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Copy Number Variants in Schizophrenia Converge on the Synapse

4 June 2010. A new study fingers a diverse array of copy number variants (CNVs)—duplications or deletions of DNA—in schizophrenia. According to the report published online in PNAS on May 20, many of these CNVs disrupt genes involved in synaptic transmission.

The results put the spotlight on the interacting network of molecules that contribute to communication between neurons, and suggest that problems with any one of these molecules may disrupt brain signaling and confer susceptibility to schizophrenia. Although previous studies of schizophrenia have turned up genes for individual synaptic proteins (see SRF related news story), the new findings seem particularly enriched for neuronal signaling genes.

"Most of the interesting genes that came up had something to do with synaptic function or transmission or signaling in the brain," said senior author Hakon Hakonarson of the Children's Hospital of Philadelphia (CHOP).

Though not the first to look for CNVs in schizophrenia (see SRF related news story), the study is one of the most thorough, using a Affymetrix 6.0 gene chip that probed over 900,000 regions of the genome for deletions or duplications and over 900,000 single nucleotide polymorphisms (SNPs). This high-resolution search revealed CNVs large and small—including many under 100 kb, which have been overlooked in previous studies.

Make it a combo
First author Joseph Glessner and colleagues at CHOP combined their samples from patients and controls with those from collaborators at Mt. Sinai School of Medicine and the University of Pennsylvania to assemble the needed samples. They also included a substantial amount of data from samples that had already been genotyped with the same chip and deposited into the database of Genotype and Phenotype (dbGaP)—but whose CNVs had not yet been analyzed (O'Donovan et al., 2008). This resulted in a total of 1,735 schizophrenia samples and 3,485 controls, which were divided into a discovery and replication group.

The researchers detected CNVs in both cases and controls, and some of these CNVs overlapped in the same genome regions. Deletions or duplications in eight of these CNV-containing regions were associated with schizophrenia in both the discovery and replication cohorts. While CNVs in five of these regions occurred nearly exclusively in schizophrenia cases, with a frequency of less than 0.25 percent among controls, CNVs in the other three regions were more common, but still occurred more frequently in schizophrenia cases.

The CNVs landed either within genes, or within striking distance, where they may still do some damage to regulatory regions of the nearby gene. Of the many genes impacted by these CNVs, four were particularly interesting. CACNA1B, which encodes a subunit of a calcium channel involved in neurotransmitter release, has been associated with schizophrenia (Moskvina et al., 2009), as has its cousin, CACNA1C, which has achieved significance in a genomewide association study of bipolar disorder (see SRF related news story), and in a combined schizophrenia/bipolar sample reported at the World Congress on Psychiatric Genetics last fall (see SRF related conference story).

Other featured genes were DOC2A, which encodes a calcium sensor involved in neurotransmitter release, and RET and RIT2, Ras-related genes important for neural development and signal transduction within cells. Though Ras is best known as a tumor suppressor, studies have uncovered a role for these small GTPases in coupling calcium signals to synaptic plasticity (Finkbeiner et al., 1996).

Other genes in the study's haul included PDPR, which regulates glucose metabolism in the brain, genes related to mitochondria function, and the more familiar COMT, which controls catecholamine neurotransmitter levels.

Focus on function
To help make sense of the jumble of genes impacted by CNVs in the schizophrenia samples, the researchers investigated whether their genes belonged to any particular functional pathway. The bioinformatics tool DAVID indicated that the CNVs tended to affect genes involved in synaptic transmission.

This lends support to proposals that the myriad genes disrupted in schizophrenia have neuronal communication in common. A problem with any one component of the complicated network of molecules involved in passing signals from one neuron to the next may distort the flow of information in the brain and result in symptoms of schizophrenia. This means individuals with schizophrenia could have different genetic reasons for the disorder—a tidy solution to the ever-expanding list of genes associated with schizophrenia. "So each individual one is rare, but when you put them all together, they actually make up quite a significant component of schizophrenia," Hakonarson said.

The researchers plan to sequence the genes turned up in this study in other individuals with schizophrenia in order to identify new variants that their current methods aren't picking up. Finding multiple variants in multiple genes would swell the potential genetic reasons for schizophrenia.—Michele Solis.

Glessner JT, Reilly MP, Kim CE, Takahashi N, Albano A, Hou C, Bradfield JP, Zhang H, Sleiman PM, Flory JH, Imielinski M, Frackelton EC, Chiavacci R, Thomas KA, Garris M, Otieno FG, Davidson M, Weiser M, Reichenberg A, Davis KL, Friedman JI, Cappola TP, Margulies KB, Rader DJ, Grant SF, Buxbaum JD, Gur RE, Hakonarson H. Strong synaptic transmission impact by copy number variations in schizophrenia. Proc Natl Acad Sci U S A. 2010 May 20. Abstract

Comments on Related News

Related News: Channeling Mental Illness: GWAS Links Ion Channels, Bipolar Disorder

Comment by:  Melvin G. McInnis
Submitted 19 August 2008
Posted 19 August 2008

The work by Ferreira et al. exemplifies the growing enthusiasm for collaborative work among investigators and marks the new era of collaborative genetic research in complex disorders. The LD data found in the extant HapMap SNPs allow investigators to use sophisticated computational approaches to impute genotypes based on these HapMap data sets and the data generated from the experimental sample, thereby maximizing the utility of the actual genotyping itself. Nothing short of brilliant. Correlates between imputed and true genotypes were estimated to be 0.987, which is quite good. The significance estimates of the combined data analyses of the three data sets identifies two genes (ANK3 and CACNA1C) in the genomewide significance range with a p value of 10-8, which is most reassuring and even more so considering that the CACNA1C gene was identified previously. The humbling fact in the mix is that the odds ratios are modest, ranging from 1.2 to 1.4, which is nonetheless in a similar arena as other complex genetic disorders such as diabetes. It is further humbling (and consistent with the modest ORs) to consider that the frequency of the risk allele for the CACNA1C gene is 7.5 percent in the BP cases and 5.6 percent in the unaffected control individuals. Finally, there was no effect of the sub-diagnostic categories, age of onset, presence of psychosis, or sex. The highly encouraging point is that these genes appear to be in pathways that are affected by lithium, the gold standard of care for BP disorder. The anchorage of a genetic finding within a mechanism of an established treatment for BP disorder (lithium) lends substantial credibility to overall results. The next questions of research will relate to the efficacy of lithium relative to genotypes of these genes and others within their pathways. These findings raise several clinical questions, and integration of clinical outcome patterns with genetic data can be expected to shed further light on the etiology of the disease and the genetics of treatment response. Long live lithium.

View all comments by Melvin G. McInnis

Related News: Channeling Mental Illness: GWAS Links Ion Channels, Bipolar Disorder

Comment by:  John I. Nurnberger, Jr.
Submitted 19 August 2008
Posted 19 August 2008

Ferreira et al. propose two specific genes to be related to bipolar disorder, ANK3, which is indirectly related to sodium channels, and CACNA1C, which is a calcium channel subunit. They hypothesize that bipolar disorder is, at least in part, a channelopathy. This hypothesis is consistent with a number of physiological observations made over the past several decades, as reviewed elsewhere.

The genetic data these authors present is certainly suggestive. They have analyzed three independent data sets, STEP-UCL (Sklar et al., 2008), Wellcome Trust (Wellcome Trust Case Control Consortium, 2007), and a third set called ED-DUB-STEP2 (not yet published). Their total sample exceeds 4,000 cases and 6,000 controls. They have direct genotype data on >300,000 SNPs and have imputed nearly 1.5 million additional. Their highest significance values (10-7 to 10-9) include a combination of genotyped and imputed SNPs. For each of these, the combined p value is a product of modest but consistent associations in the three independent data sets.

ANK3 features rs10994336 at 9x10e-9 and rs1938526 at 1x10e-8. By my reading, these two polymorphisms are both slightly distal to the gene but the second is within 10-20 kB. The first of these is imputed, and thus the p value should probably be judged as more imprecise. Both of these polymorphisms are associated with an odds ratio of ~1.4 and a minor allele frequency of ~5 percent in controls.

The CACNA1C data is based on more common polymorphisms (~30 percent in controls) and an OR~1.2. Again two SNPs are featured (rs1006737 at 7x10e-8, genotyped, and rs1024582 at 2x10-7, imputed). A third region near an uncharacterized gene (on 15q14) is also featured.

Examination of available published data from STEP-UCL and WTCCC on ANK3 and CACNA1C does not show obvious evidence of association among SNPs across each of the named genes, but reasonably consistent signals of modest significance, which is what one might expect, and this does suggest that the featured SNPs are not completely anomalous, but may represent a pattern of genotype deviation across the two genes.

Needless to say, our investigators in the GAIN (Genetic Analysis Information Network) bipolar group are extremely interested in this report and are avidly following the lead provided by Ferreira to attempt to confirm these signals in our own data. I am also pleased to say that GAIN has provided the stimulus for an international consortium that includes representatives from the Ferreira group as well as many other investigators, dedicated to assembling yet larger samples of bipolar cases and controls to elucidate the genetics of this condition through genomewide methods.

This is an important report, and it may represent a breakthrough in bipolar genetic studies. The signals for ANK3 and CACNA1C appear very promising, and we hope that they prove to be consistently observed in other data sets as well. We anticipate that additional confirmed single genes will emerge soon as well, and that the genetic structure of these disorders will be elucidated using similar methods in large data sets in the coming years.

View all comments by John I. Nurnberger, Jr.

Related News: Channeling Mental Illness: GWAS Links Ion Channels, Bipolar Disorder

Comment by:  Peter P. Zandi
Submitted 21 August 2008
Posted 21 August 2008

Are we there yet? Have we in the field of bipolar genetics finally been delivered to the promised land by GWAS? For the past year or so since GWAS burst on the scene, we have had to watch with envy as an impressive list of genes were convincingly implicated in a range of other complex diseases like type 2 diabetes, the apparent poster child for GWAS. Now, is it our turn?

The first attempts at individual-level GWAS of bipolar disorder by WTCCC and STEP-UCL were exciting because of their novelty, but the results were not particularly overwhelming. None of the findings withstood correction for the massive multiple testing inherent in GWAS, and those at the top were of ambiguous relevance to bipolar disorder. Confronted with such uninspiring findings, one could not be faulted for experiencing pangs of doubt that maybe for psychiatric disorders, GWAS would prove no better than its dusty old predecessor, the genomewide linkage study, in illuminating the underlying genetic architecture.

Nevertheless, encouraged by the lessons learned from GWAS of type 2 diabetes that the road to the promised land is not paved in individual glory but in collaborations and consortiums, the investigators of WTCCC and STEP-UCL combined samples with a third previously unstudied collection (dubbed ED-DUB-STEP2) to assemble one of the largest samples in bipolar disorder yet to be analyzed by GWAS. The combined sample included 4,387 cases and 6,209 controls genotyped at 325,690 overlapping SNPs, which after imputation yielded data on 1.8 million variants. The results from this effort were recently reported in a manuscript by Ferreira and colleagues published in the latest edition of Nature Genetics.

Despite potential concerns about the genetic and/or clinical heterogeneity of the combined sample (e.g., the genomic inflation factor was estimated to be 1.11, even after controlling for two quantitative indices of population ancestry, which might suggest residual stratification or other unaccounted biases), the results from this effort are encouraging and provide some hope to those who may have been losing their faith. The most notable findings were in ANK3 on chromosome 10q21 and CACNA1C on chromosome 12p13. Multiple SNPs were associated across a 195-kb region of ANK3, and the top SNPs had p-values <5 x 10-8 which is often invoked as an appropriate threshold for genomewide significance in GWAS. Multiple nearby SNPs were also reassuringly associated in CACNA1C, although the top SNP just missed the threshold for genomewide significance. In both ANK3 and CACNA1C the top SNPs were consistently associated in the same direction across all three individual samples, lending credence to the claim that these associations are real. Lending further credence is the fact that ANK3 and CACNA1C are biologically plausible candidates for bipolar disorder, and indeed highlight the possibility that this disorder is an ion channelopathy. Interestingly, the associations with ANK3 and CACNA1C in each of the individual samples were relatively modest and became remarkable only in the combined sample, thus providing support for the rationale to combine samples in order to increase the power to detect those more modest signals that are presumably real but buried amidst the noise of a single study.

Although the evidence is promising, more samples will be needed to confirm the findings before we can say with confidence that we have in hand our first real bipolar susceptibility genes. Fortunately, several such samples have been or will soon be GWASed, including one from GAIN, and it will be of great interest to see whether the current findings are sustained in these new samples. Moreover, there are plans to combine all existing GWAS of bipolar disorder, as part of the initiative referred to as the Psychiatric GWAS Consortium, which should provide an even more definitive picture of the role of ANK3 and CACNA1C, as well as reveal other genes with more modest relative risks whose identities up until now have been obscured.

So, are we there yet? Maybe not just yet, but we are headed in the right direction and I think I can see the promised land.

View all comments by Peter P. Zandi