Channeling Mental Illness: GWAS Links Ion Channels, Bipolar Disorder

19 August 2008. A collaborative study of bipolar disorder that combines data from two previously published genomewide association studies (GWASs) with a new GWAS associates two genes that encode components of voltage-gated ion channels with the illness. These findings raise the possibility that bipolar disorder may partly result from channelopathies, disruptions in ion channel subunits or other channel-related proteins. Such molecular lesions underlie a diverse group of disorders, including central nervous system disorders such as epilepsy, migraine, and ataxia.

The results of the study will be viewed with interest by researchers in schizophrenia genetics, as the bright diagnostic line between schizophrenia and bipolar disorder, first drawn by Emil Kraepelin in the late nineteenth century, is increasingly called into question. As has been pointed out by Cardiff, Wales-based Nick Craddock and Michael Owen, both authors on the new study, the emergence of overlapping candidate genes (e.g., DISC1, DTNBP1, NRG1) and the similarity between the psychotic phenotype seen in the manic phase of bipolar disorder and in schizophrenia suggest that there will be a growing cross-fertilization in genetic research in these two arenas (see Craddock and Owen, 2005, and SRF live discussion).

In the new multicenter study, bipolar cases and controls from the Wellcome Trust Case Control Consortium (the WTCCC study; Wellcome Trust Case Control Consortium, 2007; see SRF related news story) were added to those from the recent GWAS by Pamela Sklar and colleagues (the STEP-UCL study; Sklar et al., 2008, first reported in an SRF meeting report from the 2007 WPCG meeting), as well as a new sample of 1,098 cases and 1,267 controls, including some from the University of Edinburgh and Trinity College Dublin (the new data set was hence dubbed ED-DUB-STEP2), for a combined sample of 4,387 cases and 6,209 controls. In the case sample, 81 percent had been diagnosed with bipolar 1, and 16 percent with bipolar 2.

This large sample was directly genotyped on more than 325,000 overlapping SNPs, a number that was greatly increased, to about 1.8 million SNPs, when imputed HapMap SNPs were added using PLINK, a GWAS tool developed by study author Shaun Purcell of the Broad Institute of MIT and Harvard and colleagues (Purcell et al., 2007). “Applying a leave-out-one procedure for every genotyped SNP,” the authors write, “we estimated concordance between imputed and true genotypes as 0.987.”

Although there had been no overlap in the “top hits” identified in the WTCCC and the STEP-UCL studies—the former identified a gene-rich locus on chromosome 16, while the latter’s strongest SNPs were in MYO5B, TSPAN8, and EGFR—a post hoc comparison of the two data sets by the Sklar group found concordant SNP signals in CACNA1C, a gene on chromosome 12 that encodes the alpha 1C subunit (Cav 1.2) of the L-type voltage-dependent calcium channel.

In an initial analysis of the ED-DUB-STEP2 dataset alone, none of 14 chromosomal regions showing associations exceeded the researchers’ genomewide significance threshold of 5 x 10^-8. However, one of these regions spanned the CACNA1C association found in the Sklar team’s analysis of the WTCCC and STEP-UCL samples, providing further support for the hypothesis that mutations in CACNA1C may contribute to bipolar disorder.

In the combined WTCCC/STEP-UCL/ED-DUB-STEP2 sample, the strongest association (P = 9.1 x 10^-9) was found in ANK3 (Ankyrin-G) on chromosome 10q21, a gene that is required for the clustering of voltage-gated sodium channels at axon initial segments (Zhou et al., 1998) and nodes of Ranvier (Poliak and Peles, 2003), a configuration that underlies the rapid and efficient propagation of action potentials along myelinated axons. The second-strongest association was at rs1006737, in the third intron of CACNA1C (P = 7.0 x 10^-8). A third association was found near C15orf53, a gene on 15q14 of unknown significance. No differential associations for these three regions were found across bipolar disorder subtypes, presence of psychosis, age of onset, sex, or response to treatment.

The ANK3 and CACNA1C associations reported in the new study are particularly intriguing in light of other recent work by study coauthor Hugh Gurling and colleagues (McQuillin et al., 2007), in which lithium carbonate, the gold standard for treatment of bipolar disorder, was shown to downregulate both ANK3 and subunits of the calcium channel. In addition, many antiepileptic drugs also used to manage symptoms in bipolar disorder and schizophrenia are known to affect voltage-gated sodium or calcium channels (Johannessen Landmark, 2008).

Though channelopathies have long been known to contribute to cystic fibrosis as well as several heritable cardiac (e.g., Brugada syndrome, Long QT syndrome) and motor (e.g., myasthenia gravis, periodic paralysis) disorders, the exploration of ion channel mutations in psychiatric disorders is a fairly recent development. However, an association between a missense mutation in CACNA1C and Timothy syndrome, a complex disorder that includes cardiac abnormalities, webbing of the fingers and toes, and autism, was recently reported (Splawski et al., 2004). KCNQ5, which encodes a potassium channel crucial for the M-current, an important regulator of neuronal excitability, maps to risk loci for attention-deficit hyperactivity disorder, bipolar disorder, and schizophrenia. Another channel gene, SK3, which encodes a small-conductance calcium-activated potassium channel, has also been implicated in schizophrenia (see Gargus, 2006, for a review of ion channel candidate genes in psychiatric disease).—Peter Farley.

Reference:
Ferreira MAR and 62 others. Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder. Nat. Genet., published online 17 August, 2008.

Q&A With Nick Craddock. Questions by Hakon Heimer.

Q: This a rather large sample compared to previous ones, yet only three regions were found to have genomewide significance. Is that a disappointing result, and does it argue against either the multigenic theory of mental illness causation, on the one hand, or the use of 10^-8 as a cut-off for determining genomewide significance on the other?
A: The findings of our analysis are entirely consistent with the existence of many genes each having a small effect on susceptibility to bipolar disorder. It is important to think beyond the top few “hits.” The pattern that we see in bipolar disorder is very much the same sort of pattern that we see in studies of non-psychiatric disorders. When we have access to even larger samples, more loci will achieve genomewide significance.

Q: Do you think there is validity to the argument that large-scale studies such as this may “wash out” signals of genes that contribute to disease in certain populations but not others?
A: Large-scale studies like ours provide optimal power to detect genetic variants that influence susceptibility broadly across the bipolar phenotype and across populations. It is, however, true that they may not be the optimal approach for detecting susceptibility variants that are either specific to certain populations or confer risk specifically to certain aspects of the clinical phenotype. In such situations, specific signals could be washed out.

Q: Is there any evidence from other sources to suggest that channelopathies might play a role in schizophrenia as well (or any evidence that they probably do not)?
A: This is an issue that has not previously received much attention—but obviously now warrants specific investigation.

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. McInnisComment 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.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

Terrific update and summary for those of us not attending the meeting.

View all comments by William Carpenter

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