Li–Fraumeni cancer predisposition syndrome
Adam Shlien*
†
, Uri Tabori*
‡§
, Christian R. Marshall*
¶
, Malgorzata Pienkowska*, Lars Feuk*
¶
, Ana Novokmet*
§
,
Sonia Nanda
§
, Harriet Druker
§
, Stephen W. Scherer*
¶
, and David Malkin*
†‡§
**
*Program in Genetics and Genome Biology, and Departments of
†
Medical Biophysics,
‡
Pediatrics, and
Molecular Genetics,
¶
The Centre for Applied
Genomics, and
§
Division of Hematology/Oncology, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada M5G 1X8
Edited by Joseph F. Fraumeni, Jr., National Institutes of Health, Bethesda, MD, and approved May 27, 2008 (received for review March 26, 2008)
DNA copy number variations (CNVs) are a significant and ubiqui-
tous source of inherited human genetic variation. However, the
importance of CNVs to cancer susceptibility and tumor progression
has not yet been explored. Li–Fraumeni syndrome (LFS) is an
autosomal dominantly inherited disorder characterized by a strik-
ingly increased risk of early-onset breast cancer, sarcomas, brain
tumors and other neoplasms in individuals harboring germline
TP53
mutations. Known genetic determinants of LFS do not fully
explain the variable clinical phenotype in affected family members.
As part of a wider study of CNVs and cancer, we conducted a
genome-wide profile of germline CNVs in LFS families. Here, by
examining DNA from a large healthy population and an LFS cohort
using high-density oligonucleotide arrays, we show that the num-
ber of CNVs per genome is well conserved in the healthy popula-
tion, but strikingly enriched in these cancer-prone individuals. We
found a highly significant increase in CNVs among carriers of
germline
TP53
mutations with a familial cancer history. Further-
more, we identified a remarkable number of genomic regions in
which known cancer-related genes coincide with CNVs, in both LFS
families and healthy individuals. Germline CNVs may provide a
foundation that enables the more dramatic chromosomal changes
characteristic of TP53-related tumors to be established. Our results
suggest that screening families predisposed to cancer for CNVs
may identify individuals with an abnormally high number of these
events.
per population is necessary for the characterization of rare disease-
associated regions, while knowledge of the baseline number of
CNVs per person will aid in identifying individuals with particularly
unstable genomes.
The importance of acquired chromosomal changes in tumori-
genesis has been established; for example, amplification of the
MYCN
oncogene and deletions of chromosome 1p are major
prognostic indicators in neuroblastoma (7). Higher resolution anal-
yses have recently provided clues into the etiology of lung adeno-
carcinoma and acute lymphoblastic leukemia (8, 9) and exciting new
data from genome-wide association studies have implicated specific
single nucleotide polymorphisms to susceptibility of many diseases,
including prostate and breast cancer (10–14). However, the role of
constitutional CNVs in cancer predisposition has not yet been
explored. We set out to study the frequency of CNVs per person in
apparently healthy individuals and in the LFS cancer-prone pop-
ulation. To our knowledge, this is the first reported genome-wide
study of CNVs and genetic susceptibility to cancer.
Results
A cohort of individuals including 500 of European descent and the
multiethnic 270 person HapMap collection has been previously
assembled and used for studies of copy number variation (3, 15). We
used this cohort to establish whether a baseline CNV frequency
existed in a healthy population. In our independent analysis, we
identified 3,884 CNVs in genomic DNA from these 770 reportedly
healthy individuals using Affymetrix GeneChip 250K Nsp microar-
rays. The European cohort was analyzed on blood-derived DNA
and the HapMap cohort on lymphoblastoid cell line derived DNA.
Samples were grouped by microarray facility and normalized
against members of their group to reduce batch effects. CNVs were
then determined using dChip (16). To minimize false positives, we
only counted CNVs on autosomal chromosomes comprised of 2 or
more underlying single nucleotide polymorphism (SNP) probes.
Many CNVs were found in single individuals while others,
such as the CNV at chromosome 10q11.22 identified in 63
persons, were found in numerous individuals, demonstrating the
variability of the CNV population frequency. In contrast, the
frequency of CNVs per genome appears to be highly conserved:
the median number of CNVs detected per person was 3, with
75% of the population having 4 or fewer CNVs (Fig. 1
A
).
Moreover, CNV frequency appeared to be independent of
ethnicity, as a separate analysis of the Yorubans, Chinese,
Japanese and individuals of European descent revealed a similar
cancer genetics
p53
genomic instability
microarray
L
i–Fraumeni syndrome (LFS) is a clinically and genetically
heterogeneous familial cancer syndrome associated with a di-
verse spectrum of germline
TP53
mutations (1, 2). In contrast to
other familial cancer syndromes, LFS-affected families display a
wide array of tumors, including sarcomas of the bone and soft
tissue, carcinomas of the breast and adrenal cortex, brain tumors
and acute leukemias, among others. The spectrum of reported
germline
TP53
mutations is equally diverse and this has compli-
cated efforts to derive a clear genotype-phenotype model for the
syndrome. Indeed, even in the same LFS family, affected individ-
uals sharing an identical germline
TP53
mutation develop tumors
of varying severity, at different anatomical sites and at different ages
(1). This heterogeneity is thought to be due in part to additional
germline genetic variations present within and among LFS families.
With this in mind, we undertook a genome-wide characterization
study of the constitutional genetic variation of LFS family members.
A CNV is a segment of DNA 1 kb or larger that is present in
variable copy number in the genomes of humans, primates and
potentially many other species (3, 4). Despite efficient repair
machinery, CNVs still occur 100 to 10,000 times more frequently
than point mutations in the human genome (5). While the precise
mechanisms that give rise to most human CNVs are not known,
nonallelic homologous recombination (NAHR) and nonhomolo-
gous end joining (NHEJ) are thought to be involved (6). A
first-generation map of CNVs in the human genome was recently
completed, revealing 1,447 variable regions in 270 individuals from
the HapMap collection (3). Knowledge of the frequency of CNVs
Author contributions: A.S., U.T., M.P., S.W.S., and D.M. designed research; A.S. performed
research; A.S., A.N., S.N., and H.D. contributed new reagents/analytic tools; A.S., U.T.,
C.R.M., L.F., A.N., and D.M. analyzed data; and A.S., S.W.S., and D.M. wrote the paper.
Conflict of interest statement: A.S. and D.M. have submitted a patent application on a portion
of these findings.
This article is a PNAS Direct Submission.
**To whom correspondence should be addressed. E-mail: david.malkin@sickkids.ca.
This
article
contains
supporting
information
online
at
© 2008 by The National Academy of Sciences of the USA
11264 –11269
PNAS
August 12, 2008
vol. 105
no. 32
www.pnas.org
cgi
doi
10.1073
pnas.0802970105
result [
. Despite conserved
CNV frequencies, the varying size of these deletions and dupli-
cations could still result in individuals with different amounts of
copy number-variable DNA. To investigate this real possibility
we created a simple metric, termed total structural variation,
defined as the CNV frequency multiplied by the individual’s
average CNV size (in bp). The median total structural variation
showed a similar degree of conservation and was calculated to
be 395 kb, with 75% of the healthy population having 1.1 Mb or
less copy variable DNA (
. Therefore, this is the first
analysis establishing a baseline CNV frequency in the general
population.
Having established the distribution and frequency of CNVs in a
large reference population, we studied deviations from the global
norm in 11 well characterized cancer predisposed LFS families.
Inherited
TP53
mutations were observed in 9 families and
de novo
TP53
mutations in the other two (
. Forty-five family
members were evaluated. Eight additional unrelated
TP53
muta-
tion carriers were included for whom DNA samples were unavail-
able from other family members (
. Of these 53 individuals,
33 were
TP53
mutation carriers and 20 harbored wild-type
TP53
.In
addition, 70 unrelated healthy controls were evaluated for CNVs.
Both Affymetrix GeneChip 250K Nsp and Sty microarrays were
used for all analyses, and validation was performed using two
additional CNV detecting algorithms and quantitative PCR
(qPCR) (
.
Similar to the large reference population, our controls displayed
a median of 2 CNVs per genome, with 75% of the population
having 4 or fewer CNVs (mean
2.93). Additionally, we saw no
significant difference in CNV frequency between controls and the
TP53
wild-type group (median
2, 75th percentile
3, mean
3.4). In contrast, the
TP53
mutation carriers displayed a significant
increase in CNVs (
P
0.01). This cancer-prone group displayed a
mean of 12.19 CNVs per genome with 75% having 10 or fewer
CNVs (median
3; Fig. 1
B
). Of the 33 carriers, 17 exhibited more
alterations than the baseline (
3). Remarkably, every LFS family
with an inherited
TP53
mutation, except one, contained individuals
with CNV counts above the global norm of 3. The majority of
specific CNVs in LFS family trios were acquired and not found in
either parent (on average twice as common than inherited CNVs)
and, among families with a history of cancer, offspring were
significantly more likely to have an increase in CNVs when com-
pared with their mutation carrier parent (
P
A
B
C
0.015 by Fisher’s
exact test, observed/expected ratios: 2.0 for carriers and 0.0 for their
wild-type siblings).
Eight of the 11 families studied had histories of cancer. The only
families that did not have high CNV frequencies were those that did
not have a family history of cancer (3 of 11 families). Of these, two
had a single affected proband with a
de novo TP53
mutation
(Tyr163Cys and His193Pro). The other family had a single affected
child who harbored an extremely rare paternally inherited
TP53
mutation (Phe134Tyr).
Many of the
TP53
mutation carriers also had higher total
structural variation scores than
TP53
wild-type individuals, which is
as one would expect given their numerous CNVs. Less anticipated
were individuals found to have few CNVs but high total structural
variation scores, as a consequence of exceptionally large deletions
or duplications. The most dramatic example found was a paternally
inherited 6.1-Mb deletion encompassing 13% of chromosome 21
(21q21.1-q21.2) in an LFS family (shown below the pedigree of
family 1 in Fig. 2 as a contiguous faintly-colored vertical bar). The
deletion was confirmed by qPCR of DNA derived from blood or
normal paraffin-embedded tissue in the absence of available blood
(
P
Fig. 1.
Increased CNV frequency in LFS. (
A
) To establish a baseline CNV fre-
quency (CNVs per genome), genomic DNA from a large healthy population (
n
770) was assessed for CNVs by using the Affymetrix Nsp SNP microarray. The
distribution of CNV frequencies in the normal population is shown. Most indi-
viduals have few CNVs (median
3). Seventy-five percent of the healthy popu-
lation have four or fewer CNVs. (
B
) A significant increase in CNVs was observed in
TP53
mutation carriers as compared with controls (
P
0.01). The
TP53
wild-type
group displayed no significant increase in CNV frequency (
P
0.994). As shown,
the mean CNV frequencies are 2.93, 3.40, and 12.19 CNVs per genome in the
control (
n
31)
groups, respectively. Error bars represent SEM. (
C
) Bar graph of CNV frequency in
controls (
n
70),
TP53
wild type (
n
20), and
TP53
mutation carrier (
n
70),
TP53
wild-type individuals (
n
20),
TP53
mutation carriers
unaffected by cancer (
n
23). Both the unaffected and affected groups had significantly increased CNV
frequencies as compared with controls (
P
8), and
TP53
mutation carriers affected by cancer (
n
0.01 in all cases;
. Furthermore, we examined the SNP
genotypes in the same region and identified a 6-Mb stretch of
homozygosity, which is as expected since the individual has only one
allele at this locus. Despite the presence of a germline
TP53
mutation (codon R273S), the hallmarks of the syndrome (strong
0.046, respectively).
There is also an increase in CNVs in the affected group as compared with the
unaffected
TP53
mutation carriers, although not meeting statistical significance
because of the loss of power caused by subdividing the group into small cohorts.
Error bars represent SEM.
0.009 and
P
Shlien
et al.
PNAS
August 12, 2008
vol. 105
no. 32
11265
Fig. 2.
Inherited deletions and duplications in four LFS
families. Three examples of CNVs found in LFS families
are shown. The upper portion shows pedigrees for four
LFS families, and the lower portion shows the chromo-
somal size and relative microarray hybridization intensity
of each CNV and the family member in whom that CNV
was identified. It was not possible to evaluate all mem-
bers in every pedigree. However, in each of the four
families, an affected member, usually designated as the
proband (arrow), harbored both the displayed CNV and
a
TP53
mutation. In these pedigrees: open circles and
squares, healthy females and males; black circles and
squares, females or males affected with cancer, respec-
tively; dotted circles or squares,
TP53
mutation carriers
who have not yet developed cancer. Oblique lines indi-
cate that the person is deceased. Arrows point to the
proband in each family. The lower portion of the figure
highlights chromosomal regions of interest undergoing
copy number alteration in these four families. In copy
number analysis, faint coloring indicates deletions, while
duplications are colored more intensely. Vertical columns
represent a single individuals’ copy number for the re-
gion. Individuals of interest from the pedigree are numbered. (
A
) Paternally inherited 6.1-Mb deletion on chromosome 21q21.1-q21.2 (13% of the chromosome), the
largest deletion seen in the 893 genomes assessed in this study. Of the three children, two inherited both the deletion and
TP53
mutation (II-1 and II-2). Each developed
two neoplasms, first diagnosed at ages 6 and 7. The remaining child (II-3) harbored neither the deletion nor the mutation and is unaffected. The mother (I-2), carrying
only the
TP53
mutation, developed a single tumor (fibrous histiocytoma) at age 27. This exceptional deletion is inherited from an as-yet-unaffected individual andis
associated with a worsening of the clinical phenotype between generation I and II: the second generation displays the hallmarks of the syndrome (multiple cancers
in multiple offspring), whereas the first generation does not. From left to right: the copy number deletion (red), the SNP genotype calls (red, blue, and yellow squares),
and loss of heterozygosity (LOH) analysis (blue and yellow) in the same region. There is a concomitant region of homozygous genotype calls for individuals I-1 and II-1.
For the same individuals, SNP genotypes are colored red or blue if homozygous, yellow if heterozygous, or white if not called. An extended region of LOH is shown
in blue at the right. (
B
) An inherited 240-kb duplication at 6q27, overlapping the leukemia gene
MLLT4
, in four individuals from two LFS families: in B-I, the proband
transmitted the duplication of
MLLT4
to her son (V-1, as-yet unaffected carrier), and in B-II the proband inherited the same duplication of
MLLT4
from his mother (II-2,
affected and carrier), although this inheritance is presumptive because it could not be ascertained directly in the mother but was found in her sister (II-4, unaffected
and noncarrier). The frequency of this CNV is significantly enriched in LFS probands (
P
0.006, Fisher’s exact test;
. (
C
) A 574-kb duplication, overlapping
the cancer-related gene
ADAM12
, inherited through three generations of an LFS family (
. The proband’s brother (II-2, affected) harbored the paternally
inherited duplication at
ADAM12
(I-1, as-yet unaffected carrier), which he transmitted to his daughter (III-1, unaffected and noncarrier).
family history, multiple early onset tumors) are conspicuously
absent in the first generation. However, the second generation of
the family prominently displays these hallmarks. The full presen-
tation of the syndrome is therefore associated with an increase in
copy number variable DNA, although in this instance from an
apparently healthy individual (the father I-1). While it is possible
that the accelerated clinical phenotype in the affected mutant
TP53
carrier children (II-1 and II-2) and the presence of the 6.1 Mb
deletion inherited from the
TP53
wild-type father may be coinci-
dental, an alternative explanation is that the phenotype may have
resulted from the effect of an additional genetic modifier effect
conferred by the presence of this exceptionally large deletion. The
conf luence of these two genetic events, high total structural vari-
ation and a germline
TP53
mutation, thus correlates with the
increase in cancer incidence observed in the family.
Increased CNV frequency was found by comparing individuals at
elevated risk for cancer to those at normal risk (
TP53
mutation
carriers versus
TP53
wild type). We found no increase in cancer in
those individuals that are
TP53
wild type. Although nearly all
mutant
TP53
carriers will develop cancer in their lifetime (17), we
sought to determine whether CNV frequency may also explain the
clinical variability within the
TP53
mutant (at-risk) group. We
examined the CNV frequency of
TP53
mutation carriers affected by
cancer separately from the unaffected carriers. The unaffected and
affected groups each had significantly increased CNV frequencies
as compared with controls (
P
0.009 and 0.046, respectively). Of
particular interest is the presence of an even greater number of
CNVs present in those affected by cancer, when compared with
those who have not as yet developed cancer. Although not meeting
the threshold for statistical significance because of the loss of power
caused by splitting this group into small cohorts, this trend suggests
a dose-response relationship between CNV frequency and severity
of the LFS phenotype (Fig. 1
C
). Whether exposure to chemother-
apy inf luences accumulation of germline structural alterations is
not known. However, the fact that blood was drawn before starting
therapy in almost all of the patients in this study, and the obser-
vation of increased germline CNVs even in those mutant
TP53
carriers who are not yet affected with cancer, suggest that therapy
does not contribute to accumulation of germline DNA structural
variations (Fig. 1
C
).
We next examined the effect of germline CNVs on the devel-
opment of somatic chromosomal alterations in paired tumor tissue
(Fig. 3
A
). In a separate analysis, DNA was extracted from four
frozen tumor samples, taken from individuals whose constitutional
CNVs were known, and hybridized on the same platform. Choroid
plexus tumors were selected since they frequently occur in the
context of LFS. As expected, the tumor DNA contained many
structural changes, however, we focused only on those changes that
were previously found in normal tissue from the same person.
Three of four tumors had loci where germline hemizygous deletions
progressed into homozygous deletions in the tumor or where
germline duplications became larger in the tumor. Fifteen of 21 loci
overlapping germline CNVs became substantially larger (
50%) in
paired tumors and in all cases the new somatic alteration was of the
same orientation as the germline CNV (i.e., a deletion became a
larger deletion in the tumor and an amplification, a yet larger
amplification). Because the presence of gross tumor chromosome
changes could artificially inf late the observed number of such
events, we only selected regions undergoing discrete changes lo-
calized to the underlying CNV. This phenomenon was also vali-
dated by comparing SNP genotype homozygosity between blood
and tumor at these loci (
. One such CNV, a loss at
22q11.23, underwent an additional somatic deletion while the rest
of the chromosome maintained diploidy (Fig. 3
B
). Paired blood
tumor analysis also revealed a deletion in the tumor sample,
indicating that the deletion is located at the same locus and is
11266
www.pnas.org
cgi
doi
10.1073
pnas.0802970105
Shlien
et al.
Fig. 3.
Progression of germline chromo-
somal alterations in paired tumor DNA.
Shown are copy number alterations in blood
DNA and in paired tumor DNA for two indi-
viduals (
A
and
B
). Germline CNVs are dis-
played plotted across all autosomal chromo-
somes. Immediately below is the copy number
of the tumor, which was biopsied or resected
from the same person. (
A
) A CNV region (de-
letion) at 6q16.1 is highlighted on chromo-
some 6 and enlarged at the right. An arrow
points to the patient of interest, and their
deletion is indicated by a fainter color. The
neighboring column represents the patient’s
sister, who also has this CNV. Although the
patient’s tumor genome displays a high level
of instability, a deletion identical to the germ-
line CNV was found in both the tumor biopsy
and resection as shown below. (
B
) In this per-
son, a 70-kb hemizygous deletion in blood
DNA at 22q11.23 is highlighted and displayed
enlarged at the right. The same CNV was
found to be further deleted in the patient’s
paired tumor. That is, the remaining allele
was lost, as indicated by the yet fainter color,
which represents a reduction in the array’s
signal intensity in the same region. (
C
) The
copy number of genomic DNA at 22q11.23
was confirmed by qPCR to be both specific to
the underlying CNV and complete. From left:
a diploid control, patient blood DNA with a
hemizygous deletion, and tumor DNA from
the same patient showing a further deletion.
The difference in mean copy number be-
tween reference, blood, and tumor DNA are
highly significant (
P
0.01). Error bars repre-
sent
2 SEM. qPCR shows that the relative
copy number in tumor DNA is 0.1, meaning
that
80% of the tumor specimen is homozy-
gous for the deletion (zero copies). We can
approximate that only 20% of remaining cells
have the germline hemizygous deletion (one
copy).
deleted beyond that observed in the patient’s blood. qPCR con-
firmed a one copy loss in the germline as compared with a diploid
reference, and at the same locus, a one copy loss in tumor DNA as
compared with the germline (Fig. 3
C
). It therefore appears that
germline CNVs can act as a basis for more dramatic tumor-specific
changes.
DNA rearrangements, such as CNVs, can predispose to or cause
disease when they encompass, overlap or disrupt dosage-sensitive
genes (6, 18). CNVs can also unmask recessive mutations at
dosage-insensitive loci (18). We sought to determine which cancer-
related genes fall within copy number variable regions. In both the
large reference population as well as the LFS cohort, we observed
copy number variability in cancer-related genes. We observed
inherited duplications at
MLLT4
and
ADAM12
in LFS families
(Fig. 2
B
and
C
, and
.
MLLT4
is a target of
Ras
and
is fused with
MLL
in the common leukemia translocation t(6,
11)(q27;q23) and the frequency of this CNV is significantly en-
riched in LFS probands (
P
tionately large impact on our understanding of cancer biology in
general (1, 19–21). The primary reason for this is that defects of
TP53
, the most frequent genetic alteration in LFS, are the most
commonly acquired genetic alteration in sporadic human cancer.
Non-transformed fibroblasts and lymphocytes from
TP53
mutation
carriers display aberrant growth characteristics when passaged in
culture, spontaneously acquire properties of a transformed and
ultimately immortalized ‘tumor cell’, and ultimately display mass
chromosomal aneuploidy (22). While defective TP53 function is
known to cause increased copy number variation and instability in
tumors (23–25), our observations in primary non-cultured lympho-
cytes of
TP53
mutation carriers suggest a new model of carcino-
genesis wherein the existence of excessive submicroscopic copy
number alterations represent early germline events that may inform
the progressive changes required for neoplastic transformation
(Fig. 4). These subtle changes are likely the earliest manifestations
of instability conferred by the constitutional
TP
53 mutation, which
then progress in complexity into events that can be seen by
conventional cytogenetic techniques. TP53, as guardian of the
genome, actively suppresses cell cycle advance and DNA replication
after dsDNA damage, and is involved in the very processes known
to give rise to CNVs, including suppression of homologous recom-
bination (26). While the ubiquity and non-random distribution of
CNVs in humans highlights genomic regions that are intrinsically
unstable, their increased abundance in LFS
TP53
mutation carriers
can be explained by germline
TP53
haploinsufficiency. Genomic
0.006, Fisher’s exact test; see
.
ADAM12
is disintegrin-metalloproteinase, whose dys-
regulation has been reported in brain, breast, liver, stomach, and
colon cancer. The contribution of these CNVs to tumor predispo-
sition, initiation or progression will require further investigation (
and
.
Discussion
LFS is an ideal model for the discovery of genetic modifiers of
cancer and research on these rare families has had a dispropor-
Shlien
et al.
PNAS
August 12, 2008
vol. 105
no. 32
11267
Fig. 4.
Proposed model for the progression of copy
number variable DNA regions in the Li–Fraumeni cancer
predisposition syndrome. Shown is a model of copy num-
ber variable DNA regions in patients with sporadic (top
row) or inherited cancer (bottom row). (
A
) The total
number of CNVs in the genomes of healthy individuals is
similar. Non-cancer predisposed individuals have intact
DNA repair mechanisms that maintain the number of
CNVs close to this baseline (Fig. 1
A
). Despite efficient
repair machinery, CNVs still occur 100 –10,000 times more
frequently than point mutations in the human genome
(5). This is largely facilitated by the genomic sequence
architecture. The precise mechanisms that give rise to
most human CNVs are not known; however, nonallelic
homologous recombination (NAHR) and nonhomolo-
gous end joining (NHEJ) are thought to be involved (6).
Both NHEJ and NAHR are processes by which double-
strand (ds) DNA breaks are repaired. The ubiquity and
nonrandom distribution of CNVs in humans highlights
genomic regions that are intrinsically unstable. (
B
) CNVs
are more abundant in Li–Fraumeni cancer predisposed
TP53
mutation carriers because of germline
TP53
haploinsufficiency. TP53, as the ‘‘guardian of the genome’’ (26), suppresses cell cycle advance and DNA replication
after dsDNA damage. Furthermore, TP53 is involved in the very processes known to give rise to CNVs, including suppressing the level of homologous recombination.
Although defective TP53 is known to cause increased copy number variation and instability in tumors (23–25), our data suggests a new model wherein these alterations
arise much earlier in cancer-prone individuals. We have observed this increase of CNVs in primary LFS lymphocyte DNA, but this effect may be more dramatic in other
cells undergoing rapid remodeling, replicative stress, or in the normal tissue of patients with other cancer predisposition disorders. (
C
) CNVs become fertile ground for
changes in cancer. Genomic instability may be preferentially directed toward CNV regions that are hotspots for recombination as suggested by our observation (Fig.
3) that CNVs can act as the genetic foundation on which larger somatic chromosomal deletions and duplications develop in tumors (shown here as arrows from CNVs
in blood to those in tumor DNA). Tumor changes may be secondary to an underlying nontumor CNV or arise
de novo
at the same locus. It is likely that the sequence
architecture of genomic regions that gives rise to CNVs also facilitates large somatic alterations. In this model, CNVs are seen as crucial regions in both sporadic and
inherited tumors. Furthermore, the early age of onset of inherited tumors might be explained by the patient’s increased CNV frequency. CNVs should therefore be
viewed as important contributors to the inborn and acquired genetic changes that give rise to cancer. CNVs are shown as
(one copy loss),
(two copy loss), or
(one copy gain). Inherited CNVs are represented in black, acquired CNVs are in red, and tumor-specific CNVs
are in blue.
instability is a feature of all cancers (27) but it may be preferentially
directed toward CNV regions that are hotspots for recombination.
As we have observed, this can be accomplished in the tumor
genome by expansion of the linear extent of the same allele or by
loss of the opposite allele (Fig. 3
B
and
C
). Our observation that
CNVs can act as the genetic foundation on which larger somatic
chromosomal deletions and duplications develop in tumors, suggest
that CNVs are fertile ground for subsequently acquired changes in
cancer cells. Thus, the sequence architecture of genomic regions
that give rise to CNVs may facilitate both acquired constitutional
as well as tumor-specific genetic changes.
In this study, mutation carriers found to not have high CNV
frequencies were also those who did not have family histories of
cancer, either because their cancers arose as a consequence of
de
novo TP53
mutations or a low-penetrant mutation. It therefore
appears that CNV frequency, or another high-resolution measure
of instability, may help to define the nature and severity of the
germline
TP53
mutations found in LFS families. It will be important
in the future to determine the exact patterns of Mendelian inher-
itance of CNVs in a larger cohort of complete LFS families. It
appears that the reason that LFS offspring have a greater CNV
frequency is because not only do they inherit CNVs from their
parents, but they also acquire
de novo
CNVs. Therefore, the total
number of CNVs can be greater than in either parent. Because
mutation carriers affected with cancer had more CNVs than
unaffected carriers (while both groups harbored more CNVs than
individuals carrying wild-type
TP53
), it is tempting to speculate that
CNV frequency might also help to categorize
TP53
mutation
carriers into ‘‘risk groups’’ and provide a more rational basis for
screening and genetic counseling.
With respect to the role of CNVs and sporadic cancer, while
similar genome-wide analyses are now routinely performed on
tumor samples (8, 9), the frequent lack of matched constitutional
DNAmeans that the germline contribution to the detected somatic
alteration cannot be known. Our observation of a surprising
number of genomic regions where cancer-related genes coincide
with CNVs suggesting that germline CNVs can provide the foun-
dation for somatic chromosomal changes, in both LFS families and
healthy individuals, highlights the need for matched analyses in
cancer studies and for the establishment of a baseline for structural
variation in healthy human genomes.
Our data demonstrate that the CNV frequency is remarkably
similar among healthy individuals, but significantly increased in
individuals with germline
TP53
mutations. In addition, LFS family
members can contain exceptionally large deletions or duplications,
as identified by their total structural variation scores. This consti-
tutional structural dynamism may act as the genetic foundation on
which larger somatic chromosomal deletions and duplications build,
leading to the development of cancer. These findings also establish
a method for identifying individuals with constitutional chromo-
somal instability and inherent susceptibility to cancer.
Materials and Methods
Subject Recruitment.
After obtaining written informed consent, DNA was ex-
tracted from peripheral blood leukocytes of 53 individuals from families with a
germline
TP53
mutation and from 70 unrelated controls. These included 20
TP53
wild type and 33
TP53
mutation carriers. Of these, one individual had been
diagnosed as a
TP53
mosaic and was grouped with the
TP53
mutation carriers in
the CNV analysis. In addition, genomic DNA from five frozen choroid plexus
tumors was extracted. DNA was quantified by using a NanoDrop spectropho-
tometer, and quality was assessed by agarose gel electrophoresis. This study was
approved by the Research Ethics Board at the Hospital for Sick Children in
Toronto. Subject recruitment for the 500 individuals of European descent and the
270 individuals from the HapMap collection are described elsewhere (28, 29).
DNA Microarray Analysis.
Genomic DNA was genotyped with Affymetrix Gene-
Chip Human Mapping 250K arrays (
(30). Samples were restriction enzyme
digested, amplified, purified, labeled, fragmented, and hybridized according to
the manufacturer’s protocol. For the reference samples (
n
770), DNA copy
number analysis was performed with dChip (16) by using Affymetrix Nsp CEL files.
The LFS case-control cohort (
n
123) was assessed with dChip, CNAG (31), and
GEMCA (32) by using Affymetrix Nsp and Sty CEL files. The average call rate in the
11268
www.pnas.org
cgi
doi
10.1073
pnas.0802970105
Shlien
et al.