Findings relating to the CACNA1C gene

Schizophrenia, major depressive disorder and bipolar disorder are three severe mental disorders. They not only affect individuals and their families but also challenge society and health services worldwide. A high degree of heritability has indicated that genetic factors play an important role in their aetiologies. The CACNA1C gene was reported to have a nominal association with bipolar disorder in a family-based association study.^{Reference Sklar, Gabriel, McInnis, Bennett, Lim and Tsan27} Combined with data from the Wellcome Trust Case-Control Consortium GWAS,^{Reference Burton, Clayton, Cardon, Craddock, Deloukas and Duncanson28} Sklar et al reported that rs1006737, within the CACNA1C gene, was associated with bipolar disorder (P = 1×10^–4) in a GWAS of 1461 people with bipolar disorder and 2008 controls.^{Reference Sklar, Smoller, Fan, Ferreira, Perlis and Chambert9} A subsequent study with a larger sample size confirmed that rs1006737, in CACNA1C, was associated with bipolar disorder (P = 7.0×10^-8, OR = 1.181) in a White population.^{Reference Ferreira, O'Donovan, Meng, Jones, Ruderfer and Jones2} The CACNA1C gene has also been found to be associated with other psychiatric disorders. Genetic studies have discovered that the genotype of CACNA1C was also associated with schizophrenia^{Reference Nyegaard, Demontis, Foldager, Hedemand, Flint and Sorensen5,Reference Bigos, Mattay, Callicott, Straub, Vakkalanka and Kolachana29} and major depressive disorder.^{Reference Shi, Potash, Knowles, Weissman, Coryell and Scheftner7,Reference Casamassima, Huang, Fava, Sachs, Smoller and Cassano30} Green et al reported in a GWAS that the risk SNP (rs1006737) for bipolar disorder was also a conferred risk for schizophrenia and early recurrent major depressive disorder.^{Reference Green, Grozeva, Jones, Jones, Kirov and Caesar3} A meta-analysis of two separate GWASs of people with bipolar disorder and major depressive disorder revealed that rs1006737 reached genome-wide significance after combining bipolar with unipolar mood disorders.^{Reference Liu, Blackwood, Caesar, de Geus, Farmer and Ferreira4} The consistent findings in genetic studies indicates that the CACNA1C gene belongs to a class of shared susceptibility factors for major psychiatric disorders and has likely played an important role in the pathogenesis of them.

Nevertheless, the mechanisms underlying how genetic changes in CACNA1C modify risk for developing psychiatric disorders are still unclear. This gene encodes the alpha-1C subunit of the L-type voltage-dependent calcium channel Cav1.2. Calcium channels are involved in various aspects of neuronal development and in the establishment of maintenance of connectivity during development and throughout adulthood.^{Reference Spitzer31} Previous studies of people with bipolar disorder have consistently reported elevated basal and stimulated intracellular calcium levels in peripheral blood cells.^{Reference Emamghoreishi, Schlichter, Li, Parikh, Sen and Kamble32,Reference Perova, Wasserman, Li and Warsh33} The evidences suggested that calcium signalling may play a role in bipolar disorder. Cav1.2 participates in the proper function of numerous neurological circuits in the hippocampus, amygdala and mesolimbic reward and motivation systems.^{Reference Leitch, Szostek, Lin and Shevtsova34,Reference Nestler and Carlezon35} These are strongly implicated in psychiatric disease pathophysiology.

The CACNA1C gene has also been found to be expressed in many tissues in the human body across various developmental stages.^{Reference Pang, Koren, Wang and Nattel36} And, it is widely expressed in the brain, especially hippocampus and thalamus.^{Reference Striessnig, Koschak, Sinnegger-Brauns, Hetzenauer, Nguyen and Busquet15} The relevance of CACNA1C to bipolar disorder was supported by the observed downregulation of messenger (m)RNA transcripts in mouse brain in response to lithium.^{Reference McQuillin, Rizig and Gurling37} Notably, a missense mutation in CACNA1C can cause Timothy syndrome, which is characterised by multi-organ dysfunction such as cardiac arrhythmias and cognitive abnormalities.^{Reference Splawski, Timothy, Sharpe, Decher, Kumar and Bloise38} The heterozygous knockout adult mice of CACNA1C were found expressing decreased Cav1.2 protein levels and L-type calcium channel current, and protecting against depression-like phenotypes.^{Reference Dao, Mahon, Cai, Kovacsics, Blackwell and Arad39} A larger effect-size study clearly indicated that genetic variant at SNP rs1006737 was associated with changes in brain structure and function in normal controls.^{Reference Rose and Donohoe40} Genetic variation at rs1006737 may be involved in modulating gene expression, whereas the risk allele of rs1006737 is associated with increased expression levels of CACNA1C.^{Reference Bigos, Mattay, Callicott, Straub, Vakkalanka and Kolachana29} Three studies found the rs1006737 risk allele was associated with increased brain grey matter, either total grey matter volume or in specific brain regions in healthy controls. ^{Reference Kempton, Ruberto, Vassos, Tatarelli, Girardi and Collier41-Reference Wang, McIntosh, He, Gelernter and Blumberg43}

In our study, the main finding was a significant association between the CACNA1C gene and both schizophrenia and major depressive disorder in the Han Chinese population. We have confirmed that rs1006737 in CACNA1C was significantly associated with schizophrenia (P _allele = 0.0014, P _genotype = 0.006, OR = 1.384, MAF = 0.101) and major depressive disorder (P _allele = 0.0007, P _genotype = 0.003, OR = 1.425, MAF = 0.103) in a case-control study with a large sample size in the Han Chinese population. Even when Bonferroni correction (it is considered the most conservative method) was used, we still found that rs1006737 was associated with schizophrenia (P _allele = 0.028) and major depressive disorder (P _allele = 0.014). At the same time, we found another SNP (rs2239015) was significantly associated with schizophrenia (after Bonferroni correction: P _allele = 0.006, P _genotype = 0.030, OR = 1.249, MAF = 0.355) (Table DS4).

Nyegaard et al reported that rs1006737 was associated with schizophrenia (P = 0.015, OR = 1.16, MAF = 0.361) in a case-control study of 976 people with schizophrenia and 1489 healthy controls of European origin.^{Reference Nyegaard, Demontis, Foldager, Hedemand, Flint and Sorensen5} Green et al have reported that rs1006737 is associated with major depressive disorder (P = 0.013, OR = 1.15, MAF = 0.363) in recurrent major depressive disorder (n = 1196) and UK non-psychiatric comparison groups (n = 15 316).^{Reference Green, Grozeva, Jones, Jones, Kirov and Caesar3} Comparing our results with theirs, the MAFs for the SNPs were quite different but the ORs are all in the same direction (for schizophrenia, OR: 1.384 v. 1.16, MAF: 0.101 v. 0.361; for major depressive disorder, OR 1.425 v. 1.15, MAF 0.103 v. 0.363).

Avoiding the potential influence of population stratification in our samples was important. We therefore used the software STRUCTURE 2.3.4 and data from 79 additional random SNPs dispersed on different chromosomes to analyse the potential population stratification in our samples. We did not detect any population stratification, and therefore, the results could not be affected by this confounding factor.

Future directions for research

Given rs1006737 and rs2239015 were both located in introns of CACNA1C, they were not expected to directly interfere with properties relating to the structure and function of Cav1.2. We know that intronic variation is likely to be involved in regulating gene expression and that extensive alternative splicing exists in the CACNA1C transcripts. It may potentially generate thousands of splice variants. Now, it is important to confirm the consequences of changes in splice variation of CACNA1C on neurophysiology. If we can find relevant answers, it might be very helpful for deciding novel therapeutic targets and approaches. In addition, it is also necessary to test its association with bipolar disorder in a large sample of individuals of Han Chinese origin in the future.