Using deep long-read RNAseq in Alzheimer’s disease brain to assess medical relevance of RNA isoform diversity

Abstract

Due to alternative splicing, human protein-coding genes average over eight RNA isoforms, resulting in nearly four distinct protein coding sequences per gene. Long-read RNAseq (IsoSeq) enables more accurate quantification of isoforms, shedding light on their specific roles. To assess the clinical relevance of measuring RNA isoform expression, we sequenced 12 aged human frontal cortices (6 Alzheimer's disease cases and 6 controls, 50% female) using one Oxford Nanopore PromethION flow cell per sample. Our study uncovered 53 new high-confidence RNA isoforms in clinically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. Specific examples include WDR4 (61%; microcephaly), MYL3 (44%; hypertrophic cardiomyopathy), and MTHFS (25%; major depression, schizophrenia, bipolar disorder). Other notable genes with new high-confidence isoforms include CPLX2 (10%; schizophrenia, epilepsy) and MAOB (9%; targeted for Parkinson's disease treatment). We identified 1,917 clinically relevant genes expressing multiple isoforms in human frontal cortex, where 1,018 had multiple isoforms with different protein coding sequences, demonstrating the need to better understand how individual isoforms from a "single" gene are involved in human health and disease. Exactly 98 of the 1,917 genes are implicated in brain-related diseases, including Alzheimer's disease genes such as APP (A-beta; precursor protein; five), MAPT (tau protein; four), and BIN1 (eight). We also found 99 differentially expressed RNA isoforms between Alzheimer's cases and controls, despite the genes themselves not exhibiting differential expression. Our findings highlight the significant knowledge gaps in RNA isoform diversity and their clinical relevance. Deep long-read RNA sequencing will be necessary going forward to fully comprehend the clinical relevance of individual isoforms for a "single" gene. Key words: RNA isoforms, Clinical relevance, Long reads, Nanopore sequencing, Alzheimer's disease, Human brain.

Surveying the landscape of RNA isoform diversity and expression across 9 GTEx tissues using long-read sequencing data

“…Here, we provided a broad survey of the RNA isoform landscape, demonstrating the isoform diversity across nine tissues and emphasizing the need to better understand how individual isoforms from a single gene body contribute to human health and disease. We found genes whose isoform expression patterns differed in interesting and potentially significant ways and we validated isoforms recently discovered in Aguzzoli-Heberle et al 2 . We also identified isoforms that exhibit patterns consistent with preferential expression for a given set of tissues, and others that behaved as prospective housekeeping isoforms.…”

“…Starting with Ensembl v76 (released August 2014), there were 58,764 gene annotations present, compared to 62,710 by Ensembl v109 (released February 2023; 3,946 increase; Figure 2a). We observed a sharp increase in new gene body annotations in 2015 (1,792), followed by a sharper decrease in 2016 (2,505). Between 2016 and 2023, there was a general increase in gene annotations, including large increases in 2020 and 2023.…”

“…Here, we present results beginning with a broad overview of gene body and RNA isoform discovery in the past ten years, followed by a more in-depth analysis of RNA isoform expression across nine tissue types, using long-read data generated by Glinos et al 15 combined with results from our recent study of deep long-read cDNA sequencing in human prefrontal cortex (Brodmann 9/46) in Aguzzoli-Heberle et al 2 Also, while we report a few minor shortcomings in the Ensembl 1 tools for tracking annotation changes over the years, we emphasize that the overall quality and utility of Ensembl, their team, and their collaborators is incredible. Accurately resolving gene and isoform annotations is a Herculean task and is not a solved problem.…”

“…In our opinion, it seems unlikely that 20,000+ proteincoding genes performing a single function can enable such complex organisms 2 . Thus, here, we have provided a broad survey of the RNA isoform landscape to demonstrate both the complexity and diversity of RNA isoforms across nine human tissues from data generated by Glinos et al 15 and Aguzzoli-Heberle et al 2 . We have also tried to demonstrate how little is known about the various isoforms and why it is essential to fully characterize and quantify individual RNA isoform expression across the human cell types, tissues, and lifespan.…”

“…In their research, Glinos et al 15 initiated an investigation into tissue-specific RNA isoform expression, but given the broad scope of their article and the crucial importance of this subject, we believe a more thorough exploration is warranted. To provide a broad survey and to characterize the RNA isoform landscape across the human genome and emphasize the importance of alternative splicing in complex organisms, including humans, our aims are three-fold; (1) briefly characterize the history of gene body and RNA isoform discovery since 2014 by comparing Ensembl 1 gene annotations from 2014 to 2023 to assess changes over time; (2) characterize individual isoform expression across nine GTEx tissues (58 samples total) using data generated by Glinos et al 15 ; and (3) verify the existence and quantify the expression of new RNA isoforms, including those from new gene bodies, that we previously discovered in Aguzzoli-Heberle et al 2 across the nine GTEx tissues. We further provide a user-friendly website to explore the RNA isoform expression data for the GTEx samples (https://ebbertlab.com/gtex_rna_isoform_seq.html) and for our own RNA isoform expression data from prefrontal cortex (https://ebbertlab.com/brain_rna_isoform_seq.html) 2 .…”