Researchers uncover new disrupted genes linked to fragile X

Gene editing length of abnormal DNA repeats could help restore silenced genes

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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Excessive DNA repeats in the FMR1 gene — the cause of fragile X syndrome — lead to structural changes in other parts of the DNA that disrupt the activity of genes necessary for nerve cell function, according to a study using samples from fragile X patients.

This damaging effect was found to be dependent on the number of repeats on the FRM1 gene, with a reduction in DNA repeats reversing many of the changes in other genes.

“Our findings have implications for future Fragile X Syndrome treatment strategies and highlight potential mechanisms contributing to genome instability that may underlie other diseases as well,” Linda Zhou, MD, PhD, co-first author of the study at the University of Pennsylvania (Penn), said in a university press release.

The study, “Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome,” was published in Cell.

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FMR1 gene mutation leads to severely reduced production of FMRP protein

The FMR1 gene, located in the sex-determining X chromosome, provides instructions to make FMRP, a protein that regulates the production of proteins that support nerve cell communication.

This gene normally contains up to 40 repeats of three nucleotides, or the building blocks of DNA: one cytosine and two guanines, denoted CGG. The abnormal presence of 61 to 199 CGG repeats is classified as a premutation. More than 200 is called a full mutation and leads to fragile X.

This high number of repeats results in certain chemical changes that trigger heterochromatinization. Heterochromatin refers to a tightly packed form of DNA that limits accessibility to genes, thereby silencing their activity.

In fragile X, packing FMR1 in this way leads to severely reduced production of the FMRP protein.

The classic understanding of fragile X has been that severe FMRP deficiency is ultimately what drives abnormalities in brain development that give rise to disease symptoms. However, this has mainly been based off of animal models where the FMR1 gene is entirely deleted, as this is a much easier approach than engineering animals to have a longer CGG repeat.

In this study, scientists led by Jennifer Phillips-Cremins, PhD, an associate professor and member of the Epigenetics Institute at Penn Medicine, performed detailed genetic analyses of brain samples and cells derived from people with fragile X.

In line with earlier findings, they found the FMR1 gene containing the CGG repeat expansion was packed into heterochromatin, effectively turning off the gene. Unexpectedly, however, FMR1 wasn’t the only gene silenced in this manner.

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Nearby genes on X chromosome also affected

Results indicated, in addition to FMR1, a nearby stretch of genes on the X chromosome (containing about 5,000 nucleotides in total) was also packed into heterochromatin.

The team found 10 additional genomic locations on non-sex chromosomes, called autosomes, that were packed in a similar manner. Within the nucleus (the cellular compartment that houses DNA), the heterochromatin-packed parts of autosomes were in close physical proximity to the heterochromatin sections of the X chromosome.

The scientists dubbed these regions BREACHes, short for Beacons of Repeat Expansion Anchored by Contacting Heterochromatin.

While heterochromatin sections on autosomes weren’t always consistent from sample to sample in the experiments, they often contained genes involved in guiding nerve cell development and maintaining healthy connections between nerve cells.

As such, it’s plausible that disruption of these genes, in addition to FMR1 itself, may contribute to driving fragile X.

Notably, the researchers found that when they engineered fragile X cells to have fewer CGG repeats, some of the genes silenced by BREACHes were re-activated.

“When we cut CGG to a shorter length called premutation (100-190 triplets), we observed that many of the large swaths of silencing heterochromatin were reversed, and multiple chromosomes spatially disconnected from FMR1,” said Ken Chandradoss, PhD, and Ravi Boya, PhD, the study’s co-lead-authors and post-doctoral researchers in Phillips-Cremins’ lab.

While more work will be needed to validate these data and better understand the biochemical mechanisms at play, these findings “raise a working model for future testing in which BREACHes might be a generalized phenomenon linked to multiple pathways underlying genome instability beyond FXS [fragile X syndrome],” the team wrote.

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‘Repeat engineering alone can potentially be used as a therapeutic approach’

“The broad impact of our finding that the mutation-length CGG expansion is necessary for the maintenance of BREACHes is that repeat engineering alone can potentially be used as a therapeutic approach to reverse genome-wide silencing of multiple critical genes potentially contributing to FXS clinical presentations,” said Thomas Malachowski, a study co-author and PhD student at Penn.

The researchers also speculated that similar mechanisms might be at play in other disorders caused by repeat expansion mutations such as Huntington’s disease.

“Our results suggest that BREACHes may be found in the future to have broader impact on gene silencing in other diseases with genome instability, including certain cancers and other repeat expansion disorders,” Phillips-Cremins said.