This study represents the first genetic association study of BPD I and BPD II using dozens of genes comprising the cAMP signaling pathway. Collectively, our results indicate that some of the analyzed SNPs in several genes of the pathway are in LD with causal variants for BPD I and BPD II. Furthermore, the SNV discovery aspect of our study has identified new variants in the PDE10A gene of BPD subjects that may be causally related to the disorder. Our genetic studies are consistent with prior biochemical studies implicating cAMP signaling in BPD, and highlight specific genes and SNVs that could be of major importance for these disorders.
We employed three noteworthy strategies for elucidating the genetic basis for susceptibility to BPD. First, our association study utilized 29 candidate genes representing a single-signaling pathway. The hypothesis underlying this strategy was that if one gene in the signaling pathway is involved, then casual variants in other genes of the same pathway are likely involved as well. We recognize that there exist known and obvious intersections and cross-talk between the cAMP signaling pathway and other signaling pathways, but we selected genes that are widely recognized as comprising the canonical cAMP signaling pathway in order to sample a defined and limited set of genes. A ‘molecular pathway hypothesis' as contrasted with a ‘single gene hypothesis,' may account for the genetic heterogeneity of neuropsychiatric disorders in general and the marked difficulty in discovering genetic variants that have prominent roles in these disorders. It can also explain heterogeneity in clinical phenotypes or severity within a disorder, since a variant in an AC
gene, for instance, might have largely overlapping but some distinct phenotypic effects compared with a variant in cAMP PDE. The interaction analyses were motivated by the ‘molecular pathway hypothesis' as functional variants in two molecular functions of a pathway could produce a more pronounced effect than either single functional variant. For instance, the relatively large OR produced by considering an interaction between PDE4D
for BPD I (Supplementary Table 4
) could be due to a functional variant that decreases PDE4D
activity synergistically interacting with one that increases ADCY8
activity. Similarly, an interaction between variants in PDE4B
for BPD II could be because of two separate functional variants that have synergistic effects on signaling. These ideas remain speculative and depend on the sign and magnitude that the functional variants have on the molecular factors involved.
Second, the choice of candidate molecular pathways considered the known and major role of the signaling pathway in the regulation of behavior in model systems. Genetic studies of learning and memory, circadian rhythms and other behaviors in model systems like Drosophila
and the mouse have identified hundreds of genes that have behavioral roles. A good example of this is the cAMP PDE encoded by the Drosophila dunce
gene, which is involved in learning and memory in Drosophila
but whose human counterparts are involved in mood regulation48
and schizoaffective disorders.67
Candidate genes selected from the human homologs of genes with known behavioral roles in model systems should provide an extremely effective filter in gene discovery for neuropsychiatric disorders.
Third, we followed-up the initial association study with SNV discovery in the vicinity of the associated PDE10A tagSNPs that identified additional variants that may be causally involved in BPD. This required the development of a new method for integrating information from sequencing projects in a cost effective way. Using this method, named SEQCHIP, we were able to combine the sequencing data from 30 BPD I patients used for discovery with the data from the follow-up genotyping in a larger subset while accounting for potential bias. This approach was used to identify potentially causal variants that are tagged by the genotyped SNPs. When the data are integrated by SEQCHIP, all rare variant tests have correct type I errors. The genetic information from both sequence and genotype data were efficiently utilized, and therefore, more significant association signals were identified.
Suggestive evidence from other studies is consistent with the possibility that these newly discovered SNVs are involved in BPD. A recent RNA sequencing study employing human prefrontal cortex suggested that two of these SNVs (chr6:166052752 C>G and chr6: 166046780_166046778 delATT) may be associated with novel PDE10A transcripts.68
Another study performing chromatin immunoprecipitation sequencing on neuronal cells from human prefrontal cortex tissue showed that SNVs, chr6:166058713 C>T, chr6:166056798 A>T and chr6: 166046780_166046778 delATT are associated with an enriched H3K4me3 signal, suggesting that they participate in the transcriptional regulation of PDE10A.69
Although this preliminary evidence must be confirmed in human tissue expressing PDE10A, it does suggest that these variants may have a role in PDE10A protein expression.
Our study highlights the association of PDE10A
with BPD I. Of related and high interest is the observation that the PDE10A
gene resides at 6q26, within one of the four leading linkage regions for BPD.2
The PDEs degrade cAMP and/or cyclic guanosine monophosphate to their 5′-AMP and 5′-GMP counterparts and members of this large enzyme family are known to have broad roles in controlling behavioral output in diverse organisms from flies to humans.66, 70
The family member PDE10A
codes for an 89-kDA protein capable of hydrolyzing both cAMP and cyclic guanosine monophosphate that is expressed in the brain and preferentially in the basal ganglia.71, 72, 73, 74
This expression pattern paired with basal ganglia dysfunction in schizophrenia have led to robust efforts in identifying drugs that target PDE10A for the treatment of schizophrenia.75, 76, 77
The association of PDE10A
with BPD I expands the potential range over which PDE10A drugs might exhibit efficacy.
The gene disrupted in schizophrenia 1 (DISC1
) was first implicated in psychiatric disorders after a t(1;11) translocation was discovered in a large Scottish family cosegregating with schizophrenia, BPD and recurrent major depression.78, 79
region has subsequently been implicated with bipolar spectrum traits in several candidate gene association studies.80, 81, 82, 83, 84, 85
The selection of tagSNPs has varied from study to study so we were unable to compare the precise haplotype windows between this and other studies. In addition, the majority of studies failed to control for population admixture and in most cases termed a P
-value of less than nominal significance (P
-value <0.05) as significant. Relative to individual tagSNP associations that have been reported, Perlis et al.86
observed an association between rs1934909 and BPD. We observed an association between this tagSNP and BPD II (OR=0.8, P
=0.01) but not BPD I cases (Supplementary Table 1
). Similarly, Schosser et al.83
observed an increased prevalence of BPD for carriers of the minor allele of rs2492367. We observed an association between this SNP and BPD II but not BPD I (OR=0.8, P
It seems highly likely given the complexity of the DISC1 protein and it's multiple molecular roles87
that there exist multiple functional variants linked to the various tagSNPs used in this and other studies. DISC1 interacts with several key proteins in pathways that have important roles in neuronal migration, dendritic organization and neurogenesis in addition to cAMP signaling.67, 87, 88, 89, 90, 91, 92
The binding sites for interacting proteins are plausible regions for multiple susceptibility regions as are regions of the protein that modify binding. This could underlie the DISC1
tagSNP interactions observed to be significantly associated with BPD I and BPD II in this study. Furthermore, DISC1
variants in intronic regions may influence DISC1
mRNA and subsequent protein levels that also could interfere with key pathways. Indeed, Maeda et al.93
demonstrated reduced DISC1
mRNA levels in lymphoblasts from BPD subjects compared with unaffected family controls. Thus, it is plausible that multiple genetic variants in LD with DISC1
tagSNPs exist that alter protein function or mRNA levels that influence susceptibility to neuropsychiatric disorders. Identifying these variants and characterizing their effect on DISC1 molecular functions may provide useful information for the development of novel therapeutics for bipolar spectrum traits.
The guanine nucleotide-binding protein, α-stimulating polypeptide 1 (GNAS
) encodes the Gαs subunit of heterotrimeric G-proteins with Gαs activating AC for increased synthesis of cAMP. Although the exact mode of action of lithium in treating BPD remains elusive, there exists evidence that lithium decreases G-protein subunit mRNA levels.35
Moreover, it has been speculated that lithium functions to stabilize the inactive conformation of G-proteins.94
variants, like rs6064714 and rs6026565, may be tagging functional variants in BPD I patients that interfere with Gαs' role in regulating cAMP signaling.
It is of interest that SNPs representing certain genes, such as PDE4B and PDE10A, were found as associated with BPD I but not BPD II. It is possible that this represents an authentic genetic difference between the two BPD subtypes. However, it could also be that the power to detect the association was stronger with the BPD I population, given that the BPD I population was twice the size of the BPD II population. If the data reflect authentic genetic differences between the two subtypes, they could be important to the pharmacogenetics of BPD treatment. For instance, perhaps an altered regulation of PDE10A, specific to BPD I, is most related to full-blown mania associated with BPD I. Drugs with PDE10A action may then have a primary effect on mania.
Overall, this study provides insight into the variation that exists in genes in the cAMP signaling pathway and the association of that variation with BPD I and BPD II. Key regions were identified in PDE10A, GNAS and DISC1 that are associated with BPD I and BPD II. The effect size for these associations was modest, as observed in most other genetic association studies for neuropsychiatric disorders. Our study also illustrates the feasibility of performing large candidate gene or GWAS (genome-wide associated studies) studies followed by targeted direct sequencing to uncover novel, rare SNVs that may be associated with disease.