An unexpected molecular pattern, referred to as BREACHes, has been discovered by researchers studying fragile X syndrome (FXS), a genetic disorder that affects approximately 1 in 7,000 males and 1 in 11,000 females, according to the Centers for Disease Control and Prevention. The researchers, from the Perelman School of Medicine at the University of Pennsylvania, used cells and brain tissue donated by FXS patients to identify disrupted genes and investigate the underlying cause of the disorder. The study, published in the journal Cell, also found that editing the length of the abnormal repetitive pattern could restore the silenced genes on multiple chromosomes.
The research team, led by senior author Jennifer Phillips-Cremins, Ph.D., an associate professor in Bioengineering and Genetics, focused on FXS, the most common form of inherited intellectual disability, to gain a better understanding of the disorder. The prevailing model of FXS attributes it to the silencing of a single gene, FMR1, and the loss of the protein FMR1 encodes, Fragile X Messenger Ribonucleoprotein (FMRP). However, the team found that the model was incomplete as it did not account for the critical genetic driver of FXS: a mutation called a repeat expansion.
In FXS, the repeat expansion occurs in a three-letter sequence—CGG—at one end of the FMR1 gene. While a normal version of FMR1 has 40 or fewer CGG triplets in the repeat tract, an FXS patient will have 200 triplets or more. This abnormality leads to the cell silencing FMR1 and FMRP as a defense mechanism. Small animal models of FXS that lack the repeat tract have not been able to demonstrate important aspects of the role of repetitive DNA in mechanisms underlying FXS.
To uncover new patterns of genome disruption in FXS, the research team used advanced sequencing and imaging techniques along with human cell lines and brain tissue with the CGG repeat expansion. They discovered that large portions of multiple chromosomes in FXS patient samples, including the CGG repeat, exhibited silencing heterochromatin. These regions were referred to as BREACHes, which stands for Beacons of Repeat Expansion Anchoring Contacting Heterochromatin. These BREACHes formed clusters in the nucleus and silenced genes associated with neuron synaptic connections and genes tied to the integrity of connective tissue.
The researchers then tested whether the repeat expansion was directly linked to BREACHes by using CRISPR-Cas gene-editing technology to shorten the CGG expansion to a non-FXS-causing length. They found that cutting the CGG repeat to a shorter length reversed the silencing heterochromatin and caused multiple chromosomes to spatially disconnect from FMR1. The genes silenced by BREACHes were re-expressed in FXS cells with the shortened CGG repeat.
This finding suggests that repeat engineering alone may be a potential therapeutic approach for reversing genome-wide silencing of critical genes associated with FXS. Future treatments for FXS might consider targeting the silenced genes identified in the study, in addition to FMR1. Another strategy could involve reducing the excessively long CGG repeat expansion at a defined time in development to prevent or reverse the effects of silencing heterochromatin, although the positive effects of re-activating important genes would need to be carefully balanced with the protective role of heterochromatin against instability of the repetitive genome.
The researchers also noted that their findings could have implications for other disorders affected by repeat expansions, such as Huntington’s disease and amyotrophic lateral sclerosis (Lou Gehrig’s disease). These disorders, which belong to the same broader class as FXS, are thought to be driven by mutations of repetitive tracts in the DNA. Additionally, the presence of BREACHes in other cellular models of genome instability suggests that they may have a broader impact on gene silencing in diseases with genome instability, including certain cancers and other repeat expansion disorders.