Single-nuclei RNA sequencing detects gene splicing defect known as “cryptic exons” in ALS

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurodegenerative diseases with mean survival of 2-5 years and 6-8 years, respectively. ALS is a neurodegenerative disease that mostly affects the motor neurons of the spinal cord and brain, leading to complete paralysis. FTD is a neurodegenerative disease leading to cognitive impairments.

In some cases, the same genetic mutations occur in both. One of those mutations is a repetitive expansion in the DNA of a gene called C9ORF72. A repetitive expansion is like having a word or phrase repeated over and over again in a sentence. One of the significant changes that happens in the majority of cases of ALS and around half of the cases of FTD, is when a protein called TAR DNA binding protein (TDP-43) clumps together and stops working correctly. When TDP-43 doesn’t function properly, it puts incorrect genetic instructions into the RNA, which are called “cryptic exons.”

Now, a team of scientists from the Translational Genomics Research Institute (TGen), part of the City of Hope, and Barrow Neurological Institute have detected these cryptic exons in specific brain cells from ALS and FTD patients carrying a mutation in the C9ORF72 gene.

As they report in Acta Neuropathologica, this is the first time that these disease-linked cryptic exons have been identified in single cells. The findings could help researchers learn more about exactly how cryptic exons affect the cells’ function in ALS and FTD, and about which cells are most vulnerable to TDP-43 dysfunction.

“By examining single cells from individuals diagnosed with ALS, FTD or both, we are able to investigate which cells are most vulnerable to the genetic changes that cause disease, and which cells are more protected. This can help us understand the underlying cell processes involved in these diseases more clearly” said Kendall Van Keuren-Jensen, Ph.D., professor in the Neurogenomics Division and Deputy Director of scientific resources at TGen.

Previous researchers have described these cryptic exons in spinal cord and brain tissues from people with either or both diseases, but did not know which cells contained these cryptic exons, Van Keuren-Jensen said.

To pinpoint the cellular identity, the researchers examined post-mortem brain tissue samples from C9ORF72 ALS and FTD patients. They performed single-nuclei RNA sequencing on the samples to identify cryptic exons in specific cells. The study contained deeper sequencing than most studies, which aided the hunt for the cryptic exons, said study co-author and TGen Computational Scientist Eric Alsop, Ph.D.

The team found the cryptic exons in two genes–STMN2 and KALRN–in the frontal cortex of the brain in patients with both diseases, with the highest frequency of the cryptic exons for both genes in individuals that had FTD.

Some of the cell types identified had previously been linked to FTD, so finding the cryptic exons in them using a different approach “may further confirm them as a vulnerable cell type in the disease,” said Rita Sattler, Ph.D., a Barrow professor in the Department of Translational Neuroscience and study co-author.

The researchers also uncovered some intriguing differences in terms of how likely brain cells were to contain cryptic exons. These differences might be related to the different trajectories of developing either ALS or FTD or both, although the team stressed that it is not possible to know at this stage.

“If someone is a carrier of a gene mutation associated with ALS or FTD, such as C9ORF72, they have about a 90% chance of developing either ALS or FTD or both, but we cannot determine yet if and which disease they are going to develop,” said Sattler.

The team also noted some signs of gene dysregulation in cryptic exon-bearing cells. “But all we have to look at is post-mortem tissue, so this is the end stage of disease for these individuals,” Sattler cautioned. “We see these changes, but are they related to dying cells, or real transcriptional changes that are associated with those cells with TDP-43 pathology?”

Sattler said that study co-author Lauren M. Gittings, Ph.D., a postdoctoral fellow in the Sattler Lab, would be studying just that question.

“She’s going to dive deeper into that biology by looking at the presence of these cryptic exons in the [KALRN] gene, and whether it plays a role in the degeneration that occurs in ALS and FTD.” To accomplish this, the researchers are using patient-derived cells to create cortical neurons, Gittings said. “We’re going to look for the presence of the cryptic exons in these cells over time, to complement the post-mortem studies.”


Gittings LM, Alsop EB, Antone J, Singer M, Whitsett TG, Sattler R, Van Keuren-Jensen K. (2023) Cryptic exon detection and transcriptomic changes revealed in single-nuclei RNA sequencing of C9ORF72 patients spanning the ALS-FTD spectrum. Acta Neuropathol [Epub ahead of print]. [article]

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