NEW YORK – Long somatic expansion of the CAG repeat in the Huntingtin (HTT) gene appears to be responsible for the onset of Huntington’s disease (HD), according to a new study by researchers from the Broad Institute, Harvard Medical School, and McLean Hospital.
Published in Cell on Thursday, the study uncovered a surprising mechanism for the etiology of HD — rather than the abnormal number of CAG repeats a patient inherits, it is the expansion of this repeat beyond a certain threshold that triggers the disease.
The finding could also inform therapeutic development for HD, proposing that slowing down DNA repeat expansion could be an effective strategy to curb the disease.
"We think that at any point in time, most living cells [in an HD patient's brain] do not have a toxic protein, they just have a ticking DNA clock," said Steve McCarroll, a geneticist at the Broad and a corresponding author of the study, during a press briefing. "These results were really surprising even to us at the time."
A fatal neurodegenerative disorder, Huntington’s disease, which currently affects roughly 30,000 patients in the US, is known to be associated with an abnormal number of CAG repeats within exon 1 of the HTT gene. While healthy individuals have 15 to 30 consecutive CAGs in their alleles, people with HD are found to typically inherit at least 40 repeats.
Even with the existing knowledge, for decades, the exact mechanism by which the HTT repeats can lead to HD remained nebulous.
According to McCarroll, there were three major questions that had always been baffling to researchers: Why do people with more than 40 CAG repeats develop HD while others with 10 to 35 CAGs do not? Why does the HTT protein only destroy certain types of brain cells while sparing others? Lastly, why do HD symptoms take decades to appear in a patient?
To answer these questions, he and his collaborators developed a workflow that can concurrently measure CAG repeat length and profile genome-wide RNA expression in the same cell. To do so, they leveraged Drop-seq, a droplet-based single-cell RNA-seq method developed by McCarroll's team almost a decade ago that enables gene expression analysis in thousands of cells.
More specifically, from each set of nuclei, the researchers created two molecular libraries — one for measuring genome-wide RNA expression and another for long-read sequencing that captures the 5' region of HTT transcripts for measuring the CAG repeats. The two libraries share the same cellular barcodes, linking the CAG-length measurement to the transcriptome profile for each cell.
As part of the study, the researchers "deeply sampled" the nuclei from the anterior caudate — the brain region most affected in HD — of six deceased patients, whose samples were collected and preserved by the Harvard Brain Tissue Resource Center.
In their analysis, the authors found that different cell types had varying degrees of HTT CAG repeat expansion. For instance, astrocytes, oligodendrocytes, polydendrocytes (OPCs), microglia, and interneurons exhibited modest changes in the number of CAG repeats.
Meanwhile, striatal projection neurons (SPNs), the principal striatal cells that die in HD, showed "profound somatic expansion" of the HD-causing repeat, with some cells exhibiting an expansion to more than 800 CAGs.
To the researchers' surprise, expansions from 36 to 150 CAGs did not result in apparent toxicity in SPNs. However, once the number of CAG repeats surpassed a threshold of 150, they started to disrupt the expression of hundreds of genes, including those with important physiological functions such as genes encoding potassium channel subunits.
Furthermore, the researchers identified a set of more than 100 genes that were de-repressed in SPNs as the CAG repeat number grew beyond 150. Two of the most strongly induced genes were CDKN2A and CDKN2B, which encode proteins that promote senescence and apoptosis and could lead to cell death.
To extrapolate the temporal dynamics of CAG repeat expansion in SPNs, McCarroll and his team also built a computer model using the data generated in their study. Their model proposed a five-phase process for HD pathogenesis, involving cell autonomous events that are driven by a neuron’s expanding HTT allele.
In the first phase, an SPN undergoes decades of "slow but accelerating" CAG repeat expansion. During this phase, HTT CAGs can take, on average, 50 years to expand from 40 to 60, followed by another 12 years to expand from 60 to 80, though the expansion will vary from cell to cell and person to person.
After that, an SPN enters the second phase, where the rate of repeat expansion greatly accelerates, reaching 150 CAGs in just a few years.
Once an SPN enters the third phase, when CAG repeat units exceed 150, hundreds of genes begin to change in expression levels. This, in turn, ushers the cell into the fourth phase, where many genes are de-repressed, leading to the cell's senescence and apoptosis. Lastly, in the final phase, an SPN gets eliminated.
Based on this model, the researchers believe that an individual SPN spends more than 95 percent of its life in a period of gradual DNA repeat expansion with a biologically harmless HTT gene. It is only in the later phase that individual neurons start to asynchronously experience HTT toxicity with the rapid expansion of CAG repeats.
"This is a biological discovery rather than a therapeutic study, but it has implications for thinking about where the biological leverage for therapeutics for Huntington's disease is," said McCarroll.
Currently, almost all HD therapies in advanced clinical development focus on suppressing HTT expression, but they have largely been unsuccessful, according to the study authors. However, given that HTT toxicity is now believed to be brief, asynchronous, and intense rather than lifelong, synchronous, and indolent, it might be more successful to slow down somatic expansion of CAG repeats, McCarroll said.
According to the paper, the Broad Institute and Harvard have filed patent applications related to the study, with some coauthors named as inventors. In an email, a Broad spokesperson said the filed patent application, titled "Methods and compositions for analysis and treatment of repeat expansion disorders," is currently "open for licensing."
Moving forward, McCarroll said the team will continue to address some of the open questions. For instance, they are working to understand exactly what is changing in a cell beyond 150 CAG repeats that induces changes in gene expression, ultimately resulting in neuronal death.
Additionally, the researchers also want to understand why the CAG repeats expand more aggressively in SPNs versus other cell types.
"That is particularly important because it may actually give us a key to understand how we could possibly slow down the expansion," said Sabina Berretta, a psychiatry professor at McLean Hospital and another corresponding author of the study.