Functional characterization of schizophrenia-associated variation in CACNA1C

human genetics;schizophrenia;gene expression;gene regulation;Human Genetics and Molecular Biology

Eckart, Nicole E.McCallion, Andrew S. ;

Johns Hopkins University

关键词: human genetics;    schizophrenia;    gene expression;    gene regulation;    Human Genetics and Molecular Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39722/ECKART-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

BackgroundSchizophrenia is a complex psychiatric disorder with heritability estimated to be around 80%. Genetic studies have identified over 100 schizophrenia risk loci. These risk loci most often lie in non-coding sequences and are enriched for expression quantitative trait loci (eQTLs), suggesting that dysregulation of transcriptional control plays a role in complex disease pathogenesis. Of particular interest are non-coding variants in calcium channel subunits, which have been associated with multiple psychiatric disorders. Specifically, genome wide association studies (GWAS) have repeatedly identified the single nucleotide polymorphism (SNP) rs1006737 in the third intron CACNA1C to be strongly associated with schizophrenia. Methods We genotyped schizophrenia-associated variants and measured gene expression by qPCR in human post mortem brain samples. We looked for statistically significant genotype-expression correlations to identify eQTLs. We further investigated the putative eQTLs with dual luciferase reporter assays by transfecting reporter constructs into relevant cell lines for all variants in high linkage disequilibrium (LD) with the schizophrenia-associated variant. Furthermore, we investigated allele-specific protein binding of variant sequences in the putative eQTL through electrophoretic mobility shift assays (EMSAs) with nuclear extract from two cell lines is incubated with radiolabeled DNA probes, and a larger molecular weight band is produced if the probe binds to a protein or protein complex from the nuclear extract. With protein microarrays, proteins are immobilized on a glass slide and fluorescently labeled DNA probes are hybridized to the microarray, producing a signal when they bind a protein, we identified specific DNA-protein interactions. Lastly, we identified potential regulatory elements that interact with the promoters of our genes of interest through circular chromatin conformation capture with next generation sequencing (4C-seq). In this assay, cells were cross-linked to capture protein-mediated DNA-DNA interactions. After digestion, interacting DNA fragments were ligated together to form small hybrid circular molecules. Then, for a known viewpoint of interest, the unknown interacting fragments were identified through next generation sequencing.Results and ConclusionsHere, we showed that rs1006737 marks an eQTL for CACNA1C transcript levels in human post mortem brain tissue. We tested 16 SNPs in high LD with rs1007637 and found that one, rs4765905, consistently showed allele-dependent regulatory function in reporter assays. We found allele-specific protein binding for 13 SNPs including rs4765905 and using protein microarrays, we identified several proteins binding more than three SNPs, but not control sequences, suggesting possible functional interactions and combinatorial haplotype effects. Finally, using 4C-seq, we showed interaction of the CACNA1C promoter with the eQTL and other potential regulatory regions. Our results elucidate the pathogenic relevance of one of the best-supported risk loci for schizophrenia.