New Understanding of How Genetic Mutation Causes Huntington’s Disease

“It’s only when the repeat becomes extremely long that it begins to cause harm,” said co-senior author Steve McCarroll, an institute member and director of genomic neurobiology at the Stanley Center for Psychiatric Research at the Broad Institute, the Dorothy and Milton Flier Professor of Biomedical Science and Genetics in the Blavatnik Institute at HMS, and an investigator of the Howard Hughes Medical Institute. “This is a really different way of thinking not only about how Huntington’s disease develops but also about how a mutation brings about a disease. We think that it will apply in DNA-repeat disorders beyond Huntington’s disease.”

The findings also suggest avenues researchers could pursue to delay or even prevent Huntington’s.

For instance, candidate Huntington’s drugs that aim to reduce activity of the mutated gene (or the protein it codes for) have struggled in clinical trials. The new study implies that because very few cells have the toxic version of the gene and its proteins at a given time, the treatments may be helping only a small fraction of cells.

Developing a method that stops or slows the CAG-repeat expansion in the HTT gene might postpone toxicity in a far larger number of cells, delaying or even preventing the onset of the disease, the authors propose.

“The point of our work — what we all do — is relieving suffering caused by disease,” said co-senior author Sabina Berretta, HMS associate professor of psychiatry at McLean Hospital and director of the Harvard Brain Tissue Resource Center (HBTRC), an NIH NeuroBioBank center at McLean Hospital.

Repeat expansion

Most people inherit versions of the HTT gene with 15 to 35 consecutive CAGs and never develop Huntington’s, while those who inherit a version with 40 or more consecutive CAGs almost always develop the illness later in life. The longer the stretch of repeats, the younger a person tends to be when symptoms appear. The tract of repeated CAGs has been shown to be unstable, resulting in a variety of lengths in different tissues.

McCarroll, Berretta, and colleagues built on a technology the McCarroll Lab developed a decade ago called droplet single-cell RNA-sequencing (Drop-seq) to analyze gene expression, cell identity, and the length of CAG repeats in thousands of single cells.

“The ability to take a particular cell and measure both the CAG length and the transcriptional profile — that’s a really important underpinning that’s allowed for really powerful analysis,” said Seva Kashin, senior principal software engineer in the McCarroll Lab and co-first author with Broad senior principal software engineer Bob Handsaker and former research associate Nora Reed.

Studying 500,000 individual cells in brain tissue donated by 53 people with Huntington’s and 50 people without the disease, collected and preserved by the HBTRC, the team found that most cell types from people with Huntington’s still had essentially the same number of CAG repeats they’d inherited.

However, striatal projection neurons, the primary cells in the brain’s striatum that die in the disease, had greatly expanded CAG-repeat tracts. In fact, the study showed that some of the neurons had as many as 800 CAGs, confirming a 20-year-old observation.

(The striatum is responsible for movement, many cognitive functions, and motivation. When large numbers of striatal cells die, patients develop involuntary movements in the arms, legs, and face, and many also develop cognitive problems. These progress to more severe cognitive problems and difficulty moving or swallowing.)

Most surprisingly, the team found that expansion from 40 CAGs to 150 CAGs had no apparent effect on the neurons’ health, but neurons with more than 150 CAGs showed greatly distorted gene expression, losing activity of critical genes and then dying.

Computational extrapolation

McCarroll’s team used computer modeling of the experimental data to estimate the rate and timing of CAG-repeat expansion in striatal projection neurons.

They found that CAG-repeat tracts initially grow slowly, expanding less than once a year during the first two decades of life. Then, when a cell’s repeat tract reaches about 80 CAGs — usually after several decades — its rate of expansion accelerates dramatically, and it expands to 150 CAGs in only a few more years. The cell dies just months later.

The model thus suggests that a striatal projection neuron spends more than 95 percent of its life with an innocuous HTT gene. Because CAG repeats in different cells cross the toxicity threshold at different times, the cells as a group disappear slowly over a long period, starting about 20 years before symptoms appear and more quickly as symptoms commence.

Advancing science and health

In addition to opening a new window into how human genetic mutations can operate, the researchers hope that their insights lead to new strategies that improve or ultimately save the lives of people who inherit mutations in the HTT gene.

“A lot was known about Huntington’s disease before we started this work, but there were gaps and inconsistencies in our collective understanding,” Handsaker said. “We’ve been able to piece together the full trajectory of the pathology as it unfolds over decades in individual neurons, and that gives us potentially many different time points at which we can intervene therapeutically.”

Previous genetic studies of Huntington’s, including studies led by Vanessa Wheeler and Ricardo Mouro Pinto at HMS and Massachusetts General Hospital, hint at ways to slow the expansion of CAG repeats by targeting proteins involved in maintaining and repairing DNA. For example, the MSH3 protein helps cells monitor DNA for mutations, but DNA loops formed by extra CAGs can confuse this protein into further expanding the CAG repeats.

An international team found that common variations in the genes encoding these DNA-repair proteins can hasten or delay onset of symptoms in Huntington’s patients — findings that McCarroll says inspired his team’s focus. He adds that slowing certain DNA-maintenance processes might also slow CAG repeat expansion by allowing other, less error-prone DNA-repair mechanisms to resolve these loops.

In the meantime, the researchers are working to understand how CAG repeats above 150 lead to neuronal impairment and death and why repeats expand more in some kinds of neurons than others. They are also investigating the connection between DNA-repeat expansion and cell changes in other genetic disorders involving DNA repeats and late onset.

“It’s going to take much scientific work by many people to get to treatments that slow the expansion of DNA repeats,” McCarroll said. “But we’re hopeful that understanding this as the central disease-driving process leads to deep focus and new options.”

Berretta emphasized that the contribution of brain tissue by Huntington’s patients was critical for the work.

“Our gratitude is with the families that chose to do something that is very difficult to do,” she said. “This would not have been possible without the altruism of many brain donors who have left a legacy of knowledge that will last and benefit many other people.”

Adapted from a Broad Institute and McLean news release.