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Abstract:
Psychiatric diseases are a major public health burden worldwide, yet little is known about their underlying molecular mechanisms. Among those highly polygenic diseases, schizophrenia is characterized by a heritability above 80 %. However, the ability to translate statistically associated variants from genome ? wide association studies into insights on disease mechanisms has been severely hampered by the facts that 1) the vast majority of the associated single nucleotide polymorphisms (SNPs) reside in non-coding regions of the genome, 2) the molecular causal genetic variants cannot be annotated among the statistically associated variants in linkage disequilibrium, 3) the variants act cell type and condition specific, and, 4) the effect of those non-coding functional variants and 5) the corresponding target genes of the variants are unknown.
In order to address these questions in a disease relevant human in vitro cellular model, several patient and healthy control primary human cells were successfully reprogrammed into induced pluripotent stem cells during the time of this thesis. Immunocytochemistry, bulk and single cell ribonucleic acid sequencing (RNA-Seq) of Neurogenin-2 (Ngn2) induced neurons (iNeurons) confirmed excitatory neuronal identity of the peripheral and central nervous system. To pinpoint the effect of schizophrenia associated genetic variants on gene regulation and expression, assay for transposase-accessible chromatin sequencing (ATAC-Seq), single cell and bulk RNA-Seq, massively parallel reporter assay (MPRA), and clustered regularly interspaced short palindromic repeats interference (CRISPRi) analyses were performed on induced pluripotent stem cells (iPSCs) derived iNeurons and primary mouse neurons. High potassium induced depolarization of iNeurons altered the chromatin state of 7,635 regions and the expression levels of 3,055 genes independently of their disease status. These generated data sets of the iNeurons and other publicly available data sets were used to preselect enhancer regions containing schizophrenia associated variants to test for their activity with MPRA in different cell types and states. From the tested regions, 13 % (620) single nucleotide variants were identified to impact the activity of the tested enhancer fragment. Intersection of those allele specific enhancer regions with postmortem brain derived expression quantitative trait loci (eQTLs) and chromatin conformation data sets as well as CRISPRi interference at the TCF4 gene locus linked several genes per variant and several genetic variants per gene.
The annotation of those disease associated functional variants and their linked genes, therefore, offers a comprehensive molecular insight into development and stimulation dependent molecular processes modulated by schizophrenia associated genetic variation. In the future, this strategy can be applied to improve the targeted development of new treatments to attenuate or even abolish progression of other psychiatric and in general complex diseases.
