Hong Chang1, Xin Cai2, Hui-Juan Li2, Wei-Peng Liu2, Li-Juan Zhao2, Chu-Yi Zhang2, Jun-Yang Wang2, Jie-Wei Liu1, Xiao-Lei Ma1, Lu Wang1, Yong-Gang Yao3, Xiong-Jian Luo4, Ming Li5, Xiao Xiao6. 1. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China. 2. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; Kunming College of Life Science, University of Chinese Academy of Sciences, Shanghai, China. 3. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China. 4. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, Shanghai, China. 5. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China; CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China. Electronic address: limingkiz@mail.kiz.ac.cn. 6. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China. Electronic address: xiaoxiao2@mail.kiz.ac.cn.
Abstract
BACKGROUND: Genome-wide association studies (GWASs) have reported hundreds of genomic loci associated with schizophrenia, yet identifying the functional risk variations is a key step in elucidating the underlying mechanisms. METHODS: We applied multiple bioinformatics and molecular approaches, including expression quantitative trait loci analyses, epigenome signature identification, luciferase reporter assay, chromatin conformation capture, homology-directed genome editing by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9), RNA sequencing, and ATAC-Seq (assay for transposase-accessible chromatin using sequencing). RESULTS: We found that the schizophrenia GWAS risk variations at 16p11.2 were significantly associated with messenger RNA levels of multiple genes in human brain, and one of the leading expression quantitative trait loci genes, MAPK3, is located ∼200 kb away from these risk variations in the genome. Further analyses based on the epigenome marks in human brain and cell lines suggested that a noncoding single nucleotide polymorphism, rs4420550 (p = 2.36 × 10-9 in schizophrenia GWAS), was within a DNA enhancer region, which was validated via in vitro luciferase reporter assays. The chromatin conformation capture experiment showed that the rs4420550 region physically interacted with the MAPK3 promoter and TAOK2 promoter. Precise CRISPR/Cas9 editing of a single base pair in cells followed by RNA sequencing further confirmed the regulatory effects of rs4420550 on the transcription of 16p11.2 genes, and ATAC-Seq demonstrated that rs4420550 affected chromatin accessibility at the 16p11.2 region. The rs4420550-[A/A] cells showed significantly higher proliferation rates compared with rs4420550-[G/G] cells. CONCLUSIONS: These results together suggest that rs4420550 is a functional risk variation, and this study illustrates an example of comprehensive functional characterization of schizophrenia GWAS risk loci.
BACKGROUND: Genome-wide association studies (GWASs) have reported hundreds of genomic loci associated with schizophrenia, yet identifying the functional risk variations is a key step in elucidating the underlying mechanisms. METHODS: We applied multiple bioinformatics and molecular approaches, including expression quantitative trait loci analyses, epigenome signature identification, luciferase reporter assay, chromatin conformation capture, homology-directed genome editing by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9), RNA sequencing, and ATAC-Seq (assay for transposase-accessible chromatin using sequencing). RESULTS: We found that the schizophrenia GWAS risk variations at 16p11.2 were significantly associated with messenger RNA levels of multiple genes in human brain, and one of the leading expression quantitative trait loci genes, MAPK3, is located ∼200 kb away from these risk variations in the genome. Further analyses based on the epigenome marks in human brain and cell lines suggested that a noncoding single nucleotide polymorphism, rs4420550 (p = 2.36 × 10-9 in schizophrenia GWAS), was within a DNA enhancer region, which was validated via in vitro luciferase reporter assays. The chromatin conformation capture experiment showed that the rs4420550 region physically interacted with the MAPK3 promoter and TAOK2 promoter. Precise CRISPR/Cas9 editing of a single base pair in cells followed by RNA sequencing further confirmed the regulatory effects of rs4420550 on the transcription of 16p11.2 genes, and ATAC-Seq demonstrated that rs4420550 affected chromatin accessibility at the 16p11.2 region. The rs4420550-[A/A] cells showed significantly higher proliferation rates compared with rs4420550-[G/G] cells. CONCLUSIONS: These results together suggest that rs4420550 is a functional risk variation, and this study illustrates an example of comprehensive functional characterization of schizophrenia GWAS risk loci.
Authors: Ammar J Alsheikh; Sabrina Wollenhaupt; Emily A King; Jonas Reeb; Sujana Ghosh; Lindsay R Stolzenburg; Saleh Tamim; Jozef Lazar; J Wade Davis; Howard J Jacob Journal: BMC Med Genomics Date: 2022-04-01 Impact factor: 3.063