Variants in CHEK2 Other than 1100delC Do Not Make a Major Contribution to Breast Cancer Susceptibility
2003; Elsevier BV; Volume: 72; Issue: 4 Linguagem: Inglês
10.1086/373965
ISSN1537-6605
AutoresMieke Schutte, Sheila Seal, Rita Barfoot, Hanne Meijers‐Heijboer, Marijke Wasielewski, D. Gareth Evans, Diana Eccles, Carel Meijers, Frans P. Lohman, Jan G.M. Klijn, Ans van den Ouweland, P. Andrew Futreal, Katherine L. Nathanson, Barbara L. Weber, Douglas F. Easton, Michael R. Stratton, Nazneen Rahman,
Tópico(s)CRISPR and Genetic Engineering
ResumoWe recently reported that a sequence variant in the cell-cycle–checkpoint kinase CHEK2 (CHEK2 1100delC) is a low-penetrance breast cancer–susceptibility allele in noncarriers of BRCA1 or BRCA2 mutations. To investigate whether other CHEK2 variants confer susceptibility to breast cancer, we screened the full CHEK2 coding sequence in BRCA1/2-negative breast cancer cases from 89 pedigrees with three or more cases of breast cancer. We identified one novel germline variant, R117G, in two separate families. To evaluate the possible association of R117G and two germline variants reported elsewhere, R145W and I157T with breast cancer, we screened 737 BRCA1/2-negative familial breast cancer cases from 605 families, 459 BRCA1/2-positive cases from 335 families, and 723 controls from the United Kingdom, the Netherlands, and North America. All three variants were rare in all groups, and none occurred at significantly elevated frequency in familial breast cancer cases compared with controls. These results indicate that 1100delC may be the only CHEK2 allele that makes an appreciable contribution to breast cancer susceptibility. We recently reported that a sequence variant in the cell-cycle–checkpoint kinase CHEK2 (CHEK2 1100delC) is a low-penetrance breast cancer–susceptibility allele in noncarriers of BRCA1 or BRCA2 mutations. To investigate whether other CHEK2 variants confer susceptibility to breast cancer, we screened the full CHEK2 coding sequence in BRCA1/2-negative breast cancer cases from 89 pedigrees with three or more cases of breast cancer. We identified one novel germline variant, R117G, in two separate families. To evaluate the possible association of R117G and two germline variants reported elsewhere, R145W and I157T with breast cancer, we screened 737 BRCA1/2-negative familial breast cancer cases from 605 families, 459 BRCA1/2-positive cases from 335 families, and 723 controls from the United Kingdom, the Netherlands, and North America. All three variants were rare in all groups, and none occurred at significantly elevated frequency in familial breast cancer cases compared with controls. These results indicate that 1100delC may be the only CHEK2 allele that makes an appreciable contribution to breast cancer susceptibility. DNA damage results in activation of cell-cycle checkpoints that block proliferation and initiate DNA repair processes. Defects of these checkpoint pathways can lead to genomic instability and susceptibility to cancer. Cell-cycle–checkpoint kinase 2 (CHEK2, also known as "CHK2" [MIM 604373]), is a key mediator of cellular responses to DNA damage (Zhou and Elledge Zhou and Elledge, 2000Zhou BB Elledge SJ The DNA damage response: putting checkpoints in perspective.Nature. 2000; 408: 349-433Crossref PubMed Scopus (102) Google Scholar; Bartek et al. Bartek et al., 2001Bartek J Falck J Lukas J CHK2 kinase: a busy messenger.Nat Rev Mol Cell Biol. 2001; 2: 877-886Crossref PubMed Scopus (309) Google Scholar). Following double-strand DNA breaks, CHEK2 is activated through phosphorylation by ATM (MIM 208900) (Matsuoka et al. Matsuoka et al., 1998Matsuoka S Huang M Elledge SJ Linkage of ATM to cell cycle regulation by the Chk2 protein kinase.Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1048) Google Scholar, Matsuoka et al., 2000Matsuoka S Rotman G Ogawa A Shiloh Y Tamai K Elledge SJ Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro.Proc Natl Acad Sci USA. 2000; 97: 10389-10394Crossref PubMed Scopus (661) Google Scholar). Activated CHEK2 phosphorylates critical cell-cycle proteins, including p53 (MIM 191170), Cdc25C (MIM 157680), Cdc25A (MIM 116947), and BRCA1 (MIM 113705), which promote cell-cycle arrest and activation of DNA repair (Zeng et al. Zeng et al., 1998Zeng Y Forbes KC Wu Z Moreno S Piwnia-Worms H Enoch T Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1.Nature. 1998; 395: 507-510Crossref PubMed Scopus (301) Google Scholar; Chehab et al. Chehab et al., 2000Chehab NH Malikzay A Appel M Halazonetis TD Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.Genes Dev. 2000; 14: 278-288PubMed Google Scholar; Lee et al. Lee et al., 2000Lee JS Collins KM Brown AL Lee C-H Chung JH hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response.Nature. 2000; 404: 201-204Crossref PubMed Scopus (443) Google Scholar; Falck et al. Falck et al., 2001Falck J Mailand N Syljusen RG Bartek J Lukas J The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (831) Google Scholar). We recently reported that CHEK2 1100delC, a truncating variant that abrogates the kinase activity of the protein, is a low-penetrance breast cancer–susceptibility allele (The CHEK2-Breast Cancer Consortium The CHEK2-Breast Cancer Consortium, 2002The CHEK2-Breast Cancer Consortium Low-penetrance susceptibility to breast cancer due to CHEK2*1100delC in noncarriers of BRCA1 or BRCA2 mutations.Nat Genet. 2002; 31: 55-59Crossref PubMed Scopus (838) Google Scholar). CHEK2 1100delC was present in 1.1% of healthy control subjects, compared with 5.1% of subjects with breast cancer from BRCA1/2-negative families, including 13.5% of subjects from families with male breast cancer. We estimated that CHEK2 1100delC confers an ∼2-fold increased breast cancer risk in women and 10-fold risk in men (The CHEK2-Breast Cancer Consortium The CHEK2-Breast Cancer Consortium, 2002The CHEK2-Breast Cancer Consortium Low-penetrance susceptibility to breast cancer due to CHEK2*1100delC in noncarriers of BRCA1 or BRCA2 mutations.Nat Genet. 2002; 31: 55-59Crossref PubMed Scopus (838) Google Scholar). A study of Finnish breast cancer cases reported a CHEK2 1100delC frequency of 1.4% in controls and 5.5% in BRCA1/2-negative familial breast cancer cases, independently supporting this observation (Vahteristo et al. Vahteristo et al., 2002Vahteristo P Bartkova J Eerola H Syrjäkoski K Ojala S Kilpivaara O Tamminen A Kononen J Aittomäki K Heikkilä P Holli K Blomqvist C Bartek J Kallioniemi OP Nevanlinna H A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer.Am J Hum Genet. 2002; 71: 432-438Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). CHEK2 1100delC was originally reported in a family with Li-Fraumeni syndrome (LFS [MIM 151623]) that included three cases of breast cancer (Bell et al. Bell et al., 1999Bell DW Varley JM Szydlo TE Kang DH Wahrer DC Shannon KE Lubratovich M Verselis SJ Isselbacher KJ Fraumeni JF Birch JM Li FP Garber JE Haber DA Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (723) Google Scholar). Screening of cancer cases from LFS and families afflicted with Li-Fraumeni–like syndrome (LFL) revealed two additional CHEK2 germline sequence variants in individuals with breast cancer (Bell et al. Bell et al., 1999Bell DW Varley JM Szydlo TE Kang DH Wahrer DC Shannon KE Lubratovich M Verselis SJ Isselbacher KJ Fraumeni JF Birch JM Li FP Garber JE Haber DA Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (723) Google Scholar; Lee et al. Lee et al., 2001Lee SB Kim SH Bell DW Wahrer DC Schiripo TA Jorczak MM Sgroi DC Garber JE Li FP Nichols KE Varley JM Godwin AK Shannon KM Harlow E Haber DA Destabilisation of CHK2 by a missense mutation associated with Li-Fraumeni syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar). R145W was initially reported in the HCT15 colorectal cancer cell line and, subsequently, in the germline of an individual with sarcoma at 20 years of age, breast cancer at 42 years of age, and a family history consistent with LFL (Bell et al. Bell et al., 1999Bell DW Varley JM Szydlo TE Kang DH Wahrer DC Shannon KE Lubratovich M Verselis SJ Isselbacher KJ Fraumeni JF Birch JM Li FP Garber JE Haber DA Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (723) Google Scholar; Lee et al. Lee et al., 2001Lee SB Kim SH Bell DW Wahrer DC Schiripo TA Jorczak MM Sgroi DC Garber JE Li FP Nichols KE Varley JM Godwin AK Shannon KM Harlow E Haber DA Destabilisation of CHK2 by a missense mutation associated with Li-Fraumeni syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar). The breast tumor showed loss of the wild-type CHEK2 allele. CHEK2 R145W has been shown to be deficient in kinase activity, binding and phosphorylation of Cdc25A, and ATM-dependent phosphorylation and, thus, is plausibly associated with the cancer susceptibility in this individual (Wu et al. Wu et al., 2001Wu X Webster SR Chen J Characterization of tumor-associated Chk2 mutations.J Biol Chem. 2001; 276: 2971-2974Crossref PubMed Scopus (134) Google Scholar; Li et al. Li et al., 2002Li J Williams BL Haire LF Goldberg M Wilker E Durocher D Yaffe MB Jackson SP Smerdon SJ Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). A second reported variant in CHEK2, I157T, has wild-type kinase activity but is deficient in binding and phosphorylation of Cdc25A and in binding to BRCA1 and p53 (Falck et al. Falck et al., 2001Falck J Mailand N Syljusen RG Bartek J Lukas J The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (831) Google Scholar; Wu et al. Wu et al., 2001Wu X Webster SR Chen J Characterization of tumor-associated Chk2 mutations.J Biol Chem. 2001; 276: 2971-2974Crossref PubMed Scopus (134) Google Scholar; Li et al. Li et al., 2002Li J Williams BL Haire LF Goldberg M Wilker E Durocher D Yaffe MB Jackson SP Smerdon SJ Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). I157T has been reported in families with LFS, LFL, and breast cancer (Bell et al. Bell et al., 1999Bell DW Varley JM Szydlo TE Kang DH Wahrer DC Shannon KE Lubratovich M Verselis SJ Isselbacher KJ Fraumeni JF Birch JM Li FP Garber JE Haber DA Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (723) Google Scholar; Allinen et al. Allinen et al., 2001Allinen M Huusko P Mäntyniemi S Launonen V Winqvist R Mutation analysis of the CHK2 gene in families with hereditary breast cancer.Br J Cancer. 2001; 85: 209-212Crossref PubMed Scopus (67) Google Scholar; Bougeard et al. Bougeard et al., 2001Bougeard G Limacher JM Martin C Charbonnier F Killian A Delattre O Longy M Jonveaux P Fricker JP Stoppa-Lyonnet D Flaman JM Frebourg T Detection of 11 germline inactivating TP53 mutations and absence of TP63 and HCHK2 mutations in 17 French families with Li-Fraumeni or Li-Fraumeni-like syndrome.J Med Genet. 2001; 38: 253-257Crossref PubMed Google Scholar; Lee et al. Lee et al., 2001Lee SB Kim SH Bell DW Wahrer DC Schiripo TA Jorczak MM Sgroi DC Garber JE Li FP Nichols KE Varley JM Godwin AK Shannon KM Harlow E Haber DA Destabilisation of CHK2 by a missense mutation associated with Li-Fraumeni syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar; Vahteristo et al. Vahteristo et al., 2001Vahteristo P Tamminen A Karvinen P Eerola H Eklund C Aaltonen LA Blomqvist C Aittomaki K Nevanlinna H p53, CHK2, and CHK1 genes in Finnish families with Li-Fraumeni Syndrome.Cancer Res. 2001; 61: 5718-5722PubMed Google Scholar; Sullivan et al. Sullivan et al., 2002Sullivan A Yuille M Repellin C Reddy A Reelfs O Bell A Dunne B Gusterson BA Osin P Farrell PJ Yulug I Evans A Ozcelik T Gasco M Crook T Concomitant inactivation of p53 and Chk2 in breast cancer.Oncogene. 2002; 21: 1316-1324Crossref PubMed Scopus (72) Google Scholar). However, I157T has also been detected in control populations at frequencies of 0%–6.5% (Bell et al. Bell et al., 1999Bell DW Varley JM Szydlo TE Kang DH Wahrer DC Shannon KE Lubratovich M Verselis SJ Isselbacher KJ Fraumeni JF Birch JM Li FP Garber JE Haber DA Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (723) Google Scholar; Allinen et al. Allinen et al., 2001Allinen M Huusko P Mäntyniemi S Launonen V Winqvist R Mutation analysis of the CHK2 gene in families with hereditary breast cancer.Br J Cancer. 2001; 85: 209-212Crossref PubMed Scopus (67) Google Scholar; Lee et al. Lee et al., 2001Lee SB Kim SH Bell DW Wahrer DC Schiripo TA Jorczak MM Sgroi DC Garber JE Li FP Nichols KE Varley JM Godwin AK Shannon KM Harlow E Haber DA Destabilisation of CHK2 by a missense mutation associated with Li-Fraumeni syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar; Vahteristo et al. Vahteristo et al., 2001Vahteristo P Tamminen A Karvinen P Eerola H Eklund C Aaltonen LA Blomqvist C Aittomaki K Nevanlinna H p53, CHK2, and CHK1 genes in Finnish families with Li-Fraumeni Syndrome.Cancer Res. 2001; 61: 5718-5722PubMed Google Scholar). Thus, it is unclear whether I157T is a neutral polymorphism or confers a small increased risk of cancer. To identify additional CHEK2-sequence variants that may confer susceptibility to breast cancer, we screened the full coding sequence of CHEK2 in one case subject with breast cancer and negative for mutations in BRCA1/2 from each of 89 families (from the United Kingdom, the Netherlands, or North America) with at least three cases of breast cancer. These families are referred to as the Variant Ascertainment Set. The families with breast cancer were ascertained from genetics clinics, and samples were obtained with approval of the Local Ethics Research Committee/Institutional Review Board. The 76 families with breast cancer from the United Kingdom and the United States each consisted of a minimum of three cases of breast cancer in first- or second-degree relatives who were diagnosed prior to age 60 years. The 13 families with breast cancer from the Netherlands consisted of a minimum of three cases of breast cancer in first- or second-degree relatives, of whom at least one was diagnosed prior to age 60 years. Nine Dutch families included at least three cases of breast cancer diagnosed prior to age 60 years. At least one affected individual from each of the 89 families was screened by Conformation Sensitive Gel Electrophoresis (CSGE) (Ganguly et al. Ganguly et al., 1993Ganguly A Rock MJ Prockop DJ Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments.Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (604) Google Scholar) for the full coding sequence and intron-exon boundaries of BRCA1 and BRCA2 and was negative for mutations. In addition, families from the Netherlands were shown to be negative for rearrangements found elsewhere in the Dutch population (Petrij-Bosch et al. Petrij-Bosch et al., 1997Petrij-Bosch A Peelen T van Vliet M van Ejik R Olmer R Drusedau M Hogervorst FB Hageman S Arts PJ Ligtenberg MJ Meijers-Heijboer H Klijn JG Vasen HF Cornelisse CJ van't Veer LJ Bakker E van Ommen GJ Devilee P BRCA1 genomic deletions are major founders in Dutch breast cancer patients.Nat Genet. 1997; 17: 341-345Crossref PubMed Scopus (357) Google Scholar). For the CHEK2 whole gene screen, a single genomic DNA sample from each family was analyzed by CSGE. Exons 10–14 were amplified in a long-range PCR to avoid the partial nonprocessed genomic copies of CHEK2 (Sodha et al. Sodha et al., 2002Sodha N Houlston RS Williams R Yuille MA Mangion J Eeles RA A robust method for detecting CHK2/RAD53 mutations in genomic DNA.Hum Mutat. 2002; 19: 173-177Crossref PubMed Scopus (40) Google Scholar). The long-range PCR was variably successful in these analyses, but complete data was obtained from a minimum of 76 families for each of exons 10–13 and from 59 families for exon 14. In total, we identified three sequence variants of CHEK2 in the 89 cases. Five cases carried the synonymous A252G (E84E) variant that has been reported in several other CHEK2 screens and is a likely neutral polymorphism with no elevated risk of breast cancer (Bell et al. Bell et al., 1999Bell DW Varley JM Szydlo TE Kang DH Wahrer DC Shannon KE Lubratovich M Verselis SJ Isselbacher KJ Fraumeni JF Birch JM Li FP Garber JE Haber DA Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (723) Google Scholar; Hofmann et al. Hofmann et al., 2001Hofmann WK Miller CW Tsukasaki K Tavor S Ikezoe T Hoelzer D Takeuchi S Koefller HP Mutation analysis of the DNA-damage checkpoint gene CHK2 in myelodysplastic syndromes and acute myeloid leukaemia.Leuk Res. 2001; 25: 333-338Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar; Ingvarsson et al. Ingvarsson et al., 2002Ingvarsson S Sigbjornsdottir BI Huiping C Hafsteinsdottir SH Ragnarsson G Barkardottir RB Arason A Egilsson V Bergthorsson JT Mutation analysis of the CHK2 gene in breast carcinoma and other cancers.Breast Cancer Res. 2002; 4: R4Crossref PubMed Scopus (44) Google Scholar; Reddy et al. Reddy et al., 2002Reddy A Yuille M Sullivan A Repellin C Bell A Tidy JA Evans DJ Farrell PJ Gusterson B Gasco M Crook T Analysis of CHK2 in vulval neoplasia.Br J Cancer. 2002; 86: 756-760Crossref PubMed Scopus (30) Google Scholar; Sullivan et al. Sullivan et al., 2002Sullivan A Yuille M Repellin C Reddy A Reelfs O Bell A Dunne B Gusterson BA Osin P Farrell PJ Yulug I Evans A Ozcelik T Gasco M Crook T Concomitant inactivation of p53 and Chk2 in breast cancer.Oncogene. 2002; 21: 1316-1324Crossref PubMed Scopus (72) Google Scholar). Six cases harbored CHEK2 1100delC and have been reported elsewhere (The CHEK2-Breast Cancer Consortium The CHEK2-Breast Cancer Consortium, 2002The CHEK2-Breast Cancer Consortium Low-penetrance susceptibility to breast cancer due to CHEK2*1100delC in noncarriers of BRCA1 or BRCA2 mutations.Nat Genet. 2002; 31: 55-59Crossref PubMed Scopus (838) Google Scholar). One novel variant, R117G, was detected in two separate families, one from the United Kingdom and one from the Netherlands (fig. 1; table 1).Table 1CHEK2 Variants in BRCA1/2-Negative Breast Cancer Cases, BRCA1/2-Positive Breast Cancer Cases, and ControlsNumber ofSubjects Positive forSet, Group, and Geographic RegionSubjectsFamiliesR117GR145WI157TVariant Ascertainment SetaFull screen of CHEK2 coding sequence.: Families negative for BRCA1/BRCA2 in United Kingdom7272100 North America44000 Netherlands1313100 Total8989200Variant Evaluation SetbAllele-specific hybridization analysis of variant.: Families negative for BRCA1/BRCA2cThe subjects negative for BRCA1/2 in the Variant Evaluation Set do not include any cases from families that were screened in the Variant Ascertainment Set. in United Kingdom2411212dBoth individuals positive for R117G were from the same family.00 North America284272002 Netherlands212212000 Total737605202 Families positive for BRCA1/BRCA2 in United Kingdom8347000 North America235147000 Netherlands141141000 Total459335000 Control individuals in United Kingdom448…000 North America94…001 Netherlands181…100 Total723101a Full screen of CHEK2 coding sequence.b Allele-specific hybridization analysis of variant.c The subjects negative for BRCA1/2 in the Variant Evaluation Set do not include any cases from families that were screened in the Variant Ascertainment Set.d Both individuals positive for R117G were from the same family. Open table in a new tab To evaluate the breast cancer risk associated with R117G and two variants, I157T and R145W, reported elsewhere in the germline of breast cancer cases, we used a study design similar to that employed in our previous analysis of CHEK2 1100delC, in which the frequency of the variant in case subjects with a family history of the disease was compared with that in control subjects. Although conventional association studies to detect low-penetrance susceptibility alleles compare population-based series of case with control subjects, use of familial cases markedly improves the power to detect an effect. For each variant, we screened 737 breast cancer cases from 605 families negative for BRCA1/2 mutations, 459 cases from 335 families positive for BRCA1/2, and 723 control subjects from the United Kingdom, North America, and the Netherlands by means of high stringency, allele-specific oligonucleotide (ASO) hybridization, with sequencing of positive cases (table 1). These series are referred to as "the Variant Evaluation Set" and did not include any cases from the families screened in the Variant Ascertainment Set. The families with breast cancer each contained at least two individuals with breast cancer or one individual with breast cancer and one individual with ovarian cancer who were first- or second-degree relatives of one another. At least one of the individuals with breast cancer was diagnosed before age 60 years. Families were ascertained through cancer genetics clinics in the relevant countries. The United Kingdom control subjects were children from the North Cumbria Community Genetics Project study from the northwest of the United Kingdom (Chase et al. Chase et al., 1998Chase DS Tawn EJ Parker L Jonas P Parker CO Burn J The North Cumbria Community Genetics Project.J Med Genet. 1998; 35: 413-416Crossref PubMed Scopus (40) Google Scholar). The Dutch control subjects were spouses of cystic fibrosis heterozygotes from the southwest of the Netherlands. The North American control subjects were neighborhood control subjects from a breast cancer case-control study in the Philadelphia area or spouses marrying-in to families with breast cancer ascertained for linkage analysis from the same area. Each variant was detected by PCR amplification of the relevant exon, application of PCR products to nylon filters, and hybridization under high stringency of 32P end-labeled oligonucleotides complementary to mutant and the wild-type sequence (table 2). Every filter contained both a negative and a positive control and was scored independently by three individuals, as described elsewhere (The CHEK2-Breast Cancer Consortium The CHEK2-Breast Cancer Consortium, 2002The CHEK2-Breast Cancer Consortium Low-penetrance susceptibility to breast cancer due to CHEK2*1100delC in noncarriers of BRCA1 or BRCA2 mutations.Nat Genet. 2002; 31: 55-59Crossref PubMed Scopus (838) Google Scholar).Table 2Germline CHEK2 Variants Identified in Subjects with Breast Cancer and Probe Sequences Used for Their Detection in ASO AnalysesProtein ChangeNucleotide changeWild-Type SequenceProbe SequenceR117G349A→GggtttgggagggacaaaagctggtttgggggggacaaaagctR145W433C→TaaacactttcggattttcagaaacacttttggattttcagI157T470T→Caaaaactcttacattgcatacataaaaactcttacactgcatacat Open table in a new tab In our analyses of the Variant Evaluation Set, we identified both R117G and I157T in 2/737 BRCA1/2-negative familial case subjects and 1/723 control subjects and R145W in none of the case or control subjects (table 1). Codon 117 is within the forkhead-associated (FHA) domain of CHEK2. FHA protein motifs are phosphopeptide recognition domains (Durocher et al. Durocher et al., 2000Durocher D Taylor IA Sarbassova D Haire LF Westcott SL Jackson SP Smerdon SJ Yaffe MB The molecular basis of FHA domain: phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.Mol Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), and the CHEK2 FHA domain mediates ATM-dependent CHEK2 phosphorylation and targeting of CHEK2 to in vivo binding partners, such as BRCA1 (Li et al. Li et al., 2002Li J Williams BL Haire LF Goldberg M Wilker E Durocher D Yaffe MB Jackson SP Smerdon SJ Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The FHA domain is also implicated in CHEK2 oligomerization, which, in turn, is involved in regulation of CHEK2 activation, signal amplification, and transduction in DNA damage checkpoint pathways (Xu et al. Xu et al., 2002Xu X Tsvetkov LM Stern DF Chk2 activation and phosphorylation-dependent oligomerisation.Mol Cell Biol. 2002; 22: 4419-4432Crossref PubMed Scopus (154) Google Scholar). Arg117 is a functionally important and highly conserved residue in the FHA domain (identical in both Saccharomyces cerevisiae and Schizosaccharomyces pombe). It anchors peptides via hydrogen bonding interactions with the phosphothreonine side chain, and disruption by an artificially introduced Arg117→Ala substitution (R117A) severely reduces phosphopeptide binding (Li et al. Li et al., 2002Li J Williams BL Haire LF Goldberg M Wilker E Durocher D Yaffe MB Jackson SP Smerdon SJ Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Furthermore, there is no detectable binding of BRCA1 by R117A, in contrast to wild-type CHEK2 (Li et al. Li et al., 2002Li J Williams BL Haire LF Goldberg M Wilker E Durocher D Yaffe MB Jackson SP Smerdon SJ Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Thus, it is biologically plausible that the R117G variant, which is likely to have similar functional defects to R117A, confers susceptibility to breast cancer. However, only 2/737 BRCA1/2-negative case subjects in the Variant Evaluation Set carried R117G, and these were from the same family. One of 723 control subjects carried R117G (table 1). The three pedigrees afflicted with breast cancer in either set in which R117G was found are shown in figure 1. In one (EMC8138), all four tested individuals affected with breast cancer carried the variant. In the two other families (B546, B556), the variant was present in only two of three tested individuals with breast cancer. The probability that five out of the seven secondary cases of breast cancer in these families would carry the R117G variant, assuming that it is not associated with an increased risk of breast cancer, is 0.13. Codon 157 is also in the FHA domain, though remote from the site of phosphopeptide binding (Li et al. Li et al., 2002Li J Williams BL Haire LF Goldberg M Wilker E Durocher D Yaffe MB Jackson SP Smerdon SJ Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2.Mol Cell. 2002; 9: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). I157T was reported elsewhere to be present in 8.9% (7/79) of Finnish pedigrees with hereditary breast cancer and 6.5% (13/200) of control individuals, suggesting that the breast cancer risk, if any, is weak (Allinen et al. Allinen et al., 2001Allinen M Huusko P Mäntyniemi S Launonen V Winqvist R Mutation analysis of the CHK2 gene in families with hereditary breast cancer.Br J Cancer. 2001; 85: 209-212Crossref PubMed Scopus (67) Google Scholar). Our data are consistent with this, as we identified I157T in 2/737 BRCA1/2-negative familial cases and in 1/723 control subjects. Samples were not available to assess segregation with the disease in either family positive for I157T. Although none of the three variants we identified were associated with a statistically significantly increased risk, the CIs for the relative risk associated with these variants are wide. For all three variants combined, the crude 95% CIs, on the basis of the observed frequency in case and control subjects from the Variant Evaluation Set, are (0.21–21.3). The relative risks obtained from this data set are an overestimate of the "true" relative risk, because the frequency of a susceptibility allele will be higher in familial cases than unselected cases. As a comparison, in the analysis of CHEK2 1100delC, the estimated true relative risk was approximately twofold, but the crude relative risk on the basis of a similar series of familial cases was approximately fivefold (5% in case subjects vs. 1% in control subjects). Thus, the data on the three new CHEK2 variants are consistent either with no effect or of a relative risk comparable with that conferred by CHEK2 1100delC. Although there is considerable uncertainty about the level of risk associated with these three CHEK2 variants, we can be more certain about their overall contribution to breast cancer incidence. The upper 95% CI on the combined frequency of these variants in breast cancer cases with a comparable family history is 1.5% (as compared with 5% for CHEK2 1100delC). We have estimated elsewhere that ∼1% of breast cancer incidence and 1/2% of the familial aggregation of the excess breast cancer risk in first-degree relatives of case subjects is likelyto be due to CHEK2 1100delC. Even if the variants evaluated in this report are associated with a risk comparable with CHEK2 1100delC, much <1% of the familial aggregation of breast cancer or of breast cancer incidence overall is likely to be attributable to them. At least two other nonsynonymous germline CHEK2 variants have been reported in breast cancer since our analyses were performed: a R3W variant in an individual with breast cancer and a family history of LFL (Lee et al. Lee et al., 2001Lee SB Kim SH Bell DW Wahrer DC Schiripo TA Jorczak MM Sgroi DC Garber JE Li FP Nichols KE Varley JM Godwin AK Shannon KM Harlow E Haber DA Destabilisation of CHK2 by a missense mutation associated with Li-Fraumeni syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar), and T59K, which was reported in four Icelandic individuals with breast cancer (Ingvarsson et al. Ingvarsson et al., 2002Ingvarsson S Sigbjornsdottir BI Huiping C Hafsteinsdottir SH Ragnarsson G Barkardottir RB Arason A Egilsson V Bergthorsson JT Mutation analysis of the CHK2 gene in breast carcinoma and other cancers.Breast Cancer Res. 2002; 4: R4Crossref PubMed Scopus (44) Google Scholar). It is possible that these or other as-yet-unidentified CHEK2 variants may confer susceptibility to breast cancer, and additional studies in other familial breast cancer series will be of interest. However, we identified only two individuals with variant sequences in the 89 cases that were fully screened for mutations, compared with six occurrences of CHEK2 1100delC in the same family set. The low frequency of CHEK2 variants other than 1100delC in familial breast cancer cases indicates that, even if some are associated with an increased risk, their overall contribution to breast cancer susceptibility is likely to be very low. The relatively high prevalence of CHEK2 1100delC in both the British and Dutch populations suggests that this variant is relatively old and subject to little selective pressure. The absence of any other protein-truncating alterations with significant frequencies could simply be due to chance but might reflect some functional constraints on the viability of individuals with alternative mutations. Since we screened familial pedigrees with breast cancer from three specific populations, we cannot exclude the possibility that the contribution of CHEK2 variants to breast cancer susceptibility differs in other populations. However, our results indicate that in North European populations, 1100delC is responsible for almost all of the contribution to breast cancer susceptibility made by sequence variants of CHEK2. We thank all individuals and families who have participated in these studies and the North Cumbria Community Genetics Project for providing control individuals. We thank A. Hall and K. Redman for family ascertainment. This research was funded by grants from the Erasmus MC Revolving Fund and Cancer Research U.K. D.F.E. is a principal research fellow of Cancer Research U.K. K.L.N. and B.L.W. are supported by grants from the National Cancer Institute and funds from the Abramson Family Cancer Research Institute. The following members of the Breast Cancer Linkage Consortium contributed samples for this study: A. Brady, T. Cole, A. Collins, H. Cox, A. Donaldson, D. F. Easton, D. Eccles, R. Eeles, G. Evans, H. Gregory, J. Gray, R. Houlston, J. Klijn, F. Lalloo, A. Lucassen, J. Mackay, H. Meijers-Heijboer, G. Mitchell, P. Morrison, V. Murday, S. Narod, K. L. Nathanson, J. Patterson, T. Peretz, C. M. Phelan, N. Rahman, M. Rogers, A. Schofield, M. R. Stratton, P. Tonin, B. Weber, and W. Weber.
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