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Long-read RNA sequencing reveals synaptic genes with differential isoform usage in schizophrenia [long-read RNAseq ]

ABSTRACT: Over the past 15+ years, genetic studies have been notably successful in revealing the architecture of multiple psychiatric disorders, including schizophrenia. It is now widely acknowledged that schizophrenia is highly polygenic, consisting of both common variants of unknown significance, identified through genome-wide association studies (GWAS), and rare CNVs and coding variants. Despite these advances, the underlying mechanisms of specific genes implicated by GWAS are not well understood. Multiple lines of evidence implicate alternative splicing in the pathophysiology of schizophrenia. Single-nucleotide polymorphisms (SNPs) within implicated schizophrenia loci could alter isoform diversity and abundances which may not be reflected in a typical differential expression study. Hence, we generated a comprehensive isoform survey of postmortem human dorsolateral prefrontal cortex (DLPFC) from schizophrenia cases and neurotypical controls to identify case-control isoform-level differences. We developed an analysis pipeline that combines the strengths of PacBio SMRT long-read RNA sequencing in conducting a detailed isoform census with the capacity of short-read RNA sequencing for the quantification of isoform abundances. From several hundred thousand discovered long-read isoforms we curated a transcriptome with tens of thousands of high confidence novel isoforms. We then identified differential isoform usage (DIU) genes using a combination of established and in-house pipelines that enables case-control comparisons. Many of these novel isoforms are differentially expressed in schizophrenia DLPFC vs neurotypical controls. Differentially expressed genes are enriched in gene sets related to synaptic structure and function, RNA binding and splicing, as well as cell types previously implicated in schizophrenia, including cortical excitatory neurons, medium spiny neurons, and pyramidal CA1 neurons. Publicly available splicing data, genotyping, proteomics, and single nucleus sequencing results verify and support our results.

ORGANISM(S): Homo sapiens

PROVIDER: GSE282551 | GEO | 2025/12/01

REPOSITORIES: GEO

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Project description:Over the past 15+ years, genetic studies have been notably successful in revealing the architecture of multiple psychiatric disorders, including schizophrenia. It is now widely acknowledged that schizophrenia is highly polygenic, consisting of both common variants of unknown significance, identified through genome-wide association studies (GWAS), and rare CNVs and coding variants. Despite these advances, the underlying mechanisms of specific genes implicated by GWAS are not well understood. Multiple lines of evidence implicate alternative splicing in the pathophysiology of schizophrenia. Single-nucleotide polymorphisms (SNPs) within implicated schizophrenia loci could alter isoform diversity and abundances which may not be reflected in a typical differential expression study. Hence, we generated a comprehensive isoform survey of postmortem human dorsolateral prefrontal cortex (DLPFC) from schizophrenia cases and neurotypical controls to identify case-control isoform-level differences. We developed an analysis pipeline that combines the strengths of PacBio SMRT long-read RNA sequencing in conducting a detailed isoform census with the capacity of short-read RNA sequencing for the quantification of isoform abundances. From several hundred thousand discovered long-read isoforms we curated a transcriptome with tens of thousands of high confidence novel isoforms. We then identified differential isoform usage (DIU) genes using a combination of established and in-house pipelines that enables case-control comparisons. Many of these novel isoforms are differentially expressed in schizophrenia DLPFC vs neurotypical controls. Differentially expressed genes are enriched in gene sets related to synaptic structure and function, RNA binding and splicing, as well as cell types previously implicated in schizophrenia, including cortical excitatory neurons, medium spiny neurons, and pyramidal CA1 neurons. Publicly available splicing data, genotyping, proteomics, and single nucleus sequencing results verify and support our results.

Project description:Genome-wide association studies (GWASs) have revealed thousands of associations in many complex traits and diseases. Previous studies suggest that a subset of associations are due to alterations in splicing; however, interpreting the effects of splicing on protein isoforms is hindered by limitations in defining full-length transcript isoforms using short-read RNA-seq data. Long-read RNA-seq represents a powerful approach to define and quantify transcript isoforms. In this study, we developed a novel approach that integrates information from GWAS, splicing QTL (sQTL), and PacBio long-read RNA-seq in a disease relevant model to infer the effects of sQTL on the ultimate protein isoform products they encode. Such information enables identification of genes potentially responsible for GWAS associations. As a proof-of-concept, we generated deep coverage (N=~22 million full-length reads) PacBio long-read RNAseq data on human fetal osteoblasts (hFOBs), a cell-line of relevance to the regulation of bone mineral density (BMD). We identified 68,326 protein-coding isoforms, including 17,375 (25%) which were novel. Next, we used Bayesian colocalization to identify 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes which colocalized with BMD associations (H4PP > 0.75). A total of 836 junctions with colocalizing sQTLs in 459 (of the 732) genes were expressed in hFOB long-read RNA-seq data. With these data, we formulated hypotheses regarding the potential mechanism of action of each sQTL. For example, we identified 7 junctions with colocalizing sQTLs (maximum H4PP = 0.98-0.99) in TPM2 for splice junctions between two nearly mutually exclusive exons, and two different transcript termination sites, making it impossible to interpret without long-read RNA-seq data. siRNA mediated knockdown in hFOBs showed two TPM2 isoforms with opposing effects on mineralization. Our results suggest that splicing is a major mechanism underlying GWAS associations and long-read proteogenomics data is critical to precisely define the protein isoforms that are produced from splicing alterations.

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Long-read RNA sequencing reveals synaptic genes with differential isoform usage in schizophrenia [long-read RNAseq ]

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