Repeat-induced mutagenesis in Huntington's disease
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Medicine
Abstract
Huntington's disease (HD) is a brain disorder in which brain cells (neurons) are lost. This loss of neurons causes problems with movement, memory, thinking, and behaviour. These symptoms gradually worsen over time, leading to death 15-20 years after the first symptoms are seen. No treatment is available that can slow or stop this from happening.
HD is caused by an increase in the number of repeats of a specific DNA sequence, within a gene called huntingtin. The repeating DNA sequence motif is CAG. In unaffected people, the number of CAG repeats is small (less than 36). In people with HD, there are more repeats (over 39), with longer repeats leading to worse symptoms that appear at a younger age. In other words, the disease is more severe when a person has more repeats. Importantly, the number of repeats in the DNA sequence can increase in size over time. This process is affected by other small DNA sequences close to the CAG repeat sequence and can therefore play a part in disease severity. We do not currently understand how changes in these additional sequences are generated in people. This study aims to find the answer to these questions.
To do this, we will use state of the art techniques that allow us to investigate the DNA sequences we are interested in, in cells collected from people affected by HD, in cells grown in the laboratory, and using purified proteins and DNA in a test tube. We will collect cells from people to see if we can find the same results in people affected by HD that we find in cells grown in the laboratory. We will also mutate specific sequences in cells and study how they affect the appearance of mutations around the repeat tract.
The results we will obtain will improve our understanding of how sequences near repeats are generated and whether they correlate with disease symptoms.
HD is caused by an increase in the number of repeats of a specific DNA sequence, within a gene called huntingtin. The repeating DNA sequence motif is CAG. In unaffected people, the number of CAG repeats is small (less than 36). In people with HD, there are more repeats (over 39), with longer repeats leading to worse symptoms that appear at a younger age. In other words, the disease is more severe when a person has more repeats. Importantly, the number of repeats in the DNA sequence can increase in size over time. This process is affected by other small DNA sequences close to the CAG repeat sequence and can therefore play a part in disease severity. We do not currently understand how changes in these additional sequences are generated in people. This study aims to find the answer to these questions.
To do this, we will use state of the art techniques that allow us to investigate the DNA sequences we are interested in, in cells collected from people affected by HD, in cells grown in the laboratory, and using purified proteins and DNA in a test tube. We will collect cells from people to see if we can find the same results in people affected by HD that we find in cells grown in the laboratory. We will also mutate specific sequences in cells and study how they affect the appearance of mutations around the repeat tract.
The results we will obtain will improve our understanding of how sequences near repeats are generated and whether they correlate with disease symptoms.
Technical Summary
Huntington's disease is caused by the expansion of a CAG/CTG repeat at the Huntingtin locus. It is part of a family of at least 15 different human diseases that remain without a curative treatment. The size of the expansion accounts for about 60% of the variability in phenotype severity, the remainder of which is partly determined by sequences flanking the repeat tract. For instance, most individuals have a CAACAG motif downstream of the repeat tract. Individuals lacking this motif tend to develop the disease earlier in life compared to those with a canonical allele with the same repeat size. Despite their role in disease severity and development, how these sequences change over time in the germline or in somatic tissues is unknown.
We have recently developed a bioinformatics pipeline to identify structural rearrangements using targeted long-read sequencing from expanded repeat loci. We found that rearrangements occurred in blood and post-mortem samples from Huntington's disease as well as in mice and in cellular systems. In this proposal, we aim to address how rearrangements occur using patient-samples, genetic disruption in cells, as well as a biochemically defined system. Specifically we will ask:
1. What rearrangements occur in HD patient-derived germlines, blood, and brain tissues?
2. What are the genetic factors involved in rearrangements at CAG/CTG repeats?
3. How does sequence context affect repeat-induced mutagenesis at CAG/CTG repeats?
We have recently developed a bioinformatics pipeline to identify structural rearrangements using targeted long-read sequencing from expanded repeat loci. We found that rearrangements occurred in blood and post-mortem samples from Huntington's disease as well as in mice and in cellular systems. In this proposal, we aim to address how rearrangements occur using patient-samples, genetic disruption in cells, as well as a biochemically defined system. Specifically we will ask:
1. What rearrangements occur in HD patient-derived germlines, blood, and brain tissues?
2. What are the genetic factors involved in rearrangements at CAG/CTG repeats?
3. How does sequence context affect repeat-induced mutagenesis at CAG/CTG repeats?