Studying Stutters: What 900,000 genomes teach us about growing DNA repeats

Scientists have intensely studied the genetics of Huntington’s disease (HD) since the gene responsible was discovered in 1993. Unlike many other genetic diseases, where a change in one or a few DNA letters can be enough to cause disease, HD results from an expansion of a three-letter sequence (CAG) that repeats over and over in the huntingtin (HTTHTT one abbreviation for the gene that causes Huntington’s disease. The same gene is also called HD and IT-15) gene. When this genetic ‘stutter’ gets too long, usually 40 or more CAGs, it sets HD into motion. More recently, scientists discovered that the CAG repeatCAG repeat The stretch of DNA at the beginning of the HD gene, which contains the sequence CAG repeated many times, and is abnormally long in people who will develop HD can continue to expand in people’s cells throughout life. This ongoing growth of HTT’s CAG stutter is likely a key step in determining when the disease begins. 

But HTTHTT one abbreviation for the gene that causes Huntington’s disease. The same gene is also called HD and IT-15 is not the only gene containing an unstable genetic stutter. A team led by Dr. Po-Ru Loh at Harvard University analyzed the genomes of more than 900,000 people to investigate the behavior of other DNA repeats. Their findings revealed that repeat expansions are widespread and may be controlled by similar molecular pathways. 

Are genetic stutters common?

Although scientists know that the CAG repeatCAG repeat The stretch of DNA at the beginning of the HD gene, which contains the sequence CAG repeated many times, and is abnormally long in people who will develop HD in HTTHTT one abbreviation for the gene that causes Huntington’s disease. The same gene is also called HD and IT-15 can grow during a person’s life, and change number between generations, less is known if this is common for other repeats. The human genomegenome the name given to all the genes that contain the complete instructions for making a person or other organism contains hundreds of thousands of short repetitive DNA sequences. Do these repeats also grow (or shrink) with age or between generations? If they do, could they be associated with other human diseases? Could there be a common underlying cellular process that is malfunctioning and causing these genetic stutters? These are the questions Dr. Loh and colleagues set out to investigate. 

Searching for repeats among almost a million genomes

Measuring genetic stutters sounds simple in principle, but accurately identifying repeats across nearly a million people is technically extremely difficult. Imagine trying to count how many times the same word appears in a sentence like “the the the the the the”. Now imagine the font is size 0.0001 (molecule font!). If one “the” is added or removed, it’s surprisingly difficult to tell where it occurred or exactly how many copies are present.

Studying DNA requires researchers to prepare it using a series of chemical reactions, including reactions involving enzymes that make many copies of the DNA. This process poses another problem: the copying enzymes commonly add extra repeats that were not present in the original sample. To overcome these challenges, Dr. Loh and his team developed sophisticated computational and statistical methods to identify and filter out these ‘false’ repeats. They then used the remaining sample to estimate repeat lengths for around 350,000 locations in the genomegenome the name given to all the genes that contain the complete instructions for making a person or other organism. 

CAG repeats across the genomegenome the name given to all the genes that contain the complete instructions for making a person or other organism

The team began by searching for CAG repeats. Although many other types of repeating DNA exist, CAG repeats are already known to cause several diseases, including HD. The researchers identified 18 locations where some people had unusually long CAG repeats, possibly indicating an expansion had occurred. One of the most common expansions was in a gene called TCF4, where an expanded repeat was found in almost 9% of study participants. Finding a long repeat doesn’t necessarily mean it causes disease, but it does show that CAG repeats like to grow. The team also found 15 locations where CAG repeats changed length between generations, similar to HD. These results suggest that the genetic stuttering observed in HD might be part of a broader genetic pattern. 

Having established that some CAG repeats are longer than expected and can lengthen between generations, the next big question was whether any of them grow within a person’s lifetime. This is a key question because the growth of CAG repeats over time is thought to contribute to the onset of HD. Of the 15 CAG repeats they examined, 4 showed this “age-dependent” expansion. One finding was especially unexpected: people carrying expanded CAG repeats in a gene called GLS had an approximately 14 times increased risk of having severe kidney disease. This suggests that repeat expansions might contribute to more diseases than we currently know about, and we are only just finding them because repeats are difficult to spot in DNA. 

Why do genetic stutters grow? 

Imagine a broken printer that struggles with sentences containing repeated words (“can can can can can can”). Each time the sentence is reprinted, additional ‘cans’ are added – CAG repeats behave similarly. Because each CAG looks identical to the next and the one before, cellular machines sometimes slip, lose their spot, and add additional copies, causing the genetic stutter to change length over time. 

The researchers wanted to understand why some people’s repeats expanded faster than others. They focused on expanded repeats in a gene called TCF4 because they were common and varied widely in blood cells. By searching across the genomegenome the name given to all the genes that contain the complete instructions for making a person or other organism, the team found 7 genetic changes that seemed to track with TCF4 expansion. Excitingly, four of these locations were in DNA repair genes (MSH3, FAN1, ATAD5, and PMS2) that had already been linked to CAG repeat expansion in HTTHTT one abbreviation for the gene that causes Huntington’s disease. The same gene is also called HD and IT-15. These findings strengthen the evidence that DNA repair genes play a key role in controlling repeat expansions. 

A speed bump for repeat expansion

The research also found that a tiny interruption in the genetic stutter could slow the CAG repeatCAG repeat The stretch of DNA at the beginning of the HD gene, which contains the sequence CAG repeated many times, and is abnormally long in people who will develop HD from growing. Imagine the sequence “can can can can can.” Because every word is the same, the DNA copying machinery easily loses its place. Now insert another word “can can yes can can”. This tiny interruption acts like a speed bump, breaking the repetitive pattern and helping the DNA machinery keep track of its location. For example, in the TCF4 gene, 18 CAG repeats with one tiny interruption expanded 135 times slower than 18 CAG repeats without an interruption. 

Sequence interruptions like this are highly relevant to HD. Though it’s super rare, people with CAG repeatCAG repeat The stretch of DNA at the beginning of the HD gene, which contains the sequence CAG repeated many times, and is abnormally long in people who will develop HD interruptions have a significantly later age of disease onset than expected. And scientists are already working hard to develop repeat-busting genetic therapies. 

What does this mean for HD? 

One major takeaway from this research is that CAG repeats have a natural tendency to grow, and some of the same DNA repair machinery influences this expansion across different genes. The involvement of DNA repair machinery in both TCF4 and HTTHTT one abbreviation for the gene that causes Huntington’s disease. The same gene is also called HD and IT-15 (along with many other repeats) strongly supports the idea that these pathways are central to repeat expansion – and perhaps to disease onset. We still don’t know whether repeat expansion with age triggers HD, but this growing evidence suggests an intriguing connection. 

One important limitation is that this study primarily measured expansion in blood cells. Repeat growth in blood cells does not necessarily mean that the same repeats are growing in the brain, because each cell accumulates expansions at different rates and in ways that depend on complex cellular processes. 

Nevertheless, this study highlights the need for repeat-busting genetic therapies and treatments to modify the activity of DNA repair pathways. Both approaches are already under investigation, but more research is needed to determine whether they will be safe and effective in people. 

Summary

• Scientists studied repetitive DNA sequences in more than 900,000 people.
• They found that CAG repeats can expand across generations and within a person’s lifetime.
• Some of the same DNA repair genes influence repeat expansion in both TCF4 and the HD gene.
• Tiny interruptions within CAG repeats dramatically slowed their growth, supporting efforts to develop repeat-busting therapies.
• Most measurements came from blood, so further research is needed to understand how these findings apply to the brain.