CHEK2 variants: linking functional impact to cancer risk
2022; Elsevier BV; Volume: 8; Issue: 9 Linguagem: Inglês
10.1016/j.trecan.2022.04.009
ISSN2405-8033
AutoresRick A.C.M. Boonen, Maaike P.G. Vreeswijk, Haico van Attikum,
Tópico(s)Genetic factors in colorectal cancer
ResumoFunctional assays have been developed that can determine the impact of missense variants of uncertain significance (VUS) on CHK2 protein function.Functional analyses of CHEK2 missense VUS reveal an association between impaired protein function and increased breast cancer risk.Damaging CHEK2 missense VUS may be associated with a risk of breast cancer similar to that of protein-truncating variants.A comprehensive functional characterization of CHEK2 missense VUS is needed to determine the associated cancer risk.Functional analysis of missense VUS in CHEK2 will improve the clinical management of carriers and their family members. Protein-truncating variants in the breast cancer susceptibility gene CHEK2 are associated with a moderately increased risk of breast cancer. By contrast, for missense variants of uncertain significance (VUS) in CHEK2 the associated breast cancer risk is often unclear. To facilitate their classification, functional assays that determine the impact of missense VUS on CHK2 protein function have been performed. Here we discuss these functional analyses that consistently reveal an association between impaired protein function and increased breast cancer risk. Overall, these findings suggest that damaging CHEK2 missense VUS are associated with a risk of breast cancer similar to that of protein-truncating variants. This indicates the urgency of expanding the functional characterization of CHEK2 missense VUS to further understand the associated cancer risk. Protein-truncating variants in the breast cancer susceptibility gene CHEK2 are associated with a moderately increased risk of breast cancer. By contrast, for missense variants of uncertain significance (VUS) in CHEK2 the associated breast cancer risk is often unclear. To facilitate their classification, functional assays that determine the impact of missense VUS on CHK2 protein function have been performed. Here we discuss these functional analyses that consistently reveal an association between impaired protein function and increased breast cancer risk. Overall, these findings suggest that damaging CHEK2 missense VUS are associated with a risk of breast cancer similar to that of protein-truncating variants. This indicates the urgency of expanding the functional characterization of CHEK2 missense VUS to further understand the associated cancer risk. The checkpoint kinase 2 (CHK2; see Glossary) protein kinase was initially identified as the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases [1.Matsuoka S. et al.Linkage of ATM to cell cycle regulation by the Chk2 protein kinase.Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar]. Its characterization revealed an important role in cell-cycle control and apoptosis following exposure of cells to DNA damaging agents [1.Matsuoka S. et al.Linkage of ATM to cell cycle regulation by the Chk2 protein kinase.Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar,2.Chehab N.H. et al.Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53.Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar]. The ataxia-telangiectasia mutated (ATM) kinase phosphorylates and activates CHK2, resulting in the modification of downstream substrates such as p53, cell division cycle (CDC) 25A and CDC25C, KAP1, and breast cancer type 1 susceptibility protein (BRCA1). Collectively, this signaling cascade may prevent genome instability and cancer development by instructing cells to stop proliferating and repair the DNA damage, or promote apoptosis as a response to inefficient or improper repair (Figure 1). Shortly after its identification, frameshift variants such as the well-known c.1100del;p.T367Mfs variant were identified in the CHEK2 gene and linked to a cancer susceptibility disorder called Li–Fraumeni syndrome (LFS) [3.Bell D.W. et al.Heterozygous germ line hCHK2 mutations in Li–Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (746) Google Scholar]. LFS is a rare hereditary autosomal dominant disorder that is characterized by a wide range of malignancies that appear at an unusually early age [4.Schneider K. et al.Li–Fraumeni syndrome.GeneReviews. 1999; (Published online January 19, 1999)https://www.ncbi.nlm.nih.gov/books/NBK1311/Google Scholar]. Similarly to CHK2, the well-described tumor-suppressor protein p53 halts cell division following DNA damage and inherited mutations in the corresponding gene (tumor protein 53, TP53) account for most cases of LFS [5.McBride K.A. et al.Li–Fraumeni syndrome: cancer risk assessment and clinical management.Nat. Rev. Clin. Oncol. 2014; 11: 260-271Crossref PubMed Scopus (158) Google Scholar]. A link between CHK2 and p53 became evident when it was shown that CHK2 phosphorylates p53 at S20, resulting in dissociation of preformed p53–Mdm2 complexes and leading to p53 stabilization [2.Chehab N.H. et al.Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53.Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar]. These observations suggested that CHK2 is a tumor-suppressor protein that acts within the p53 signaling pathway. In recent years, several studies have confirmed the tumor-suppressive function of CHK2 by showing that truncating variants in the CHEK2 gene (e.g., c.1100del;p.T367Mfs) are associated with a moderate risk of breast cancer (two- to threefold increased risk) [6.Breast Cancer Association Consortium et al.Breast cancer risk genes – association analysis in more than 113,000 women.N. Engl. J. Med. 2021; 384: 428-439Crossref PubMed Scopus (191) Google Scholar, 7.Couch F.J. et al.Associations between cancer predisposition testing panel genes and breast cancer.JAMA Oncol. 2017; 3: 1190-1196Crossref PubMed Scopus (364) Google Scholar, 8.Decker B. et al.Rare, protein-truncating variants in ATM, CHEK2 and PALB2, but not XRCC2, are associated with increased breast cancer risks.J. Med. Genet. 2017; 54: 7327-7341Crossref Scopus (49) Google Scholar, 9.Hauke J. et al.Gene panel testing of 5589 BRCA1/2-negative index patients with breast cancer in a routine diagnostic setting: results of the German Consortium for Hereditary Breast and Ovarian Cancer.Cancer Med. 2018; 7: 1349-1358Crossref PubMed Scopus (83) Google Scholar, 10.Meijers-Heijboer H. et al.Low-penetrance susceptibility to breast cancer due to CHEK2*1100delC in noncarriers of BRCA1 or BRCA2 mutations.Nat. Genet. 2002; 31: 555-559Google Scholar, 11.Weischer M. et al.Increased risk of breast cancer associated with CHEK2*1100delC.J. Clin. Oncol. 2007; 25: 57-63Crossref PubMed Scopus (96) Google Scholar]. For heterozygous female carriers of CHEK2 truncating variants, this finding translates to a lifetime risk of ~25% of developing breast cancer before the age of 80 years [6.Breast Cancer Association Consortium et al.Breast cancer risk genes – association analysis in more than 113,000 women.N. Engl. J. Med. 2021; 384: 428-439Crossref PubMed Scopus (191) Google Scholar]. Furthermore, CHEK2 has been characterized as a multi-organ cancer susceptibility gene [12.Cybulski C. et al.CHEK2 is a multiorgan cancer susceptibility gene.Am. J. Hum. Genet. 2004; 75: 1131-1135Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar], a finding that was confirmed by other studies (reviewed in [13.Stolarova L. et al.CHEK2 germline variants in cancer predisposition: stalemate rather than checkmate.Cells. 2020; 9: 2675Crossref Scopus (38) Google Scholar]). These findings have led to a significant increase in genetic testing for CHEK2 and the identification of many rare missense variants whose clinical relevance is unclear. In addition to the high-risk breast cancer susceptibility genes BRCA1, BRCA2, and partner and localizer of BRCA2 (PALB2), it is now evident that CHEK2, together with ATM, is the most commonly mutated gene in the germline of breast cancer patients [6.Breast Cancer Association Consortium et al.Breast cancer risk genes – association analysis in more than 113,000 women.N. Engl. J. Med. 2021; 384: 428-439Crossref PubMed Scopus (191) Google Scholar]. Currently 1148 distinct missense VUS in CHEK2 have been reported in ClinVar [14.Landrum M.J. et al.ClinVar: public archive of relationships among sequence variation and human phenotype.Nucleic Acids Res. 2014; 42: D980-D985Crossref PubMed Scopus (1525) Google Scholar] (as of February 2022). In aggregate, many of these rare missense variants, also termed missense VUS, are also associated with breast cancer [odds ratio (OR) 1.42; 95% confidence interval (CI), 1.28–1.58; P = 2.5 × 1011] [6.Breast Cancer Association Consortium et al.Breast cancer risk genes – association analysis in more than 113,000 women.N. Engl. J. Med. 2021; 384: 428-439Crossref PubMed Scopus (191) Google Scholar]. This association appears to be independent of their position within the gene and thus their impact on any of the functional domains of CHK2 – a N-terminal SQ/TQ cluster domain (SCD) [amino acids (aa) 19–69], a Forkhead-associated (FHA) domain (aa 92–205), a serine/threonine kinase domain (aa 212–501), and a nuclear localization signal (NLS; aa 515–522) (Figure 2). Knowing which missense variants impact on protein function, and to what extent, can help to determine which variants are associated with increased breast cancer risk. To this end, the outcomes of quantitative and well-validated functional assays for CHEK2, in line with American College of Medical Genetics and Genomics (ACMG) guidelines [15.Brnich S.E. et al.Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework.Genome Med. 2019; 12: 3Crossref PubMed Scopus (133) Google Scholar], can help to guide the clinical classification of genetic variants in this gene, thereby improving the counseling of carriers. Indeed, several recent studies have described the functional characterization of CHEK2 variants. We provide an overview of these studies, the different approaches and outcomes, the potential pitfalls of functional assays, and the association between the functional outcomes and breast cancer risk. Numerous studies have set out to test the functional consequences of rare variants in the CHEK2 gene to aid clinical interpretation (Table 1) [16.Bell D.W. et al.Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts.Int. J. Cancer. 2007; 121: 2661-2667Crossref PubMed Scopus (62) Google Scholar, 17.Boonen R. et al.Functional analysis identifies damaging CHEK2 missense variants associated with increased cancer risk.Cancer Res. 2022; 82: 615-631Crossref PubMed Google Scholar, 18.Chrisanthar R. et al.CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer.PLoS One. 2008; 3e3062Crossref PubMed Scopus (70) Google Scholar, 19.Cuella-Martin R. et al.Functional interrogation of DNA damage response variants with base editing screens.Cell. 2021; 184: 1081-1097Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20.Delimitsou A. et al.Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system.Hum. Mutat. 2019; 40: 631-648Crossref PubMed Scopus (15) Google Scholar, 21.Falck J. et al.The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (861) Google Scholar, 22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar, 23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar, 24.Roeb W. et al.Response to DNA damage of CHEK2 missense mutations in familial breast cancer.Hum. Mol. Genet. 2012; 21: 2738-2744Crossref PubMed Scopus (53) Google Scholar, 25.Shaag A. et al.Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population.Hum. Mol. Genet. 2005; 14: 555-563Crossref PubMed Scopus (94) Google Scholar, 26.Tischkowitz M.D. et al.Identification and characterization of novel SNPs in CHEK2 in Ashkenazi Jewish men with prostate cancer.Cancer Lett. 2008; 270: 173-180Crossref PubMed Scopus (14) Google Scholar, 27.Wang N. et al.A novel recurrent CHEK2 Y390C mutation identified in high-risk Chinese breast cancer patients impairs its activity and is associated with increased breast cancer risk.Oncogene. 2015; 34: 5198-5205Crossref PubMed Scopus (20) Google Scholar, 28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Ideally, a functional assay for a cancer predisposition gene measures a function that has been linked to the cancer phenotype. Although it is known that CHK2 phosphorylates a wide spectrum of substrates involved in cell-cycle regulation, DNA repair, and apoptosis [29.Ahn J. et al.The Chk2 protein kinase.DNA Repair (Amst). 2004; 3: 1039-1047Crossref PubMed Scopus (207) Google Scholar, 30.Bartek J. Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer.Cancer Cell. 2003; 3: 421-429Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar, 31.Kastan M.B. Bartek J. Cell-cycle checkpoints and cancer.Nature. 2004; 432: 316-323Crossref PubMed Scopus (2138) Google Scholar, 32.Li J. et al.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 (186) Google Scholar, 33.Zhang J. et al.Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair.Mol. Cell. Biol. 2004; 24: 708-718Crossref PubMed Scopus (252) Google Scholar, 34.Hu C. et al.Roles of Kruppel-associated box (KRAB)-associated co-repressor KAP1 Ser-473 phosphorylation in DNA damage response.J. Biol. Chem. 2012; 287: 18937-18952Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar], precisely which modifications are relevant for cancer development is largely unclear. Nonetheless, the ability of CHK2 to phosphorylate any of these substrates may reflect its activity towards all other substrates and thus inform on its functionality in general. In the following, we discuss the different functional assays and readouts that have been used for the classification of missense VUS in CHEK2 (Table 1).Table 1List of functional studies for variants in the CHEK2 geneaAbbreviations; aa, amino acids, KO, knockout; mES cells, mouse embryonic stem cells; MMS, methyl methanesulfonate; N/A, not applicable.TrackbTracks correspond to rings in the Circos plot (see Figure 2 in the main text). Track numbers only apply to a functional readout that resulted in a functional classification by the authors (i.e., functional, intermediate, and damaging).StudyModel systemFunctional assayNumber of variantscThe number of variants indicates the number of unique variants that were assessed in a model system with a specific functional readout.N/ACuella-Martin et al. [19.Cuella-Martin R. et al.Functional interrogation of DNA damage response variants with base editing screens.Cell. 2021; 184: 1081-1097Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar]MCF7 and MCF10A cellsGrowth after DNA damage induction using cisplatin, olaparib, doxorubicin, or camptothecin~1592Delimitsou et al. [20.Delimitsou A. et al.Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system.Hum. Mutat. 2019; 40: 631-648Crossref PubMed Scopus (15) Google Scholar]RAD53-null yeast strainsGrowth after DNA damage induction using MMS1223Boonen et al. [17.Boonen R. et al.Functional analysis identifies damaging CHEK2 missense variants associated with increased cancer risk.Cancer Res. 2022; 82: 615-631Crossref PubMed Google Scholar]Chek2 KO mES cellsKap1 S473 phosphorylation63N/ABoonen et al. [17.Boonen R. et al.Functional analysis identifies damaging CHEK2 missense variants associated with increased cancer risk.Cancer Res. 2022; 82: 615-631Crossref PubMed Google Scholar]Chek2 KO mES cellsProtein stability30N/ABoonen et al. [17.Boonen R. et al.Functional analysis identifies damaging CHEK2 missense variants associated with increased cancer risk.Cancer Res. 2022; 82: 615-631Crossref PubMed Google Scholar]Chek2 KO mES cellsGrowth after DNA damage induction using phleomycin84Kleiblova et al. [22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar]CHEK2 KO RPE1 cellsKAP1 S473 phosphorylation285Kleiblova et al. [22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar]In vitroPhosphorylation of KAP1 peptide (aa 467–478)286Kleiblova et al. [22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar]In vitroOmnia kinase assay287Roeb et al. [24.Roeb W. et al.Response to DNA damage of CHEK2 missense mutations in familial breast cancer.Hum. Mol. Genet. 2012; 21: 2738-2744Crossref PubMed Scopus (53) Google Scholar]RAD53-null yeast strainsGrowth after DNA damage induction using MMS268Bell et al. [16.Bell D.W. et al.Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts.Int. J. Cancer. 2007; 121: 2661-2667Crossref PubMed Scopus (62) Google Scholar]In vitroPhosphorylation of BRCA1 peptide (aa 758–1064)9N/ABell et al. [16.Bell D.W. et al.Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts.Int. J. Cancer. 2007; 121: 2661-2667Crossref PubMed Scopus (62) Google Scholar]In vitroProtein stability9Lee et al. [23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar]In vitroPhosphorylation of CDC25C peptide (aa 200–256)6N/ALee et al. [23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar]In vitroProtein stability10Chrisanthar et al. [18.Chrisanthar R. et al.CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer.PLoS One. 2008; 3e3062Crossref PubMed Scopus (70) Google Scholar]In vitroPhosphorylation of CDC25C peptide4N/AChrisanthar et al. [18.Chrisanthar R. et al.CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer.PLoS One. 2008; 3e3062Crossref PubMed Scopus (70) Google Scholar]In vitroAutophosphorylation11Wu et al. [28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]In vitroPhosphorylation of CDC25C peptide (aa 200–256)4N/AWu et al. [28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]In vitroCHK2 T68 phosphorylation12Tischkowitz et al. [26.Tischkowitz M.D. et al.Identification and characterization of novel SNPs in CHEK2 in Ashkenazi Jewish men with prostate cancer.Cancer Lett. 2008; 270: 173-180Crossref PubMed Scopus (14) Google Scholar]RAD53-null yeast strainsGrowth413Shaag et al. [25.Shaag A. et al.Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population.Hum. Mol. Genet. 2005; 14: 555-563Crossref PubMed Scopus (94) Google Scholar]RAD53-null yeast strainsGrowth414Falck et al. [21.Falck J. et al.The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (861) Google Scholar]In vitroPhosphorylation of CDC25A peptide315Wang et al. [27.Wang N. et al.A novel recurrent CHEK2 Y390C mutation identified in high-risk Chinese breast cancer patients impairs its activity and is associated with increased breast cancer risk.Oncogene. 2015; 34: 5198-5205Crossref PubMed Scopus (20) Google Scholar]Eμ–Myc p19Arf−/− B cellsGrowth after DNA damage induction using cisplatin, olaparib, or doxorubicin1N/AWang et al. [27.Wang N. et al.A novel recurrent CHEK2 Y390C mutation identified in high-risk Chinese breast cancer patients impairs its activity and is associated with increased breast cancer risk.Oncogene. 2015; 34: 5198-5205Crossref PubMed Scopus (20) Google Scholar]Eμ–Myc p19Arf−/− B cellsp53 S20 and CDC25A phosphorylation1N/AWang et al. [27.Wang N. et al.A novel recurrent CHEK2 Y390C mutation identified in high-risk Chinese breast cancer patients impairs its activity and is associated with increased breast cancer risk.Oncogene. 2015; 34: 5198-5205Crossref PubMed Scopus (20) Google Scholar]Eμ–Myc p19Arf−/− B cellsp53 protein levels1a Abbreviations; aa, amino acids, KO, knockout; mES cells, mouse embryonic stem cells; MMS, methyl methanesulfonate; N/A, not applicable.b Tracks correspond to rings in the Circos plot (see Figure 2 in the main text). Track numbers only apply to a functional readout that resulted in a functional classification by the authors (i.e., functional, intermediate, and damaging).c The number of variants indicates the number of unique variants that were assessed in a model system with a specific functional readout. Open table in a new tab Shortly after the identification of the CHK2 protein [1.Matsuoka S. et al.Linkage of ATM to cell cycle regulation by the Chk2 protein kinase.Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar], the effect of the first reported missense variants identified in patients were tested in functional assays [3.Bell D.W. et al.Heterozygous germ line hCHK2 mutations in Li–Fraumeni syndrome.Science. 1999; 286: 2528-2531Crossref PubMed Scopus (746) Google Scholar,21.Falck J. et al.The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (861) Google Scholar,23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar,28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. This work identified the first damaging missense variants in CHEK2 (e.g., p.R145W) by showing a profound impact on CHK2 protein stability and/or kinase activity, as measured by in vitro kinase assays using CDC25A [21.Falck J. et al.The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (861) Google Scholar] or CDC25C peptides [23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar,28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar] as substrates. Three later studies similarly employed in vitro assays using CDC25C [18.Chrisanthar R. et al.CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer.PLoS One. 2008; 3e3062Crossref PubMed Scopus (70) Google Scholar], BRCA1 [16.Bell D.W. et al.Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts.Int. J. Cancer. 2007; 121: 2661-2667Crossref PubMed Scopus (62) Google Scholar], and KAP1 peptides [22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar] as substrates. These studies mostly relied on the immunoprecipitation of activated and tagged CHK2 from cells (i.e., after induction of DNA damage) [16.Bell D.W. et al.Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts.Int. J. Cancer. 2007; 121: 2661-2667Crossref PubMed Scopus (62) Google Scholar,18.Chrisanthar R. et al.CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer.PLoS One. 2008; 3e3062Crossref PubMed Scopus (70) Google Scholar,21.Falck J. et al.The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (861) Google Scholar,23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar,28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar] or purification of recombinant CHK2 [22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar]. Overall, these studies resulted in the functional characterization of 39 distinct variants in the CHEK2 gene (Figure 2, Table 1, and see Table S1 in the supplemental information online) [16.Bell D.W. et al.Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts.Int. J. Cancer. 2007; 121: 2661-2667Crossref PubMed Scopus (62) Google Scholar,18.Chrisanthar R. et al.CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer.PLoS One. 2008; 3e3062Crossref PubMed Scopus (70) Google Scholar,21.Falck J. et al.The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.Nature. 2001; 410: 842-847Crossref PubMed Scopus (861) Google Scholar, 22.Kleiblova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797PubMed Google Scholar, 23.Lee S.B. et al.Destabilization of CHK2 by a missense mutation associated with Li–Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar,28.Wu X. et al.Characterization of tumor-associated Chk2 mutations.J. Biol. Chem. 2001; 276: 2971-2974Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. A second system used for functional analysis of CHEK2 variants relies on budding yeast S. cerevisiae strains that are null for RAD53 (and SML1 to rescue viability), which is the homolog of human CHEK2 [1.Matsuoka S. et al.Linkage of ATM to cell cycle regulation by the Chk2 protein kinase.Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar] and the functional analog of CHEK1 [35.Lanz M.C. et al.DNA damage kinase signaling: checkpoint and repair at 30 years.EMBO J. 2019; 38e101801Crossref PubMed Scopus (94) Google Scholar]. Expressing human wild-type CHEK2 cDNA in RAD53-null yeast strains rescued their slow growth phenotype, likely by restoring its functions in cell-cycle checkpoints [36.Zhao X. et al.The ribonucleotide reductase inhibitor Sml1 is a new target of the Mec1/Rad53 kinase cascade during growth and in response to DNA damage.EMBO J. 2001; 20: 3544-3553Crossref PubMed Scopus (225) Google Scholar]. Accordingly, this system efficiently distinguished the damaging effect of the truncating c.1100del;p.T367Mfs variant from wild-type CHEK2 because expression of the variant resulted in reduced growth compared with the wild-type control [25.Shaag A. et al.Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population.Hum. Mol. Genet. 2005; 14: 555-563Crossref PubMed Scopus (94) Google Scholar,26.Tischkowitz M.D. et al.Identification and characterization of novel SNPs in CHEK2 in Ashkenazi Jewish men with prostate cancer.Cancer Lett. 2008; 270: 173-180Crossref PubMed Scopus (14) Google Scholar]. This system was later adapted by treating the cells with the DNA damaging agent methyl methanesulfonate (MMS) [20.Delimitsou A. et al.Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system.Hum. Mutat. 2019; 40: 631-648Crossref PubMed Scopus (15) Google Scholar,24.Roeb W. et al.Response to DNA damage of CHEK2 missense mutations in familial breast cancer.Hum. Mol. Genet. 2012; 21: 2738-2744Crossref PubMed Scopus (53) Google Scholar], which results in cell-cycle arrest caused by the induction of stalled replication forks. Using this approach, two independent studies reported on the functional characterization of 132 distinct CHEK2 variants (Figure 2, Table 1, and Table S1). Specifically, 35 missense VUS which were identified in patients, two control deletion variants (p.E107_K197del and p.D265_H282del), and a catalytically dead variant (p.D347A) that impairs kinase activity [20.Delimitsou A. et al.Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system.Hum. Mutat. 2019; 40: 631-648Crossref PubMed Scopus (15) Google Scholar,24.Roeb W. et al.Response to DNA damage of CHEK2 missense mutations in familial breast cancer.Hum. Mol. Genet. 2012; 21: 2738-2744Crossref PubMed Scopus (53) Google Scholar] were classified as damaging. A third system for functional analysis relies on mammalian cell lines de
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