Heterozygous Germ Line hCHK2 Mutations in Li-Fraumeni Syndrome

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Science  24 Dec 1999:
Vol. 286, Issue 5449, pp. 2528-2531
DOI: 10.1126/science.286.5449.2528


The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in theTP53 gene. These observations suggest that hCHK2is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.

Li-Fraumeni syndrome (LFS) is a rare familial multicancer syndrome characterized by the occurrence of sarcomas, breast cancer, brain tumors, leukemia, and adrenal cortical tumors in multiple relatives (1). In most cases, LFS results from inheritance of a mutantTP53 allele, followed by somatic loss of the remaining wild-type allele, which thus constitutes the primary initiating event leading to cancer (2, 3). The central role ofTP53 mutation during cancer progression is also demonstrated by its inactivation in ∼50% of all sporadic human cancers (4). p53 is thought to mediate a cell cycle checkpoint, whose activation following ionizing radiation (IR) is dependent on the ataxia telangiectasia (ATM) gene product and leads to either G1 arrest or apoptosis (5).

In addition to this G1 checkpoint, which prevents the replication of damaged DNA, chromosomal integrity before mitosis is monitored by the G2 checkpoint, a pathway that has been best studied in yeast (6). InSchizosaccharomyces pombe, IR triggers activation of the Chk1 and Cds1 kinases, which is dependent on the ATM homolog Rad3 (7). Mutation of CHK1 is sufficient to prevent activation of this checkpoint in response to DNA damage, but disruption of the CHK1 and CDS1 genes is required to abrogate the response to hydroxyurea (HU), a ribonucleotide reductase inhibitor that triggers a Rad3-independent DNA replication checkpoint (8). Activation of Chk1 and Cds1 leads to the inhibitory phosphorylation of the phosphatase Cdc25, excluding it from the nucleus and preventing it from activating Cdc2, hence blocking entry of the cell into mitosis (9).

Mammalian homologs of the yeast G2 checkpoint genes show sequence conservation and, in some cases, partial functional rescue of a null phenotype in yeast. In human cells, hCHK1 phosphorylates Cdc25C at Ser216, leading to its binding and cytoplasmic sequestration by 14-3-3 proteins (10). The p53 target gene product 14-3-3σ also sequesters Cdc2–cyclin B1, suggesting a role for p53 in the G2 checkpoint (11).hCHK2 encodes a kinase with ∼30% amino acid identity to both the S. pombe Cds1 and its Saccharomyces cerevisiae homolog, Rad53 (12). Like hCHK1, hCHK2 is phosphorylated in an ATM-dependent manner in response to DNA damage and can phosphorylate Cdc25C on Ser216. Activation of the DNA replication checkpoint by treatment of cells with HU leads to the ATM-independent phosphorylation of hCHK2 (12).

To determine whether disruption of the G2 checkpoint is associated with human cancer, we first screened for heterozygous germ line mutations in hCHK1 or hCHK2 in four cases of classical LFS [see (13) for clinical criteria] that lacked mutations in TP53. The respective cDNAs, derived from either Epstein-Barr virus (EBV)–immortalized lymphoblasts or primary cultured fibroblasts, were amplified in overlapping fragments by reverse transcriptase– polymerase chain reaction (RT-PCR), and uncloned products were screened for mismatches by denaturing high-performance liquid chromatography (DHPLC) (14), followed by nucleotide sequence analysis (15). Mutations were confirmed by analysis of genomic DNA. A heterozygous germ line mutation in hCHK2 was detected in kindred MA81, cosegregating with the cancer predisposition phenotype (Fig. 1). In this family, remarkable for the incidence of rhabdomyosarcoma, brain tumors, and multiple cases of early-onset and bilateral breast cancer, a single nucleotide deletion was present within the kinase domain [deletion of C at nucleotide 1100 (1100delC)], resulting in premature termination. The mutation was present in three affected family members but it was absent from an unaffected sibling. The 1100delC mutation was not detected in 50 control individuals (100 alleles).

Figure 1

Germ line truncating mutation inhCHK2 in a classical LFS family with wild-type p53. (A) Pedigree of kindred MA81 with classical LFS. Solid circles (females) or squares (males) denote affected individuals. Slashed symbols indicate deceased individuals. EBV-immortalized lymphoblasts from one affected family member, peripheral blood mononuclear cells from two other affected family members, and one unaffected individual were available for analysis (denoted by arrowheads). Type of cancer and age of diagnosis are noted. The case of colon cancer is presumably sporadic. (B) Deletion of C at nucleotide 1100, resulting in premature truncation within the kinase domain of hCHK2. Sequence analysis of uncloned PCR products demonstrates the single nucleotide deletion (short arrows), which results in unsynchronized sequence, shown in the plus strand and minus strand (converted to sense sequence). This heterozygous germ line mutation was present in all three affected family members but was absent from the unaffected family member and from 100 control alleles. Red, T; green, A; blue, C; and black, G.

We extended our germ line mutational analysis to individuals with LFS-variant. These probands belong to families with multiple cancers that do not meet the clinical criteria for classical LFS (13). Although ∼70% of classical LFS kindreds are due to germ line TP53 mutations, these are detected in only ∼20% of LFS-variant (3), suggesting that these more moderate phenotypes are linked to other genes. Germ line specimens were available from 18 cases of LFS-variant in which TP53mutations were excluded. A single nucleotide deletion within the kinase domain of hCHK2 (deletion of T at nucleotide 1422) leading to a frameshift was detected in LFS-variant DF593 (Table 1). The proband had multiple colonic polyps, colorectal cancer, and bilateral ocular melanomas and had a family history of sarcomas and breast, colorectal, gastric, and lung cancers. A second mutation, an Ile → Thr missense mutation at codon 157 of hCHK2 was present in LFS-variant MGH005, in which the proband had developed three primary tumors: early-onset breast cancer, melanoma, and lung cancer (Fig. 2). This nonconservative substitution is within the forkhead homology-associated (FHA) domain of hCHK2, a highly conserved protein-interaction domain of 60 amino acids that is essential for activation of the yeast homolog Rad53 in response to DNA damage (16). The mutation was not detected in 100 alleles from 50 control individuals of comparable ethnic background, indicating that it is not a polymorphic variant that is prevalent in the population. The germ line Ile157 → Thr157 mutation in hCHK2 is therefore likely to be responsible for this case of LFS-variant.

Figure 2

Missense mutations in the hCHK2 FHA domain. (A) Pedigree of LFS-variant family MGH005. The proband, with three primary cancers, was a cigarette smoker; hence, the development of lung cancer may reflect this exposure as well as the germ line hCHK2 mutation. Labeling is as in Fig. 1A. (B) Heterozygous T → C missense mutation at nucleotide 470, resulting in an Ile → Thr substitution within the critical FHA domain of hCHK2, detected in EBV-immortalized lymphoblasts from proband MGH005. (C) Heterozygous C → T missense mutation at nucleotide 433, leading to an Arg → Trp substitution within the FHA domain, detected in the colon cancer cell line HCT15. Short arrows in (B) and (C) indicate the single nucleotide deletion.

Table 1

hCHK1 and hCHK2 sequence variations in LFS kindreds and in cancer cell lines; nt, nucleotide.

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To identify common sequence variations in hCHK2 within the population, we analyzed the entire coding sequence in 28 EBV-immortalized lymphoblasts from healthy controls. A silent polymorphism, Ala252 → Gly252, was detected in 4 of 28 individuals, but no amino acid substitutions in thehCHK2 coding sequence were observed in control specimens (Table 1). Given the similar function of hCHK1 and hCHK2, we also analyzed our cohort of LFS and LFS-variants for mutations inhCHK1. No germ line mutations in hCHK1 were detected in individuals with these familial cancer syndromes. Two silent polymorphisms in hCHK1 were identified, each present in 1 of 23 individuals (Table 1).

To screen for somatic inactivation of hCHK2, we analyzed 49 cancer cell lines, derived from a wide variety of sporadic human cancers (Table 1). We detected a mutation in only one: The colon cancer cell line HCT15 contained a heterozygous Arg → Trp missense mutation at codon 145, within the FHA domain (Fig. 2). This mutation may contribute to tumorigenesis either as a result of reduced gene dosage or through a dominant negative effect, as has recently been proposed for heterozygous mutations in the spindle-assembly checkpoint kinase gene hBUB1 (17). The observation thathCHK2 mutations are rare in cell lines derived from sporadic tumors is unexpected given the striking phenotype associated with its inactivation in the germ line. Conceivably, the high frequency of G1 checkpoint disruption in sporadic tumors, either by mutation of TP53 itself or by destabilization of p53 protein (18), may preclude the need for simultaneous disruption of the G2 checkpoint though hCHK2 mutation. In this context, it is of interest that the HCT15 colon cancer cell line contains one wild-type allele of TP53 and has homozygous inactivation of MSH6, resulting in a defect in the repair of single point mutations (19). Analysis of thehCHK1 transcript in our panel of cancer cell lines did not reveal any sequence variations, other than the silent polymorphisms noted above. Alterations in a polyadenosine stretch within thehCHK1 coding region have been reported in a few colon cancers with mismatch repair defects (20), but we did not detect such mutations in sporadic cancer cell lines.

Our observation that LFS and LFS-variant kindreds with wild-type p53 harbor mutations in the G2 checkpoint kinasehCHK2 suggests that germ line mutations in these two genes have similar consequences. Sarcomas and breast and brain tumors, the most common cancers seen in LFS associated with mutant p53 (1), were also prevalent in carriers of germ linehCHK2 mutations. However, LFS is a rare syndrome, and only a few cases with wild-type p53 were available for analysis. Gene-specific differences in tumor spectrum may emerge from the study of additional families. Similarly, more extensive studies will be required to determine whether the FHA domain of hCHK2 is a mutational hotspot and whether truncating and missense mutations in this gene are associated with distinct phenotypes.

The comparable clinical features of TP53 andhCHK2 germ line inactivation have implications for our understanding of the critical functional pathways for these two gene products. It is possible that disruption of either the G1or G2 checkpoints may be distinct but functionally equivalent in triggering genomic instability. Alternatively, the shared phenotype of TP53 and hCHK2 inactivation may underscore the contribution of p53 itself to maintenance of the G2 checkpoint (11, 21). Finally, there may be a direct functional interaction between hCHK2 and p53. Activation of p53 and hCHK2 after DNA damage is ATM-dependent, raising the possibility that hCHK2 may be an intermediate kinase in the phosphorylation of p53 [reviewed in (22)].

The identification of hCHK2 as a human tumor suppressor gene is noteworthy in that the gene was isolated and its presumed function was determined by virtue of its homology to yeast genes known to function in a critical DNA damage response pathway (12). A comparable degree of evolutionary conservation has been observed for the mismatch repair genes, inactivated in hereditary nonpolyposis colon cancer (23). The presence of hCHK2 mutations in a human cancer syndrome highlights the importance of cell cycle checkpoints in tumorigenesis. However, inactivation of the G2 checkpoint may also have therapeutic implications (6), because it may render cancer cells particularly sensitive to DNA damaging agents, triggering a mitotic catastrophe (24) as they attempt segregation of damaged chromosomes.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: Haber{at}


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