Next-Generation Bisulfite Sequencing Of Cacna1c With Illumina Miseq

Background The complex etiology of psychiatric illness involves both genetic and environmental factors. The latter may act via epigenetic processes such as DNA methylation. Studies suggest that epigenetic regulation of gene expression may mediate the effects of early life experiences on adult behavior and susceptibility to psychiatric illness. CACNA1C is an epigenetically regulated gene implicated in the etiology of psychiatric illness. Here, the third CpG island located in intron 3 is of particular interest since: (1) differential methylation is reported between bipolar patients and controls, with higher methylation levels being observed in patients; (2) CpG sites in this CpG island show blood/brain correlation; (3) individual methylation level predict more of the variance than tissue methylation level; and (4) it is a significant meQTL, as it is located in close proximity (5kb) to rs1006737, which shows a genome wide significant association to bipolar disorder, schizophrenia, and major depression. The aim of the present study was to test the feasibility of next generation bisulfite sequencing in the methylation analysis of CpG sites of the third CACNA1C CpG island. Methods Next generation sequencing (NGS) analysis of the CACNA1C region of Intron 3 was performed. A total of 63 CpG sites from three bisulfite converted whole blood DNA samples were analyzed. Both single- and multiplex-PCRs were used. Results Singleplex-PCR generated 8,000 to 36,000 sequence reads, with a sequence identity of 99.0 to 99.9%. The standard deviation of the methylation level per CpG of the three samples ranged from 0.3 to 6.4%. Multiplex-PCR generated variable methylation values, with lower sequence identity and fewer sequence reads. Discussion This is the first comprehensive analysis of methylation levels of the CACNA1C third intronic region to use NGS. The results indicate that multiplex PCR is not an appropriate method for methylation analysis of bisulfite converted DNA. Now it is planed to perform simultaneous sequencing of 384 samples using singleplex-PCR.