Fragile X chromosome, illustration

Researchers at the Perelman School of Medicine at the University of Pennsylvania have discovered new disrupted genes and an unexpected molecular pattern in Fragile X Syndrome (FXS), a genetic disorder that causes the most common form of intellectual disability. The researchers showed that editing the length of the unexpected molecular pattern could restore the silenced genes on multiple chromosomes.

“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,” said Linda Zhou, MD, PhD, a clinical resident of dermatology at Penn Medicine and co-first author of the study published this week in Cell.

Prior models of FXS have associated the genetic condition with the silencing of a single gene, FMR1 and the loss of the protein it encodes FMRP (Fragile X Messenger Ribonucleoprotein). The common concept of FXS is that the loss of FMRP causes severe synaptic dysregulation of synapses and disruption of how genes are expressed on the neurons’ nuclei. The leading model of FXS was created from studies using transgenic mice with the FMR1 gene knocked out. But this model did not include the repeat expansion—the repetitive sequence of two or more DNA letters that grows unstable and unusually long.

In the case of FXS the repeat expansion is a repetition of the three-letter sequence CGG that occurs at the end of the FMR1 gene. When FMR1 is normal the CGG repeat expansion comprises 40 or fewer repetitions. In a person with FXS there are more than 200 repeats of the CGG triplet. This abnormal pattern triggers a defensive response in the cell, which silences FMR1 and FMRP.

Engineering this repeat expansion in difficult so small animal models of FXS do not have these repeats with the result being these models not exhibiting important aspects of the role of repetitive DNA in mechanisms underlying FXS.

In this new study, led by senior author Jennifer Phillips-Cremins, PhD, an associate professor in bioengineering and genetics, and a member of the Epigenetics Institute at Penn Medicine, the investigators used advanced sequencing and imaging to analyze brain tissue and cell lines with the CGG repeat expansion and discovered new patterns of genome disruption. The researchers discovered that large swaths of multiple chromosomes in FXS patient samples—which include the CGG repeat—are marked with the silencing heterochromatin. These heterochromatin “domains” are coined BREACHes (Beacons of Repeat Expansion Anchoring Contacting Heterochromatin).

BREACHes contact clusters in the nucleus and silence genes involved in neuron synaptic connections, along with genes tied to the integrity of connective tissue such as skin and joints—both of which have been observed clinically in patients with FXS. Having the ability to identify BREACHes could potentially allow researchers to discover important disrupted genes other than FMR1.

In another phase of the study, the Penn team tested whether the repeat could be directly linked to BREACHes by using CRISPR-Cas gene-editing technology to cut the CGG expansion down to a non-FXS-causing length.

“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 co-lead-authors Ken Chandradoss, PhD, and Ravi Boya, PhD, post-doctoral researchers in Phillips-Cremins’ lab.

The team’s experiments demonstrated that genes originally silenced by BREACHes were re-expressed in FXS cells with the CRISPR-shortened CGG repeat. These findings indicate that mutation-length CGG expansion is necessary for the maintenance of BREACHes and that repeat engineering could be employed as a therapeutic approach to reverse the genome-wide silencing of not just FMR1, but multiple genes that may contribute to FXS.

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