A hitch in the stitch reveals why DNA sequence patterns matter in Huntington’s disease  

For individuals in the HD “grey zone” between 36-39 repeats, an understanding of when symptoms may arise and what kind of symptoms is critical. A new study sheds light on different sequence patterns present that may help explain why some individuals with the same CAG repeat size have such vastly different disease trajectories. Let’s get into what they found. 

HD genetics 101

In humans, everyone has a stretch of the genetic letters C-A-G within the huntingtin (HTT) gene. In Huntington’s disease (HD), when this stretch expands beyond 40 repeats, symptoms of HD will usually develop at some point in that person’s life. 

There is also a puzzling grey zone of repeats between 36 and 40 where some individuals go on to develop HD, while others may never show clear symptoms. Adding to the complexity, it’s not only the number of C-A-G repeats that matter for when individuals start developing symptoms but also the exact sequence of the genetic letters itself! A team of scientists from Vancouver and in Paris studied the DNA letter sequence of individuals with CAG repeats in the 36-42 range and discovered how both the repeat number and sequence pattern can shape HD onset and progression.  

A hitch in the stitch: how sequence patterns shape HD

Imagine the C-A-G repeat sequence as a stitched pattern, each “stitch” or knit represents DNA letters (A, C, G and T) repeated to form a long, uniform thread. However, the HTT gene isn’t only made of just C-A-G stitches. Right next to the C-A-G stretch, a second stitched region is knit using a different pattern, using C-C-G repeats. Together, these form a combined pattern rather than a single, uninterrupted block. These patterns are knitted together to make the first part of the HTT gene. Small variations or “hitches” are often woven in, sort of like using a differently coloured thread for an occasional stitch. These variations don’t ultimately alter the final material (HTT protein), but they do have massive implications as to when people start symptoms.  Over the years, scientists have unravelled some common patterns by studying many HD individuals. Let’s take a closer look at the patterns we already know. 

Regular: This is the most common pattern, where the C-A-G stretch is interrupted by a C-A-A hitch. The neighbouring C-C-G stretch is also interrupted by a C-C-A hitch. These hitches or interruptions break up the pattern into shorter segments and are generally associated with later and more predictable onset of symptoms for a given C-A-G repeat length. 

Loss of interruption (LOI): In this pattern, both the C-A-A and C-C-A repeats are missing their interruptions. There is a long uniform uninterrupted thread of C-A-G stretch followed by an uninterrupted C-C-G stretch. This stitched pattern becomes more prone to fraying and as the cell tries to fix these issues, it can become longer and longer as the DNA thread comes apart more easily. People with this pattern develop symptoms years earlier than expected based on their C-A-G repeat length alone. 

C-C-G Loss of interruption (CCG-LOI): In this pattern, the C-A-G repeat stretch still has a single C-A-A hitch, but the rest of the C-C-G sequence is uninterrupted.  This shows that changes don’t have to affect both parts of the overall thread equally. This pattern has also been linked to earlier symptom onset. Altering just one section or having just one hitch can still have big impacts! 

Reading the pattern, not just counting stitches

For decades, genetic testing for HD has focused on counting the number of repeats, essentially measuring the length of the stitched pattern without looking at how the pattern itself is woven. The current tests can miss interruptions and variations in both the C-A-G and neighbouring C-C-G regions, meaning that two individuals with the same repeat count may have completely different patterns influencing the ages of symptom onset and the progression of their disease.  

To truly understand how these stitching patterns affect HD, the researchers looked closely at the exact HTT repeat sequence pattern in the DNA of 328 people with CAG repeat lengths between 36 and 42. They combined data from two large cohorts: 162 individuals from France and 166 from Vancouver and affiliated international centres. Together, this created one of the largest datasets to focus specifically on this so-calledCAG grey zone and smaller CAG repeats.  

The team used high resolution sequencing technology. Think of it like unravelling an entire knit sweater or hat and checking what pattern of stitch was present when it was made. Sequencing can reveal interruptions and variations within both the C-A-G and C-C-G stretches. When the researchers looked at individuals from the French cohort in the grey zone (36-39 repeats, total of 68 individuals), nearly 27% (19 out of 68) of the individuals carried a irregular stitching pattern rather than the standard design. 

Existing methods can underestimate the length of uninterrupted CAG repeats so whether a hitch stich is present or missing has consequences. In 11 of the 19 cases, knowing the true pattern would have shifted individuals from the grey zone category to a higher risk zone in terms of symptom and disease onset. 

A new pattern reveals itself

The researchers also identified a new pattern and sequence they termed CAG loss of interruption (CAG-LOI).  5 individuals had an interruption in the C-C-G repeat stretch but not in the CAG stretch meaning the CAG repeat portion doesn’t have hitches. This is the first time this pattern was found and highlights the importance of looking at the repeat sequence directly!   

What the stitching patterns reveal about symptoms

Those carrying the loss of interruption (no hitches) patterns developed movement symptoms much earlier than people with the regular sequence. When compared with common prediction models based on repeat length alone, symptom onset in these individuals occurred nearly 13 years earlier than expected. However, this effect was stronger for larger CAG lengths (CAG 39-42) and nearly half of the individuals in the 36-38 CAG range were symptomatic regardless of their sequence pattern.  

The study did not stop at when symptoms begin. By following people over time, the researchers showed that variations in the sequence also influences how the disease progresses. Individuals with the loss-of-interruption patterns experienced a faster worsening of movement symptoms, with motor scores increasing almost twice as quickly as in people with the regularsequence. Measures of thinking speed also declined more rapidly in this group, suggesting that the DNA sequence differences affect more than just movement. 

Why the pattern of the stitch matters for testing, genetic counselling and clinical trials

For people living in the HD CAG grey zone, uncertainly has long been part of the experience. A result showing 36-39 CAG repeats can raise difficult questions: Will symptoms develop? If so, when? And what might the disease look like? This study helps explain why those questions are often so hard to answer. Standard genetic tests focus on counting repeats, but they do not reveal how the sequence is stitched together. As this study shows, people in the grey zone can carry sequence patterns that can shift symptom onset earlier and influence how quickly the disease progresses. 

For genetic counselling, particularly for people undergoing predictive testing, knowing when they might potentially get symptoms could be critical for planning life. Two individuals with the same CAG number may face very different risks depending on their underlying sequence pattern. Incorporating sequence information could help provide clearer, more personalized discussions about risk, timing and expectations especially near traditional cutoff ranges. 

These findings also could have important implications for clinical trials. Often, studies rely on CAG repeat length to define eligibility and estimate disease stage. If sequence patterns affect both onset and progression, then grouping participants by repeat count alone may unintentionally mix people with very different disease trajectories. Reading the CAG stitch pattern more carefully could improve how trials are designed, interpreted and compared. 

The biology of HD is complex! 

Finally, this work reinforces a broader message emerging across HD research. The biology of the HTT gene, it’s sequence pattern and how to predict when someone will start symptoms are all extremely complex factors that are under investigation. Importantly, the HTT gene is not the be all, end all of when an individual will get symptoms. Decades of research have shown that other genes involved in DNA repair and known as  genetic modifiers can affect the age of onset. Genetic modifiers are a factor that this study does not assess. It is not only the pattern or sequence variation in the HTT gene that are important but also the pattern other genes that make DNA repair proteins! 

As tools for sequencing become more accessible and cheaper, moving beyond simple repeat counts towards a better understanding of sequence patterns may help reduce uncertainty, improve prediction and ultimately lead to better care for people navigating a diagnosis of Huntington’s disease.   

The major takeaways

• The CAG “grey zone”: Individuals with 36-39 CAG repeats sit in a Huntington’s disease “grey zone” and can have very different ages of onset and symptoms. 
• The method: Researchers from Vancover and France used DNA sequencing to read the exact pattern or sequence from individuals with 36-42 CAG’s, revealing changes that standard repeat counting tests usually miss. 
• The sequence pattern matters: Interruptions or “hitches” and loss of these within the C-A-G repeat and C-C-G repeat stretches play a major role as to when symptoms start with loss of interruptions leading to earlier age of onset.
• Why look at the pattern?: Individuals in the 36-39 CAG repeat range can carry sequence variants that standard genetic tests miss. Individuals who did not have the interruptions showed faster worsening of movement symptoms and more rapid decline in thinking speed after symptoms began. 
• Putting it all together: Reading the full DNA sequence could improve genetic counselling, refine disease prediction and help design better clinical trials.