A process as intricate as brain development requires precisely controlled expression of crucial genes, but it’s the different flavors of a protein, or isoforms, that the genes encode that do the heavy lifting.
“One thing that hadn't been systematically studied is how those genes [involved in brain development] are spliced into different transcripts,” said Michael Gandal, a physician scientist at the University of Pennsylvania. “We think of genes as the main output of our genome, but they really aren't. It's the specific transcript of a gene and how it gets spliced together that ultimately is what makes a functional protein or a regulatory molecule.”
While researchers have shown how isoform diversity regulates synapse formation in the brain and contributes to the risk of developing neuropsychiatric disorders, less is known about their role in shaping neurodevelopment.1,2 Now, in a paper published in Science, Gandal and his team sequenced regions of the developing brain and discovered a number of different types of isoforms linked with neurodevelopmental disorders.3 These findings provide a framework that allows scientists to understand how isoforms and the way they change could potentially orchestrate and influence neurodevelopment.
“We were quite interested in understanding how genes regulate [development], how that process may go awry in individuals with neurodevelopmental disorders, and whether we can connect the two together through this study,” said Gandal.
Previous attempts to study isoforms in neurodevelopment used a technique called short-read sequencing. But this approach failed to provide a complete picture of the isoforms since they showed up as small fragments that the researchers had to stitch together into a meaningful picture. Gandal and his team bypassed this tedious task by using long-read sequencing, which gives researchers the ability to sequence entire, uninterrupted genetic sequences. With this approach, the researchers set out to get a better picture of the different isoforms during a critical window of development.
The team looked at tissue from the human cortex collected at midgestation, around 15 to 17 weeks postconception and a critical time window of development during which most of the newborn neurons in the brain are generated. Specifically, they investigated the germinal zone and the cortical plate, where cells are born and migrate out of to populate the brain.
Using long-read sequencing, the researchers identified more than 200,000 distinct isoforms in the brain tissue, but one observation caught Gandal’s attention. “The most unexpected finding was just the sheer number of new transcripts that we found,” said Gandal. When they compared their results with established gene databases like GENCODE, they found that around 70 percent of the isoforms were novel and had not been identified previously.
Peter Choi, a cancer biologist also at the University of Pennsylvania who was not involved with the study, said, “The study demonstrates that with long-read technology, you're actually picking up a lot of novel transcripts, which we have previously not appreciated.” He added, "It's a really good assessment of what we're actually missing.
“One major question in the field of developmental biology is how does the genome encode for something as complex as the human brain,” said Gandal. In addition to levels of gene activity, the findings suggest that alternative splicing varies across development, which switches up isoform expression over time, a process which likely regulates the birth and maturation of brain cells. Many of the isoforms that they linked with this process were in proteins that bind RNA, proteins involved in synaptic transmission, and neuronal cell adhesion molecules.
Neurodevelopment disorders like autism spectrum disorder are characterized by dysfunctional alternative splicing, so the team wanted to study their data set and identify features in the isoforms associated with these conditions. When the researchers examined gene properties, they found that genes with more exons and more transcripts, evidence of more splicing events, were more likely to be associated with neurodevelopmental disorders.
Finally, Gandal and his team looked at over 200,000 genetic variants that other researchers had previously identified in individuals with neurodevelopmental disorders but had determined were noncoding variants and unlikely to influence the condition. In this case, they found that around one percent of the variants previously classified as harmless overlapped with or were near the newly identified isoforms in the genome, suggesting these could potentially influence neurodevelopmental disorders.
The team compiled their isoform sequences into an open access database that they hope will help other researchers more accurately study isoforms, particularly in the context of neurodevelopment and psychiatric conditions. “[The resource] helps prioritize what we should look into more,” said Choi.
There is still more work to be done. For example, Choi pointed out that researchers still need to understand which of these isoforms matter biologically. “Which of these represent noise in the cell versus actually having a function?” he wondered.
Gandal said that he hopes that their findings might help inform genetic treatment of neurodevelopmental conditions. “This has the potential to really move the needle for families who are affected by neurodevelopmental disorders in terms of better understanding risk mechanisms and genetic contribution to these disorders.”
- Zhang X, et al. Cell-type-specific alternative splicing governs cell fate in the developing cerebral cortex. Cell. 2016;166(5):1147-1162.e15.
- Takata A, et al. Genome-wide identification of splicing QTLs in the human brain and their enrichment among schizophrenia-associated loci. Nat Commun. 2017;8:14519.
- Patowary A, et al. Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms. Science. 2024;384(6698):eadh7688.
