Analysis of Biased Allelic Enhancer Activity of Schizophrenia-Linked Common Variants

Schizophrenia (SCZ) is a neuropsychiatric disorder with heritability estimates between 60% and 80%. Although genome-wide association studies have identified many genetic loci linked to the disorder, most of these are noncoding variants whose functional impacts are not well understood. To bridge this gap, we prioritized potential functional variants linked to SCZ by utilizing a human brain epigenomic roadmap. We assessed the regulatory activity of these variants using an adapted STARR-seq screening method across four cell lines: Neuro-2a, SH-SY5Y, HEK-293T, and PC-12. Furthermore, we pinpointed candidate target genes through functional characterization and investigated their roles using zebrafish models. Our study identified 351 candidate single nucleotide polymorphisms (SNPs). Among these, 46 SNPs exhibited biased allelic enhancer activity, termed baaSNPs, with notable cell-type specificity. Chromatin interaction profiling and expression quantitative trait loci analyses linked these baaSNPs to 217 candidate target genes, and pathway enrichment analysis indicated that these genes are involved in critical neurological processes such as synaptic transmission and GABAergic signaling. One baaSNP in particular, rs13072690, showed regulatory effects across all examined cell lines and was associated with reduced expression of the PCCB gene in multiple brain regions. Heterozygous pccb knockout zebrafish exhibited abnormal behaviors, including hyperactivity, increased anxiety-like responses, and social deficits. In conclusion, our study presents an approach for functionally annotating putative causative variants. These findings enhance our understanding of the genetic factors underlying the disorder. We employed a modified STARR-seq approach to assess the regulatory activity of SNPs across four cell lines: HEK-293T, SH-SY5Y, Neuro-2a, and PC-12. These lines were selected for their relevance to neuronal biology and experimental utility: HEK-293T cells for high transfection efficiency and robust reporter assays; SH-SY5Y cells for neuronal characteristics; Neuro-2a cells for studying neurodevelopmental signaling; and PC-12 cells for differentiation and neuroendocrine studies.