mediates the effects of maternal immune activation (e.g., cytokines, antibodies, corticosteroids), and do they act directly on the fetus or via the placenta? Can imaging be used with the rodents to explore dopamine receptor occupancy? Which of the observed pathologies are most relevant for each of the behavioral abnormalities?

References:
Babulas V, Factor-Litvak P, Goetz R, Schaefer CA, Brown AS. Prenatal exposure to maternal genital and reproductive infections and adult schizophrenia. Am J Psychiatry. 2006 May;163(5):927-9. Abstract Borrell J, Vela JM, Arevalo-Martin A, Molina-Holgado E, Guaza C. Prenatal immune challenge disrupts sensorimotor gating in adult rats. Implications for the etiopathogenesis of schizophrenia. Neuropsychopharmacology. 2002 Feb;26(2):204-15. Abstract Brown AS. Prenatal infection as a risk factor for schizophrenia. Schizophr Bull. 2006 Apr;32(2):200-2. Epub 2006 Feb 9. Abstract Buka SL, Tsuang MT, Torrey EF, Klebanoff MA, Bernstein D, Yolken RH. Maternal infections and subsequent psychosis among offspring. Arch Gen Psychiatry. 2001 Nov;58(11):1032-7. Abstract Fatemi SH, Earle J, Kanodia R, Kist D, Emamian ES, Patterson PH, Shi L, Sidwell R. Prenatal viral infection leads to pyramidal cell atrophy and macrocephaly in adulthood: implications for genesis of autism and schizophrenia. Cell Mol Neurobiol. 2002 Feb;22(1):25-33. Abstract Meyer U, Feldon J, Schedlowski M, Yee BK. Towards an immuno-precipitated neurodevelopmental animal model of schizophrenia. Neurosci Biobehav Rev. 2005;29(6):913-47. Abstract Meyer U, Nyffeler M, Engler A, Urwyler A, Schedlowski M, Knuesel I, Yee BK, Feldon J. The time of prenatal immune challenge determines the specificity of inflammation-mediated brain and behavioral pathology. J Neurosci. 2006 May 3;26(18):4752-62. Abstract Ozawa K, Hashimoto K, Kishimoto T, Shimizu E, Ishikura H, Iyo M. Immune activation during pregnancy in mice leads to dopaminergic hyperfunction and cognitive impairment in the offspring: a neurodevelopmental animal model of schizophrenia. Biol Psychiatry. 2006 Mar 15;59(6):546-54. Epub 2005 Oct 26. Abstract Shi L, Fatemi SH, Sidwell RW, Patterson PH. Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring. J Neurosci. 2003 Jan 1;23(1):297-302. Abstract Zuckerman L, Rehavi M, Nachman R, Weiner I. Immune activation during pregnancy in rats leads to a postpubertal emergence of disrupted latent inhibition, dopaminergic hyperfunction, and altered limbic morphology in the offspring: a novel neurodevelopmental model of schizophrenia. Neuropsychopharmacology. 2003 Oct;28(10):1778-89. Abstract

View all comments by Paul Patterson

Related News: Bad Timing: Prenatal Exposure to Maternal STDs Raises Risk of Schizophrenia

Comment by:  Jürgen Zielasek
Submitted 3 June 2006
Posted 3 June 2006

Meyer and coworkers provide interesting new data on the role of the immune system in mediating the damage caused by viral infections during pregnancy on the developing nervous system of the fetus. Not just the timing of the infection appears to be critical, but the developing fetal immune system appears to play a role, too.

Polyinosinic-polycytidylic acid (polyI:C), which was employed by Meyer et al., is frequently used to mimic viral infections. It is a synthetic double-stranded RNA and has adjuvant-effects (Salem et al., 2005). PolyI:C binds to target cells via the "Toll-like receptor 3" (TLR3). TLR3 serves as a receptor in trophoblast cells and uterine epithelial cells mediating local immune activation at the maternal-fetal interface after viral infections (Abrahams et al., 2005; Schaefer et al., 2005). Glial cells like microglia and astrocytes also express functional TLR3 (Farina et al., 2005; Park et al., 2006; Town et al., 2006). Thus, TLR3 plays an important role in immune responses, and its natural function appears to be immune activation in addition to cross-priming the immune system to virus-infected cells (Schulz et al., 2005). Given the expression of TLR3 at the maternal-fetal interface and on glial cells, the polyI:C-TLR3-model appears to be useful to study the basic mechanisms of viral infections and their consequences for brain development in animal models.

However, several limitations are evident: PolyI:C is not a virus, and different immunological pathways may be activated by intact viruses after binding to their appropriate receptors. Findings from the immune system of rodents cannot be directly transferred to humans, and it may be difficult to dissect—on a molecular level—the protective aspects of an immune response against a viral infection from its putative detrimental effects on human neurodevelopment. Still, such mechanisms may now be studied in the rodent models used by Meyer and coworkers and other groups, and this will help to pave the way for future studies in humans. This will hopefully lead to a better understanding of the role of the immune system and viral infections in the pathogenesis of schizophrenia.

References:

Abrahams VM, Visintin I, Aldo PB, Guller S, Romero R, Mor G. A role for TLRs in the regulation of immune cell migration by first trimester trophoblast cells. J Immunol. 2005 Dec 15;175(12):8096-104. Abstract

Farina C, Krumbholz M, Giese T, Hartmann G, Aloisi F, Meinl E. Preferential expression and function of Toll-like receptor 3 in human astrocytes. J Neuroimmunol. 2005 Feb;159(1-2):12-9. Epub 2004 Nov 11. Abstract

Park C, Lee S, Cho IH, Lee HK, Kim D, Choi SY, Oh SB, Park K, Kim JS, Lee SJ. TLR3-mediated signal induces proinflammatory cytokine and chemokine gene expression in astrocytes: differential signaling mechanisms of TLR3-induced IP-10 and IL-8 gene expression. Glia. 2006 Feb;53(3):248-56. Abstract

Salem ML, Kadima AN, Cole DJ, Gillanders WE. Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity. J Immunother. 2005 May-Jun;28(3):220-8. Abstract

Schaefer TM, Fahey JV, Wright JA, Wira CR. Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C). J Immunol. 2005 Jan 15;174(2):992-1002. Abstract

Schulz O, Diebold SS, Chen M, Naslund TI, Nolte MA, Alexopoulou L, Azuma YT, Flavell RA, Liljestrom P, Reis e Sousa C. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature. 2005 Feb 24;433(7028):887-92. Epub 2005 Feb 13. Abstract

Town T, Jeng D, Alexopoulou L, Tan J, Flavell RA. Microglia recognize double-stranded RNA via TLR3. J Immunol. 2006 Mar 15;176(6):3804-12. Abstract

View all comments by Jürgen Zielasek

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Dan Javitt, SRF Advisor
Submitted 3 September 2007
Posted 3 September 2007

A toast to success, or new wine in an old skin?
Patil et al. present a landmark study. It is the kind of study that represents the best of how science should work. It pulls together the numerous strands of schizophrenia research from the last 50 years, from the development of PCP psychosis as a model for schizophrenia in the late 1950s, through the links to glutamate, the discovery of metabotropic receptors, and the seminal discovery in 1998 by Moghaddam and Adams that metabotropic glutamate 2/3 receptor (mGluR2/3) agonists reverse the neurochemical and behavioral effects of PCP in rodents (Moghaddam and Adams, 1998. The story would not be possible without the elegant medicinal chemistry of Eli Lilly, which provided the compounds needed to test the theories; the research support of NIMH and NIDA, who have been consistent supporters of the “PCP theory”; or the hard work of academic investigators, who provided the theories and the platforms for testing. The study is large and the effects robust. Assuming they replicate (and there is no reason to suspect that they will not), this compound, and others like it, will represent the first rationally developed drugs for schizophrenia. Patients will benefit, drug companies will benefit, and academic investigators and NIH can feel that they have played their role in new treatment development.

Nevertheless, it is always the prerogative of the academic investigator to ask for more. In this case, we do not yet know if this will be a revolution in the treatment of schizophrenia, or merely a platform shift. What is striking about the study, aside from the effectiveness of LY2140023, is the extremely close parallel in both cross-sectional and temporal pattern of response between it and olanzapine. Both drugs change positive and negative symptoms in roughly equal proportions, despite their different pharmacological targets. Both drugs show approximately equal slopes over a 4-week period. There is no intrinsic reason why symptoms should require 4 or more weeks to resolve, or why negative and positive symptoms should change in roughly the same proportion with two medications from two such different categories, except that evidently they do.

There are many things about mGluR2/3 agonists that we do not yet know. The medication used here was administered at a single, fixed dose. It is possible that a higher dose might have been better, and that optimal results have not yet been achieved. The medications were used in parallel. It is possible that combined medication might be more effective than treatment with either class alone. The study was stopped at 4 weeks, with the trend lines still going down. It is possible that longer treatment duration in future studies might lead to even more marked improvement and that the LY and olanzapine lines might separate. No cognitive data are reported. It is possible that marked cognitive improvement will be observed with these compounds when cognition is finally tested, in which case a breakthrough in pharmacotherapy will clearly have been achieved.

If one were to look at the glass as half empty, then the question is why the metabotropic agonist did not beat olanzapine, and why the profiles of response were so similar. If these compounds work, as suggested in the article by modulating mesolimbic dopamine, then it is possible that metabotropic agonists will share the same therapeutic limitations as current antipsychotics—good drugs certainly and without the metabolic side effects of olanzapine, but not “cures.” The recent study with the glycine transport inhibitor sarcosine by Lane and colleagues showed roughly similar overall change in PANSS total (-17.1 pts) to that reported in this study, but larger change in negative symptoms (-5.5 pts), and less change in positive symptoms (-2.3 pts) in a similar type of patient population. Onset of effect in the sarcosine study also appeared somewhat faster. The sarcosine study was smaller (n = 20) and did not include a true placebo group. As with the Lilly study, it was only 4 weeks in duration, and did not include cognitive measures. It also included only two, possibly non-optimized doses. As medications become increasingly available to test a variety of mechanisms, side-by-side comparisons will become increasingly important.

There are also causes for concern and effects to be watched. For example, a side effect signal was observed for affect lability in the LY group, at about the same prevalence rate as weight increase in the olanzapine group. What this means for the mechanism and how this will effect treatment remains to be determined. Since these medications are agonists, there is concern that metabotropic receptors may downregulate over time. Thus, whether treatment effects increase, decrease, or remain constant over the course of long-term treatment will need to be determined. Nevertheless, 50 years since the near-contemporaneous discovery of both PCP and chlorpromazine, it appears that glutamatergic drugs for schizophrenia may finally be on the horizon.

References:

Moghaddam B, Adams BW. Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonist in rats. Science. 1998 Aug 28;281(5381):1349-52. Abstract

View all comments by Dan Javitt

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Gulraj Grewal
Submitted 4 September 2007
Posted 4 September 2007
  I recommend the Primary Papers

Related News: 5HT and Glutamate Receptors—Unique Complex Linked to Psychosis

Comment by:  Brian Dean
Submitted 20 March 2008
Posted 20 March 2008

Altered receptor dimerization: a new paradigm in the pathology of schizophrenia
Understanding the pathology of complex diseases such as schizophrenia requires the use of the full arsenal at the disposal of medical research. Such an approach has been used to make an exciting new discovery that suggests that abnormal dimerization between the serotonin 2A receptor (2AR) and the metabotropic glutamate 2 receptor(mGluR2) may underlie some of the symptoms of schizophrenia (González-Maeso et al., 2008).

This discovery is based on an initial finding that 2AR is coexpressed with mGluR2 in layer 5 of the mouse somatosensory cortex (SCx) and that levels of mGluR2 were decreased in the cortex of 2AR-/- mice, suggesting a relationship between the expression of the two genes. This hypothesis was further supported by data showing that expression of mGluR2 was selectively restored in mice where 2AR expression had been re-established in layer 5 of the SCx. From these data, and data from other studies suggesting G protein-coupled receptors (GPCRs) can form heterodimers (Angers et al., 2002), the authors began to test the hypothesis that 2AR and mGluR2 could form heterodimers.

Using human cortical samples and an anti-2AR antibody, the authors showed that they could immunoprecipitate an immunogenic band with a molecular weight that matches a 2AR/mGluR2 receptor dimer complex if an anti-GluR2 antibody was used with Western blotting. Significantly, that heterodimer complex could not be visualized in Western blots using anti-mGluR3 antibody instead of an anti-mGluR2 antibody. This reinforces the notion that it is mGluR2 that dimerizes with 2AR. Finally, a close interaction between the two receptors was demonstrated using fluorescence resonance energy transfer in transfected HEK-293 cells.

The authors then used molecular chimaeras to localize the site on mGluR2 that was a requirement for heterodimerization with 2AR and showed that the transmembrane helices 4 and 5 were required for this interaction. The authors then tested the posit that the interaction between 2AR and mGluR2 served to integrate cross-talk between the serotonergic and glutamatergic pathways in the CNS. To this end they showed that activation of Gαq/11 by 2AR was reduced in cells coexpressing mGluR2 and that this effect was lessened by mGluR2 receptor agonists. Significantly, this activity was dependent on the 4 and 5 transmembrane domain of the mGluR2, the domain required to form heterodimers.

Having demonstrated an impact of receptor dimerization on G protein signaling, the authors then investigated whether dimerization affected either receptor-modulated changes in c-fos, which is a marker of the signal-transduction stimulated by non-hallucinogenic 2AR agonists, or on levels of egr-2, which is induced by hallucinogens such as lysergic acid diethylamide (González-Maeso et al., 2007). The authors showed that stimulating mGluR2 with an mGluR2/3 agonist only affected the ability of hallucinogens to induce egr-2 in mouse SCx, suggesting the 2AR/mGluR2 dimers were involved in modulating hallucinogenic pathways of the CNS. To confirm this finding might have functional consequences. The authors then showed that the mGluR2/3 agonist suppressed the induction of hallucinogen-induced head twitches in the mice. These data supported the notion that receptor heterodimers are active in appropriate pathways in the CNS that have been used to model hallucinogenic effects. To extend this behavioral data, the authors also showed that mGluR2/3 agonist-induced locomotion and vertical activity were attenuated in 2AR-/- mice.

The authors had amassed a large quantity of data to suggest that 2AR/mGluR2 dimers may be important in generating hallucinogenic activity, which raised the possibility that altered levels of such dimers may be altered in the CNS of subjects with schizophrenia. To address this issue, the authors used radioligand binding to show that expression levels of 2AR and mGluR2/3 receptors were increased and decreased, respectively, in the dorsolateral prefrontal cortex (DLPFC) from untreated subjects with schizophrenia. In addition, the authors showed that the level of mGluR2, but not mGluR3, mRNA was decreased in the same CNS regions from the subjects with schizophrenia. These differences were not apparent in the same CNS regions from subjects with schizophrenia who had been treated with antipsychotic drugs. This raised the possibility that antipsychotic drug treatment may affect levels of 2AR/mGluR2 dimerization, and therefore the authors went on to show that clozapine downregulated levels of the mRNA for the two receptors in mouse cortex. The 2AR was critical in this process as clozapine did not downregulate mGluR2 mRNA in 2AR-/- mice. Haloperidol treatment had no effect on the expression of either 2AR or mGluR2. Finally, it was shown that levels of receptor binding to both receptors were reduced with aging.

From this large amount of data, the authors could conclude that they had shown that 2AR/mGluR2 heterodimers are important in hallucinogenic pathways of the CNS, using both cellular and animal models. They also argue that increased expression of 2AR and decreased expression of mGluR2 in the cortex of subjects with schizophrenia predispose these individuals to hallucinations. Presumably, therefore, the reduction in 2AR caused by certain antipsychotic drugs would be a mechanism by which a potential imbalance in heterodimer formation could be reversed to lessen hallucinations. Finally, the authors argue that the propensity for antipsychotic drugs and age to decrease levels of 2AR is why 2AR levels are reported as decreased in the majority of prior studies in schizophrenia (Dean, 2003), which mainly used cohorts made up of either treated or older subjects with schizophrenia.

As is often the case, the proposed link of a clear finding of 2AR/mGluR2 heterodimers in the mammalian cortex to hallucinations and then schizophrenia is dependent on data from the CNS of subjects with the disorder. Like many novel and compelling discoveries, the data from animal and cellular models appear clear-cut. However, there are some issues that leave in doubt the link between changes in receptor dimerization and schizophrenia. In particular, the authors did not demonstrate altered levels of dimerized receptors using the co-immunoprecipitation/Western blot approach; rather, they rely on inferences from the measurement of the two receptors separately using radioligand binding. In addition, the authors have not addressed the fact that the majority of imaging studies, many using young drug naïve subjects, did not find changes in levels of the 2AR in subjects with the disorder (Verhoeff et al., 2000; Lewis et al., 1999; Okubo et al., 2000; Trichard et al., 1998). The argument that findings in postmortem studies showing decreased levels of 2AR were due to studies being completed on treated or older subjects with the disorder is also not supported by neuroimaging studies showing decreased levels of 2AR in subjects with schizophrenia who were younger than 29 years of age (Ngan et al., 2000) or who were at high risk for the disorder (Hurlemann et al., 2005). These later studies suggest that low levels of 2AR may be more apparent earlier in the disease progression.

It is clear that the report of increased levels of 2AR with schizophrenia in the paper reporting the discovery of the 2AR/mGluR2 heterodimers (González-Maeso et al., 2008) is at odds with other postmortem (Dean, 2003) and neuroimaging studies (see above). This raises the possibility that the postmortem findings are in some way unique to the tissue collection used in the study. One difference in the postmortem tissue used in the study is that 85 percent of the subjects with schizophrenia had died by suicide. This would be higher than in most other studies of schizophrenia using postmortem CNS. Significantly, a number of studies have reported an increase in 2AR in the cortex of subjects that had died by suicide (Pandey et al., 2002; Mann et al., 1986; Hrdina et al., 1993; Escribá et al., 2004). This means the increased levels of 2AR reported in the study on heterodimers may be associated with suicide within schizophrenia, rather than schizophrenia per se.

In conclusion, like any novel finding, there are a number of important issues that will need addressing in future testing of the hypothesis that altered 2AR/mGluR2 heterodimerization is involved in the pathology of schizophrenia. However, the idea that changes in receptor heterodimerization could be involved in the pathology of schizophrenia is an exciting new direction arising from what is an excellent broad-based approach to understanding this complex disorder.

References:

González-Maeso J, Ang RL, Yuen T, Chan P, Weisstaub NV, López-Giménez JF, Zhou M, Okawa Y, Callado LF, Milligan G, Gingrich JA, Filizola M, Meana JJ, Sealfon SC. Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature. 2008 Mar 6;452(7183):93-7. Abstract

Angers S, Salahpour A, Bouvier M. Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol. 2002 Jan 1;42():409-35. Abstract

González-Maeso J, Weisstaub NV, Zhou M, Chan P, Ivic L, Ang R, Lira A, Bradley-Moore M, Ge Y, Zhou Q, Sealfon SC, Gingrich JA. Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior. Neuron. 2007 Feb 1;53(3):439-52. Abstract

Dean B. The cortical serotonin2A receptor and the pathology of schizophrenia: a likely accomplice. J Neurochem. 2003 Apr 1;85(1):1-13. Abstract

Verhoeff NP, Meyer JH, Kecojevic A, Hussey D, Lewis R, Tauscher J, Zipursky RB, Kapur S. A voxel-by-voxel analysis of [18F]setoperone PET data shows no substantial serotonin 5-HT(2A) receptor changes in schizophrenia. Psychiatry Res. 2000 Oct 30;99(3):123-35. Abstract

Lewis R, Kapur S, Jones C, DaSilva J, Brown GM, Wilson AA, Houle S, Zipursky RB. Serotonin 5-HT2 receptors in schizophrenia: a PET study using [18F]setoperone in neuroleptic-naive patients and normal subjects. Am J Psychiatry. 1999 Jan 1;156(1):72-8. Abstract

Okubo Y, Suhara T, Suzuki K, Kobayashi K, Inoue O, Terasaki O, Someya Y, Sassa T, Sudo Y, Matsushima E, Iyo M, Tateno Y, Toru M. Serotonin 5-HT2 receptors in schizophrenic patients studied by positron emission tomography. Life Sci. 2000 Jan 1;66(25):2455-64. Abstract

Trichard C, Paillère-Martinot ML, Attar-Levy D, Blin J, Feline A, Martinot JL. No serotonin 5-HT2A receptor density abnormality in the cortex of schizophrenic patients studied with PET. Schizophr Res. 1998 May 4;31(1):13-7. Abstract

Ngan ET, Yatham LN, Ruth TJ, Liddle PF. Decreased serotonin 2A receptor densities in neuroleptic-naive patients with schizophrenia: A PET study using [(18)F]setoperone. Am J Psychiatry. 2000 Jun 1;157(6):1016-8. Abstract

Hurlemann R, Boy C, Meyer PT, Scherk H, Wagner M, Herzog H, Coenen HH, Vogeley K, Falkai P, Zilles K, Maier W, Bauer A. Decreased prefrontal 5-HT2A receptor binding in subjects at enhanced risk for schizophrenia. Anat Embryol (Berl). 2005 Dec 1;210(5-6):519-23. Abstract

Pandey GN, Dwivedi Y, Rizavi HS, Ren X, Pandey SC, Pesold C, Roberts RC, Conley RR, Tamminga CA. Higher expression of serotonin 5-HT(2A) receptors in the postmortem brains of teenage suicide victims. Am J Psychiatry. 2002 Mar 1;159(3):419-29. Abstract

Mann JJ, Stanley M, McBride PA, McEwen BS. Increased serotonin2 and beta-adrenergic receptor binding in the frontal cortices of suicide victims. Arch Gen Psychiatry. 1986 Oct 1;43(10):954-9. Abstract

Hrdina PD, Demeter E, Vu TB, Sótónyi P, Palkovits M. 5-HT uptake sites and 5-HT2 receptors in brain of antidepressant-free suicide victims/depressives: increase in 5-HT2 sites in cortex and amygdala. Brain Res. 1993 Jun 18;614(1-2):37-44. Abstract

Escribá PV, Ozaita A, García-Sevilla JA. Increased mRNA expression of alpha2A-adrenoceptors, serotonin receptors and mu-opioid receptors in the brains of suicide victims. Neuropsychopharmacology. 2004 Aug 1;29(8):1512-21. Abstract

View all comments by Brian Dean

Related News: 5HT and Glutamate Receptors—Unique Complex Linked to Psychosis

Comment by:  Gerard J. Marek (Disclosure)
Submitted 21 March 2008
Posted 21 March 2008

Another bicycle trip?
Ever since dopamine was first implicated in the therapeutic effects of antipsychotic drugs by Arvid Carlsson and colleagues over 50 years ago, and then dopamine D2 receptors were implicated in the Parkinsonian side effects and late-evolving movement disorders, an intense search has been underway for antipsychotic drugs that might act through other mechanisms. In parallel with this search, drugs with psychotomimetic effects in healthy volunteers or exacerbating psychosis have also been used to discover new antipsychotic drugs. With an evolving understanding of the neuropharmacology underlying ketamine or PCP, amphetamines, and serotonergic hallucinogens (LSD, mescaline, and psilocybin), glutamatergic, dopaminergic, and serotonergic theories of psychotic pathophysiology have been advanced. Converging evidence points to activation of 5-HT2A receptors as a necessary action in the psychotomimetic effects of the serotonergic “hallucinogens.” The recent description of a proof-of-concept clinical study where a prodrug for a metabotropic glutamate2/3 (mGlu2/3) receptor agonist exerted therapeutic effects in schizophrenic patients may be the most promising report for an elusive antipsychotic medication generally viewed as lacking direct effects on dopamine D2 receptors (Patil et al., 2007). More recently, a report has appeared which raises the possibility that glutamate and serotonin may be involved in the therapeutic effects of mGlu2/3 receptors by virtue of a molecular complex between mGlu2 and 5-HT2A receptors (González-Maeso et al., 2008). Beyond replication of these effects in other laboratories, several fundamental questions have been raised that should be addressed.

First, does this type of interaction occur in the prefrontal cortex, which (through cortico-thalamo-striatal loops) is more closely related to the core symptoms of schizophrenia than the somatosensory cortex? Second, are the therapeutic actions of mGlu2/3 receptors mediated through activation of postsynaptic mGlu2 receptors, rather than presynaptic mGlu2 receptors (Marek et al., 2001)? Third, do other G protein-coupled receptors similarly act through complexes with 5-HT2A receptors?

Further research will be required to address this first question, especially since both mGlu2/3 agonists and NMDA receptor antagonists appear to have more potent or efficacious effects in the prefrontal cortex than the somatosensory cortex under either in vitro or in vivo conditions. The second question will be important to address at a fundamental level, since “simple” intra-cortical processes invoke different levels of analyses than do hypotheses that presynaptic mGlu2 receptors on long-loop afferents may play key roles as therapeutic targets. In fact, previous experiments involving rescue of 5-HT2A receptors in the cortex or thalamus appear to be compromised by confounds. Namely, the cortical rescue of 5-HT2A receptors in the htr2A-/- mice using the Emx1 promoter does not rule out an involvement of afferents to the cortex from a poorly understood region involved in integrating multi-modal associations, the claustrum. 5-HT2A receptor expression was also restored to the claustrum with this rescue strategy (Weisstaub et al., 2006). The thalamic rescue of 5-HT2A receptors, which generally fails to reprise the effects seen in the cortical rescue preparation, may be problematic in that the promoter utilized expresses SERT in thalamocortical projections from primary sensory relay neurons rather than the midline and intralaminar thalamic neurons intimately involved in arousal and stress-related biology (Lebrand et al., 1996; Van der Werf et al., 2002). The relatively dense expression of cortical 5-HT2A and mGlu2 receptor expression in layers I and Va of the prefrontal cortex is an excellent match for the laminar distribution of afferents from the midline and intralaminar thalamic nuclei (Marek et al., 2001). Further work is required to understand the magnitude of the involvement of thalamic afferents from the posterior thalamic nucleus to the somatosensory cortex vs. involvement of the afferents from midline and intralaminar thalamic nuclei throughout the prefrontal cortex. Third, do other Gi/Go-coupled GPCRs form complexes with 5-HT2A receptors? Other, much stronger candidates for such a role than mGlu3 receptors would be μ-opioid receptors and adenosine A1 receptors. The physiology of both μ-opioid receptors and adenosine A1 receptors share a striking degree of similarity with mGlu2 receptors ranging from regulating excitatory synaptic afferents to the prefrontal cortex in slice preparations to in vivo modulation of the three major classes of psychotomimetic drugs.

Both the replication of the exciting basic findings reported by the Gingerich and Sealfon laboratories and answers to these questions above should add another chapter to the story that began in earnest over 60 years ago with a bicycle ride by the Sandoz chemist Albert Hoffman following the ingestion of the twenty-fifth lysergic diethylamide that he had synthesized.

References:

Patil ST, Zhang L, Martenyi F, Lowe SL, Jackson KA, Andreev BV, Avedisova AS, Bardenstein LM, Gurovich IY, Morozova MA, Mosolov SN, Neznanov NG, Reznik AM, Smulevich AB, Tochilov VA, Johnson BG, Monn JA, Schoepp DD. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial. Nat Med. 2007 Sep 1;13(9):1102-7. Abstract

González-Maeso J, Ang RL, Yuen T, Chan P, Weisstaub NV, López-Giménez JF, Zhou M, Okawa Y, Callado LF, Milligan G, Gingrich JA, Filizola M, Meana JJ, Sealfon SC. Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature. 2008 Mar 6;452(7183):93-7. Abstract

Marek GJ, Wright RA, Gewirtz JC, Schoepp DD. A major role for thalamocortical afferents in serotonergic hallucinogen receptor function in the rat neocortex. Neuroscience. 2001 Jan 1;105(2):379-92. Abstract

Weisstaub NV, Zhou M, Lira A, Lambe E, González-Maeso J, Hornung JP, Sibille E, Underwood M, Itohara S, Dauer WT, Ansorge MS, Morelli E, Mann JJ, Toth M, Aghajanian G, Sealfon SC, Hen R, Gingrich JA. Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science. 2006 Jul 28;313(5786):536-40. Abstract

Lebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P. Transient uptake and storage of serotonin in developing thalamic neurons. Neuron. 1996 Nov 1;17(5):823-35. Abstract

Van der Werf YD, Witter MP, Groenewegen HJ. The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence for participation in processes of arousal and awareness. Brain Res Brain Res Rev. 2002 Sep 1;39(2-3):107-40. Abstract

View all comments by Gerard J. Marek

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Shoreh Ershadi
Submitted 8 June 2008
Posted 9 June 2008
  I recommend the Primary Papers

Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Todd Lencz, Anil Malhotra (SRF Advisor)
Submitted 3 July 2009
Posted 3 July 2009

The three companion papers published in Nature provide important new evidence for a role of the MHC complex and common variation across the genome in risk for schizophrenia. These studies have exploited the availability of comprehensive genotyping technologies, coupled with large cohorts of cases and controls, to identify candidate loci for disease susceptibility.

A notable feature of these papers is the clear willingness of each of the groups to share its data, and to provide overlapping presentations of each others’ results. The combination of datasets permitted the statistical significance of the MHC findings to emerge, thereby increasing confidence in results. The implication that immune processes may interact with genetic risk to influence schizophrenia risk is consistent with several lines of evidence, including our own small GWAS study (Lencz et al., 2007) implicating cytokine receptors in schizophrenia susceptibility.

Perhaps most intriguing is the finding from the International Schizophrenia Consortium demonstrating that a “score” test—combining information from many thousands of common variants—can reliably differentiate patients and controls across multiple psychiatric cohorts. These results indicate that hundreds, if not thousands, of genes of small effect may contribute to schizophrenia risk. Moreover, these same genes were shown to contribute to bipolar risk (but not risk for non-psychiatric disorders such as diabetes).

Much more work remains to be done in psychiatric genetics. While the score test accounted for about 3 percent of the observed case-control variance, statistical modeling suggested that common variation could explain as much as one-third or more of the total risk. Nevertheless, there remains a substantial proportion of genetic “dark matter” (unexplained variance), given the high heritability of a disorder such as schizophrenia. Complementary approaches are needed to further parse the source of the common genetic variance, as well as to identify rare yet highly penetrant mutations. Additional techniques, such as pharmacogenetic studies and endophenotypic research, will help to explicate the functionality and clinical significance of observed risk alleles.

View all comments by Todd Lencz
View all comments by Anil Malhotra

Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Daniel Weinberger, SRF Advisor
Submitted 3 July 2009
Posted 3 July 2009

The three Nature papers reporting GWAS results in a large sample of cases of schizophrenia and controls from around Western Europe and the U.S. are decidedly disappointing to those expecting this strategy to yield conclusive evidence of common variants predicting risk for schizophrenia. Why has this extensive and very costly effort not produced more impressive results? There are likely to be many explanations for this, involving the usual refrains about clinical and genetic heterogeneity, diagnostic imprecision, and technical limitations in the SNP chips. But the likely, more fundamental problem in psychiatric genetics involves the biologic complexity of the conditions themselves, which renders them especially poorly suited to the standard GWAS strategy. The GWA analytic model assumes fixed, predictable relationships between genetic risk and illness, but simple relationships between genetic risk and complex pathophysiological mechanisms are unlikely. Many biologic functions show non-linear relationships, and depending on the biologic context, more of a potential pathogenic factor, can make things worse or it can make them better. Studies of complex phenotypes in model systems illustrate that individual gene effects depend upon non-linear interactions with other genes (Toma et al., 2002; Shaoa et al, 2008). Similar observations are beginning to emerge in human disorders, e.g., in risk for cancer (Lo et al., 2008) and depression (Pezawas et al., 2008).

The GWA approach also assumes that diagnosis represents a unitary biological entity, but most clinical diagnoses are syndromal and biologically heterogeneous, and this is especially true in psychiatric disorders. Type 2 diabetes is the clinical expression of changes in multiple physiologic processes, including in pancreatic function, in adipose cell function, as well as in eating behavior. Likewise, hypertension results from abnormalities in many biologic processes (e.g., vascular reactivity, kidney function, CNS control of blood pressure, metabolic factors, sodium regulation), and even a large effect on any specific process within a subset of individuals will seem small when measured in large unrelated samples (Newton-Cheh et al., 2009). In the case of the cognitive and emotional problems associated with psychiatric disorders, the biologic pathways to clinical manifestations are probably much more heterogeneous. While the results of GWAS in disorders like type 2 diabetes and hypertension have been more informative than in the schizophrenia results so far, they, too, have been disappointing, considering all the fanfare about their expectations. But given the pathophysiologic realities of diabetes, hypertension, or psychiatric disorders, how could the effect of any common genetic variant acting on only one of the diverse pathophysiological mechanisms implicated in these disorders be anything other than small when measured in large pathophysiologically heterogeneous populations? Other approaches, e.g., family studies, studies of smaller but much better characterized samples, and studies of genetic interactions in these samples, will be necessary to understand the variable genetic architectures of such biologically complex and heterogeneous disorders.

References:

Toma DP, White KP, Hirsch J and Greenspan RJ: Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nature Genetics 2002; 31: 349-353. Abstract

Shaoa H, Burragea LC, Sinasac DS et al : Genetic architecture of complex traits: Large phenotypic effects and pervasive epistasis. PNAS 2008 105: 19910–19914. Abstract

Lo S-W, Chernoff H, Cong L, Ding Y, and Zheng T: Discovering interactions among BRCA1 and other candidate genes associated with sporadic breast cancer. PNAS 2008; 105: 12387–12392. Abstract

Pezawas L, Meyer-Lindenberg A, Goldman AL, et al.: Biologic epistasis between BDNF and SLC6A4 and implications for depression. Mol Psychiatry 2008;13:709-716. Abstract

Newton-Cheh C, Larson MG, Vasan RS: Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure. Nat Gen 2009; 41: 348-353. Abstract

View all comments by Daniel Weinberger

Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Irving Gottesman
Submitted 3 July 2009
Posted 3 July 2009
  I recommend the Primary Papers

The synthesis and extraction of the essence of the 3 Nature papers by Heimer and Farley represents science reporting at its best. Completion of the task while the ink was still wet shows that SRF is indeed in good hands. Congratulations on being concise, even-handed, non-judgmental, and challenging under the pressure of time.

View all comments by Irving Gottesman

Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Christopher Ross, Russell L. Margolis
Submitted 6 July 2009
Posted 6 July 2009

Schizophrenia Genetics: Glass Half Full?
While it may be disappointing that the GWAS described above did not identify more genes, they nevertheless represent a landmark in psychiatric genetics and suggest a dual approach for the future: continued large-scale genetic association studies along with alternative genetic approaches leading to the discovery of new genetic etiologies, and more functional investigations to identify pathways of pathogenesis—which may themselves suggest new etiologies.

The consistent identification of an association with the MHC locus reinforces (without proving, as pointed out in the SRF news story) long-standing interest in the involvement of infectious or immune factors in schizophrenia pathogenesis (Yolken and Torrey, 2008). Epidemiologic and neuropathological studies that include patients selected for the presence or absence of immunologic genetic risk variants could potentially clarify etiology; cell and mouse model studies could clarify pathogenesis (Ayhan et al., 2009). It is striking that a major genetic finding in schizophrenia serves to reinforce the concept of environmental risk factors.

The two specific genes identified by the SGENE consortium, NRGN and TCF4, offer intriguing new leads into schizophrenia. This should foster a number of further genetic and neurobiological studies. Deep resequencing (and CNV analysis) can detect rare causative mutations, as exemplified by TCF4 mutations leading to Pitt-Hopkins syndrome. Neurogranin already has clear connections to interesting signaling pathways related to glutamate transmission. A hope is that further studies of both gene products and their interactions will identify pathogenic pathways.

The ISC used common genetic variants “en masse” to generate a “polygene score” from discovery samples of patients; that score was able to predict case status in test populations. The success of this approach provides very strong evidence that a portion of schizophrenia risk status is attributable to common genetic variants acting in concert and that schizophrenia shares genetic factors with bipolar disorder, but not with other diseases. This analysis has multiple practical implications for the direction of research. First, since polygenic factors explain only a portion of the genetic risk, the search for other genetic factors—rare mutations of major effect detectable by deep sequencing, CNVs, variations in tandem repeats (Bruce et al., 2009, in press), and other genomic lesions—takes on new importance. Second, a meaningful integration of polygenic factors in a way that facilitates understanding of schizophrenia pathogenesis and the discovery of therapeutic targets will require identification of relevant pathways. Examination of patient-derived material—such as neurons differentiated from induced pluripotent stem cells taken from well-characterized, patient populations—may be of great value.

The remarkable overlap between the genetic factors of schizophrenia and bipolar disorder suggests the need for further and more inclusive clinical studies—not just of “endophenotypes,” but also of the phenotypes themselves, together, rather than in isolation (Potash and Bienvenu, 2009). For instance, it is only within the past few years that the importance of cognitive dysfunction in schizophrenia has been appreciated. Cognition in bipolar disorder is even less well studied.

How much is really known about the longitudinal course of both disorders? Do genetic factors predict disease outcome? It is only recently that studies have focused intensively on the early course of schizophrenia and its prodrome. Much more is still to be learned, and even less is known about bipolar disorder. In conjunction with this greater understanding of clinical phenotype, it will clearly be necessary to refine the approach to phenotype by establishing the biological framework for these diseases and by establishing biomarkers, such as disruption in white matter (Karlsgodt et al., 2009) or abnormalities in functional networks (Demirci et al., 2009), that cut across current nosological categories. In turn, longitudinal study of clinical, imaging, and functional outcomes of schizophrenia and bipolar disorders should facilitate both focused candidate genetic studies and GWAS of large populations.

References:

Yolken RH, Torrey EF. Are some cases of psychosis caused by microbial agents? A review of the evidence. Mol Psychiatry. 2008 May;13(5):470-9. Abstract

Ayhan Y, Sawa A, Ross CA, Pletnikov MV. Animal models of gene-environment interactions in schizophrenia. Behav Brain Res. 2009 Apr 18. Abstract

Potash JB, Bienvenu OJ. Neuropsychiatric disorders: Shared genetics of bipolar disorder and schizophrenia. Nat Rev Neurol. 2009 Jun;5(6):299-300. Abstract

Karlsgodt KH, Niendam TA, Bearden CE, Cannon TD. White matter integrity and prediction of social and role functioning in subjects at ultra-high risk for psychosis. Biol Psychiatry. 2009 May 6. Epub ahead of print. Abstract

Demirci O, Stevens MC, Andreasen NC, Michael A, Liu J, White T, Pearlson GD, Clark VP, Calhoun VD. Investigation of relationships between fMRI brain networks in the spectral domain using ICA and Granger causality reveals distinct differences between schizophrenia patients and healthy controls. Neuroimage. 2009 Jun;46(2):419-31. Abstract

Bruce HA, Sachs NA, Rudnicki DD, Lin SG, Willour VL, Cowell JK, Conroy J, McQuaid D, Rossi M, Gaile DP, Nowak NJ, Holmes SE, Sklar P, Ross CA, DeLisi LE, Margolis RL. Long tandem repeats as a form of genomic copy number variation: structure and length polymorphism of a chromosome 5p repeat in control and schizophrenia populations. Psychiatric Genetics, in press.

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Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  David Collier
Submitted 6 July 2009
Posted 6 July 2009
  I recommend the Primary Papers

This report is unnecessarily negative, from my point of view. The three studies show not only that GWAS can identify susceptibility alleles for schizophrenia, but that the majority of risk comes from common variants of small effect. These can be found, but as in other complex traits and diseases, such as obesity and height, considerable power is needed, because effect sizes are small, meaning greater samples sizes. This approach works: there are now almost 60 variants influencing height (Hirschhorn et al., 2009; Soranzo et al., 2009; Sovio et al., 2009). Furthermore, the genes identified so far from both traditional mapping, CNV analysis and GWAS, point to two biological pathways, the integrity of the synapse (neurexin 1, neurogranin, etc.) and the wnt/GSK3β signaling pathway (DISC1, TCF4, etc.), which is involved in functions such as neurogenesis in the brain. The identification of disease pathways for schizophrenia has major implications and should not be underestimated. It would be daft to lose nerve now and turn away from GWAS just as they are bearing fruit.

I would like to correct/expand on my comments to Peter Farley, to say that while statistical significance for some markers may be reached sooner, significance for many of the hundreds if not thousands of common schizophrenia susceptibility alleles of small effect might not emerge until samples of 100,000 cases and more than 100,000 controls have been collected. Another point is that organizations such the Wellcome Trust are already assembling case samples for schizophrenia as well as control samples.

Also, I would like to clarify that I believe the remainder of genetic variation, after common variation has been taken into account, will come from some combination of rare CNVs, other rare variants such as SNPs and other types of genetic marker such as variable number of tandem repeats (VNTRs) and of course the much neglected contribution from gene-environment interactions, in which main genetic effects may be obscured.

References:

Hirschhorn JN, Lettre G. Progress in genome-wide association studies of human height. Horm Res. 2009 Apr 1 ; 71 Suppl 2():5-13. Abstract

Soranzo N, Rivadeneira F, Chinappen-Horsley U, Malkina I, Richards JB, Hammond N, Stolk L, Nica A, Inouye M, Hofman A, Stephens J, Wheeler E, Arp P, Gwilliam R, Jhamai PM, Potter S, Chaney A, Ghori MJ, Ravindrarajah R, Ermakov S, Estrada K, Pols HA, Williams FM, McArdle WL, van Meurs JB, Loos RJ, Dermitzakis ET, Ahmadi KR, Hart DJ, Ouwehand WH, Wareham NJ, Barroso I, Sandhu MS, Strachan DP, Livshits G, Spector TD, Uitterlinden AG, Deloukas P. Meta-analysis of genome-wide scans for human adult stature identifies novel Loci and associations with measures of skeletal frame size. PLoS Genet. 2009 Apr 1 ; 5(4):e1000445. Abstract

Sovio U, Bennett AJ, Millwood IY, Molitor J, O'Reilly PF, Timpson NJ, Kaakinen M, Laitinen J, Haukka J, Pillas D, Tzoulaki I, Molitor J, Hoggart C, Coin LJ, Whittaker J, Pouta A, Hartikainen AL, Freimer NB, Widen E, Peltonen L, Elliott P, McCarthy MI, Jarvelin MR. Genetic determinants of height growth assessed longitudinally from infancy to adulthood in the northern Finland birth cohort 1966. PLoS Genet. 2009 Mar 1 ; 5(3):e1000409. Abstract

View all comments by David Collier

Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Michael O'Donovan, SRF Advisor, Nick Craddock, Michael Owen (SRF Advisor)
Submitted 9 July 2009
Posted 9 July 2009

Some commentators in their reflections take a rather negative view on what has been achieved through the application of GWAS technology to schizophrenia and psychiatric disorders more generally. We strongly disagree with this position. Below, we give examples of a number of statements that can be made about the aetiology of schizophrenia and bipolar disorder that could not be made at high levels of confidence even two years ago that are based upon evidence deriving from the application of GWAS.

1. We know with confidence that the role of rare copy number variants in schizophrenia is not limited to 22q11DS (VCFS) (reviewed recently in O’Donovan et al., 2009). We do not yet know how much of a contribution, but we know the identity of an increasing number of these. Most span multiple genes so it may prove problematic as it has in 22q11DS to identify the relevant molecular mechanisms. However, for one locus, the CNVs are limited to a single gene: Neurexin1 (Kirov et al., 2008; Rujescu et al., 2009). Genetic findings are merely the start of the journey to a deeper biological understanding, but no doubt many neurobiological researchers have already embarked on that journey in respect of neurexin1.

2. Although we have argued in this forum that some of the major pre-GWAS findings in schizophrenia very likely reflect true susceptibility genes (DTNBP1, NRG1, etc), we now have at least 4 novel loci where the evidence is more definitive (ZNF804A, MHC, NRGN, TCF4), (O’Donovan et al., 2008a; ISC, 2009; Shi et al., 2009; Stefansson et al., 2009) and two novel loci (Ferreira et al., 2008) in bipolar disorder (ANK3 and CACNA1C), at least one of which (CACNA1C) additionally confers risk of schizophrenia (Green et al., 2009). This is obviously a small part of the picture, but it is certainly better than no picture at all. These findings also offer a much more secure foundation than the earlier findings upon which to build follow up studies, for example brain imaging, and cognitive phenotypes (Esslinger et al., 2009), and even candidate gene studies. We would not regard the first convincing evidence that altered calcium channel function is a primary aetiological event in at least some forms of psychosis as a trivial gain in knowledge.

3. We can say with confidence that common alleles of small effect are abundant in schizophrenia, and that they contribute to a substantial part of the population risk (ISC, 2009). Identifying any one of these at stringent levels of statistical significance may be challenging in terms of sample sizes. As we have pointed out before, merging multiple datasets may indeed obscure some true associations because of sometimes unpredictable relationships between risk alleles and those assayed indirectly in GWAS studies (Moskvina and O’Donovan, 2007). Nevertheless, that many of the same alleles are overrepresented in multiple independent GWAS datasets from different countries (ISC, 2009) means that larger samples offer the prospect of identifying many more of these. This is not to say that large samples are the only approach; genetic heterogeneity may well underpin some aspects of clinical heterogeneity (Craddock et al., 2009a). However, with the exception of individual large pedigrees, it is not yet evident which type of clinical sample one should base a small scale study on. It should also be self-evident that the analysis of multiple samples, each with a different phenotypic structure, will pose major problems in respect of multiple testing and subsequent replication. Moreover, ascertaining special samples that represent putative subtypes of the clinical (and endophenotypic) spectrum of psychosis will first require large samples to be carefully assessed and the relevant subjects extracted. Subsequently, downstream, evaluation of specific genotype-phenotype relationships will require the remainder of the clinical population to be genotyped in a suitably powered way to show that those effects are specific to some clinical features of the disorder. Increasingly, it is ascertainment and assessment that dominate the cost of GWAS studies so it is not clear this approach will achieve any economies. We must also remember that after a GWAS study, there remains the opportunity to look in a controlled manner for relatively specific associations in the context of the heterogeneous clinical picture. For example we are aware of a number of papers in development that will exploit the sorts of multi-locus tests reported by the ISC to refine diagnostics, and no doubt many other applications of this will emerge in the next year or so.

Critics should bear in mind that the GWAS data are not just there for the ‘headline’ genome-wide findings, but that the data will be available to mine for years to come. The findings reported to date are based on only the simplest analyses.

4. If it were the case that the thousands of SNPs of small effect were randomly distributed across biological systems, none being of more relevance to pathophysiology than another, identifying them would probably be a pointless endeavour. However, there is no reason to believe this will be the case. We have recently shown that in bipolar disorder, the GWAS signals are enriched in particular biological pathways (Holmans et al., 2009) and we also published strong evidence for a relatively selective involvement of the GABAergic system in schizoaffective disorder (Craddock et al., 2009b). We are aware of an as-yet unpublished independent sample with similar findings. We would not regard the first convincing evidence that altered GABA function is a primary aetiological event in at least some forms of psychosis as a trivial gain in knowledge.

Incidentally it is a common misconception that the identification of risk alleles of small effect necessarily confers no useful insights into pathogenesis and possible drug targets. For example, common alleles in PPARG and KCNJ11 have been robustly shown to confer risk to Type 2 diabetes (T2D) but with odds ratios in the region of only 1.14 (of similar magnitude to those revealed by GWAS of schizophrenia). PPARG encodes the target for the thiazolidinedione class of drugs used to treat T2D. KCNJ11 encodes part of the target for another class of diabetes drug, the sulphonylureas (Prokopenko et al., 2008). Moreover, studies of novel associated variants identified in T2D GWAS in healthy, non-diabetic, populations have demonstrated that for most, the primary effect on T2D susceptibility is mediated through deleterious effects on insulin secretion, rather than insulin action (Prokopenko et al., 2008). Further examples of insights into the biology of common diseases coming from the identification of loci of small effect are the implication of the complement system in age-related macular degeneration and autophagy in Crohn’s disease (Hirschhorn, 2009). Already, efforts are under way to translate the new recognition of the role of autophagy in Crohn’s disease into new therapeutic leads (Hirschhorn, 2009). Of course many of the loci identified in GWAS implicate genes whose functions are as yet largely or completely unknown, and determining those functions is a prerequisite of translating those findings. Nevertheless, we believe that the greatest benefits that will accrue from the continued discovery of risk loci through GWAS will come from the assembly of that information into novel disease pathways leading to novel therapeutic targets.

5. We can say with confidence that bipolar disorder and schizophrenia substantially overlap, at least in terms of polygenic risk (ISC, 2009). As clinicians, we do not regard that knowledge as a trivial achievement.

6. We can say with confidence from studies of CNVs that schizophrenia and autism share at least some risk factors in common (O’Donovan et al., 2009). We believe that is also an important insight.

The above are major achievements in what we expect to be a long but accelerating process of picking apart the origins of schizophrenia and other psychotic disorders. We do not think that any other research discipline in psychiatry has done more to advance that knowledge in the past 100 years.

Like that other common familial diseases, the genetics of schizophrenia and bipolar disorder is a “mixed economy” of common alleles of small effect and rare alleles of large and small effects, including CNVs. Those who are concerned at the cost of collecting large samples for GWAS studies must bear in mind that the robust identification of both types of mutation will require similarly large samples; we will just have to get used to that fact if we want to make progress. Collecting samples like this may be expensive, but as clinicians, we know those costs are trivial compared with the human and economic costs of psychotic disorders.

The question of phenotype definition is one which we have repeatedly addressed (Craddock et al., 2009a). Unquestionably, if we knew the true pathophysiological basis of these disorders, we could do better. The fact is that we don’t. Given that, it must be extremely encouraging that despite the problems, risk loci can be robustly identified by GWAS using samples defined by current diagnostic criteria. Moreover, armed with GWAS data in these heterogeneous populations, additional risk genes can be identified through strategies aimed at refining the phenotype that are not constrained by the current dichotomous view of the functional psychoses. We have shown at least one way in which this might be achieved without imposing a further burden of multiple testing (Craddock et al., 2009b), and have little doubt that others will emerge. We agree that approaches to phenotyping that more directly index underlying pathophysiology are highly appealing, and will ultimately be necessary for understanding the mechanistic relationships between gene and disorder. However, the two cardinal assumptions upon which the use of endophenotypes is predicated for gene discovery are questionable. First, there is little good evidence that putative endophenotypes are substantially simpler genetically than “exophenotypes” (Flint and Munafo, 2007). Second, there is rarely good evidence that the current crop of popular putative endophenotypes lie on the disease pathway, indeed there seems to be substantial pleiotropy in the genetics of complex traits, psychosis included (Prokopenko et al., 2008; O’Donovan et al., 2008b).

Finally, we reiterate that while only small parts of the heritability of any complex disorder have been accounted for, large-scale genetic approaches have been extremely successful in studies of non-psychiatric diseases (Manolio et al., 2008) and have led to substantial advances in our understanding of pathogenesis, even for diseases like Crohn’s disease where there was already prior knowledge of pathogenesis from other research methods (Mathew, 2008).

Psychiatry starts from a situation in which there is no robust prior knowledge of pathogenesis for the major phenotypes. Recent findings suggest that mental illness may be the medical field that will actually benefit most over the coming years from application of these powerful molecular genetic technologies.

References:
Craddock N, O'Donovan MC, Owen MJ. (2009a) Psychosis Genetics: Modeling the Relationship between Schizophrenia, Bipolar Disorder, and Mixed (or "Schizoaffective") Psychoses. Schizophrenia Bulletin 35(3):482-490. Abstract

Craddock N, Jones L, Jones IR, Kirov G, Green EK, Grozeva D, Moskvina V, Nikolov I, Hamshere ML, Vukcevic D, Caesar S, Gordon-Smith K, Fraser C, Russell E, Norton N, Breen G, St Clair D, Collier DA, Young AH, Ferrier IN, Farmer A, McGuffin P, Holmans PA, Wellcome Trust Case Control Consortium (WTCCC), Donnelly P, Owen MJ, O’Donovan MC. Strong genetic evidence for a selective influence of GABAA receptors on a component of the bipolar disorder phenotype. Molecular Psychiatry advanced online publication 1 July 2008; doi:10.1038/mp.2008.66. (b) Abstract

Esslinger C, Walter H, Kirsch P, Erk S, Schnell K, Arnold C, Haddad L, Mier D, Opitz von Boberfeld C, Raab K, Witt SH, Rietschel M, Cichon S, Meyer-Lindenberg A. (2009) Neural mechanisms of a genome-wide supported psychosis variant. Science 324(5927):605. Abstract

Ferreira MAR, O’Donovan MC, Meng YA, Jones IR, Ruderfer DM, Jones L, Fan J, Kirov G, Perlis RH, Green EK, Smoller JW, Grozeva D, Stone J, Nikolov I, Chambert K, Hamshere ML, Nimgaonkar V, Moskvina V, Thase ME, Caesar S, Sachs GS, Franklin J, Gordon-Smith K, Ardlie KG, Gabriel SB, Fraser C, Blumenstiel B, Defelice M, Breen G, Gill M, Morris DW, Elkin A, Muir WJ, McGhee KA, Williamson R, MacIntyre DJ, McLean A, St Clair D, VanBeck M, Pereira A, Kandaswamy R, McQuillin A, Collier DA, Bass NJ, Young AH, Lawrence J, Ferrier IN, Anjorin A, Farmer A, Curtis D, Scolnick EM, McGuffin P, Daly MJ, Corvin AP, Holmans PA, Blackwood DH, Wellcome Trust Case Control Consortium (WTCCC), Gurling HM, Owen MJ, Purcell SM, Sklar P and Craddock NJ. (2008) Collaborative genome-wide association analysis of 10,596 individuals supports a role for Ankyrin-G (ANK3) and the alpha-1C subunit of the L-type voltage-gated calcium channel (CACNA1C) in bipolar disorder. Nature Genetics 40:1056-1058. Abstract

Flint J, Munafò MR. (2007) The endophenotype concept in psychiatric genetics. Psychological Medicine 37(2):163-180. Abstract

Green EK, Grozeva D, Jones I, Jones L, Kirov G, Caesar S, Gordon-Smith K, Fraser C, Forty L, Russell E, Hamshere ML, Moskvina V, Nikolov I, Farmer A, McGuffin P, Wellcome Trust Case Consortium, Holmans PA, Owen MJ, O’Donovan MC and Craddock N. (2009) Bipolar disorder risk allele at CACNA1C also confers risk to recurrent major depression and to schizophrenia. Molecular Psychiatry (in press).

Hirschhorn JN. (2009) Genomewide association studies--illuminating biologic pathways. New England Journal of Medicine 360(17):1699-1701. Abstract

Holmans P, Green E, Pahwa J, Ferreira M, Purcell S, Sklar P, Owen M, O’Donovan M, Craddock N. Gene ontology analysis of GWAS datasets provide insights into the biology of bipolar disorder. The American Journal of Human Genetics 2009 Jun 17 [Epub ahead of print]. International Schizophrenia Consortium. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 2009 Jul 1 [Epub ahead of print]. Abstract

Kirov G, Gumus D, Chen W, Norton N, Georgieva L, Sari M, O'Donovan MC, Erdogan F, Owen MJ, Ropers HH, Ullmann R. (2008) Comparative genome hybridization suggests a role for NRXN1 and APBA2 in schizophrenia. Human Molecular Genetics 17(3):458-465. Abstract

Manolio TA, Brooks LD, Collins FS. (2008) A HapMap harvest of insights into the genetics of common disease. Journal of Clinical Investigation 118(5):1590-1605. Abstract

Mathew CG. (2008) New links to the pathogenesis of Crohn disease provided by genome-wide association scans. Nature Review Genetics 9(1):9-14. Abstract

Moskvina V and O'Donovan MC. (2007) Detailed analysis of the relative power of direct and indirect association studies and the implications for their interpretation. Human Heredity 64(1):63-73. Abstract

O’Donovan MC, Kirov G, Owen MJ. (2008a) Phenotypic variations on the theme of CNVs. Nature Genetics 40(12):1392-1393. Abstract

O’Donovan MC, Craddock N, Norton N, Williams H, Peirce T, Moskvina V, Nikolov I, Hamshere M, Carroll L, Georgieva L, Dwyer S, Holmans P, Marchini JL, Spencer C, Howie B, Leung H-T, Hartmann AM, Möller H-J, Morris DW, Shi Y, Feng G, Hoffmann P, Propping P, Vasilescu C, Maier W, Rietschel M, Zammit S, Schumacher J, Quinn EM, Schulze TG, Williams NM, Giegling I, Iwata N, Ikeda M, Darvasi A, Shifman S, He L, Duan J, Sanders AR, Levinson DF, Gejman P, Molecular Genetics of Schizophrenia Collaboration , Cichon S, Nöthen MM, Gill M, Corvin A, Rujescu D, Kirov G, Owen MJ. (2008b) Identification of novel schizophrenia loci by genome-wide association and follow-up. Nature Genetics 40:1053-1055. Abstract

O’Donovan MC, Craddock N, Owen MJ. Genetics of psychosis; Insights from views across the genome. Human Genetics 2009 Jun 12 [Epub ahead of print]. Abstract

Prokopenko I, McCarthy MI, Lindgren CM. (2008) Type 2 diabetes: new genes, new understanding. Trends in Genetics 24(12):613-621. Abstract

Rujescu D, Ingason A, Cichon S, Pietiläinen OP, Barnes MR, Toulopoulou T, Picchioni M, Vassos E, Ettinger U, Bramon E, Murray R, Ruggeri M, Tosato S, Bonetto C, Steinberg S, Sigurdsson E, Sigmundsson T, Petursson H, Gylfason A, Olason PI, Hardarsson G, Jonsdottir GA, Gustafsson O, Fossdal R, Giegling I, Möller HJ, Hartmann AM, Hoffmann P, Crombie C, Fraser G, Walker N, Lonnqvist J, Suvisaari J, Tuulio-Henriksson A, Djurovic S, Melle I, Andreassen OA, Hansen T, Werge T, Kiemeney LA, Franke B, Veltman J, Buizer-Voskamp JE; GROUP Investigators, Sabatti C, Ophoff RA, Rietschel M, Nöthen MM, Stefansson K, Peltonen L, St Clair D, Stefansson H, Collier DA. (2009) Disruption of the neurexin 1 gene is associated with schizophrenia. Human Molecular Genetics 18(5):988-996. Abstract

Shi J, Levinson DF, Duan J, Sanders AR, Zheng Y, Pe'er I, Dudbridge F, Holmans PA, Whittemore AS, Mowry BJ, Olincy A, Amin F, Cloninger CR, Silverman JM, Buccola NG, Byerley WF, Black DW, Crowe RR, Oksenberg JR, Mirel DB, Kendler KS, Freedman R & Gejman PV. (2009) Common variants on chromosome 6p22.1 are associated with schizophrenia. Nature doi:10.1038/nature08192. Abstract

Stefansson H, Ophoff RA, Steinberg S, Andreassen OA, Cichon S, Rujescu D, Werge T, Pietiläinen OPH, Mors O, Mortensen PB, Sigurdsson E, Gustafsson O, Nyegaard M, Tuulio-Henriksson A, Ingason A, Hansen T, Suvisaari J, Lonnqvist J, Paunio T, Børglum AD, Hartmann A, Fink-Jensen A, Nordentoft M, Hougaard D, Norgaard-Pedersen B, Böttcher Y, Olesen J, Breuer R, Möller H-J, Giegling I, Rasmussen HB, Timm S, Mattheisen M, Bitter I, Réthelyi JM, Magnusdottir BB, Sigmundsson T, Olason P, Masson G, Gulcher JR, Haraldsson M, Fossdal R, Thorgeirsson TE, Thorsteinsdottir U, Ruggeri M, Tosato S, Franke B, Strengman E, Kiemeney LA, GROUP†, Melle I, Djurovic S, Abramova L, Kaleda V, Sanjuan J, de Frutos R, Bramon E, Vassos E, Fraser G, Ettinger U, Picchioni M, Walker N, Toulopoulou T, Need AC, Ge D, Yoon JL, Shianna KV, Freimer NB, Cantor RM, Murray R, Kong A, Golimbet V, Carracedo A, Arango C, Costas J, Jönsson EG, Terenius L, Agartz I, Petursson H, Nöthen MM, Rietschel M, Matthews PM, Muglia P, Peltonen L, St Clair D, Goldstein DB, Stefansson K, Collier DA & Genetic Risk and Outcome in Psychosis (GROUP). (2009) Common variants conferring risk of schizophrenia. Nature doi:10.1038/nature08186. Abstract

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Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Kevin J. Mitchell
Submitted 9 July 2009
Posted 9 July 2009

GWAS Results: Is the Glass Half Full or 95 Percent Empty?
The publication of the latest schizophrenia GWAS papers represents the culmination of a tremendous amount of work and unprecedented cooperation among a large number of researchers, for which they should be applauded. In addition to the hope of finding new “schizophrenia genes,” GWAS have been described by some of the researchers involved as, more fundamentally, a stern test of the common variants hypothesis. Based on the meagre haul of common variants dredged up by these three studies and their forerunners, this hypothesis should clearly now be resoundingly rejected—at least in the form that suggests that there is a large, but not enormous, number of such variants, which individually have modest, but not minuscule, effects. There are no common variants of even modest effect.

However, Purcell and colleagues now argue for a model involving vast numbers of variants, each of almost negligible effect alone. The authors show that an aggregate score derived from the top 10-50 percent of a set of 74,000 SNPs from the association results in a discovery sample can predict up to 3 percent of the variance in a target group. Simply put, a set of putative “risk alleles” can be defined in one sample and shown, collectively, to be very slightly (though highly significantly in a statistical sense) enriched in the test sample, compared to controls. This is consistent across several different schizophrenia samples and even in two bipolar disorder samples. The authors go on to perform a set of control analyses that suggest that these results are not due to obvious population stratification or genotype rate effects (although effects at this level are obviously prone to cryptic artifacts).

If taken at face value, what do these results mean? They imply some kind of polygenic effect on risk, but of what magnitude? The answer to that depends on the interpretation of the additional simulations performed by the authors. They argue that the risk allele set inevitably contains very many false positives, which dilute the predictive power of the real positives hidden among them. Based on this logic, if we only knew which were the real variants to look at, then the variance explained in the target group would be much greater.

To try and estimate the magnitude of the effect of the polygenic load of “true risk” alleles, the authors conducted a series of simulations, varying parameters such as allele frequencies, genotype relative risks, and linkage disequilibrium with genotyped markers. They claim that these analyses converge on a set of models that recapitulate the observed data and that all converge on a true level of variance explained of around 34 percent, demonstrating a large polygenic component to the genetic architecture of schizophrenia.

These simulations adopt a level of statistical abstraction that should induce a healthy level of skepticism or at least reserved judgment on their findings. Most fundamentally, they rely explicitly for their calculations of the true variance on a liability-threshold model of the genetic architecture of schizophrenia. In effect, the “test” of the model incorporates the assumption that the model is correct.

The liability-threshold model is an elegant statistical abstraction that allows the application of the powerful statistics of normal distributions. Unfortunately, it suffers from the fact that it has no support whatsoever and makes no biological sense. First, there is no justification for assuming a normal distribution of “underlying liability,” whatever that term is taken to mean. Second, as usual when it is invoked, the nature of this putative threshold is not explained, though it surreptitiously implies some form of very strong epistasis (to explain the difference in risk between someone with x liability alleles and someone else with x+1 alleles). If this model is not correct, then these simulations are fatally flawed.

Even if the model were correct, the calculations are far from convincing. From a starting set of 560 models, the authors arrive at seven that are consistent with the observed degree of prediction in the target samples. According to the authors, the fact that these seven models converge on a small range of values for the underlying variance explained by the markers is evidence that this value (around 34 percent) represents the true situation. What is not highlighted is the fact that the values for the actual additive genetic variance (taking into account incomplete linkage disequilibrium between the markers and the assumed causal variants) across these models ranges from 34 percent to 98 percent and that the number of SNPs assumed to be having an effect ranges from 4,625 to 74,062. This extreme variation in the derived models hardly inspires confidence in the authors’ claim that their data “strongly support a polygenic basis to schizophrenia that (1) involves common SNPs, [and] (2) explains at least one-third of the total variation in liability.” (italics added)

From a more theoretical perspective, it should be noted that a polygenic model involving thousands of common variants of tiny effect cannot explain and will not contribute to the observed heightened familial relative risks. Such risk can only be explained by a variant of large effect or by an oligogenic model involving at most two to three loci (Bodmer and Bonilla, 2008; Hemminki et al., 2008; Mitchell and Porteous, in preparation). It seems much more likely that the observed predictive power in the target samples represents a modest “genetic background” effect, which could influence the penetrance and expressivity of rare, causal mutations. However, if the point of GWAS is to find genetic variants that are predictive of risk or that shed light on the pathogenic mechanisms of the disease, then clearly, even if such variants can be found by massively increasing sample sizes, their identification alone would not achieve or even appreciably contribute to either of these goals.

References:

Hemminki K, Försti A, Bermejo JL. The “common disease-common variant” hypothesis and familial risks. PLoS ONE. 2008 Jun 18;3(6):e2504. Abstract

Bodmer W, Bonilla C. Common and rare variants in multifactorial susceptibility to common diseases. Nat Genet. 2008 Jun;40(6):695-701. Abstract

View all comments by Kevin J. Mitchell

Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  David J. Porteous, SRF Advisor
Submitted 9 July 2009
Posted 10 July 2009
  I recommend the Primary Papers

Thumbs up or down on schizophrenia GWAS?
The triumvirate of schizophrenia GWAS studies just published in Nature gives cause for thought, and bears close scrutiny and reflection. To my reading, these three studies individually and collectively lead to an unambiguous conclusion—there is a lot of genetic heterogeneity and not one individual variant of common ancient origin accounts for a significant fraction of the genetic liability. To put it another way, there is no ApoE equivalent for schizophrenia. Strong past claims for ZNF804A and others look to have fallen by the statistical wayside. Putting the results of all three studies together does appear to provide support for a long known, pre-GWAS association with HLA, but otherwise it is hard to give a strong "thumbs up" to any specific result, not least because of the lack of replication between studies. The results are nevertheless important because the common disease, common variant model, on which GWAS are based and the associated cost justified, is strongly rejected as the main contributor to the genetic variance.

The ISC proposes a highly polygenic model with thousands of variants having an additive effect on both schizophrenia and bipolar disorder. I find no fault with their evidence, but its meaning and interpretation remains speculative. Simply consider the fact that SNPs carefully selected to tag half the genome account for about a third of the variance. It follows that the lion's share has gone undetected and will, by design and limitation, remain impervious to the GWAS strategy.

Part of the GWAS appeal is that the genotyping is technically facile and it is easier to collect lots of cases than it is families, but for as long as a diagnosis of schizophrenia or BP depends upon DSM-IV or ICD-10 classification, then diagnostic uncertainty will have a major effect on true power and validity of statistical association, both positive or negative. Indeed, the longstanding evidence from variable psychopathology amongst related individuals, the recent epidemiology evidence for shared genetic risk for schizophrenia and BP, and the further evidence supporting this from the ISC GWAS, all suggest that we should be returning more to family-based studies as a strategy to reduce genetic heterogeneity and find explanatory genetic variants. Plainly, adding ever more uncertainty through ever larger sample sizes is neither smart nor efficient.

I would certainly give the thumbs up to the full and unencumbered release of the primary data to the community as a whole, as this could usefully recoup some of the GWAS investment. It would facilitate a range of statistical and bioinformatics analyses and, who knows, there might be hidden nuggets of statistical support for independent genetic and biological studies.

View all comments by David J. Porteous

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Comment by:  Sagiv Shifman
Submitted 11 July 2009
Posted 11 July 2009

The main question that arises from the three large genomewide association studies published in Nature is, What should we do next?

One important way forward would be to follow up the association findings in the MHC region. We need to understand the biological mechanism underlying this association. If the association signal is indeed related to infectious diseases, this line of inquiry may lead to the highly desired development of a treatment that might prevent the diseases in some cases.

One possible explanation for the association between schizophrenia and the MHC region (6p22.1) is that infection during pregnancy leads to disturbances of fetal brain development and increases the risk of schizophrenia later in life. A possible test for the theory of infectious diseases as risk factors for schizophrenia would be to study the associated SNPs in 6p22.1 in fathers and mothers of subjects with schizophrenia relative to parents of control subjects. If the 6p22.11 region is related to the tendency of mothers to be infected by viruses during pregnancy, we would expect the SNPs in 6p22.1 to be most strongly associated with being a mother to a subject with schizophrenia.

Another broader and more complicated part of the question is: What would be the best strategy for continued study of the genetic causes of schizophrenia? There shouldn’t be only one way to proceed. Testing samples that are 10 times larger seems likely to lead to the identification of more genes, but with much smaller effect size. Testing the association of common variants with schizophrenia is unlikely to lead to the development of genetic diagnostic tools in the near future. If we want to understand the biology of the disease, it might be easier to concentrate our efforts on the identification of rare inherited and non-inherited variants with large effect on the phenotype. Such rare variants are easier to model in animals (relative to common variants with very small functional effect) and might even account for a larger proportion of cases.

View all comments by Sagiv Shifman

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Comment by:  Alan Brown, Paul Patterson
Submitted 17 July 2009
Posted 17 July 2009

The three companion papers in this week’s issue of Nature, in our view, support the case for investigating interaction between susceptibility genes and infectious exposures in schizophrenia. We and others have argued previously that genetic studies conducted in isolation from environmental factors, and studies of environmental influences in the absence of genetic data, are necessarily limited. Maternal influenza, rubella, toxoplasmosis, herpes simplex virus, and other infections have each been associated with an increased risk of schizophrenia, with effect sizes ranging from twofold to over fivefold. While these epidemiologic findings clearly require replication in independent cohorts, two new developments provide further support for the hypothesis. First, a growing number of animal studies of maternal immune activation have documented behavioral and brain phenotypes in offspring that are analogous to findings from clinical research in schizophrenia, and these findings are mediated in large part by specific cytokines (Meyer et al., 2009; Patterson, 2008). Second, recent evidence indicates that maternal infection is also related to deficits in executive and other cognitive functions and neuropathology thought to arise from disruptions in brain development (Brown et al., 2009a; Brown et al., 2009b).

While the MHC region contains genes not involved in the immune system, in light of the epidemiologic findings on maternal infection, it is intriguing to see that this region is once more implicated in genetic studies of schizophrenia as the importance of this region in the response to infectious insults cannot be ignored. Although it is heartening to see that the potential implications of these findings for infectious etiologies were raised in the article from the SGENE plus group, an analysis of the frequency of SNPs by season of birth falls well short of the type of research that will yield definitive findings on the relationships between susceptibility genes and infectious insults. Hence, we advocate a strategy aimed at large scale genetic analyses of schizophrenia cases using birth cohorts with infectious exposures documented from prospectively collected biological samples from the prenatal period. If the schizophrenia-related pathogenic mechanisms by which MHC-related genetic variants operate involve interactions with prenatal infection, we would expect that studies of gene-infection interaction will yield larger effect sizes than those found in these new papers. The evidence from these papers and the epidemiologic literature should also facilitate narrowing of the number of candidate genes to be tested for interactions with infectious insults, thereby ameliorating the potential for type I error due to multiple comparisons.

References:

Meyer U, Feldon J, Fatemi SH. In-vivo rodent models for the experimental investigation of prenatal immune activation effects in neurodevelopmental brain disorders. Neurosci Biobehav Rev . 2009 Jul 1; 33(7):1061-79. Abstract

Patterson PH. Immune involvement in schizophrenia and autism: Etiology, pathology and animal models. Behav Brain Res. 2008 Dec 24; Abstract

Brown AS, Vinogradov S, Kremen WS, Poole JH, Deicken RF, Penner JD, McKeague IW, Kochetkova A, Kern D, Schaefer CA. Prenatal exposure to maternal infection and executive dysfunction in adult schizophrenia. Am J Psychiatry . 2009a Jun 1 ; 166(6):683-90. Abstract

Brown AS, Deicken RF, Vinogradov S, Kremen WS, Poole JH, Penner JD, Kochetkova A, Kern D, Schaefer CA. Prenatal infection and cavum septum pellucidum in adult schizophrenia. Schizophr Res . 2009b Mar 1 ; 108(1-3):285-7. Abstract

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Related News: Largest GWAS Analysis to Date Offers Only Two New Candidate Genes

Comment by:  Javier Costas
Submitted 17 July 2009
Posted 17 July 2009
  I recommend the Primary Papers

Two hundred years after Darwin’s birth and 150 years after the publication of On the Origin of Species, these three papers in Nature show the important role of natural selection in shaping the genetic architecture of schizophrenia susceptibility. If we compare the GWAS results for schizophrenia with those obtained for other diseases, it seems that there are less common risk alleles and/or lower effect sizes in schizophrenia than in many other complex diseases (see, for instance, the online catalog of published GWAS at NHGRI). This fact strongly suggests that negative selection limits the spread of susceptibility alleles, as expected due to the decreased fertility of schizophrenic patients.

Interestingly, the MHC region may be an exception. This region represents a classical example of balancing selection, i.e., the presence of several variants at a locus maintained in a population by positive natural selection (Hughes and Nei, 1988). In the case of the MHC, this balancing selection seems to be related to pathogen resistance or MHC-dependent mating choice. Therefore, the presence of common schizophrenia susceptibility alleles at this locus might be explained by antagonistic pleiotropic effects of alleles maintained by natural selection.

If negative selection limits the spread of schizophrenia risk alleles, most of the genetic susceptibility to schizophrenia is likely due to rare variants. Resequencing technologies will allow the identification of many of these variants in the near future. In the meantime, it would be interesting to focus our attention on non-synonymous SNPs at low frequency. Based on human-chimpanzee comparisons and human sequencing data, Kryukov et al. (2008) have shown that a large fraction of de novo missense mutations are mildly deleterious (i.e., they are subject to weak negative selection) and therefore they can still reach detectable frequencies. Assuming that most of these mildly deleterious alleles may be detrimental (i.e., they confer risk for disease) the authors conclude that numerous rare functional SNPs may be major contributors to susceptibility to common diseases Kryukov et al., 2008. Similar conclusions were obtained by the analysis of the relative frequency distribution of non-synonymous SNPs depending on their probability to alter protein function (Barreiro et al., 2008; Gorlov et al., 2008). As shown by Evans et al. (2008), genomewide scans of non-synonymous SNPs might complement GWAS, being able to identify rare non-synonymous variants of intermediate penetrance not detectable by current GWAS panels.

References:

Barreiro LB, Laval G, Quach H, Patin E, Quintana-Murci L (2008) Natural selection has driven population differentiation in modern humans. Nat Genet 40: 340-5. Abstract

Evans DM, Barrett JC, Cardon LR (2008) To what extent do scans of non-synonymous SNPs complement denser genome-wide association studies? Eur J Hum Genet 16: 718-23. Abstract

Gorlov IP, Gorlova OY, Sunyaev SR, Spitz MR, Amos CI (2008) Shifting paradigm of association studies: value of rare single-nucleotide polymorphisms. Am J Hum Genet 82: 100-12. Abstract

Hughes AL, Nei M (1988) Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection. Nature 335: 167-70. Abstract

Kryukov GV, Pennacchio LA, Sunyaev SR (2007) Most rare missense alleles are deleterious in humans: implications for complex disease and association studies. Am J Hum Genet 80: 727-39. Abstract

View all comments by Javier Costas

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