Increased CNV frequency in LFS. (A) To establish a baseline CNV frequency (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 individuals have few CNVs (median = 3). Seventy-five percent of the healthy population 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 = 70), TP53 wild type (n = 20), and TP53 mutation carrier (n = 31) groups, respectively. Error bars represent SEM. (C) Bar graph of CNV frequency in controls (n = 70), TP53 wild-type individuals (n = 20), TP53 mutation carriers unaffected by cancer (n = 8), and TP53 mutation carriers affected by cancer (n = 23). Both the unaffected and affected groups had significantly increased CNV frequencies as compared with controls (P = 0.009 and P = 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.

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 chromosomal 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 members 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, respectively; dotted circles or squares, TP53 mutation carriers who have not yet developed cancer. Oblique lines indicate 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 region. 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 and is 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; SI Discussion). (C) A 574-kb duplication, overlapping the cancer-related gene ADAM12, inherited through three generations of an LFS family (SI Discussion). 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).

Progression of germline chromosomal alterations in paired tumor DNA. Shown are copy number alterations in blood DNA and in paired tumor DNA for two individuals (A and B). Germline CNVs are displayed plotted across all autosomal chromosomes. Immediately below is the copy number of the tumor, which was biopsied or resected from the same person. (A) A CNV region (deletion) at 6q16.1 is highlighted on chromosome 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 germline CNV was found in both the tumor biopsy and resection as shown below. (B) In this person, 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 between reference, blood, and tumor DNA are highly significant (P < 0.01). Error bars represent ± 2 SEM. qPCR shows that the relative copy number in tumor DNA is 0.1, meaning that >80% of the tumor specimen is homozygous for the deletion (zero copies). We can approximate that only 20% of remaining cells have the germline hemizygous deletion (one copy).

Proposed model for the progression of copy number variable DNA regions in the Li–Fraumeni cancer predisposition syndrome. Shown is a model of copy number 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. 1A). 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 nonhomologous 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.