In recent years, there has been increasing recognition of co-morbid conditions in mental disorders. A deeper understanding of this phenomenon and its risk factors could improve treatment and prevention. Our results indicate heterogeneity in bipolar disorder patients with regard to co-morbidity consistent with emerging evidence from other studies. In this study we have explored possible genetic risk factors that might shed some light on underlying patho-mechanisms. Latent class analysis indicated the existence of three distinct subgroups of patients characterized by co-morbidity profiles, one group with predominantly substance abuse and/or psychosis, one group in which alcohol dependence prevailed, and one group with very low probability for any co-morbid conditions. Addressing this heterogeneity led to the identification of several highly significant associations with SNPs in genome-wide association analyses. Our results suggest that phenotype heterogeneity in BPD might indicate genetic heterogeneity. However, the interpretation of our findings poses several problems. Since genome-wide association analyses can, by design, only point to regions of the genome associated with a disease phenotype, the actual functional variants often remain elusive. Genome-wide association studies are also underpowered to replicate association with rare variants. Therefore, it is now commonly agreed upon that re-sequencing approaches are necessary to follow-up on genome-wide association studies and to identify the underlying causal variants.
The associations found in this study point to several interesting genes and chromosomal regions that might justify re-sequencing approaches in order to find the underlying functional variants. The genomic variant rs1039002, which was associated with a subgroup of bipolar patients with substance abuse and/or psychotic symptoms, is located close to a transcribed genomic sequence with unknown function. The product of the closest gene sequence with known function is phosphodiesterase 10A (PDE10A
), a protein involved in the elimination of the intracellular signaling molecules cAMP and cGMP. The highest expression levels of this gene are found in heart, brain, kidney and testes. In the brain, expression is particularly high in the medium spiny neurons of the striatum. Inhibitors of this phosphodiesterase have shown therapeutic potential for the treatment of psychotic symptoms in schizophrenia, as well as the treatment of Parkinson's disease, Huntington's disease, addiction, and obsessive compulsive disorder. PDE10A inhibitors are now being tested in clinical trials 
is the closest expressed sequence to the SNP rs12563333, which is also associated with Latent Class 1 membership. This gene is located immediately 5′ to the serine/threonine-protein kinase gene MARK1
is highly expressed in brain and testes, with highest levels of expression found in the hippocampus. MARK1 phosphorylates microtubule-associated proteins and is involved in synaptic plasticity and dendritic trafficking 
. Evidence for the involvement of this gene in autism has come from gene expression studies in postmortem brains, as well as genome-wide association studies 
. Comparison of human MARK1
with the mouse sequence showed significant differences and lack of conservation. It has therefore been hypothesized that MARK1
could be involved in the development of higher cognitive functions that separate humans from mice 
. However, it remains to be determined if rs12563333 is in linkage disequilibrium with any functional variants in MARK1
Two SNPs that approached genome-wide significance are located in genes that are involved in response to environmental stresses, and therefore, might warrant further exploration. SNP rs9493867 on chromosome 6q23.2 is located within the gene serum/glucocorticoid regulated kinase 1 (Sgk1
). This gene encodes a serine/threonine-protein kinase that is involved in the activation of potassium, sodium and chloride channels 
. In rats it appears to be involved in memory consolidation, spatial learning, and cellular stress response through negative regulation of the SK1-JNK1-MEKK1 pathway 
. The second SNP is located in the 3′ region of the gene mitogen-activated protein kinase kinase kinase 7 (MAP3K7
) on chromosome 6q15. This serine/threonine protein kinase is also involved in cell response to environmental stresses in related pathways through activation of MAPK8/JNK and the MAP2K4/MKK4 protein complex 
The most significant association with Latent Class 2 was within a region that has been found to be deleted in individuals with autistic features. SNP rs2727943 is located on chromosome 3p26.3 between the genes contactin-4 precursor (BIG-2
) and the gene encoding the neural adhesion molecule contactin 6 (CNTN6
). The protein products of these genes might play a role in the formation of axon connections in the developing brain 
A limitation of our study is, foremost, the small sample size. Re-sequencing of the most significant regions in a larger sample of bipolar patients would be desirable in order to evaluate all variants in the identified genomic regions and to determine their functional consequences. The high genotype quality of the re-called and re-genotyped significant SNPs, the fact that the identified rare variants were present only in cases and not in controls and strong evidence for the therapeutic potential of PDE10A inhibitors might justify further follow-up studies. Our analysis indicates three major points. 1. Rare variants might be important pathogenic factors in BPD. Rare variants in gene regions were the most significant signals in our analysis, and these variants were exclusively present in cases and not in controls. In our small sample and without replication it is difficult to claim certainty about the disease association; however, cumulative evidence for the implication of the identified genomic regions could justify further investigation. 2. Since the associated variants were very rare, a genome-wide association design might not be the most appropriate approach for replication. Future studies could focus on re-sequencing of the chromosomal regions in a sample of BPD patients and controls in order to identify all rare and possibly coding variants in the region that might play a role in BPD pathophysiology. The fact that all of our associations were found in or near genes that have been implicated in psychiatric disorders and even in psychosis underlines the importance of these significant associations. 3. Taking the heterogeneity of the phenotype into account when performing disease association studies might increase the power of detecting significant associations.
In summary, our paper describes a novel multivariate approach to the phenotype of BPD. We identified rare variants in regions of interest that might warrant further studies. However, we were unable to find common variants with functional consequences associated with BPD. Focused or genome-wide re-sequencing might be important for the identification of genetic risk factors in bipolar disorder.