Schizophrenia Research Forum - A Catalyst for Creative Thinking

The Many Faces of CNVs: The Case of 1q21.1

22 December 2008. Earlier this year, back-to-back articles in Nature implicated rare 1q21.1 deletions and duplications in schizophrenia (see SRF related news story), but as illustrated by two new reports that tie deletions and duplications in this same genetic region to a wide array of developmental and behavioral phenotypes, the brave new world of copy number variation (CNV) research is spawning as many questions as answers.

These studies—one published in October in the New England Journal of Medicine from the laboratory of Evan Eichler at the University of Washington (Mefford et al., 2008) and the other in the December issue of Nature Genetics, by Ankita Patel and colleagues at Baylor College of Medicine (Brunetti-Pierri, 2008)—took a different approach. Instead of conducting genomewide searches for CNVs in one disease, both research groups specifically tested for 1q21.1 CNVs in groups of patients who had been referred for genetic testing with widely diverse phenotypes suggesting disruptions in neural development (the 1q21.1 region contains some 27 genes, most expressed in the brain), including mental retardation, autism, and various congenital brain anomalies.

In the earlier study from the International Schizophrenia Consortium (ISC), which compared 3,291 patients with schizophrenia with 3,181 controls, deletions in 1q21.1 were found in 10 cases and one control (ISC; International Schizophrenia Consortium, 2008). The companion work by Stefansson and colleagues identified 66 de novo CNVs in a population-based sample, which were winnowed down in two successive association studies to three CNVs, including recurrent 1q21.1 deletions, warranting further study. Ultimately, 11 of 4,718 cases (0.23 percent) were found to carry 1q21.1 deletions, compared to only eight of 41,999 controls (0.02 percent) (Stefansson et al., 2008). As the intensive discussion at SRF on these papers (see commentary on SRF related news story) and on the earlier CNVs-in-schizophrenia paper by Walsh and colleagues (see commentary on SRF related news story) attest, researchers are divided about the meaning and usefulness of these findings. In particular, the current papers recall the objections of some commentators that these deletions may generally perturb brain development, with psychosis only one of many possible manifestations.

A wide spectrum
In the New England Journal study, first authors Heather Mefford and Andrew Sharp and an international team of collaborators screened 5,218 such patients and identified a recurrent 1.35 mb deletion including at least seven genes in 25 persons (0.5 percent). The deletion was not found in a subsequent screen of 4,737 control subjects. A reciprocal duplication was found in nine patients (0.2 percent), and in only one control subject. Five patients with 1q21.1 CNVs, four with a deletion and one with a duplication, were excluded from the study because they had additional chromosomal abnormalities that could have contributed to their phenotype. Some deletions were de novo, some inherited, and some of unknown origin.

Most patients with deletions had mild to moderate developmental delay (76.2 percent) and dysmorphic features (81 percent), but five had normal cognitive development, and there were four parents of patients who carried the same deletion but were apparently unaffected. As for duplications, seven of the eight carriers had learning problems or mental retardation, and four of the eight had autism or autistic features. However, patients with 1q21.1 CNVs exhibited a range of skeletal, facial, cardiac, ocular, and neurological features with no apparent pattern. “[D]etailed clinical evaluations of affected persons disclosed a much broader spectrum of phenotypes than anticipated, dispelling any notion of syndromic disease,” the team writes. “These results. . .also further dispel the notion that rare copy-number variants will necessarily follow the one gene (or one rearrangement)-one disease model.” (See Q&A with authors Mefford and Eichler below).

A similarly broad spectrum of phenotypes associated with 1q21.1 CNVs is reported in the Nature Genetics study, led by first authors Nicola Brunetti-Pierri and Jonathan Berg. The research group examined 16,557 patient samples with comparative genomic hybridization over the course of four years, assembling what they call “the largest collection of individuals with microdeletions or microduplications in chromosome 1q21.1 reported in the literature”: 27 patients with deletions and 17 with duplications (with detailed clinical information available for 21 and 15 patients, respectively).

However, aside from consistent associations of 1q21.1 deletions with decreased head circumference (which has also been observed in schizophrenia; see, e.g., Ward et al., 1996) and duplications with increased head circumference, the range of observed phenotypes was wide and exhibited no clear syndromic pattern among patients (e.g., short stature, eye abnormalities, hallucinations, seizures, brain malformations with deletions; heart defects, scoliosis, autism, hydrocephalus with duplications). Moreover, like the Mefford and Sharp team, the group reports that 1q21.1 deletions and duplications can be carried by apparently unaffected individuals, prompting them to question “whether 1q21.1 microdeletions and microduplications are benign CNVs or are pathogenic variants with incomplete penetrance.”

In commentary accompanying the Nature Genetics paper, Michael O’Donovan, George Kirov, and Michael Owen (all of whom took part in the ISC genomewide CNV study) assess the methodological dilemma posed by CNVs thus far. “[P]henotype-led approaches have greater power to implicate specific CNVs in specific disorders,” they write, “but they are unlikely to capture the full range of phenotypic diversity. In contrast, the CNV-led approach can expand our knowledge of the range of phenotypes related to a specific CNV, but. . .we cannot be certain which specific phenotypes drive this enrichment.”—Pete Farley.

References:
Brunetti-Pierri N, Berg JS, Scaglia F, Belmont J, Bacino CA, Sahoo T, Lalani SR, Graham B, Lee B, Shinawi M, Shen J, Kang SH, Pursley A, Lotze T, Kennedy G, Lansky-Shafer S, Weaver C, Roeder ER, Grebe TA, Arnold GL, Hutchison T, Reimschisel T, Amato S, Geragthy MT, Innis JW, Obersztyn E, Nowakowska B, Rosengren SS, Bader PI, Grange DK, Naqvi S, Garnica AD, Bernes SM, Fong CT, Summers A, Walters WD, Lupski JR, Stankiewicz P, Cheung SW, Patel A. Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormalities. Nat Genet. 2008 Dec;40(12):1466-71. Abstract

Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, Buysse K, Huang S, Maloney VK, Crolla JA, Baralle D, Collins A, Mercer C, Norga K, de Ravel T, Devriendt K, Bongers EM, de Leeuw N, Reardon W, Gimelli S, Bena F, Hennekam RC, Male A, Gaunt L, Clayton-Smith J, Simonic I, Park SM, Mehta SG, Nik-Zainal S, Woods CG, Firth HV, Parkin G, Fichera M, Reitano S, Lo Giudice M, Li KE, Casuga I, Broomer A, Conrad B, Schwerzmann M, Räber L, Gallati S, Striano P, Coppola A, Tolmie JL, Tobias ES, Lilley C, Armengol L, Spysschaert Y, Verloo P, De Coene A, Goossens L, Mortier G, Speleman F, van Binsbergen E, Nelen MR, Hochstenbach R, Poot M, Gallagher L, Gill M, McClellan J, King MC, Regan R, Skinner C, Stevenson RE, Antonarakis SE, Chen C, Estivill X, Menten B, Gimelli G, Gribble S, Schwartz S, Sutcliffe JS, Walsh T, Knight SJ, Sebat J, Romano C, Schwartz CE, Veltman JA, de Vries BB, Vermeesch JR, Barber JC, Willatt L, Tassabehji M, Eichler EE. Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes. N Engl J Med. 2008 Oct 16;359(16):1685-99. Abstract

O’Donovan MC, Kirov G, Owen MJ. Phenotypic variations on the theme of CNVs. Nat Genet. 2008 Dec;40(12):1392-3. Abstract

Q&A with Evan Eichler and Heather Mefford. Questions by Pete Farley.

SRF: What led you to think about this chromosomal region to begin with?
Eichler: This is one of the regions we identified way back in 2002. We developed a duplication map of the human genome after the Genome Project was finished, and we looked for regions that we thought had an architecture, that would predispose to instability. It’s known that duplicated sequences promote genomic instability (this is some work from Lupski and others over the years), and so when we developed this map of duplications, we used it as a kind of morbidity map to predict unstable regions in the human genome. The 1q21 region was one of about 150 regions that we identified back in 2002, and in 2004 we started screening patients with mental retardation for gains and losses of these hotspots in the genome. Andy Sharp in 2006, from my group, published one patient with a 1q21 deletion, based on a small screen of about 300 children with mental retardation. What we did over the last couple of years was just take on each of these regions, from the list of about a dozen regions that we identified in 2006, and target each of those systematically in children with mental retardation and more broadly in children with autism spectrum disorder. Heather at that point picked it up and began screening larger numbers of patients for the 1q21 deletion and working with other groups to really nail down the features of this particular “syndrome.” Ironically, the point of the paper was that, in the end, the deletion eludes syndromic classification.
Mefford: The reason we focused on this was for just that reason; we saw a peppering in the literature of case reports or small series of individuals with different phenotypes who had alterations in this region. And it became very clear that people weren’t sure what to do with that information. So our goal was to really systematically look at both affected individuals and controls to first answer the question, Are alterations in this region pathogenic?
Eichler: I have to tell you, over the last two years, we’ve flip-flopped probably at least three or four times as to whether the deletion event was really disease-causing. Once we started to pick up more and more children with mental retardation with the 1q21 deletion, we started to dig into the phenotypes.

That’s when we got confused because these kids looked very different clinically. They didn’t really have strong dysmorphology. Then we would find parents that were unaffected, and we went back to clinicians and they confirmed that in many cases, about half the cases, the parents carried the event, the deletion event, but were essentially unaffected with respect to what we saw in the kids.

So we went from thinking it's just a rare polymorphism to being a disease, back to being a polymorphism, least two or three times. I think what finally cinched it for us was 1) the work Heather did for us to pull all of these patients together with the other groups where we started to see what I would call sub-phenotypes or sub-syndromes, where we saw kids having some features that were in common. So some would have cardiac defects, some would have cataracts, some would have severe mental retardation with other similarities. And then 2) the screen of more than 5,000 “normal individuals,” and finding this event essentially nonexistent, at least the deletion event, in those cases. So that convinced us that this event was pathogenic, but we knew that this would be problematic for the diagnostic community. It’s not something where you can tell the parent, Well, your child is going to have severe mental retardation, or your child could be normal but might have an increased risk for cardiac defects, or your child could grow up with a normal heart, and become schizophrenic later on in life. The schizophrenia details came from the other papers that were published, not from our group, but from the two papers that were published this summer in Nature.

SRF: Yes, the paper, as your conclusion indicates, raises as many questions as it answers, I guess.
Eichler: I think it’s important that it is actually changing the way we think about it—at least for me; I’m not going to say for all human geneticists—but it’s changing the way I was taught to think about human disease. I was taught to think that you have a lesion, and you have a phenotype, and there’s going to be some variability in that phenotype, but this is actually jumping across different diseases. So the idea is that you can have a schizophrenic, and then you can have a child with cardiac defect, and normal mentation, and then you have a child that is severely handicapped, or an autistic child that has no problem with the heart. These are such wildly diverse clinical manifestations, it makes me start to think that maybe we should be thinking about them just as cases and controls—kids with some neurologic impairment versus ones without. Maybe we should be lumping many different classes of diseases together.

SRF: In a general hypothesis that you put forward about explaining this phenotypic variation, you said that “modifiers” played quite an important role, but you seemed to eliminate the first suspects that anyone would think of: recessive variants, epigenetic effects/imprinting. So where do you think the culprits are?
Eichler: To me, these are all different types of modifiers, so I guess one question would be, Is the modifier going to be located in cis, so in the region of the microdeletion itself, or is it going to be somewhere else in the genome? And I think the most logical place to begin would be in the region itself, right? To look at the genes that are underneath the deletion or the duplication, look for what haplotypes there are there. The other thing to consider, which I think is probably important, are the environmental influences when these conceptuses are formed. There may be different disease courses determined not just by the genetic background, but also by the environmental insults that this embryo or this conceptus is exposed to. I think we’re really quite ignorant about that aspect.
Mefford: In the case of 1q21, we only looked at two obvious genes for two of the phenotypes. One for heart disease and one for cataracts. And the bigger question is why are some of these kids severely mentally retarded and why do some people grow up and have schizophrenia? Although we made an initial attempt of look at some of these things, we really haven’t gotten anywhere with respect to looking globally at modifiers that might affect brain development and cognition.

I agree with Evan. I think that looking in the region is the first place to look, but we can’t forget that there may be SNPs in genes or copy number variants elsewhere in the genome that might also influence what the final phenotype is going to be and what you’ll get in the individual.
Eichler: One way to think about this is that because it’s such a large region, and it takes out so many genes, it creates such a sensitized individual for copy number imbalance, or at least in terms of gene expression. So if you can look at all of the different genes, and different processes they played in that critical region, you can imagine that any other mutation that occurs somewhere else, that in a normal individual would not have a huge effect, now in fact manifests itself and can have a very significant effect in terms of what the outcome is in the child. Obviously, targeting these regions and resequencing is important, and we and the other groups want to find the genes that are responsible for the different aspects of the phenotype. But I think the most important thing that we need right now is to combine sample collections with detailed clinical information. When we start looking at large numbers it’s not as confusing I think as the first blush of analysis. We start to see certain groups of patients grouped together and, say, these ones all have microcephaly, all these kids have heart defects, all these kids have cataracts.

So if we combine ours, Jim Lupski’s collection, and other collections that are being made across the world, we could in principle have a detailed dissection of the phenotype that would help find the genes that are responsible for different aspects. Down the road, we might be able to predict the outcome of some of the kids with the 1q21 deletion much better.

SRF: Just curiosity on my part, but you seem to use terminology other than CNV in the paper: things like unbalanced micro-rearrangements. Is that just a preference of nomenclature or is there some theoretical thing embedded in that?
Eichler: Actually, that stems from complaints from the cytogenetics community. There are well-established professors in cytogenetics that have criticized the use of CNV to refer to something that is pathogenic. And that’s because implied in copy number variant, at least until probably last year, was the idea that variant equals benign. Microdeletion and microduplication to cytogeneticists typically means that you have something pathogenic, but there was actually a move among a few cytogeneticists to write a white paper or a piece that would help clarify this. But I would have to say that in the last year there’s been so much interchange between the use of CNV and microdeletion and microduplication, I think it’s a foregone conclusion now that it's almost impossible to stem that tide and change the usage back to the way that cytogeneticists viewed it originally.

SRF: And one last question: your lab’s output is prolific and very diverse in terms of the sorts of questions you’re looking at including a lot of things like the platypus paper, which had an evolutionary or phylogenetic focus. The new paper in Science on 17q21, and this other paper that came out recently, by Brunetti-Pierri, on 1q21—they all touch on evolutionary questions, and I’m curious about whether you think evolutionary and clinical questions, in genomics in particular, will continue to be intertwined and what those two perspectives can bring to bear on one another.
Eichler: This has been the mission of my lab for probably 10 years. We see clinical mutations and evolutionary process as all the same, and we find that both inform upon each other. The clinical information is really valuable because it gives us real-time information on mutations in humans. The evolutionary process tells things about the mechanism and the patterns that we can use. If you think about the way that we strategized and went after these regions to begin with, it’s largely from an evolutionary perspective: finding things that were CNVs five to 10 million years ago, and using them to predict locations of new CNV’s that cause disease. So our approach has always been intertwined with evolution and disease, and I think it’s a very powerful way to go. In the future, this approach will be commonplace. I think it’s unfortunate that a lot of human geneticists do not have a strong background in or consider evolutionary biology, because I think that would give them more insights than they currently have. It’s like history is bound to repeat itself, as we say, and the genome tells us the same thing over and over again. Instability that we’ve seen happen 10 million years ago occurred in the same regions that are unstable in us now, and for good reason. I guess our twist on it, which is probably different from most other labs, is that we’re interested in finding the flip side or positive aspects of duplication architecture. We know that duplications cause disease, and we know that humans are enriched for these duplications, and we have lots of them that are interspersed. This creates long-term hotspots of instability like a series of volcanoes in our genome that are going to erupt at any moment. The question is, Why is that architecture there at all? What we have been finding, and publishing from our evolutionary analyses, is that embedded in these duplicated regions are new genes and gene families that seem to have evolved specifically in the human and great ape lineage. And so we’re very interested in these, because we think they’re going to tell us something fundamental about the evolution of our species, at the same time predicting sites of instability that cause disease.

SRF: Any parting thoughts?
Eichler: I know this is a phrase that has been used way too much in this field, but I believe this is the tip of the iceberg. I think that the way that we’ve been thinking about the common disease-common variant hypothesis has been, in part, shadowplay. Many of the substantive variants—those with high odds ratios—are coming from rare variants. The rare variant-common disease hypothesis is now gaining more and more traction. The idea that we now can explain maybe as much as 15 percent of mental retardation, autism, and other complex neurologic phenotypes by this model is the really important message. This message should reverberate to other communities that are studying other complex diseases, such as asthma, diabetes, or hypertension. I would be surprised if this model isn’t going to be important for those complex diseases as well. It will be interesting to see what the next couple of years will hold, but I would predict even more papers on more diverse phenotypes related to large copy number variants. I think what is really cool about this whole field, if you will, is that we have so much instability in our genome, and yet as a species we’re so successful. And we have a huge diversity of diseases that will be essentially evolutionarily very, very young. We’re talking about diseases that are going to be specific to specific families, a few generations old, as opposed to this idea that diseases are inherited from mutations that happened hundreds if not thousands of generations ago in populations that left Africa. So I think human disease studies and human genomics and human evolutionary work really is a personal issue, and I think it’s really the fun part. I’m still wrapping my head around the idea that we have so much variation in our genome and some of us are even going to be different from either of our parents. We’re not just the sum of both of them; we have more or less material than our parents for some small number of loci. It’s kind of a radical concept. If you had asked me even eight years ago, I would have thought the genome was kind of a sacrosanct thing. You can’t mess with it too much—small mutations, yes, but big mutations, probably not likely, not over a long haul. But our genome is really quite plastic. It’s amazing that any of us are normal at all, and maybe like I said before, none of us really are. And so that’s kind of the beauty of it.

SRF: That’s comforting in a way.
Eichler: It is. I was thinking, if I had to write a book, the title of it would be “No Perfect Genome.” Because there is no individual out there who has a perfect genome in terms of mutation, in terms of organization or structure. So we’re all screwed up a little bit, and maybe that’s why we’re human.
Mefford: I’m not sure I can top that. But one of my thoughts from a more clinical perspective is I think the nice thing about the type of study we did is we brought together a large number of groups and a large number of patients and controls. I think we picked all the low hanging fruit for some of these disorders and the obvious pathogenic events, and we’re going to see more and more cases like the 1q21 rearrangements where it’s not quite clear if there’s one phenotype or more. Really bringing together large groups of people and patients and genotypes is going to be required to understand the real phenotypic diversity and pathogenicity of some of the rearrangements. I think human genetics is going to be more and more collaborative research and large numbers are going to be important. It’s going to be important to help people on the clinical side also to be able to talk to their patients and their families about what all these findings mean, because we’re continuing to perform our ACGH and sometimes it’s not clear what it means. It’s going to take a lot of work to be able to explain that.

SRF: Yes, I thought that was quite an interesting part of the paper, about the practical ramifications for genetic counseling.
Eichler: I have talked to clinicians in diagnostic centers, and I have to say that they’re probably as flummoxed as we are, probably more so, because they have to convey this information to families and Heather actually does quite a bit of that herself. We’ve been getting quite a few e-mails from families asking us what does this mean for them and their kids.

And I have to tell you that this is quite a sobering thing because you know in many ways, not to trivialize it, but all we can tell them is there’s really a good chance that their kid is going to be sick. It doesn't really give them a lot of news. When they realize that their father, for example, carries the same deletion and is normal, how do you make a decision on the child or how do you make a decision on how to raise that child? If children are born and are normal, do you need to have someone watch them for the rest of their lives to see if they develop schizophrenia. These are really big questions that affect people in very significant ways. That aspect of it is supposed to be worrying, but also we have to be very careful in what we say and what we don’t say.

SRF: Well, I thank you very much, both of you, for your time, and thanks for the interesting paper, also.

Comments on Related News


Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  Daniel Weinberger, SRF Advisor
Submitted 27 March 2008
Posted 27 March 2008

The paper by Walsh et al. is an important addition to the expanding literature on copy number variations in the human genome and their potential role in causing neuropsychiatric disorders. It is clear that copy number variations are important aspects of human genetic variation and that deletions and duplications in diverse genes throughout the genome are likely to affect the function of these genes and possibly the development and function of the human brain. So-called private variations, such as those described in this paper, i.e., changes in the genome found in only a single individual, as all of these variations are, are difficult to establish as pathogenic factors, because it is hard to know how much they contribute to the complex problem of human behavioral variation in a single individual. If the change is private, i.e., only in one case and not enriched in cases as a group, as are common genetic polymorphisms such as SNPs, how much they account for case status is very difficult to prove.

An assumption implicit in this paper is that these private variations may be major factors in the case status of the individuals who have them. The data of this paper suggest, however, this is actually not the case, at least for the childhood onset cases. Here’s why: mentioned in the paper is a statement that only two of the CNVs in the childhood cases are de novo, i.e., spontaneous and not inherited (and one of these is on the Y chromosome, making its functional implications obscure). If most of the CNVs are inherited, they can’t be causing illness per se as major effect players because they are coming from well parents.

Also, if you add up all CNVs in transmitted and non-transmitted chromosomes of the parents, it’s something like 31 gene-based CNVs in 154 parents (i.e., 20 percent of the parents have a gene-based deletion or duplication in the very illness-related pathways that are highlighted in the cases), which is at least as high a frequency as in the adult-onset schizophrenia sample in this study…and three times the frequency as found in the adult controls. This is not to say that such variants might not represent susceptibility genetic factors, or show variable penetrance between individuals, like other polymorphisms, and contribute to the complex genetic risk architecture, like other genetic variations that have been more consistently associated with schizophrenia. However, the CNV literature has tended to seek a more major effect connotation to the findings.

View all comments by Daniel Weinberger

Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  William Honer
Submitted 28 March 2008
Posted 28 March 2008
  I recommend the Primary Papers

As new technologies are applied to understanding the etiology and pathophysiology of schizophrenia, considering the clinical features of the cases studied and the implications of the findings is of value. The conclusion of the Walsh et al. paper, “these results suggest that schizophrenia can be caused by rare mutations….“ is worth considering carefully.

What evidence is needed to link an observation in the laboratory or clinic to cause? Recent recommendations for the content of papers in epidemiology (von Elm et al., 2008) remind us of the suggestions of A.V. Hill (Hill, 1965). To discern the implications of a finding, or association, for causality, Hill suggests assessment of the following:

1. Strength of the association: this is not the observed p-value, but a measure of the magnitude of the association. In the Walsh et al. study, the primary outcome measure, structural variants duplicating or deleting genes was observed in 15 percent of cases, and 5 percent of controls. But what is the association with? The diagnostic entity of schizophrenia, or some risk factor for the illness? Of interest, and noted in the Supporting Online Material, these variants were present in 7/15 (47 percent) of the cases with presumed IQ <80, but only 15/135 (11 percent) of the cases with IQ >80. Are the structural variants more strongly associated with mental retardation (within schizophrenia 47 percent vs. 11 percent) than with diagnosis (11 percent vs. 5 percent of controls, assuming normal IQ)? This is of particular interest in the context of the speculation in the paper concerning the importance of genes putatively involved with brain development in the etiology of schizophrenia.

2. Consistency of results in the literature across studies and research groups: there are now several papers examining copy number variation in schizophrenia, including a report from our group (Wilson et al., 2006). The authors of the present paper state that each variant observed was unique, and so consistency of the specific findings could be argued to be irrelevant, if the model is of novel mutations (more on models below). Undoubtedly, future meta-analyses and accumulating databases help determine if there is anything consistent in the findings, other than a higher frequency of any abnormalities in cases rather than controls.

3. Specificity of the findings to the illness in question: this was not addressed experimentally in the paper. However, the findings of more abnormalities in the putative low IQ cases, and the similarity of the findings to reports in autism and mental retardation, suggest that this criterion for supporting causality is unlikely to be met.

4. Temporality: the abnormalities should precede the illness. If DNA from terminally differentiated neurons harbors the same variants as DNA from constantly renewed populations of lymphocytes, then clearly this condition is met. While it seems highly likely that this is the case, it is worthwhile considering the possibility that DNA structure may vary between tissue types, or between cell populations. Even within human brain there is some evidence for chromosomal heterogeneity (Rehen et al., 2005).

5. Biological gradient: presence of a “dose-response” curve strengthens the likelihood of a causal relationship. This condition is not met within cases: only 1/115 appeared to have more than one variant. However, in the presumably more severe childhood onset form of schizophrenia, four individuals carried multiple variants, and the observation of a higher prevalence of variants overall. Still, the question of what the observations of CNV are associated with is relevant, since one of the inclusion/exclusion criteria for COS allowed IQ 65-80, and it is uncertain how many of these cases had some degree of intellectual deficit.

6. Plausibility: biological likelihood—quite difficult to satisfy as a criterion, in the context of the limits of knowledge concerning the mechanisms of illness of schizophrenia.

7. Coherence of the observation with known facts about the illness: the genetic basis of schizophrenia is quite well studied, and there is no dearth of theories concerning genetic architecture. However, a coherent model remains lacking. As examples, the suggestion is made that the observations concerning inherited CNVs in the COS cases are linked with a severe family history in this type of illness. This appears inconsistent with a high penetrance model for CNVs as suggested in the opening of the paper (presuming the parents in COS families are unaffected, as would seem likely). Elsewhere, CNVs are proposed by the authors to be related to de novo events, and an interaction with an environmental modifier, folate (and exposure to famine), is posited (McClellan et al., 2006). A model of the effects of CNVs, which generates falsifiable hypotheses is needed.

8. Experiment: the ability to intervene clinically to modify the effects of CNVs disrupting genes seems many years away.

9. Analogy: the novelty of the CNV findings is both intriguing, but limiting in understanding the likelihood of causal relationships.

The intersection of clinical realities and novel laboratory technologies will fuel the need for better translational research in schizophrenia for many, many more years.

References:

von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 2008 Apr 1;61(4):344-349. Abstract

HILL AB. THE ENVIRONMENT AND DISEASE: ASSOCIATION OR CAUSATION? Proc R Soc Med. 1965 May 1;58():295-300. Abstract

Wilson GM, Flibotte S, Chopra V, Melnyk BL, Honer WG, Holt RA. DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling. Hum Mol Genet. 2006 Mar 1;15(5):743-9. Abstract

Rehen SK, Yung YC, McCreight MP, Kaushal D, Yang AH, Almeida BSV, Kingsbury MA, Cabral KMS, McConnell MJ, Anliker B, Fontanoz M, Chun J: Constitutional aneuploidy in the normal human brain. J Neurosci 2005; 25:2176-2180. Abstract

McClellan JM, ESusser E, King M-C: Maternal famine, de novo mutations, and schizophrenia. JAMA 2006; 296:582-584. Abstract

View all comments by William Honer

Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  Todd LenczAnil Malhotra (SRF Advisor)
Submitted 30 March 2008
Posted 30 March 2008

The new study by Walsh et al. (2008), as well as recent data from other groups working in schizophrenia, autism, and mental retardation, make a strong case for including copy number variants as an important source of risk for neurodevelopmental phenotypes. These findings raise several intriguing new questions for future research, including: the degree of causality/penetrance that can be attributed to individual CNVs; diagnostic specificity; and recency of their origins. While these questions are difficult to address in the context of private mutations, one potential source of additional information is the examination of common, recurrent CNVs, which have not yet been systematically studied as potential risk factors for schizophrenia.

Still, the association of rare CNVs with schizophrenia provides additional evidence that genetic transmission patterns may be a complex hybrid of common, low-penetrant alleles and rare, highly penetrant variants. In diseases ranging from Parkinson's to colon cancer, the literature demonstrates that rare penetrant loci are frequently embedded within an otherwise complex disease. Perhaps the most well-known example involves mutations in amyloid precursor protein and the presenilins in Alzheimer’s disease (AD). Although extremely rare, accounting for <1 percent of all cases of AD, identification of these autosomal dominant subtypes greatly enhanced understanding of pathophysiology. Similarly, a study of consanguineous families in Iran has very recently identified a rare autosomal recessive form of mental retardation (MR) caused by glutamate receptor (GRIK2) mutations, thereby opening new avenues of research (Motazacker et al., 2007). In schizophrenia, we have recently employed a novel, case-control approach to homozygosity mapping (Lencz et al., 2007), resulting in several candidate loci that may harbor highly penetrant recessive variants. Taken together, these results suggest that a diversity of methodological approaches will be needed to parse genetic heterogeneity in schizophrenia.

References:

Motazacker MM, Rost BR, Hucho T, Garshasbi M, Kahrizi K, Ullmann R, Abedini SS, Nieh SE, Amini SH, Goswami C, Tzschach A, Jensen LR, Schmitz D, Ropers HH, Najmabadi H, Kuss AW. (2007) A defect in the ionotropic glutamate receptor 6 gene (GRIK2) is associated with autosomal recessive mental retardation. Am J Hum Genet. 81(4):792-8. Abstract

Lencz T, Lambert C, DeRosse P, Burdick KE, Morgan TV, Kane JM, Kucherlapati R,Malhotra AK (2007). Runs of homozygosity reveal highly penetrant recessive loci in schizophrenia. Proc Natl Acad Sci U S A. 104(50):19942-7. Abstract

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Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  Ben Pickard
Submitted 31 March 2008
Posted 31 March 2008

In my mind, the study of CNVs in autism (and likely soon in schizophrenia/bipolar disorder, which are a little behind) is likely to put biological meat on the bones of illness etiology and finally lay to rest the annoyingly persistent taunts that genetics hasn’t delivered on its promises for psychiatric illness.

I don’t think it’s necessary at the moment to wring our hands at any inconsistencies between the Walsh et al. and previous studies of CNV in schizophrenia (e.g., Kirov et al., 2008). There are a number of factors which I think are going to influence the frequency, type, and identity of CNVs found in any given study.

1. CNVs are going to be found at the rare/penetrant/familial end of the disease allele spectrum—in direct contrast to the common risk variants which are the targets of recent GWAS studies. In the short term, we are likely to see a large number of different CNVs identified. The nature of this spectrum, however, is that there will be more common pathological CNVs which should be replicated sooner—NRXN1, APBA2 (Kirov et al., 2008), CNTNAP2 (Friedman et al., 2008)—and may be among some of these “low hanging fruit.” For the rarer CNVs, proving a pathological role is going to be a real headache. Large studies or meta-analyses are never going to yield significant p-values for rare CNVs which, nevertheless, may be the chief causes of illness for those few individuals who carry them. Showing clear segregation with illness in families is likely to be the only means to judge their role. However, we must not expect a pure cause-and-effect role for all CNVs: even in the Scottish t(1;11) family disrupting the DISC1 gene, there are several instances of healthy carriers.

2. Sample selection is also likely to be critical. In the Kirov paper, samples were chosen to represent sporadic and family history-positive cases equally. In the Walsh paper, samples were taken either from hospital patients (the majority) or a cohort of childhood onset schizophrenia. Detailed evidence for family history on a case-by-case basis was not given but appeared far stronger in the childhood onset cases. CNVs appeared to be more prevalent, and as expected, more familial, in the latter cohort. A greater frequency was also observed in the Kirov study familial subset.

3. Inclusion criteria are likely to be important—particularly in the more sporadic cases without family history. This is because CNVs found in this group may be commoner and less penetrant—they will be more frequent in cases than in controls but not exclusively found in cases. Any strategy, such as that used in the Kirov paper, which discounts a CNV based on its presence—even singly—in the control group is likely to bias against this class.

4. Technical issues. Certainly, the coverage/sensitivity of the method of choice for the “event discovery” stage is going to influence the minimum size of CNV detectable. However, a more detailed coverage often comes with a greater false-positive rate. Technique choice may also have more general issues. In both of the papers, the primary detection method is based on hybridization of case and pooled control genomes prior to detection on a chip. Thus, a more continuously distributed output may result—and the extra round of hybridization might bias against certain sequences. More direct primary approaches such as Illumina arrays or a second-hand analysis of SNP genotyping arrays may provide a more discrete copy number output, but these, too, can suffer from interpretational issues.

The other major implication of these and other CNV studies is the observation that certain genes “ignore” traditional disease boundaries. For example, NRXN1 CNVs have now been identified in autism and schizophrenia, and CNTNAP2 translocations/CNVs have been described in autism, Gilles de la Tourette syndrome, and schizophrenia/epilepsy. This mirrors the observation of common haplotypes altering risk across the schizophrenia-bipolar divide in numerous association studies. It might be the case that these more promiscuous genes are likely to be involved in more fundamental CNS processes or developmental stages—with the precise phenotypic outcome being defined by other variants or environment. The presence of mental retardation comorbid with psychiatric diagnoses in a number of CNV studies suggests that this might be the case. I look forward to the Venn diagrams of the future which show us the shared neuropsychiatric and disease-specific genes/gene alleles. It will also be interesting to see if the large deletions/duplications involving numerous genes give rise to more severe, familial, and diagnostically more defined syndromes or, alternatively, a more diffuse phenotype. Certainly, it has not been easy to dissect out individual gene contributions to phenotype in VCFS or the minimal region in Down syndrome.

References:

Friedman JI, Vrijenhoek T, Markx S, Janssen IM, van der Vliet WA, Faas BH, Knoers NV, Cahn W, Kahn RS, Edelmann L, Davis KL, Silverman JM, Brunner HG, van Kessel AG, Wijmenga C, Ophoff RA, Veltman JA. CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy. Mol Psychiatry. 2008 Mar 1;13(3):261-6. Abstract

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Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  Christopher RossRussell L. Margolis
Submitted 3 April 2008
Posted 3 April 2008

We agree with the comments of Weinberger, Lencz and Malhotra, and Pickard, and the question raised by Honer about the extent to which the association may be more to mental retardation than schizophrenia. These new studies of copy number variation represent important advances, but need to be interpreted carefully.

We are now getting two different kinds of data on schizophrenia, which can be seen as two opposite poles. The first is from association studies with common variants, in which large numbers of people are required to see significance, and the strengths of the associations are quite modest. These kinds of vulnerability factors would presumably contribute a very modest increase in risk, and many taken together would cause the disease. By contrast, the “private” mutations, as identified by the Sebat study, could potentially be completely causative, but because they are present in only single individuals or very small numbers of individuals, it is difficult to be certain of causality. Furthermore, since some of them in the early-onset schizophrenia patients were present in unaffected parents, one might have to assume the contribution of a common variant vulnerability (from the other parent) as well.

If a substantial number of the private structural mutations are causal, then one might expect to have seen multiple small Mendelian families segregating a structural variant. The situation would then be reminiscent of the autosomal dominant spinocerebellar ataxis, in which mutations (currently about 30 identified loci) in multiple different genes result in similar clinical syndromes. The existence of many small Mendelian families would be less likely if either 1) structural variants that cause schizophrenia nearly always abolish fertility, or 2) some of the SVs detected by Walsh et al. are risk factors, but are usually not sufficient to cause disease. The latter seems more likely.

We think these two poles highlight the continued importance of segregation studies, as have been used for the DISC1 translocation. In order to validate these very rare “private” copy number variations, we believe that it would be important to look for sequence variations in the same genes in large numbers of schizophrenia and control subjects, and ideally to do so in family studies.

One very exciting result of the new copy number studies is the implication of whole pathways rather than just single genes. This highlights the importance of a better understanding of pathogenesis. The study of candidate pathways should help facilitate better pathogenic understanding, which should result in better biomarkers and potentially improve classification and treatment. In genetic studies, development of pathway analysis will be fruitful. Convergent evidence can come from studies of pathogenesis in cell and animal models, but this will need to be interpreted with caution, as it is possible to make a plausible story for so many different pathways (Ross et al., 2006). The genetic evidence will remain critical.

References:

Ross CA, Margolis RL, Reading SA, Pletnikov M, Coyle JT. Neurobiology of schizophrenia. Neuron. 2006 Oct 5;52(1):139-53. Abstract

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Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  Michael Owen, SRF AdvisorMichael O'Donovan (SRF Advisor)George Kirov
Submitted 15 April 2008
Posted 15 April 2008

The idea that a proportion of schizophrenia is associated with rare chromosomal abnormalities has been around for some time, but it has been difficult to be sure whether such events are pathogenic given that most are rare. Two instances where a pathogenic role seems likely are first, the balanced ch1:11 translocation that breaks DISC1, where pathogenesis seems likely due to co-segregation with disease in a large family, and second, deletion of chromosome 22q11, which is sufficiently common for rates of psychosis to be compared with that in the general population. This association came to light because of the recognizable physical phenotype associated with deletion of 22q11, and the field has been waiting for the availability of genome-wide detection methods that would allow the identification of other sub-microscopic chromosomal abnormalities that might be involved, but whose presence is not predicted by non-psychiatric syndromal features. This technology is now upon us in the form of various microarray-based methods, and we can expect a slew of studies addressing this hypothesis in the coming months.

Structural chromosomal abnormalities can take a variety of forms, in particular, deletions, duplication, inversions, and translocations. Generally speaking, these can disrupt gene function by, in the case of deletions, insertions and unbalanced translocations, altering the copy number of individual genes. These are sometimes called copy number variations (CNVs). Structural chromosomal abnormalities can also disrupt a gene sequence, and such disruptions include premature truncation, internal deletion, gene fusion, or disruption of regulatory or promoter elements.

It is, however, worth pointing out that structural chromosomal variation in the genome is common—it has been estimated that any two individuals on average differ in copy number by a total of around 6 Mb, and that the frequency of individual duplications or deletions can range from common through rare to unique, much in the same way as other DNA variation. Also similar to other DNA variation, many structural variants, indeed almost certainly most, may have no phenotypic effects (and this includes those that span genes), while others may be disastrous for fetal viability. Walsh and colleagues have focused upon rare structural variants, and by rare they mean events that might be specific to single cases or families. For this reason, they specifically targeted CNVs that had not previously been described in the published literature or in the Database of Genomic Variants. The reasonable assumption was made that this would enrich for CNVs that are highly penetrant for the disorder. Indeed, Walsh et al. favor the hypothesis that genetic susceptibility to schizophrenia is conferred not by relatively common disease alleles but by a large number of individually rare alleles of high penetrance, including structural variants. As we have argued elsewhere (Craddock et al., 2007), it seems entirely plausible that schizophrenia reflects a spectrum of alleles of varying effect sizes including common alleles of small effect and rare alleles of larger effect, but data from genetic epidemiology do not support the hypothesis that the majority of the disorder reflects rare alleles of large effect.

Walsh et al. found that individuals with schizophrenia were >threefold more likely than controls to harbor rare CNVs that impacted on genes, but in contrast, found no significant difference in the proportions of cases and controls carrying rare mutations that did not impact upon genes. They also found a similar excess of rare structural variants that deleted or duplicated one or more genes in an independent series of cases and controls, using a cohort with childhood onset schizophrenia (COS).

The results of the Walsh study are important, and clearly suggest a role for structural variation in the etiology of schizophrenia. There are, however, a number of caveats and issues to consider. First, it would be unwise on the basis of that study to speculate on the likely contribution of rare variants to schizophrenia as a whole. It is likely correct that, due to selection pressures, highly penetrant alleles for disorders (like schizophrenia) that impair reproductive fitness are more likely to be of low frequency than they are to be common, but this does not imply that the converse is true. That is, one cannot assume that the penetrance of low frequency alleles is more likely to be high than low. Thus, and as pointed out by Walsh et al., it is not possible to know which or how many of the unique events observed in their study are individually pathogenic. Whether individual loci contribute to pathogenesis (and their penetrances) is, as we have seen, hard to establish. Estimating penetrance by association will require accurate measurement of frequencies in case and control populations, which for rare alleles, will have to be very large. Alternatively, more biased estimates of penetrance can be estimated from the degree of co-segregation with disease in highly multiplex pedigrees, but these are themselves fairly rare in schizophrenia, and pedigrees segregating any given rare CNV obviously even more so.

As Weinberger notes, the case for high penetrance (at the level of being sufficient to cause the disorder) is also undermined by their data from COS, where the majority of variants were inherited from unaffected parents. This accords well with the observation that 22q11DS, whilst conferring a high risk of schizophrenia, is still only associated with psychosis in ~30 percent of cases. It also accords well with the relative rarity of pedigrees segregating schizophrenia in a clearly Mendelian fashion, though the association of CNVs with severe illness of early onset might be expected to reduce the probability of transmission.

Third, there are questions about the generality of the findings. Cases in the case control series were ascertained in a way that enriched for severity and chronicity. Perhaps more importantly, the CNVs were greatly overrepresented in people with low IQ. Thus, one-third of all the potentially pathogenic CNVs in the case control series were seen in the tenth of the sample with IQ less than 80. The association between structural variants and low IQ is well known, as is the association between low IQ and psychotic symptoms, and it seems plausible to assume that forms of schizophrenia accompanied by mental retardation (MR) are likely to be enriched for this type of pathogenesis. The question that arises is whether the CNVs in such cases act simply by influencing IQ, which in turn has a non-specific effect on increasing risk of schizophrenia, or whether there are specific CNVs for MR plus schizophrenia, and some which may indeed increase risk of schizophrenia independent of IQ. In the case of 22q11 deletion, risk of schizophrenia does not seem to be dependent on risk of MR, but more work is needed to establish that this applies more generally.

Another reason to caution about the generality of the effect is that Walsh et al. found that cases with onset of psychotic symptoms at age 18 or younger were particularly enriched for CNVs, being greater than fourfold more likely than controls to harbor such variants. There did remain an excess of CNVs in cases with adult onset, supporting a more general contribution, although it should be noted that even in this group with severe disorder, this excess was not statistically significant (Fisher’s exact test, p = 0.17, 2-tailed, our calculation). The issue of age of onset clearly impacts upon assessing the overall contribution CNVs may make upon psychosis, since onset before 18, while not rare, is also not typical. A particular contribution of CNVs to early onset also appears supported by the second series studied, which had COS. However, this is a particularly unusual form of schizophrenia which is already known to have high rates of chromosomal abnormalities. Future studies of more typical samples will doubtless bear upon these issues.

Even allowing for the fact that many more CNVs may be detected as resolution of the methodology improves, the above considerations suggest it is premature to conclude a substantial proportion of cases of schizophrenia can be attributed to rare, highly penetrant CNVs. Nevertheless, even if it turns out that only a small fraction of the disorder is attributable to CNVs, as seen for other rare contributors to the disorder (e.g., DISC1 translocation), such uncommon events offer enormous opportunities for advancing our knowledge of schizophrenia pathogenesis.

References:

Craddock N, O'Donovan MC, Owen MJ. Phenotypic and genetic complexity of psychosis. Invited commentary on ... Schizophrenia: a common disease caused by multiple rare alleles.Br J Psychiatry. 2007 90:200-3. Abstract

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Related News: Copy Number Variations in Schizophrenia: Rare But Powerful?

Comment by:  Ridha JooberPatricia Boksa
Submitted 2 May 2008
Posted 4 May 2008

Walsh et al. claim that rare and severe chromosomal structural variants (SVs) (i.e., not described in the literature or in the specialized databases as of November 2007) are highly penetrant events each explaining a few, if not singular, cases of schizophrenia.

However, their definition of rareness is questionable. Indeed, it is unclear why SVs that are rare (<1 percent) but previously described should be omitted from their analysis. In addition, contrary to their own definition of rareness, the authors included in the COS sample several SVs that have been previously mentioned in the literature (e.g. “115 kb deletion on chromosome 2p16.3 disrupting NRXN1”). Furthermore, some of these SVs (entire Y chromosome duplication) are certainly not rare (by the authors’ definition), nor highly penetrant with regard to psychosis (Price et al., 1967). Finally, as their definition of rareness depends on a specific date, the results of this study will change over time.

As to the assessment of severity, it can equally be concluded from table 2 and using their statistical approach that "patients with schizophrenia are significantly more likely to harbor rare structural variants (6/150) that do not disrupt any gene compared to controls(2/268) (p = 0.03)", thus contradicting their claim. In fact, had they used criteria in the literature (Lee et al., 2007; (Brewer et al., 1999) (i.e., deletion SVs are more likely than duplications to be pathogenic) and appropriate statistical contrasts, deletions are significantly (p = 0.02) less frequent in patients (5/23) than in controls (9/13) who have SVs. In addition, the assumption of high penetrance is questionable given the high level (13 percent) of non-transmitted SVs in parents of COS patients. Is the rate of psychosis proportionately high in the parents? From the data presented, we know that only 2/27 SVs in COS patients are de novo and that “some” SVs are transmitted. Adding this undetermined number of transmitted SVs to the reported non-transmitted SVs will lead to an even larger proportion of parents carrying SVs. Disclosing the inheritance status of SVs in COS patients along with information on diagnoses in parents from this “rigorously characterised cohort,” represents a major criterion for assessing the risk associated with these SVs.

Consequently, it appears that the argument of rareness is rather idiosyncratic and contains inconsistencies, and the one of severity is very open to interpretation. Most importantly, it should be emphasized that amalgamated gene effects at the population level do not allow one to conclude that any single SV actually contributes to schizophrenia in an individual. Thus it is unclear how this study of grouped events differs from the thousands of controversial and underpowered association studies of single genes.

References:

Price WH, Whatmore PB. Behaviour Disorders and Pattern of Crime among XYY males Identified at a Maximum Security Hospital. Brit Med J 1967;1:533-6.

Lee C, Iafrate AJ, Brothman AR. Copy number variations and clinical cytogenetic diagnosis of constitutional disorders. Nat Genet 2007 July;39(7 Suppl):S48-S54.

Brewer C, Holloway S, Zawalnyski P, Schinzel A, FitzPatrick D. A chromosomal duplication map of malformations: regions of suspected haplo- and triplolethality--and tolerance of segmental aneuploidy--in humans. Am J Hum Genet 1999 June;64(6):1702-8.

View all comments by Ridha Joober
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Related News: More Evidence for CNVs in Schizophrenia Etiology—Jury Still Out on Practical Implications

Comment by:  Christopher RossRussell L. Margolis
Submitted 1 August 2008
Posted 1 August 2008

The two recent papers in Nature, from the Icelandic group (Stefansson et al., 2008), and the International Schizophrenia Consortium (2008) led by Pamela Sklar, represent a landmark in psychiatric genetics. For the first time two large studies have yielded highly significant consistent results using multiple population samples. Furthermore, they arrived at these results using quite different methods. The Icelandic group used transmission screening and focused on de novo events, using the Illumina platform in both a discovery population and a replication population. By contrast, the ISC study was a large population-based case-control study using the Affymetrix platform, which did not specifically search for de novo events.

Both identified the same two regions on chromosome 1 and chromosome 15, as well as replicating the previously well studied VCFS region on chromosome 22. Thus, we now have three copy number variants which are replicated and consistent across studies. This provides data on rare highly penetrant variants complementary to the family based study of DISC1 (Porteous et al., 2006), in which the chromosomal translocation clearly segregates with disease, but in only one family. In addition, they are in general congruent with three other studies (Walsh et al., 2008; Kirov et al., 2008; Xu et al., 2008) which also demonstrate a role for copy number variation in schizophrenia. These studies together should put to rest many of the arguments about the value of genetics in psychiatry, so that future studies can now begin from a firmer base.

However, these studies also raise at least as many questions as they answer. One is the role of copy number variation in schizophrenia in the general population. The number of cases accounted for by the deletions on chromosome 1 and 15 in the ISC and Icelandic studies is extremely small--on the order of 1% or less. The extent to which copy number variation, including very rare or even private de novo variants, will account for the genetic risk for schizophrenia in the general population is still unknown. The ISC study indicated that there is a higher overall load of copy number variations in schizophrenia, broadly consistent with Walsh et al and Xu et al but backed up by a much larger sample size, allowing the results to achieve high statistical significance. The implications of these findings are still undeveloped,

Another issue is the relationship to the phenotype of schizophrenia in the general population. Many more genotype-phenotype studies will need to be done. It will be important to determine whether there is a higher rate of mental retardation in the schizophrenia in these studies than in other populations.

Another question is the relationship between these copy number variations (and other rare events) and the more common variants accounting for smaller increases in risk, as in the recent O’Donovan et al. (2008) association study in Nature Genetics. It is far too early to know, but there may well be some combination of rare mutations plus risk alleles that account for cases in the general population. This would then be highly reminiscent of Alzheimer’s disease, Parkinson’s disease, and other diseases which have been studied for a longer period of time.

For instance, in Alzheimer’s disease there are rare mutations in APP and presenilin, as well as copy number variation in APP, with duplications causing the accelerated Alzheimer’s disease seen in Down syndrome. These appear to interact with the risk allele in APOE, and possibly other risk alleles, and are part of a pathogenic pathway (Tanzi and Bertram, 2005). Similarly in Parkinson’s disease, rare mutations in α-synuclein, LRRK2 and other genes can be causative of PD, though notably the G2019S mutation in LRRK2 has incomplete penetrance. In addition, duplications or triplications of α-synuclein can cause familial PD, and altered expression due to promoter variants may contribute to risk. By contrast, deletions in Parkin cause an early onset Parkinsonian syndrome (Hardy et al., 2006). Finally, much of PD may be due to genetic risk factors or environmental causes that have not yet been identified. Further studies will likely lead to the elucidation of pathogenic pathways. These diseases can provide a paradigm for the study of schizophrenia and other psychiatric diseases. One difference is that the copy number variations in the neurodegenerative diseases are often increases in copies (as in APP and α-synuclein), consistent with gain of function mechanisms, while the schizophrenia associations were predominantly with deletions, suggesting loss of function mechanisms. The hope is that as genes are identified, they can be linked together in pathways, leading to understanding of the neurobiology of schizophrenia (Ross et al., 2006).

The key unanswered questions, of course, are what genes or other functional domains are deleted at the chromosome 1, 15, and 22 loci, whether the deletions at these loci are sufficient in themselves to cause schizophrenia, and, if sufficient, the extent to which the deletions are penetrant. Both of the current studies identified deletions large enough to include several genes. The hope is that at least a subset of copy number variations (unlike SNP associations identified in schizophrenia to date) may be causative, making the identification of the relevant genes or other functional domains—at least in principle—more feasible.

Another tantalizing observation is that the copy number variations associated with schizophrenia were defined by flanking repeat regions. This raises the question of the extent to which undetected smaller insertions, deletions or other copy number variations related to other repetitive motifs, such as long tandem repeats, may also be associated with schizophrenia. Identification and testing of these loci may prove a fruitful approach to finding additional genetic risk factors for schizophrenia.

References:

Hardy J, Cai H, Cookson MR, Gwinn-Hardy K, Singleton A. Genetics of Parkinson's disease and parkinsonism. Ann Neurol. 2006 Oct;60(4):389-98. Abstract

Kirov G, Gumus D, Chen W, Norton N, Georgieva L, Sari M, O'Donovan MC, Erdogan F, Owen MJ, Ropers HH, Ullmann R. Comparative genome hybridization suggests a role for NRXN1 and APBA2 in schizophrenia. Hum Mol Genet . 2008 Feb 1 ; 17(3):458-65. Abstract

Porteous DJ, Thomson P, Brandon NJ, Millar JK. The genetics and biology of DISC1—an emerging role in psychosis and cognition. Biol Psychiatry. 2006 Jul 15;60(2):123-31. Abstract

Ross CA, Margolis RL, Reading SA, Pletnikov M, Coyle JT. Neurobiology of schizophrenia. Neuron. 2006 Oct 5;52(1):139-53. Abstract

Singleton A, Myers A, Hardy J. The law of mass action applied to neurodegenerative disease: a hypothesis concerning the etiology and pathogenesis of complex diseases. Hum Mol Genet. 2004 Apr 1;13 Spec No 1:R123-6. Abstract

Tanzi RE, Bertram L. Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective. Cell. 2005 Feb 25;120(4):545-55. Abstract

Walsh T, McClellan JM, McCarthy SE, Addington AM, Pierce SB, Cooper GM, Nord AS, Kusenda M, Malhotra D, Bhandari A, Stray SM, Rippey CF, Roccanova P, Makarov V, Lakshmi B, Findling RL, Sikich L, Stromberg T, Merriman B, Gogtay N, Butler P, Eckstrand K, Noory L, Gochman P, Long R, Chen Z, Davis S, Baker C, Eichler EE, Meltzer PS, Nelson SF, Singleton AB, Lee MK, Rapoport JL, King MC, Sebat J. Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science. 2008 Apr 25;320(5875):539-43. Abstract

Xu B, Roos JL, Levy S, van Rensburg EJ, Gogos JA, Karayiorgou M. Strong association of de novo copy number mutations with sporadic schizophrenia. Nat Genet. 2008 Jul;40(7):880-5. Abstract

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Related News: More Evidence for CNVs in Schizophrenia Etiology—Jury Still Out on Practical Implications

Comment by:  Daniel Weinberger, SRF Advisor
Submitted 3 August 2008
Posted 3 August 2008

Several recent reports have suggested that rare CNVs may be highly penetrant genetic factors in the pathogenesis of schizophrenia, perhaps even singular etiologic events in those cases of schizophrenia who have them. This is potentially of enormous importance, as the definitive identification of such a “causative” factor may be a major step in unraveling the biologic mystery of the condition. I would stress several issues that need to be considered in putting these recent findings into a broader perspective.

It is very difficult to attribute illness to a private CNV, i.e., one found only in a single individual. This point has been potently illustrated by a study of clinically discordant MZ twins who share CNVs (Bruder et al., AJHG, 2008). Inherited CNVs, such as those that made up almost all of the CNVs described in the childhood onset cases of the study by Walsh et al. (Science, 2008), are by definition not highly penetrant (since they are inherited from unaffected parents). The finding by Xu et al. (Nat Gen, 2008) that de novo (i.e., non-inherited) CNVs are much more likely to be associated with cases lacking a family history is provocative but difficult to interpret as no data are given about the size of the families having a family history and those not having such a history. Unless these family samples are of comparable size and obtained by a comparable ascertainment strategy, it is hard to know how conclusive the finding is. Indeed, in the study of Walsh et al., rare CNVs were just as likely to be found in patients with a positive family history. Finally, in contrast to private CNVs, recurrent (but still rare) CNVs, such as those identified on 1q and 15q in the studies of the International Schizophrenia Consortium (Nature, 2008) and Stefansson et al. (Nature, 2008), are strongly implicated as being associated with the diagnosis of schizophrenia and therefore likely involved in the causation of the illnesses in the cases having these CNVs. In all, these new CNV regions, combined with the VCFS region on 22q, suggest that approximately five to 10 patients out of 1,000 who carry the diagnosis of schizophrenia may have a well-defined genetic lesion (i.e., a substantial deletion or duplication).

The overarching question now is how relevant these findings are to the other 99 percent of individuals with this diagnosis who do not have these recurrent CNVs. Before we had the capability to perform high-density DNA hybridization and SNP array analyses, chromosomal anomalies associated with the diagnosis of schizophrenia were identified using cytogenetic techniques. Indeed, VCFS, XXX, XXY (Kleinfelter’s syndrome), and XO (Turner syndrome) have been found with similarly increased frequency in cases with this diagnosis in a number of studies. Now that we have greater resolution to identify smaller structural anomalies, the list of congenital syndromes that increase the possibility that people will manifest symptoms that earn them this diagnosis appears to be growing rapidly. Are we finding causes for the form of schizophrenia that most psychiatrists see in their offices, or are we instead carving out a new set of rare congenital syndromes that share some clinical characteristics, as syphilis was carved out from the diagnosis of schizophrenia at the turn of the twentieth century? Is schizophrenia a primary expression of these anomalies or a secondary manifestation? VCFS is associated with schizophrenia-like phenomena but even more often with mild mental retardation, autism spectrum, and other psychiatric manifestations. The same is true of the aneuploidies that increase the probability of manifesting schizophrenia symptoms. The two new papers in Nature allude to the possibility that epilepsy and intellectual limitations may also be associated with these CNVs. The diagnostic potential of any of these new findings cannot be determined until the full spectrum of their clinical manifestations is clarified.

One of the important insights that might emerge from identification of these new CNV syndromes is the identification of candidate genes that may show association with schizophrenia based on SNPs in these regions. VCFS has been an important source of promising candidate genes with broader clinical relevance (e.g., PRODH, COMT). Stefansson et al. report, however, that none of the 319 SNPs in the CNV regions showed significant association with schizophrenia in quite a large sample of individuals not having deletions in these regions. The Consortium report also presumably has the results of SNP association testing in these regions in their large sample but did not report them. It is very important to explore in greater genetic detail these regions of the genome showing association with the diagnosis of schizophrenia in samples lacking these lesions and to fully characterize the clinical picture of individuals who have them. It is hoped that insights into the pathogenesis of symptoms related to this diagnosis will emerge from these additional studies.

Anyone who has worked in a public state hospital or chronic schizophrenia care facility (where I spent over 20 years) is not surprised to find an occasional patient with a rare congenital or acquired syndrome who expresses symptoms similar to those individuals also diagnosed with schizophrenia who do not have such rare syndromes. Our diagnostic procedures are not precise, and the symptoms that earn someone this diagnosis are not specific. Schizophrenia is not something someone has; it is a diagnosis someone is given. In an earlier comment for SRF on structural variations in the genome related to autism, I suggested that, “From a genetic point of view, autism is a syndrome that can be reached from many directions, along many paths. It is not likely that autism is any more of a discrete disease entity than say, blindness or mental retardation.” These new CNV syndromes manifesting schizophrenia phenomena are probably a reminder that the same is true of what we call schizophrenia.

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Related News: Mixed Message: 15q13.3 Deletions Confer Risk, But for What?

Comment by:  Ben Pickard
Submitted 21 January 2009
Posted 21 January 2009

Before Christmas, an insightful discussion between SRF's Pete Farley and researchers Heather Mefford and Evan Eichler delved into the complex interplay between genotype (copy number variant status at 1q21.1) and phenotype (psychiatric illness, autism, mental retardation, and congenital abnormalities) (see SRF related news story). The upshot was that although deletions at this locus were statistically associated with pathologies, the severity and nature of those pathologies was extremely variable. This raised questions about whether researchers and clinicians should focus on the disease or the deletion, and what the mechanisms that determine the clinical endpoint might be. This is becoming a clear trend. Another CNV region at 16p11.2 has also been variously associated with both autism and schizophrenia. Deletions of just a single gene, CNTNAP2, as opposed to a gene cluster, have also shown this phenomenon of variable phenotype expression—deletion carriers have been diagnosed with autism, Gilles de la Tourette/obsessive compulsive disorder, schizophrenia/epilepsy, or remain entirely healthy (Bakkaloglu et al., 2008; Friedman et al., 2008; Verkerk et al., 2003; Belloso et al., 2007).

In the same vein, this new paper by Helbig and colleagues describes yet another example of a discrete copy number variant (microdeletion) that was originally linked with psychiatric phenotypes but is now also shown to give rise to idiopathic generalized epilepsy (IGE). The deletion is at 15q13.3, which encompasses the candidate neurotransmitter receptor gene, CHRNA7, among others. In fact, with a frequency of 1 percent in the IGE population and absence in controls, the deletion is the strongest genetic risk factor for this condition and is more prevalent in IGE than in either mental retardation or schizophrenia.

Although the study of CNVs has highlighted this genotype-phenotype issue, it has been observed previously in the context of the overlap of linkage hotspots between schizophrenia and bipolar disorder (Berrettini, 2003), in case-control association studies linking the same gene to multiple disorders (Chubb et al., 2008), and in the case of the Scottish family with the t(1;11) translocation disrupting DISC1, in which carrier phenotypes ranged from healthy to major depression, bipolar disorder, and schizophrenia (Blackwood et al., 2001).

So we are now faced with complex genetic disorders that really live up to their name. As such, two particular issues warrant further discussion.

The first issue is that clinicians seem to observe discrete rather than continuous disorder phenotypes. Despite the current diagnostic manuals leaving little room for diagnostic leeway, it seems that the majority of case phenotypes tend toward a limited number of outcomes such as schizophrenia, bipolar disorder, mental retardation, autism, and epilepsy. Moreover, no psychiatrist can distinguish DISC1 schizophrenia from 1q21.1 schizophrenia or NRG1 schizophrenia without recourse to genetic methodologies, suggesting that there is a positive biological drive towards the endpoint. To borrow what may be a useful analogy from physics, the system is “chaotic” (in terms of its genetic input and its effect on cellular biology) but tends toward “strange attractors” (a limited set of diagnoses) [http://en.wikipedia.org/wiki/Attractor]. Why might this be so? It may be that there are several higher order functional bottlenecks within the brain such as synaptic transmission efficiency, cortical development, astrocyte/oligodendrocyte function, hippocampal neurogenesis, higher order communication between brain regions, etc. These act to “sum” the expected environmental, genetic, and cellular complexity present within an individual and transform it into a limited set of potential outcomes—in essence, these are the strange attractors.

The next issue is how the same mutation can give rise to two (or more) different conditions. It may be useful to think of the Knudson “two-hit” hypothesis of cancer in which environment and other genetic factors act subsequent to a “deep” genetic fault (Knudson, 1971).

The CNV examples above may represent such fundamental disruptions and most probably impinge on neurodevelopmental pathways, priming the brain to be tipped over the threshold into a disease state. In fact, the t(1:11) translocation carriers present evidence for such a phenomenon as both healthy and affected carriers show abnormal P300 brain response activities suggesting this endophenotype highlights an underlying brain dysfunction (Blackwood et al., 2001).

We have to postulate that the additional genetic or environmental influences (modifiers) not only determine entry into the disease state but also dictate the final outcome. Possible candidates for modifiers of the deletions above are the remaining single copy alleles at the CNV locus—exposed recessive mutations, imprinting, or epigenetic modification could all alter expressivity and penetrance of the deletion phenotype. However, limited studies by Eichler’s group seem to discount this possibility (Mefford et al., 2008).

In any case, genomewide association and CNV studies suggest that there is plenty of scope for a sufficient burden of genetic modifiers outside the CNV region. This may also fit in with the seemingly disparate concepts of rare/familial variants exposed by linkage and common/low odds ratio variants revealed by association. Both act causally with the former potentially acting as the “first hit.”

As time progresses, we will move towards the definition of the range of phenotypes potentially resulting from each genotype and the spectrum of genotypes causing each phenotype. CNVs represent a pretty blunt tool to dissect finer relationships between genotype and phenotype, so it is to be expected that rare but penetrant point mutations that emerge from resequencing projects will be of greater use in dissecting function-phenotype links—as has been seen with the connexin gene family, for example (Rabionet et al., 2002).

In summary, it is to be hoped that the clinical and research communities are able to embrace these complexities for what they offer—a deeper understanding of these disorders, one that is intimately linked to the development and function of the brain.

References:

Bakkaloglu B, O'roak BJ, Louvi A, Gupta AR, Abelson JF, Morgan TM, Chawarska K, Klin A, Ercan-Sencicek AG, Stillman AA, Tanriover G, Abrahams BS, Duvall JA, Robbins EM, Geschwind DH, Biederer T, Gunel M, Lifton RP, State MW. Molecular cytogenetic analysis and resequencing of contactin associated protein-like 2 in autism spectrum disorders. Am J Hum Genet. 2008 Jan 1;82(1):165-73. Abstract

Friedman JI, Vrijenhoek T, Markx S, Janssen IM, van der Vliet WA, Faas BH, Knoers NV, Cahn W, Kahn RS, Edelmann L, Davis KL, Silverman JM, Brunner HG, van Kessel AG, Wijmenga C, Ophoff RA, Veltman JA. CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy. Mol Psychiatry. 2008 Mar 1;13(3):261-6. Abstract

Verkerk AJ, Mathews CA, Joosse M, Eussen BH, Heutink P, Oostra BA, . CNTNAP2 is disrupted in a family with Gilles de la Tourette syndrome and obsessive compulsive disorder. Genomics. 2003 Jul 1;82(1):1-9. Abstract

Belloso JM, Bache I, Guitart M, Caballin MR, Halgren C, Kirchhoff M, Ropers HH, Tommerup N, Tümer Z. Disruption of the CNTNAP2 gene in a t(7;15) translocation family without symptoms of Gilles de la Tourette syndrome. Eur J Hum Genet. 2007 Jun 1;15(6):711-3. Abstract

Berrettini W. Evidence for shared susceptibility in bipolar disorder and schizophrenia. Am J Med Genet C Semin Med Genet. 2003 Nov 15;123C(1):59-64. Abstract

Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. The DISC locus in psychiatric illness. Mol Psychiatry. 2008 Jan 1;13(1):36-64. Abstract

Blackwood DH, Fordyce A, Walker MT, St Clair DM, Porteous DJ, Muir WJ. Schizophrenia and affective disorders--cosegregation with a translocation at chromosome 1q42 that directly disrupts brain-expressed genes: clinical and P300 findings in a family. Am J Hum Genet. 2001 Aug 1;69(2):428-33. Abstract

Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A. 1971 Apr 1;68(4):820-3. Abstract

Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, Buysse K, Huang S, Maloney VK, Crolla JA, Baralle D, Collins A, Mercer C, Norga K, de Ravel T, Devriendt K, Bongers EM, de Leeuw N, Reardon W, Gimelli S, Bena F, Hennekam RC, Male A, Gaunt L, Clayton-Smith J, Simonic I, Park SM, Mehta SG, Nik-Zainal S, Woods CG, Firth HV, Parkin G, Fichera M, Reitano S, Lo Giudice M, Li KE, Casuga I, Broomer A, Conrad B, Schwerzmann M, Räber L, Gallati S, Striano P, Coppola A, Tolmie JL, Tobias ES, Lilley C, Armengol L, Spysschaert Y, Verloo P, De Coene A, Goossens L, Mortier G, Speleman F, van Binsbergen E, Nelen MR, Hochstenbach R, Poot M, Gallagher L, Gill M, McClellan J, King MC, Regan R, Skinner C, Stevenson RE, Antonarakis SE, Chen C, Estivill X, Menten B, Gimelli G, Gribble S, Schwartz S, Sutcliffe JS, Walsh T, Knight SJ, Sebat J, Romano C, Schwartz CE, Veltman JA, de Vries BB, Vermeesch JR, Barber JC, Willatt L, Tassabehji M, Eichler EE. Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes. N Engl J Med. 2008 Oct 16;359(16):1685-99. Abstract

Rabionet R, López-Bigas N, Arbonès ML, Estivill X. Connexin mutations in hearing loss, dermatological and neurological disorders. Trends Mol Med. 2002 May 1;8(5):205-12. Abstract

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Related News: CNV “Double Whammies” May Account for Variable Neuropsychiatric Phenotypes

Comment by:  Ben Pickard
Submitted 25 February 2010
Posted 25 February 2010

In their Nature Genetics paper, Girirajan et al. contribute to the slow shift of focus in the field of complex genetic disorders, away from population risks towards the risks specific to the individual. The driving force of this shift is the ongoing discovery of mutations more penetrant than the common single nucleotide polymorphisms (SNPs) studied in case-control association studies. Copy number variants (CNVs) and coding variants are the two principal classes of these mutations, typified by their relative rarity, frequent familiality, and generally higher odds ratio (OR) values indicative of their impact.

Considerable evidence from increased levels of comorbidity, dysmorphic features, brain structural changes, and latent endophenotypes suggests that early neurodevelopmental deficits can predispose to later neuropsychiatric conditions (Ross et al., 2006). This paper demonstrates how the phenotype in a single individual can be more closely linked with the causative genotype when the simultaneous action of two CNVs is considered. This provides some concrete evidence to explain how seemingly disparate diagnoses (for example, epilepsy, autism, schizophrenia and mental retardation) and variable penetrance can occur in different individuals who ostensibly carry the same pathogenic CNV (van Bon et al., 2009; Mefford et al., 2008)—specifically, it appears that a second CNV can translate a generalized neurodevelopmental pathology into a more specific, severe, and reproducible final clinical endpoint. Hence, the authors invoke a “two-hit” hypothesis, as originally applied by Knudson to explain the observed progression of familial forms of cancer (Knudson, 1971).

From a semantic point of view, the use of the “two-hit” terminology is a little loosely applied here. Originally, this described an inherited predisposition to cancer in the form of a tumor suppressor mutation which was coupled with a later, somatic, “second hit” oncogenic mutation that resulted in the tumor. In the current application of the term, both mutational changes are germline, present from the outset, and even a single CNV “hit” can still produce a phenotype. However, the comprehensive data in the paper and the analogy used are thought-provoking, as they highlight three etiological issues, outlined below, which may have wide applicability to complex genetic disorders.

1. A key observation from this paper is that the hypothetical Venn diagrams illustrating the overlapping relationships between the varied diagnoses and also between diagnoses and genomic variation would be highly complex. The phenotypic reach of each CNV may also be considerably greater than we currently suppose—limited by the developmental or neurological conditions that have yet to be comprehensively studied by comparative genomic hybridization. Conversely, the interpretation of shared genetic risk in association and epidemiological studies may have to be reassessed in the light of this paper’s findings. For example, the demonstration of a component of genetic contribution shared between bipolar disorder and schizophrenia may not be such a simple story (Lichtenstein et al., 2009). That component may be just a genetic contribution to generalized neurodevelopmental failure which predisposes to neuropsychiatric disorders in the context of “second hit,” disease-specific mutations (schizophrenia or bipolar disorder, in this case). Can a gene truly be called a “schizophrenia gene” if you haven’t discounted its role in other conditions first? This is reminiscent of the shared genetic contribution to underlying autoimmunity processes which is emerging from the genomewide association studies of diagnostically discrete disorders such as type I diabetes, Crohn’s disease, and rheumatoid arthritis (Baranzini, 2009).

2. The two-hit terminology and the involvement of neurodevelopmental deficits clearly imply a sequential pattern of CNV effect—early predisposition followed by later resolution of diagnosis. Mouse gene knockouts have shown that such staged action is indeed possible, and its confounding effects on animal models of disease led to the development of spatially and temporally controllable transgenic technologies (Gingrich et al., 1998). However, to prove this in the context of the CNV model will require both the individual spatiotemporal expression profiles of the deleted genes and also some measure of individual gene dosage sensitivity to be correlated with the apparent mode of CNV action (developmental or “modifier”).

3. The model’s restriction to just two contributory CNVs as presented here is, in my opinion, a by-product of CNV scarcity, visibility, and sample size, rather than a genuine biological phenomenon. As the authors state, the accuracy of genotype-phenotype correlations at the level of the individual will undoubtedly increase when common and rare SNP variation is also taken into account—potentially to the point where predictive diagnosis is realistic. Likewise, the authors’ suggestion that the second CNV has a “modifier” effect on the foundation CNV is enticing, and backed up by the ubiquity of the 16p12.1 across several genomic disorders, but it is still difficult at this stage to discount the equally compelling explanation that two CNVs just represent a simple increase in mutational load. However, this latter explanation has its own problems, as it suggests a continuum of illness dictated by additive genetic risk. Does this continuum start with mild developmental delay and end with neuropsychiatric illness—or vice versa?

Finally, it must not be forgotten that schizophrenia, bipolar, autism, and epilepsy can exist without comorbid traits and without evidence for developmental issues. Is this, then, an important dimension to be considered in the future sub-categorization of these disorders?

References:

Ross CA, Margolis RL, Reading SA, Pletnikov M, Coyle JT. Neurobiology of schizophrenia. Neuron . 2006 Oct 5 ; 52(1):139-53. Abstract

van Bon BW, Mefford HC, Menten B, Koolen DA, Sharp AJ, Nillesen WM, Innis JW, de Ravel TJ, Mercer CL, Fichera M, Stewart H, Connell LE, Ounap K, Lachlan K, Castle B, Van der Aa N, van Ravenswaaij C, Nobrega MA, Serra-Juhé C, Simonic I, de Leeuw N, Pfundt R, Bongers EM, Baker C, Finnemore P, Huang S, Maloney VK, Crolla JA, van Kalmthout M, Elia M, Vandeweyer G, Fryns JP, Janssens S, Foulds N, Reitano S, Smith K, Parkel S, Loeys B, Woods CG, Oostra A, Speleman F, Pereira AC, Kurg A, Willatt L, Knight SJ, Vermeesch JR, Romano C, Barber JC, Mortier G, Pérez-Jurado LA, Kooy F, Brunner HG, Eichler EE, Kleefstra T, de Vries BB. Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome. J Med Genet . 2009 Aug 1 ; 46(8):511-23. Abstract

Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, Buysse K, Huang S, Maloney VK, Crolla JA, Baralle D, Collins A, Mercer C, Norga K, de Ravel T, Devriendt K, Bongers EM, de Leeuw N, Reardon W, Gimelli S, Bena F, Hennekam RC, Male A, Gaunt L, Clayton-Smith J, Simonic I, Park SM, Mehta SG, Nik-Zainal S, Woods CG, Firth HV, Parkin G, Fichera M, Reitano S, Lo Giudice M, Li KE, Casuga I, Broomer A, Conrad B, Schwerzmann M, Räber L, Gallati S, Striano P, Coppola A, Tolmie JL, Tobias ES, Lilley C, Armengol L, Spysschaert Y, Verloo P, De Coene A, Goossens L, Mortier G, Speleman F, van Binsbergen E, Nelen MR, Hochstenbach R, Poot M, Gallagher L, Gill M, McClellan J, King MC, Regan R, Skinner C, Stevenson RE, Antonarakis SE, Chen C, Estivill X, Menten B, Gimelli G, Gribble S, Schwartz S, Sutcliffe JS, Walsh T, Knight SJ, Sebat J, Romano C, Schwartz CE, Veltman JA, de Vries BB, Vermeesch JR, Barber JC, Willatt L, Tassabehji M, Eichler EE. Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes. N Engl J Med . 2008 Oct 16 ; 359(16):1685-99. Abstract

Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A . 1971 Apr 1 ; 68(4):820-3. Abstract

Lichtenstein P, Yip BH, Björk C, Pawitan Y, Cannon TD, Sullivan PF, Hultman CM. Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study. Lancet . 2009 Jan 17 ; 373(9659):234-9. Abstract

Baranzini SE. The genetics of autoimmune diseases: a networked perspective. Curr Opin Immunol . 2009 Dec 1 ; 21(6):596-605. Abstract

Gingrich JR, Roder J. Inducible gene expression in the nervous system of transgenic mice. Annu Rev Neurosci . 1998 Jan 1 ; 21():377-405. Abstract

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