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In a class of approximately 30 neurological disorders including ALS, Huntington's disease and Fragile X syndrome, the relevant mutant gene has sections of repeated base pair sequences known as short tandem repeats or STR. Healthy people have normal length STRs spread across their DNA. For people with trinucleotide repeat expansion (TNR) diseases, however, the STRs in the mutant genes are unstable: the number of repetitions extends to very long lengths related to the pathology of the disease.
Researchers at the University of Pennsylvania have found another common thread for almost all diseases related to the expansion of TNRs: complex 3D models in which DNA is folded for s & # 39; integrate into the nucleus of the cell. They found that almost all STRs known to be unstable in a disease are located at the boundaries that separate neighboring folded domains.
The researchers also created high-resolution genome folding maps around the FMR1 gene in patients with Fragile X syndrome and in healthy individuals. They found that the 3D genome was misfolded in the disease; the limit was destroyed around FMR1 in all fragile X patients who also exhibited pathologic STR expansion and FMR1 extinction.
The results establish a strong correlation between misfolding of the 3D genome, repeated tandem instability and pathological genetic disruption of these deadly and debilitating diseases, suggesting new research questions whose answers could improve diagnosis or treatment .
The research was conducted by Jennifer E. Phillips-Cremins, Assistant Professor, Department of Biological Engineering, Penn Engineering, and the Department of Genetics, Perelman School of Medicine, with James Sun and Linda Zhou, members of her laboratory.
Their study was published in the journal Cell.
At the heart of the mystery behind TNR diseases lies the pathological extent of their repetitive sequences. The same exact repetitive patterns appear in hundreds of thousands of other locations along the linear genome, in genes and non-coding regions, but they are not known to become unstable.
"I wanted to work on this project because I was fascinated by the idea that folding the 3D genome – which I had barely heard before joining the Cremins lab – could be the missing piece to understand why some parts of the genome behave as they do, "says Zhou. "In this case, why can the repetitive DNA of certain genes become unstable while others do not?"
The mapping of 3D DNA folding patterns has only recently become possible, so that genes related to TNR diseases have been mainly studied via their linear sequences.
The researchers' discoveries were made possible by the Cremins Lab technique to produce genome folding maps. By fixing the DNA so that its 3D folding patterns are preserved before sequencing, two distant parts of the linear sequence will end up in the same hybrid DNA chain and will therefore be detected together when the sequence is closed. DNA will be sequenced. The statistical mapping of these associations provides a high definition image of the disparate parts of a linear sequence in physical contact – and thus can affect the gene expression of the other – when the genome is in its folded state .
Zoomed out, this map looks like a row of densely populated cities, connected by sections of highway. Each of these "cities" is a topologically related domain, or TAD, and contains sequences in physical contact with each other. Highways between cities are called limits. they contain linker sequences that physically prevent neighboring TADs from interacting with each other.
In trying to determine what makes unstable repeats associated with TNR diseases different from their stable counterparts, the researchers examined whether their location relative to the genome folding models had played a role.
"Each human individual has hundreds of thousands of short, tandemly repeated sequences in his genome, and the repeats have a large variation in sequence, location in the body of the gene, normal length ranges, and mutations. phenotypes and phenotypes that they produce ". Phillips-Cremins says. "But for the handful of short tandem repeat sequences known to be unstable in disease, almost all are specifically localized to the boundaries of genome folding."
The strength of this correlation immediately raised questions of causality for the researchers: do the boundaries provide a local environment where the genome is likely to repeat or does repetition determine the location of the genome? ;a limit?
"Recent studies have provided strong evidence that the 3D genome can be misfolded in specific cancers and limb development disorders," Phillips-Cremins explains. "As we began our studies to understand the cause-and-effect relationship between placement of boundaries and repetition of expansion, we also began to question whether we would observe topological changes in TNR diseases or whether limits remained intact. "
In collaboration with Beverly Davidson of the Children's Hospital of Philadelphia and Flora Tassone of UC Davis, Phillips-Cremins and his co-authors studied the brain tissue and B-cells donated by patients with Fragile X syndrome.
"We have discovered that in Fragile X Syndrome, the leading cause of intellectual disability in the United States, DNA is badly bent exactly the same place where genetic defects and the pathogenic gene occur. " "This discovery highlights many important questions about the relationship between genome folding, repeated expansion, and gene silencing.
The fact that the Fragile X gene limit is destroyed strongly suggests that this change is related to the gene silencing of the disease. Issues of causality will have to be addressed before researchers fully understand the role of folding. The recently developed CRISPR / Cas9 gene editing technology will allow Phillips-Cremins and his colleagues to conduct experiments exploring the potential causal role of the 3D genome in continuous expansion.
"We are extremely excited to examine whether and how the folding of the 3D genome in Fragile X syndrome is causally linked to the quenching of gene expression in this disease." In our ongoing studies, we want to determine if silencing genes disrupts the breaking of boundaries silences the gene, "says Phillips-Cremins." Mechanistic studies have focused primarily on linear DNA so far, but we can now add a third dimension to the understanding of the genetic basis of TNR diseases. "
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