A multinational research collaboration led by researchers at the University of Kentucky Sanders-Brown Center on Aging have used long-read RNA sequencing to identify 53 new RNA isoforms in medically relevant genes related to Alzheimer’s disease (AD) and other neurodegenerative diseases. Their study, published in Nature Biotechnology, is part of an effort to develop a pre-symptomatic diagnostic test for AD.
For the study, the team sequenced 12 frontal cortices of deceased subjects, six with Alzheimer’s and six controls using long-read sequencing.
The sequencing data identified 1,917 medically relevant genes expressing multiple isoforms in the frontal cortex where 1,018 had multiple isoforms with different protein-coding sequences. Of these 1,018 genes, 57 are implicated in brain-related diseases including AD, major depression, schizophrenia, and Parkinson’s disease. The study also found 53 new RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene.
“In the present study, we demonstrate that RNA isoform quantification through deep long-read sequencing can be a step toward understanding the function of individual RNA isoforms and provide insights into how they may impact human health and disease,” the researchers wrote. “Specifically, in addition to discovering new (that is, unannotated) RNA isoforms in known medically relevant genes, we also discovered new spliced mitochondria-encoded RNA isoforms and entirely new gene bodies in nuclear DNA and demonstrated the complexity of RNA isoform diversity for medically relevant genes within a single tissue.”
The researchers noted that they found five new, complex RNA variants from mitochondrial DNA and believe this is the first study to identify this genetic material in human tissue.
“Although their expression is low, these genes could serve as biomarkers for mitochondrial function, which play an important role in many age-related diseases. It’s crucial to understand the role these new isoforms play in human health and disease,” said research team leader and corresponding author Mark T. W. Ebbert, PhD, an associate professor in the department of internal medicine in the College of Medicine, University of Kentucky, with a joint appointment in the department of neuroscience.
These discoveries have the potential to both inform the development of new treatments targeting relevant RNA isoforms, but also develop new diagnostic tools.
“With this method, we’ve shown there’s potential to specifically target isoforms that are either promoting cellular health or dysfunction rather than treating a gene as a single entity,” said Ebbert. “The analysis can also help us reveal unique signatures in Alzheimer’s disease not detectable at the gene level.”
To assess RNA isoform expression for medically relevant genes in the frontal cortex, the Sanders-Brown investigators used a list of medically relevant genes defined in a prior study, while also adding genes relevant to brain-related diseases.
“Identification and quantification of all isoforms are especially important for known medically relevant genes because, for example, when clinicians interpret the consequence of a genetic mutation, it is interpreted in the context of a single isoform of the parent gene body,” the researchers noted. “That isoform may not even be expressed in the relevant tissue or cell type, however. Thus, knowledge about which tissues and cell types express each isoform will allow clinicians and researchers to better interpret the consequences of genetic mutations in human health and disease.”
While the investigators noted that their research using long-read sequencing revealed much more than would be possible with short-read RNA-seq, challenges remain to accurately quantify RNA isoforms in genes with many large and similar isoforms.
“Although this work is a substantial improvement over short-read sequencing, the data are not perfect and future improvements in sequencing, transcriptome annotation and bioinformatic quantification will continue to improve the accuracy of long-read RNA-seq,” they added.
The team said that a study limitation was the small number of frontal cortices sequenced, but that it served primarily as a proof-of-concept of the value of measuring individual RNA isoform expression in disease tissue.
Nevertheless, “our study highlights the advantage of long-read RNA-seq in assessing RNA expression patterns in complex human diseases to identify new molecular targets for treatment and diagnosis,” the investigators concluded.
