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Abigail Loszko
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The Phenomenon of Chromothripsis
Complex Chromosomal Rearrangements (CCRs) are structural rearrangements of three or more
chromosomes or involving more than 2 breakpoints. They are typically thought to occur during
spermatogenesis and may be transmitted in families through oogenesis (Patsalis 2007). However, CCRs
have been recently gaining momentum in the scientific research community and it is being shown that
they are more complex and more common than originally thought. Many molecular mechanisms have
been proposed to explain CCRs, but the prevailing idea regarding such complex chromosomal
rearrangements is that these rearrangements gradually accumulate from errors in DNA reparation or
from a defect in DNA recombination. However, a new phenomenon, called chromothripsis, has been
postulated as a cause of complex chromosomal arrangements. The term “chromothripsis,” coined in
2011 by Stephens et al., comes from the Greek words “chromos” for chromosome, and “thripsis” for
shattering into pieces. In this proposed phenomenon, tens to hundreds of genomic rearrangements are
acquired in a single, catastrophic event where a chromosome is essentially cut up into pieces and pasted
back together with some pieces thrown out (Stephens et al. 2011).
Stephens et al. (2013) proposed the idea of chromothripsis after looking at cases of Chronic
Lymphocytic Leukemia (CLL). One of the patients exhibited forty-two somatically acquired genomic
rearrangements involving the long arm of chromosome 4 which were characterized by six striking
features. The first feature that stuck out to Stephens et al. (2011) was that the rearrangements showed
geographic localization in the genome. Aside from a separate deletion on the small arm of chromosome
13, all of the rearrangements were limited to chromosome 4q (q stands for the long arm of the
chromosome) and specific focal points on chromosomes 1, 12, and 15. This is noteworthy because usual
patterns of genomic instability in breast, lung, or pancreatic cancer tend to be scattered across the
genome or are associated with extensive genomic amplification if they are localized (Stephens et al.
2011).
Another prominent feature was that many regions of the chromosomes showed copy number
changes alternating between two states. When an analysis of allelic ratios at germline single nucleotide
polymorphism (SNP) positions on chromosome 4q was performed, the results revealed that regions of
copy 1 lost heterozygosity, whereas regions of copy 2 retained it. Third, regions of single copy did not
result from single deletions but were the byproduct of complex rearrangements spanning the region.
Fourth, there was pronounced clustering of breakpoints across the chromosome 4q arm, with one
instance of 7 rearrangements occurring in just a 20 kb region. Fifth, despite the clustering of DNA
breaks, the conjoined fragments did not reside in close proximity in the original chromosomes. Lastly,
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breakpoints that involved more than one chromosome also showed clustering. There were nine
rearrangements linking chromosome 4q to other chromosomes and these breakpoints were clustered
even on the partner chromosomes (Stephens et al. 2011).
The unusual genomic landscape of this patient with CLL sparked the curiosity of Stephen et al.
(2011) to see if other cancer cell germ lines had similar features that suggested similar complex
rearrangements originating from a single, devastating incidence. This led them to analyze highresolution copy number profiles of 746 cancer cell lines obtained using SNP arrays. Of these, 96 had at
least one chromosome with more than 50 positions at which copy number changes, and 18 out of the
746 cell lines displayed the “hallmarks” of chromothripsis seen in the original CLL patient. Upon an
additional analysis of segmented SNP array, this time with data from 2792 cancers, the same proportion
of cases showed evidence for chromothripsis. Thus about 2 to 3% of cancers showed signs of
chromothripsis. Screening of rearrangements in tumor samples from 20 patients with bone cancer also
suggested that chromothripsis is even more common in cases of bone cancer (Stephens et al. 2011).
One reason that Stephens et al. (2011) suggested that complex chromosomal rearrangements
occur in a single catastrophic event as opposed to the conventional idea of resulting from a progression
of events was that the number of copy states was low. The number of different states observed would
be expected to increase as the number of breakpoints rises if sequential, independent arrangements
had occurred as seen in Figure 1 below. Another problem with a model of progressive rearrangements
is the retention of heterozygosity in regions with higher copy number. Generally speaking, once
heterozygosity is lost, it cannot be regained, and under progressive rearrangement, a deletion that
occurred early in the sequence of rearrangements would permanently remove heterozygosity between
breakpoints (Stephens et al. 2011).
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Figure 1: This model shows (a) an example of how progressive rearrangements would disrupt a model chromosome
compared to (b) a chromothripsis event. Notice the average copy number and rearrangement plot associated with
each. (Figure 5 Stephens et al. 2011)
The fact that the breakpoints show significantly more clustering along chromosomes or
chromosome arms than expected by chance also points to a single catastrophic event. With the
progression model, this type of clustering would suggest broadly varied locations of fragility among the
chromosome. Some specific regions of increased tendency to rearrange have been acknowledged
before, but never as extensively as what was observed in these studies.
A more recent study by Rausch et al. on genome sequencing of pediatric medulloblastoma
published in 2012 links chromothripsis with TP53 mutations (Rausch et al. 2012). They performed whole
genome sequencing of Sonic-Hedgehog subtype of medulloblastoma (SHH-MB) from a female patient
with Li-Fraumeni syndrome (LFS). LFS increases susceptibility to cancer and is characterized by a TP53
mutation. When Rausch et al. (2012) performed whole genome sequencing of this patient’s DNA, the
“hallmarks” of a chromothripsis event were evident. This result prompted the researchers to analyze
the germline DNA of more SHH-MB patients showing signs of chromothripsis, and 5 out of the 6 had
germline TP53 mutations, suggesting that germline mutations of TP53 in SHH-MB patients occurred
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prior to the chromothripsis event in each patient. Therefore, TP53 mutations may be either an initiating
factor or a response to chromothripsis (Maher & Wilson 2012).
One of the most striking observations found in the study by Rausch et al. (2012) was a pattern of
complex somatic chromosomal rearrangements unlike any other previously described in
medulloblastoma. They noted highly amplified genomic segments, clustered on individual chromosome
arms and resulting in frequent alternations between two copy states. Three segments were amplified
on chromosome 3, six segments were amplified on chromosome 4, and four segments were amplified
on chromosome 14. By determining physical connections linking the amplified sequences, they were
surprised to find that the amplified fragments from chromosomes 4 and 14 were fused together to form
a 1.2 Mb extra-chromosomal structure (also known as a “double-minute” structure) consisting of a
complex mix of inter- and intrachromosomal junctions. The formation of the double-minute structure
as well as the other trademarks of chromothripsis made the explanation of progressive genomic
alterations very unlikely (Rausch et al. 2012).
Although there is much evidence to support the idea of a single event which causes the
shattering and unorderly reassembly of chromosomes during chromothripsis, the causative agent of this
phenomenon is still unknown. Two theories of causative agents were proposed by Stephen et al. (2011)
but neither was proven in their research. One of the agents proposed to cause a chromothripsis event is
a pulse of ionizing radiation. A pulse of this type of energy has the potential to damage everything in its
way, and may just damage a single chromosome or just part of a chromosome depending on the size of
the pulse and the direction at which the path of the radiation hits a cell’s nucleus. This theory could be
tested by in vitro cells surviving radiation and by analysis of genomes from cancer patients previously
exposed to radiation (Stephens et al. 2011).
Another proposed causative agent of chromothripsis is that the breakage-fusion-bridge cycle
associated with telomere erosion could induce the damage. As the telomere (cap at the end of
chromosome) gets worn away, this increases the probability of chromosome end-to-end fusions and
could ultimately cause intense breakage during the stretching and pinching of the chromosome bridge
during the end of cytokinesis. In order to test this theory, models could be analyzed in cancer genomes
from genetically engineered mice (Stephens et al. 2011).
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Rausch et al. added a third model suggesting that premature chromosome compaction could be
a causative agent due to the link between TP53 mutations and chromothripsis (Figure 2). In this model,
chromosomes that condense before all DNA is replicated may shatter (Rausch et al. 2012).
Figure 2: This model links TP53 Mutation status to catastrophic DNA rearrangements (Figure 6. Rausch et al. 2012).
Although the causative agent of this chromothripsis is unclear, the distinctive signature of the
process does give some clues as to when it may happen. Stephens et al. (2011) suggest that
chromothripsis seems likely to happen during mitosis while chromosomes are condensed for mitosis
because of the intense clustering of breaks that results. According the Stephens et al. (2011), it is hard
to imagine such clustering and specific localization during interphase while chromosomes are relaxed
with long loops of DNA winding throughout the nucleus. However, despite the claims of Stephens et al.
that interphase would be an unlikely time for chromothripsis to occur, Rausch et al. (2012) do not rule it
out. They note that the known spatial organization of chromosomes maintained during interphase may
also provide conditions for the local occurrence of DNA shattering (Rausch et al. 2012)
Chromothripsis has also been presumed to bring about complex rearrangements that don’t lead
to cancer. In a case study of a boy exhibiting 16q22 deletion syndrome, a close look at his chromosomes
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suggested a CCR possibly resulting from a chromothripsis event. Through G-banding, Genesio et al
(2013), discovered a pericentric (around the centromere) inversion of one chromosome 12 from band
p12 to q13, an interstitial deletion from 12q14 to 12q21, and additional material on chromosome 16
within q21q22. Dual color FISH analysis using green and red probes showed that the additional material
from the small arm on of chromosome 16 came from chromosome 12 and confirmed the pericentric
inversion on one copy of chromosome 12. High resolution multicolor banding (MCB) was also performed
to compare the normal chromosome 12 to the rearranged chromosome 12. Figure 3 below shows the
pericentric inversion extending from the band 12p12 to the band 12p14 and an inverted insertion of the
12q14q21 region on chromosome 16. Lastly, Oligo-array CGH analysis was also performed and
identified the 16q21q22.1 deletion where the 12q14q21.1 tract was inserted (Genesio et al. 2013).
Figure 3: High resolution multicolor banding (MCB) compares the normal chromosome 12 to the chromosome 12
with a pericentric inversion. Also note the additional material on chromosome 16 from Chromosome 12 (Figure 4.
Genesio 2013).
Through these tests, Genesio et al. (2013) concluded that the case, characterized by 5
breakpoints, one inversion, 2 deletions and the inverted insertion of one of the deleted tracts within a
non-homologous chromosome, had undergone a complex chromosomal rearrangement due to
chromothripsis. Figure 4 below hypothesizes a schematic representation of the mechanism of
chromothripsis, but confirmation of this hypothesis can only come with further analysis of breakpoint
sequencing (Genesio et al. 2013).
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Figure 4:The double strand breaks of DNA (black triangles) are formed as a result of "chromosome shattering" with
the subsequent formation of chromosomal fragments that are haphazardly pasted back together through
chromosome reassembly. This results in inversions (“inv,” blue arrows), deletions (the black arrow) and inserted
translocation (“IT,” green dashed arrow) that create the two derivative chromosomes 12 and 16. The new
chromosome 12 exhibits a pericentric inversion and deletion of 12q14q21.1 and chromosome 16 with the insertion
of the inverted 12q14q21.1 region in place of the deleted 16q21-22.1 region (Figure 7 Genesio et al. 2013).
Despite the complex chromosomal rearrangement, the proband in the study by Genesio et al.
(2013) only exhibited typical signs of a single syndrome. Thus, it is important to note, that if the complex
chromosomal rearrangement was indeed due to a chromothripsis event, this example suggests that
chromothripsis events may promote a wide variety of diseases. So far, major studies of chromothripsis
have focused on cancer genomes, but even in the case of classical phenotypes, very complex
rearrangements cannot be excluded (Genesio et al. 2013).
Although many modern studies are already contributing several complex chromosomal
rearrangements to chromothripsis, such as the study by Genesio et al. (2013), not everyone agrees that
chromothripsis is legitimate. Shattered and Stitched Chromosomes—Chromothripsis and
Chromoanasynthesis—Manifestations of a New Chromosome Crisis? by Christiaan Righolt and Sabine
Mai discussed the problem with declaring that chromothripsis is due to a single event. Righolt and Mai
pointed out that such an event was not documented in the original study by Stephens et al. (2011) nor
has there been any experimental confirmation published since then. To prove the theory that CCRs can
happen in one single event, repeated instances of chromothripsis must occur in animal models or
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patients. Furthermore, they argued that if such complex genomic rearrangements were to happen in
one cell cycle, then the typical dynamic progression of tumors would not be seen and there would be no
genetic distinction between tumor stages (Righolt & Mai 2012).
Righolt and Mai (2012) presented other retorts to the study by Stephens et al. (2011) but they
kept returning to the fact that a “complete shattering and stitching together of individual chromosomes
as a result of DNA damage” has not yet been observed nor have “hot spots” for radiation-induced
double-strand breaks been identified. They discredit the methods of SNP array analysis and whole
genome sequencing that were used by Stephens et al., claiming these “approaches of a heterogeneous
population of tumor cells do not explain what happens in a single cell.” Alternative terms such as
punctuated rearrangements or localized complex rearrangements may more accurately reflect genetic
alteration in tumors than “chromothripsis.” “A term that postulates a time frame during which genomic
changes occur needs to prove that this time frame actually occurs before its general use can be
recommended” (Righolt & Mai 2012).
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Abigail Loszko
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Literature Cited
Genesio, Rita, et al. "Pure 16q21q22.1 Deletion in a Complex Rearrangement Possibly Caused by a
Chromothripsis Event." MOLECULAR CYTOGENETICS 6(2013): N. pag. Web.
http://apps.webofknowledge.com.pallas2.tcl.sc.edu/full_record.do?product=WOS&search_mod
e=GeneralSearch&qid=1&SID=2B7eXXDbc3mWCTzGyhG&page=1&doc=1
Maher, Christopher, and Richard Wilson. "Chromothripsis and Human Disease: Piecing Together the
Shattering Process." Cell. 148 (2012): 29-32. Web.
http://www.sciencedirect.com/science/article/pii/S0092867412000074
Patsalis PC. Complex chromosomal rearrangements. Genet Couns. 2007;18(1):57–69
http://www.ncbi.nlm.nih.gov/pubmed/17515301
Rausch, Tobias, David Jones, et al. "Genome Sequencing of Pediatric Medulloblastoma Links
Catastrophic DNA Rearrangements with TP53 Mutations." Cell. 148 (2012): 59-71. Web.
http://www.sciencedirect.com.pallas2.tcl.sc.edu/science/article/pii/S0092867411015169#
Righolt, Christiaan, and Sabine Mai. "Shattered and Stitched Chromosomes— Chromothripsis and
Chromoanasynthesis— Manifestations of a New Chromosome Crisis?." GENES, CHROMOSOMES
& CANCER. (2012): 975-981. Web.
http://onlinelibrary.wiley.com.pallas2.tcl.sc.edu/doi/10.1002/gcc.21981/pdf
Stephens, Philip, Chris Greenman, et al. "Massive Genomic Rearrangement Acquired in a Single
Catastrophic Event during Cancer Development." Cell. 144 (2011): 27-40. Web.
http://www.sciencedirect.com.pallas2.tcl.sc.edu/science/article/pii/S0092867410013772#