NEW YORK (GenomeWeb News) – The chromosomes of individuals with genomic disorders such as developmental delay can harbor dramatic chromosomal rearrangements similar to those seen in cancer genomes that have undergone chromothripsis, researchers reported today in Cell.
An international research team described the genomic strategies it used to characterize complex genomic rearrangements in clinical samples from 17 individuals with developmental delays or related conditions. Based on the patterns detected in the rearrangements, which involve at least two breakpoint junctions each, the researchers were able to track down the DNA replication mechanisms underlying these catastrophic rearrangements.
The findings suggest that mechanisms producing these complex rearrangements are the same regardless of whether they occur in a cancer cell, a germ cell, or during early development, corresponding author James Lupski, vice chair of molecular and human genetics at the Baylor College of Medicine, told GenomeWeb Daily News.
"The mechanisms are all the same," he said. "These are mechanisms of genomic change."
Since around 2004, Lupski and his colleagues have been using high-resolution arrays to assess clinical samples from patients referred for various developmental problems or birth defects. In the process, he explained, they have unearthed some rearrangements, including DNA insertions from nearby sequences in the genome, which were far more complicated than they appeared by lower resolution copy number analyses.
"We started to see some massively complex rearrangements," Lupski said. "When they occurred and we checked both parents, neither parent had this complexity."
"These were occurring as de novo events," he added, "and they seemed to be cataclysmic change in the structure of the genome, usually localized to one region of the genome or one chromosome."
When a Sanger Institute-led team published a sequencing-based study in Cell earlier this year showing that a subset of cancer harbor large rearrangements caused by a single catastrophic event, Lupski and his co-workers suspected that these complex somatic rearrangements might share mechanistic roots with the complicated genomic rearrangements that they were finding in patient samples.
"[G]enomic studies of rearrangements associated with cancer and genomic disorders reveal unanticipated complexities," the study authors wrote. "Elucidating the mechanism underlying such apparent 'one-off' events is essential to understanding mutational processes."
To look at this in more detail, researchers did copy number and breakpoint analyses on samples from 10 males and seven females with complex genomic rearrangements and developmental delay or cognition problems who had been referred by Baylor College of Medicine's Medical Genetics Laboratories.
The researchers did comparative genomic hybridization and SNP analyses with several Agilent, NimbleGen, and Illumina arrays, as well as fluorescence in situ hybridization, to look at these rearrangements at increasingly refined resolutions.
Once they had narrowed in on breakpoint regions using the arrays, they also used PCR amplification and Sanger sequencing to sequence these breakpoints.
The rearrangements, while extremely complex, were not quite as complicated as those found in some cancer samples with chromothripsis, Lupski explained, possibly due to selection against the most calamitous rearrangements during development.
"At an organismal level, the rearrangements acquired in cancers differ from the ones in genomic disorders in the time they arise during the life cycle," he and his co-authors explained. "Genomic disorders frequently result from 'constitutional' germline rearrangements that occur during gametogenesis or early post-zygotic development, whereas rearrangements acquired in cancer involve 'somatic' differentiated cells."
All of the rearrangements seem to have sprung up spontaneously, Lupski explained, noting that "the parents had none of these changes." SNP array and other data suggested that these de novo rearrangements had occurred either within germline cells or early in post-zygotic development.
In a few cases, the team did find parents who were mosaic carriers for cells with complex rearrangement, but these changes could only be detected by PCR once the breakpoints had already been mapped in their affected children, since they were present in a small subset of the parents' cells.
If such mosaicism exists in germline cells, Lupski noted, it might be important for clinical management and determining the risk of recurrence in families affected by complex genomic rearrangements. For example, he explained, quickly mapping breakpoints and using a PCR assay to look for mosaicism in blood or other tissue samples could theoretically offer clues about whether either parent is mosaic for complex rearrangements in their germ cells.
Overall, the breakpoint and insertion patterns that they found in the affected individuals, including a high incidence of microhomology regions, resembled those that occur during fork stalling and template switching — a phenomenon the team first identified in 2007 — and microhomology-mediated break-induced replication events, a related mechanism that was defined more recently.
The genomic changes introduced by this replication-related mechanism may impact not only human health, by producing clinically relevant rearrangements, the researchers reasoned, but could also influence species evolution over time.
"The mechanism can happen in the germ cell, it can happen during embryological development, it can happen in a cancer," Lupski said. "But these replicative mechanisms are potentially going to turn out to be extremely important for disease and for evolution of the human genome."