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Study Finds Replication-Related Rearrangements Influence Copy Number

NEW YORK (GenomeWeb News) – Stalled replication and related mitotic processes can contribute to copy number variation in human cells, according to a paper appearing online last night in Nature Genetics.

Researchers from Texas, Missouri, and Massachusetts used high-density oligonucleotide array comparative genomic hybridization, or array CGH, to investigate whether a process called fork stalling and template switching, or FoSTeS, influences copy number variation in human cells. They found that FoSTeS, which is similar to the microhomology-mediated break-induced replication, or MMBIR, process in simpler organisms, can generate genomic rearrangements involving hundreds to millions of DNA bases.

"[W]e show that FoSTeS/MMBIR-mediated rearrangements can occur mitotically and can result in duplication or triplication of individual genes or even rearrangements of single exons," senior author James Lupski, a researcher affiliated with the Baylor College of Medicine and Texas Children's Hospital, and his co-authors wrote. "The FoSTeS/MMBIR mechanism can explain both the duplication-divergence hypothesis and exon shuffling, suggesting an important role in both genome and single-gene evolution."

Fork stalling and template switching refers to the situation in which DNA replication stalls and replication enzymes jump to another template to begin copying DNA elsewhere. In a paper published online in Cell last January, Lupski and his team identified the process, demonstrating that it is a factor in genomic rearrangements associated with a rare central nervous system condition called Pelizaeus-Merzbacher.

"The involved forks can be separated by sizeable linear distances but may be adjacent or in close proximity in three-dimensional space, perhaps within replication factories," the authors explained. "The mechanism enables the joining or template-driven juxtaposition of different sequences from discrete genomic positions and can result in complex rearrangements."

Based on these results, the researchers hypothesized that FoSTeS might explain other complex human genome rearrangements as well. To test this, they evaluated non-recurrent rearrangements associated with two conditions: a chromosome 17 micro-duplication condition called Potocki-Lupski syndrome and the neurological disorder called Charcot-Marie-Tooth disease type 1 that's been linked to copy number changes in the chromosome 17 gene PMP22.

The team did oligonucleotide array CGH with DNA samples from 14 individuals with Potocki-Lupski syndrome and seven individuals with Charcot-Marie-Tooth disease using Agilent high-density oligonucleotide-based microarrays. They also looked at parental DNA from six Potocki-Lupski syndrome cases, one Charcot-Marie-Tooth disease family member, and two healthy controls.

Among the 14 individuals with Potocki-Lupski syndrome, eight had duplications and deletions beyond those previously associated with the condition — a pattern that the researchers attributed to numerous FoSTeS/MMBIR-type events.

When they compared the patterns to rearrangement profiles in parental DNA from six of the Potocki-Lupski individuals, the team found evidence that these rearrangements had occurred de novo, likely during mitosis, consistent with a role for FoSTeS in the process.

Their subsequent breakpoint analyses uncovered a variety of rearrangements — from simple to complex — in the Potocki-Lupski cases tested.

Together, the researchers concluded that their results "show that complex genomic rearrangements are frequent, can be explained by the FoSTeS/MMBIR mechanism, and may require different levels of resolution to fully visualize the complexity."

The team then turned their attention from the chromosome 17 region associated with Potocki-Lupski to a specific chromosome 17 gene: the Charcot-Marie-Tooth disease-related gene PMP22.

When the researchers investigated DNA samples from individuals with Charcot-Marie-Tooth disease, they confirmed seven previously identified rearrangements in the gene. But their breakpoint analyses of these rearrangements suggested that FoSTeS led to exon shuffling within the gene.

When the team looked more closely at a family with non-recurrent PMP22 rearrangements, they found a situation in which two affected siblings had a shared deletion that was present in some, but not all, of their mother's cells.

"These observations indicate that this complex deletion rearrangement is mitotic, consistent with a replicative error during mitotic DNA synthesis, as proposed for the FoSTeS/MMBIR model," the authors noted.

And, they added, the finding raises the possibility that replication-related arrangements may offer insights into recurrence risk assessments in genetic counseling programs.

FoSTeS seems to influence copy number in other genes as well, the researchers noted. When they searched the Human Gene Mutation Database, the team found 17 genes that had breakpoint patterns consistent with some sort of replication-based genetic rearrangement.

Together, the results suggest that FoSTeS/MMBIR and other replication-related processes may have a previously unappreciated role in copy number variation across the genome, contributing to small and large rearrangements affecting everything from a lone exon to millions of DNA bases. That, in turn, suggests the process may affect the evolution of both genes and the genome.

"I think this is going to make people think very hard about copy number variation with respect to genome evolution, gene evolution, and exon shuffling," Lupski said in a statement.

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