Springer 2005
Familial Cancer (2005) 4: 177–181 DOI: 10.1007/s10689-004-1946-5
The TP53 mutational spectrum and frequency of CHEK2*1100delC in Li–Fraumeni-like kindreds Rina Siddiqui1, Kenan Onel3, Flavia Facio1, Kedoudja Nafa1, Louis Robles Diaz2, Noah Kauff1, Helen Huang1, Mark Robson1, Nathan Ellis1 and Kenneth Offit1 1
Memorial Sloan Kettering Cancer Center, Clinical Genetics Service, Department of Medicine, New York, USA; Hospital Universitario Doce de Octubre, Madrid, Spain; 3Department of Pediatrics, University of Chicago, Chicago, Illinois, USA
2
Received 27 July 2004; accepted in revised form 27 July 2004
Key words: CHEK2*1100delC, germline mutations, Li–Fraumeni syndrome, polymorphism, TP53
Abstract Li–Fraumeni syndrome (LFS) is a dominantly inherited cancer predisposition syndrome characterized by a wide spectrum of neoplasms occurring at young age. Germline mutations in the TP53 tumor suppressor gene have been identified in approximately 71% of LFS patients and 22% of Li–Fraumeni-like (LFL) patients. Mutations within the cell cycle checkpoint gene CHEK2 have also been reported in some patients with LFS, LFL, and phenotypically suggestive of LFS (PS-LFS) not carrying a TP53 mutation. In this study, we show that 7 of the 23 patients with LFS/LFL tested positive for deleterious mutations in p53. Fifteen of the remaining sixteen were not found to carry the CHEK2* 1100delC mutation. These results indicate that CHEK2*1100delC is not a common cause of LFS, LFL, or PS-LFS in North American kindreds not carrying a TP53 mutation. Of note, two patients were found to carry p53* R72P, which is of unknown clinical significance. Lack of segregation of this allele in one of these kindreds provides strong evidence that the R72P allele is not disease-causing. While mutations in p53 account for a proportion of patients with LFS/LFL, future studies are needed to determine if other genes are responsible for LFS/ LFL families not carrying germline p53 mutations. Abbreviations: LFL – Li–Fraumeni-like; LFS – Li–Fraumeni syndrome; PS-LFS – phenotypically suggestive of Li–Fraumeni syndrome; SNP – single nucleotide polymorphism
Introduction Li–Fraumeni syndrome (LFS MIM# 151623) is an autosomal dominant familial cancer predisposition syndrome resulting from the inactivation of the TP53 tumor suppressor gene [1, 2]. It is characterized by an increased propensity for the development of a wide spectrum of neoplasms, including soft-tissue and bone sarcomas, brain tumors, adrenocortical tumors, breast cancers and leukemia [1, 3]. In affected individuals, these tumors typically occur at a young age, and multiple primary malignancies are common [1]. Clinically, LFS is diagnosed in an individual with a sarcoma diagnosed before 45 years of age, a first-degree
relative with any cancer under 45 years of age, and a first- or second-degree relative with either any cancer under 45 years of age or a sarcoma at any age [1]. Criteria for Li–Fraumeni-like (LFL) syndrome are similar, but do not encompass all of the features listed for LFS. Birch’s criteria include: a proband with any childhood cancer or a sarcoma, brain tumor, or adrenal cortical tumor diagnosed before 45 years of age, and a first- or second-degree relative with a typical LFS cancer (sarcoma, breast cancer, brain tumor, adrenal cortical tumor, or leukemia) at any age, and a first- or seconddegree relative with any cancer under the age of 60 years [4]. Eeles’ criteria include two first- or second- degree relatives with LFS-related malignancies at any age [5].
Correspondence to: Kenneth Offit, Clinical Genetics Service, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Tel: +1-212-434-5151; Fax: +1-212-434-5166; E-mail: offi
[email protected]
178 Molecularly, germline mutations in the TP53 tumor suppressor have been identified in approximately 71% of patients with LFS, 22% of LFL-Birch patients [6], and approximately 8% of LFL-Eeles patients [7]. Inactivating mutations of the cell cycle checkpoint gene, CHEK2, which encodes a kinase that phosphorylates and activates TP53 in response to DNA damage [8], have also been reported in some patients with LFS/LFL not carrying a TP53 mutation. Germline mutations in CHEK2 have been described in families with LFS/LFL [9, 10]. 1100delC is a frameshift mutation that introduces a premature stop codon in CHEK2, resulting in a truncated and nonfunctional protein [9, 10]. More recently, the 1100delC mutation was identified in 2 out of 44 (4.5%) unrelated Finnish families with LFS/LFL/phenotypically suggestive (PS) of LFS (3 LFS, 7 LFL, 34 PS-LFS), whereas it is present in only 0.5%–1.5% of unaffected controls [11]. The purpose of this study is to describe the spectrum of TP53 mutations in a cohort of 23 patients with LFS/ LFL, and to evaluate whether the CHEK2*1100delC mutation is disease-causing in patients with LFS/LFL, who were shown not to carry a TP53 mutation.
Materials and methods Patients Twenty-three probands from families meeting either LFS criteria or LFL criteria (as defined by Birch or Eeles) were ascertained for this study. All patients were seen at the Clinical Genetics Service at MSKCC in New York City from 1994 to 2003. One of 23 patients was self-referred, the remaining were referred by their physicians. All patients had their personal cancer history confirmed by pathology reports and medical records. Patients were of various ethnic backgrounds (Scottish/ Irish, n ¼ 1; Lebanon Christian, n ¼ 1; French Christian, n ¼ 1; Filipino, n ¼ 1; German Catholic, n ¼ 1; Greek Orthodox, n ¼ 2; Puerto-Rican, n ¼ 2; Cuban, n ¼ 1; Italian, n ¼ 1; Syrian Catholic, n ¼ 1; Eastern European Jewish, n ¼ 5; Irish, n ¼ 2; Scottish/Polish, n ¼ 1; and unknown ethnicity, n ¼ 3). Three of 23 patients (13%) met LFS criteria; 15/23 (65%) met Eeles criteria for LFL; 3/23 met Birch criteria for LFL; and 2 out of 23 (9%) did not meet any criteria but were PS of LFS. Of these 2 patients, one had metachronous bilateral breast cancer diagnosed at ages 31 and 34 and thyroid cancer diagnosed at age 35. The other was diagnosed with sarcoma at age 9 and had a family history of sarcoma and gastrointestinal cancers at ages less than 60 in distant relatives. Of the 23 patients in this study, 7 (30%) developed a second primary cancer, 6 (26%) had a third primary, and 2 (9%) eventually developed a fourth primary. Patient characteristics are described in Table 1. For samples tested for the CHEK2*1100delC mutation, genotyping was done on anonymized specimens
R. Siddiqui et al. Table 1. Characteristics of probands. Patients characteristics
N = 23
Median age M:F Malignancies Breast cancer Sarcoma Hematological malignancies Brain tumor Melanoma Others Patients with 2 primary malignancies Patients with 3 primary malignancies Patients with 4 primary malignancies
30 (2–63 yrs) 5:18 37 14 7 5 4 3 4 7 (30%) 6 (26%) 2 (9%)
according to a study protocol approved by institutional IRB and human tissue utilization committee. Samples tested for TP53 were performed in a clinical laboratory with genetic counseling and informed consent. Mutation analysis Germline genomic DNA was extracted from peripheral blood lymphocytes using Puregene kit (Gentra Systems). Samples were sent to outside laboratories to determine the TP53 status for all patients. These laboratories performed mutation analysis for TP53 by direct DNA sequencing. Exons 4–10 were sequenced in 19 patients; exons 5–9 in three patients; and exons 3–9 were sequenced in one patient. Of the patients wild-type for TP53, 15 out of 16 were tested for the CHEK2*1100delC mutation. Testing for the CHEK2*1100delC mutation was done by direct sequencing of exon 10 (224 bp) following PCR amplification. PCR was performed to generate a 1280 bp fragment encompassing 33 nucleotides from exons 9–10 of CHEK2 using forward primer 9Fe 50 -CTG TCATCTCAAGAAGAGGAC-30 (this forward oligonucleotide corresponds to a sequence within the nonduplicated exon 9 to ovoid amplification of the nonfunctional exon 10) and reverse primer 10R 50 -CAT TTGTGACTTCATC TAATCACCTCC-30 [12]. The PCR amplification was performed in a final volume of 50 ll containing 50 ng of DNA, 2.5 mM dNTPs, 15 pmol of each primer, and 1.25 U of DNA polymerase (AmpliTaq Gold, Applied Biosystem). The PCR amplification consisted of 10 min at 95 C followed by 35 cycles of 45 s at 94 C, 45 s at the annealing temperature, and 1.30 minutes at 72 C; a final extension of 7 min ended the reaction. PCR products were purified with the QIAquick PCR purification Kit (Qiagen) and quantified by agarose gel electrophoresis. The purified PRC products were sequenced using internal forward primer 10Fe: 50 -CTGATCTAGCCTACGTGTCTTC-30 and Big Dye Terminator chemistry, and then run on an ABI 3100 DNA sequencer (Applied Biosystem). Two probands were found to carry the R72P polymorphism of unknown clinical significance upon TP53 clinical testing. Segregation analysis was carried out in
179
Li–Fraumeni-like kindreds one kindred for this polymorphism by testing three other family members. After DNA extraction from the whole blood, exon 4 of TP53 was amplified to generate a 433bp fragment using forward primer TP53/E4F 50 -ACG TTCTGGTAAGGACAAGG-30 and reverse primer TP53/E4R 50 -GATACGGCCAGGCATTGAAG-30 . The PCR conditions were the same as described above. The purified PCR products were sequenced using the same PCR primers and Big Dye Terminator chemistry, and then run on an ABI 3100 DNA sequencer (Applied Biosystem).
Results Seven patients were identified with inactivating mutations of TP53 (Table 2). CHEK2*1100delC status was determined in 15 of the remaining 16 patients. None were found to carry the deleterious allele. TP53 germline mutations were identified in 2 out of 3 LFS patients, 4 out of 18 LFL patients, and 1 out of 2 PS-LFS patients (Table 2). Of the seven TP53 germline mutations identified, six were in exons 5–8, and one at the intron 9/exon 10 splice junction site (Table 3). Five exonic mutations were missense, four involving hotspot codons. Of note, although the mutation at codon 172 in proband 1 has Table 2. TP53 mutation status and cancer status of individuals as a function of clinical group. Patients
N (%)
Positive for TP53 mutation (%)
Negative for TP53 mutation (%)
TP53 variant R72P (%)
All patients LFSa LFL Birch LFL Eeles PS-LFSb
23 3 3 15 2
7 2 1 3 1
14 1 2 10 1
2 (9) 0 0 2 (13) 0
a b
(13) (13) (65) (9)
(30) (67) (33) (20) (50)
(61) (33) (67) (67) (50)
LFS – Li–Fraumeni syndrome. PS – phenotypically suggestive of LFS.
been reported as a somatic mutation in TP53 tumors, it has not previously been observed as a germline mutation in LFS[13]. Of the remaining two mutations, one was a frameshift mutation in exon 6 resulting in a premature stop at codon 222. The mutation in proband 7 was a single base substitution (G to C) that disrupts the intron 9/exon 10 splice junction. The result of this mutation is an alternatively spliced TP53 mRNA lacking the oligomerization domain of TP53 protein. The two patients carrying R72P were females of Puerto Rican descent. One was affected with breast cancer at age 29, and the other developed acute lymphocytic leukemia (ALL) at age 2. To test the hypothesis that the proline allele at codon 72 can be associated with LFS/LFL, the kindred of the latter patient (Figure 1) was chosen for biallelic genotyping at this site. The proband was found to be homozygous polymorphic for proline. One brother, who was also diagnosed with childhood ALL, was heterozygous for R72P, as were both parents, neither of whom is affected. The paternal family history was significant for cancer whereas the maternal was not.
Discussion The TP53 tumor suppressor is the most frequently mutated gene in human cancer [14]. Exons 4–8 in the TP53 gene, which encompass the highly conserved DNA binding domain [15], is the region in TP53 that contains the known mutational hotspots. Germline mutations in TP53 are frequently, but not always, observed in patients with LFS or LFL. The observation that TP53 is not mutated in a substantial number of patients with LFS/LFL or who are PS of LFS, however, suggests that germline mutations in other genes in the TP53 pathway may also confer the same cancer phenotype. CHEK2 is a kinase that signals to TP53 and activates it by phosphorylation following DNA damage. Associ-
Table 3. TP53 mutations identified in LFS/LFL/PS. Proband Mutations
Total # of tumors in proband
Types of tumor
Age at first primary (gender)
Criteria met
Brain, breast Breast, kidney Sarcoma Bilateral breast, thyroid Bilateral breast Breast, sarcoma, leukemia Bilateral breast, melanoma
28 39 2 31
LFL-Birch LFL-Eeles LFS None
Type
Exon
Codon
Nucleotide change
Effect on protein
1 2 3 4
Missense Missense Missense Missense
5 8 5 5
172 273 175 175
G-T G-A G-A G-A
V172F R273H R175H R175H
2 2 2 3
5 6
Missense Frameshift
7 6
245 Deletion 218–222
G-A Stop codon
G245S
2 3
7
Single base substitution in splice site of TP53
Intron9/Exon10 junction
AG-AC
Alternate splicing in pre RNA
3
(F) (F) (M) (F)
24 (F) 34 (F)
LFL-Eeles LFS
27 (F)
LFL-Eeles
180
R. Siddiqui et al.
n 6 Brain
n 17 Leuk - NOS
Arg72Pro Arg72Pro 22 (heterozygote) (heterozygote) Leuk - ALL 1
7 Leuk - ALL 1
13 13 Leuk - ALL 8 Leuk - ALL 4
9 Leuk - ALL 2
4
Arg72Pro Arg72Pro (homozygote) (heterozygote)
Figure 1. Kindred with R72P polymorphism.
ations have also been reported between the CHEK2*1100delC mutation and familial cancer syndromes. CHEK2*1100delC is associated with an increased risk of breast cancer [16]. In one study of familial breast cancer patients who were not BRCA1 or BRCA2 mutation carriers, the frequency of the CHEK2*1100delC mutation was approximately 5% [17]. Further, it was estimated that in these families, the presence of the CHEK2*1100delC variant correlates with a 2-fold increased risk of breast cancer in females and a 10-fold increased risk in males [17]. In one New York based study, we estimated the frequency of the 1100delC to be 1.0% in cases with a family history of breast cancer or a personal history of breast cancer compared to a frequency of 0.3% in healthy controls [12]. CHEK2*1100delC has also been associated with hereditary prostate cancer [18]. Mutations in CHEK2 have also been described in families meeting LFS/LFL-Birch criteria [9,10]. In particular, CHEK2*1100delC has been identified in one classical LFS family [9], and in two Finnish families with a cancer phenotype suggestive, but not meeting, LFS/LFL criteria [11]. Taken together, these data suggest that the CHEK2*1100delC mutation may be the mechanism underlying the phenotype in a subset of LFS, LFL, PS-LFS families. To test this hypothesis, patients in our cohort who were found not to carry a TP53 deleterious mutation were tested for CHEK2*1100delC. Out of these 15 patients, none were found to carry the deleterious allele. Our results do not support a role of the CHEK2*1100delC mutation as a common cause of LFS, LFL, or PSLFS in North American kindreds not carrying a TP53 mutation.
Of interest, 2 out of 23 patients, both meeting LFLEeles criteria and testing negative for a deleterious TP53 mutation, were found to carry a variant in exon 4 of TP53, Arg72Pro. This polymorphism is located within the proline rich DNA binding domain of TP53. It might be predicted that the effect of substituting a ringed amino acid, proline, for arginine in this region would alter the tertiary structure of TP53, and therefore, its function. Indeed, in vitro, this SNP has been shown to markedly attenuate the efficiency of TP53-mediated apoptosis [19]. Whether this variant influences cancer risk in vivo, however, remains controversial. To test the hypothesis that this polymorphism can be associated with LFS/ LFL, one kindred was chosen for biallelic genotyping. The segregation of the allele in this kindred does not suggest that the R72P polymorphism is disease-causing. In this cohort of 23 patients, 2 out of 3 with LFS had mutations in TP53, and 6 out of 7 met criteria for LFS/ LFL. Hence, as has previously been shown, the loss of TP53 is associated with the classical LFS/LFL phenotype. Although no patient in this cohort carried the CHEK2*1100delC allele, it is possible that some patients had as yet unidentified germline mutations elsewhere in the CHEK2 gene. While mutations in TP53 account for a proportion of patients with LFS/LFL, future studies are needed to determine if other genes are responsible for LFS/LFL families not carrying a TP53 deleterious mutation.
Acknowledgements This study was supported by a gift from the Lymphoma Foundation and the Koodish Fellowship.
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