Association of CACNA1C Variants with Bipolar Disorder in the Korean Population

Objective Previous studies have suggested an association between CACNA1C and susceptibility of bipolar disorder. In this study, we examined the association of CACNA1C variants with bipolar disorder in the Korean population. Methods We selected 2 CACNA1C single nucleotide polymorphisms (SNPs), namely, rs723672 and rs1051375, based on their functions and minor allele frequencies described in previous studies. Genotypes of these 2 SNPs were analyzed by extracting DNA from blood samples collected from 287 patients with bipolar disorder and 340 healthy controls. Results Genotype frequencies of both rs723672 and rs1051375 SNPs were significantly different in patients and controls (p=0.0462 and 1.732E-14, respectively). Dominant, recessive, and allele models showed significant differences between patients and controls with respect to the rs1051375 SNP (p=1.72E-11, 4.17E-10, 4.95E-16, respectively). Conclusion Our results suggested that CACNA1C SNPs rs723672 and rs1051375 were associated with bipolar disorder in the Korean population. In addition, our results highlighted the importance of CACNA1C in determining susceptibility to bipolar disorder.


Patients with bipolar disorder
All patients who met DSM-IV-TR criteria for bipolar disorder type 1 or 2 were recruited from Korea University Hospital, Seoul, between March 2009 and August 2015. All the patients were ethnically homogeneous Koreans and were examined by trained psychiatrists by using the Korean version of Structured Clinical Interview for DSM-IV, leading to a diagnosis based on DSM-IV criteria. 22 Written informed consent was obtained from each patient, and study protocols were approved by the Ethics Committee of the Korea University Hospital. None of the patients included in the study had significant comorbid neurological illnesses, mental retardation, or history of substance abuse.

Healthy controls
In all, 340 community-based healthy adults were recruited through a newspaper advertisement. The age of healthy controls ranged from 23 to 83 years. Absence of lifetime or current psychiatric disorders in healthy controls was confirmed by an experienced psychiatrist by using Mini-International Neuropsychiatric Interview. Subjects with a family history of substance abuse or major psychiatric disorders (e.g., schizophrenia or mood disorders) were excluded. All the healthy controls were unrelated Koreans who did not have any major medical problems and who lived in Seoul, Korea. Informed consent was obtained from all the healthy controls.

Statistical analysis
Conformance with Hardy-Weinberg equilibrium was determined using chi-square (χ 2 ) test for goodness of fit. Probability value of <0.05 was considered statistically significant. Power calculation was performed using G*Power 3.1.9 (Dusseldorf, Germany). 23 In all, 429 samples (df=2) were required to detect an SNP of the effective size and power (80%). Statistical analyses were performed using SPSS 18.0 for Windows (SPSS Inc., Chicago, IL, USA) and SNPAlyze version 7 (Dynacom, Chiba, Japan).

Demographic data
Demographic information of patients with bipolar disorder and healthy controls is presented in Table 1. The study included 627 subjects, of which 287 were patients and 340 were healthy controls. Of the 287 patients, 131 were men and 169 were women, with an average age of 37.02 years. The mean age of disease onset among the patients was 24.24 years, and the average number of total episodes was 9.18. Of the 340 healthy controls, 155 were men and 171 were women, with an average age of 33.55 years (Table 1).

Genotype distribution and allele frequencies of rs723672 and rs1051375
Genotype distribution of rs723672 and rs1051375 is shown in Table 2. Genotype distribution of both rs723672 and rs1051375 followed the Hardy-Weinberg equilibrium. In patients with bipolar disorder, frequencies of rs723672 genotypes T/T, T/C, and C/C were 64.5%, 30.7%, and 4.9%, respectively, and frequencies of T and C alleles were 79.8% and 20.2%, respectively. In healthy controls, frequencies of rs723672 genotypes T/T, T/C, and C/C were 66.8%, 31.8%, and 1.5%, respectively, and frequencies of T and C alleles were 82.6% and 17.4%, respectively. Further, in patients with bipolar disorder, frequencies of rs1051375 genotypes A/A, A/G, and G/ G were 41.5%, 43.2%, and 15.3%, respectively, and frequencies of A and G alleles were 63.1% and 36.9%, respectively. In healthy controls, frequencies of rs1051375 genotypes A/A, A/G, and G/G were 68.2%, 30.0%, and 1.8% and frequencies of A and G alleles were 83.2% and 16.8%, respectively.
Results of HRM analysis were validated by randomly selecting blood samples for direct sequencing. Results of direct sequencing were parallel to those of HRM analysis.
Genotype model showed statistically significant differences between patients with bipolar disorder and healthy controls with respect to the rs723672 and rs1051375 SNPs (pGeno= 0.0462 and pGeno=1.732E-14, respectively). In addition, both recessive and dominant allele models showed statistically significant differences between patients with bipolar disorder and controls with respect to the rs1051375 SNP, indicating that the frequency of the A/G heterotype of the rs1051375 SNP was higher in patients with bipolar disorder than in controls (pRec=4.172E-10 and pDom=1.72E-11, respectively). More patients with bipolar disorder harbored the C allele of the rs723672 SNP than controls (pRec=0.0131) ( Table 3).

DISCUSSION
As an L-type voltage-gated Ca 2+ channel, CACNA1C contributes to the depolarization of neurons through calcium ion influx. 24 Variations in SNPs in CACNA1C affect both the structure and function of the nervous system, 25 including synaptic plasticity and neuron viability. 13 Association of CAC-NA1C SNPs with Timothy syndrome, fear conditioning, major depression, and schizophrenia 26-29 has been found, and correlation with bipolar disorder was also reported through GWAS study. 12 The association between bipolar disorder and CACNA1C SNPs has been examined in Han Chinese population (HCP). 30 However, only the association between schizophrenia and CACNA1C SNPs has been examined in the Korean population. 31 Therefore, our study is the first to directly analyze the association between CACNA1C SNPs and bipolar disorder.
Although the genotype frequencies of rs1051375 in our control group showed statistically different frequencies compared to those observed in Han Chinese population (HCP) in the  However, further studies should be performed to determine the mechanisms underlying the effect of CACNA1C rs723672 and rs1051375 SNPs on the expression and function of the Ltype voltage-gated Ca 2+ channel. Particularly, the G allele, which is dominant in the rs1051375 SNP, creates a CpG site containing a cytosine nucleotide adjacent to a guanine nucleotide. Although the rs1051375 SNP does not directly affect the primary structure of the L-type voltage-gated Ca 2+ channel, it may serve as a methylation site (Figure 1). Although it is unclear whether this CpG site undergoes methylation, a study showed that 70-80% cytosines in CpG sites of mammalian genomes are methylated. 32 Therefore, it is highly likely that the rs1051375 SNP contributes to epigenetic regulation through the methylation of CACNA1C. Moreover, a study showed that DNA methylation pattern of CACNA1C is specific in neuronal cells, thus reinforcing that changes in methylation patterns of specific sites in CACNA1C can induce psychiatric disorders by modulating neuronal activity.
However, our study has some limitations. First, the average age of healthy controls was slightly lower than that of patients with bipolar disorder, suggesting that the healthy controls could develop bipolar disorder as they aged. Although this possibility bridged the difference between the 2 groups, a meaningful difference was still observed in our study. Second, genotype distribution of the rs723672 SNP slightly deviated from the Hardy-Weinberg equilibrium in healthy controls. However, this deviation was very small to refute the assumption that the rs723672 SNP is underlying the Hardy-Weinberg equilibrium. These data were verified by performing repeated HRM analyses. Third, haplotype combinations were not analyzed because LD value was not significant between the rs723672 and rs1051375 SNPs (D'=-9.609E-3).
In conclusion, the results of our study suggested the association of CACNA1C rs723672 and rs1051375 SNPs with bipolar disorder in the Korean population, thus supporting the hypothesis that CACNA1C is involved in the pathogenesis of bipolar disorder. Our data together with those of other studies highlight an association between CACNA1C SNPs and bipolar disorder. these results will provide useful insights for performing further research to determine genetic causes underlying bipolar disorder.