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CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function

2024; Elsevier BV; Volume: 300; Issue: 3 Linguagem: Inglês

10.1016/j.jbc.2024.105767

ISSN

1083-351X

Autores

Carolina Pires, Inês J. Marques, Mariana Valério, Ana Saramago, Paulo E. Santo, Sandra P. Santos, Margarida Silva, Margarida M. Moura, João Matos, Teresa Pereira, Rafael Cabrera, Diana Lousa, Valeriano Leite, Tiago M. Bandeiras, João B. Vicente, Branca Cavaco,

Tópico(s)

Genetic factors in colorectal cancer

Resumo

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation. Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation. Thyroid cancer (TC) is the most common endocrine malignancy, accounting for ∼3% of all cancer diagnoses worldwide (1Ferlay J. Colombet M. Soerjomataram I. Parkin D.M. Piñeros M. Znaor A. et al.Cancer statistics for the year 2020: an overview.Int. J. Cancer. 2021; 149: 778-789Crossref Scopus (2431) Google Scholar). It continues to be the most rapidly increasing cancer type, with a male/female ratio of 1:3 (2Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2017.CA Cancer J. Clin. 2017; 67: 7-30Crossref PubMed Scopus (13851) Google Scholar). Most thyroid tumors (approximately 95%) are derived from the thyroid follicular cells and are designated as nonmedullary thyroid cancers (NMTC) (3Moretti F. Nanni S. Pontecorvi A. Molecular pathogenesis of thyroid nodules and cancer.Baillieres Best Pract. Res. Clin. Endocrinol. Metab. 2000; 14: 517-539Crossref PubMed Scopus (55) Google Scholar). Although the majority of these tumors are sporadic, 5 to 15% of all follicular cell-derived thyroid tumors are familial, being designated as familial nonmedullary thyroid cancers (FNMTC). In FNMTC families, patients frequently present TC together with benign lesions [e.g., thyroid follicular nodular disease (FND) and follicular thyroid adenomas], with papillary thyroid cancer (PTC) being the most frequent subtype (4Pinto A.E. Silva G.L. Henrique R. Menezes F.D. Teixeira M.R. Leite V. et al.Familial vs sporadic papillary thyroid carcinoma: a matched-case comparative study showing similar clinical/prognostic behaviour.Eur. J. Endocrinol. 2014; 170: 321-327Crossref PubMed Scopus (40) Google Scholar). FNMTC can be divided into two groups based on clinical characteristics: syndromic and nonsyndromic. Despite being rare, the genetic alterations underlying syndromic FNMTC are well-defined (5Klubo-Gwiezdzinska J. Kushchayeva Y. Gara S.K. Kebebew E. Familial non-medullary thyroid cancer.in: Mallick U.K. Harmer C. Practical Management of Thyroid Cancer. Springer International Publishing, Cham, Switzerland2018: 241-270Crossref Scopus (6) Google Scholar). Conversely, the genetic background of nonsyndromic FNMTC and its association with clinical behavior is currently controversial and not well understood (6Guilmette J. Nosé V. Hereditary and familial thyroid tumours.Histopathology. 2018; 72: 70-81Crossref PubMed Scopus (61) Google Scholar). Nonsyndromic FNMTC has been clinically defined in the World Health Organization classification by the presence of follicular cell-derived TC in at least three first-degree relatives, or by the existence of PTC in two or more first-degree relatives, in the absence of a history of previous radiation or inherited cancer syndrome (7Nosé V. Gill A. Teijeiro J.M.C. Perren A. Erickson L. Overview of the 2022 WHO classification of familial endocrine tumor syndromes.Endocr. Pathol. 2022; 33: 197-227Crossref PubMed Scopus (23) Google Scholar). In recent years, several approaches have been undertaken to better understand FNMTC's etiology. Genome-wide association studies, linkage analyses, targeted, and whole exome/genome sequencing led to the identification of several chromosomal loci [MNG1 (14q32), TCO (19p13.2), NMTC1 (2q21), FTEN (8p23.1-p22), PRN (1q21), 6q22, 8q24, and 12q14] (8Navas-Carrillo D. Ríos A. Rodríguez J.M. Parrilla P. Orenes-Piñero E. Familial nonmedullary thyroid cancer: screening, clinical, molecular and genetic findings.Biochim. Biophys. Acta. 2014; 1846: 468-476PubMed Google Scholar), as well as predisposing risk variants in genes such as DICER1, SRGAP1, NKX2-1, FOXE1, SRRM2, RTFC, HABP2, MYO1F, MAP2K5 and, more recently, SPRY4 (9Rio Frio T. Bahubeshi A. Kanellopoulou C. Hamel N. Niedziela M. Sabbaghian N. et al.DICER1 mutations in familial multinodular goiter with and without ovarian Sertoli-Leydig cell tumors.JAMA. 2011; 305: 68-77Crossref PubMed Scopus (251) Google Scholar, 10He H. Bronisz A. Liyanarachchi S. Nagy R. Li W. Huang Y. et al.SRGAP1 is a candidate gene for papillary thyroid carcinoma susceptibility.J. Clin. Endocrinol. Metab. 2013; 98: 973-980Crossref PubMed Scopus (66) Google Scholar, 11Diquigiovanni C. Bergamini C. Evangelisti C. Isidori F. Vettori A. Tiso N. et al.Mutant MYO1F alters the mitochondrial network and induces tumour proliferation in thyroid cancer.Int. J. Cancer. 2018; 143: 1706-1719Crossref PubMed Scopus (31) Google Scholar, 12Ngan E.S. Lang B.H. Liu T. Shum C.K. So M.T. Lau D.K. et al.A germline mutation (A339V) in thyroid transcription factor-1 (TITF-1/NKX2. 1) in patients with multinodular goiter and papillary thyroid carcinoma.J. Natl. Cancer Inst. 2009; 101: 162-175Crossref PubMed Scopus (95) Google Scholar, 13Pereira J.S. da Silva J.G. Tomaz R.A. Pinto A.E. Bugalho M.J. Leite V. et al.Identification of a novel germline FOXE1 variant in patients with familial non-medullary thyroid carcinoma (FNMTC).Endocrine. 2015; 49: 204-214Crossref PubMed Scopus (52) Google Scholar, 14Gara S.K. Jia L. Merino M.J. Agarwal S.K. Zhang L. Cam M. et al.Germline HABP2 mutation causing familial nonmedullary thyroid cancer.N. Engl. J. Med. 2015; 373: 448-455Crossref PubMed Scopus (114) Google Scholar, 15Tomsic J. Fultz R. Liyanarachchi S. He H. Senter L. de la Chapelle A. HABP2 G534E variant in papillary thyroid carcinoma.PLoS One. 2016; 11e0146315Crossref PubMed Scopus (29) Google Scholar, 16Liu C. Yu Y. Yin G. Zhang J. Wen W. Ruan X. et al.C14orf93 (RTFC) is identified as a novel susceptibility gene for familial nonmedullary thyroid cancer.Biochem. Biophys. Res. Commun. 2017; 482: 590-596Crossref PubMed Scopus (14) Google Scholar, 17Ye F. Gao H. Xiao L. Zuo Z. Liu Y. Zhao Q. et al.Whole exome and target sequencing identifies MAP2K5 as novel susceptibility gene for familial non-medullary thyroid carcinoma.Int. J. Cancer. 2018; 144: 1321-1330Crossref PubMed Scopus (34) Google Scholar, 18Marques I.J. Gomes I. Pojo M. Pires C. Moura M.M. Cabrera R. et al.Identification of SPRY4 as a novel candidate susceptibility gene for familial non-medullary thyroid cancer.Thyroid. 2021; 31: 1366-1375Crossref PubMed Scopus (8) Google Scholar). Additionally, germline variants in DNA repair related-genes, namely BRCA1/2, ATM, CHEK2, and MSH6 have been recently reported in cases with FNMTC and NMTC (19Yu Y. Dong L. Li D. Chuai S. Wu Z. Zheng X. et al.Targeted DNA sequencing detects mutations related to susceptibility among familial non-medullary thyroid cancer.Sci. Rep. 2015; 516129Google Scholar, 20Fahiminiya S. de Kock L. Foulkes W.D. Biologic and clinical perspectives on thyroid cancer.N. Engl. J. Med. 2016; 375: 2306-2307Crossref PubMed Scopus (52) Google Scholar, 21Wang Y. Liyanarachchi S. Miller K.E. Nieminen T.T. Comiskey Jr., D.F. Li W. et al.Identification of rare variants predisposing to thyroid cancer.Thyroid. 2019; 29: 946-955Crossref PubMed Scopus (37) Google Scholar, 22Zhao Y. Yu T. Chen L. Xie D. Wang F. Fu L. et al.A germline CHEK2 mutation in a family with papillary thyroid cancer.Thyroid. 2020; 30: 924-930Crossref PubMed Scopus (17) Google Scholar, 23Pires C. Marques I.J. Dias D. Saramago A. Leite V. Cavaco B.M. A pathogenic variant in CHEK2 shows a founder effect in Portuguese Roma patients with thyroid cancer.Endocrine. 2021; 73: 588-597Crossref PubMed Scopus (3) Google Scholar, 24Srivastava A. Giangiobbe S. Skopelitou D. Miao B. Paramasivam N. Diquigiovanni C. et al.Whole genome sequencing prioritizes CHEK2, EWSR1, and TIAM1 as possible predisposition genes for familial non-medullary thyroid cancer.Front. Endocrinol. 2021; 12600682Crossref PubMed Scopus (13) Google Scholar, 25Kamihara J. Zhou J. LaDuca H. Wassner A.J. Dalton E. Garber J.E. et al.Germline pathogenic variants in cancer risk genes among patients with thyroid cancer and suspected predisposition.Cancer Med. 2022; 11: 1745-1752Crossref PubMed Scopus (3) Google Scholar), suggesting a role for these genes in FNMTC pathogenesis. Despite these findings, the molecular basis of FNMTC remains largely unknown, since these genes are only mutated in a small fraction of the families. As such, for the nonsyndromic families, genetic testing is still not covered by guidelines for diagnostic, therapeutic, and follow-up decisions (26de Mello L.E.B. Carneiro T.N.R. Araujo A.N. Alves C.X. Galante P.A.F. Buzatto V.C. et al.Identification of NID1 as a novel candidate susceptibility gene for familial non-medullary thyroid carcinoma using whole-exome sequencing.Endocr. Connect. 2022; 11e210406Crossref PubMed Scopus (2) Google Scholar, 27Kamani T. Charkhchi P. Zahedi A. Akbari M.R. Genetic susceptibility to hereditary non-medullary thyroid cancer.Hered. Cancer Clin. Pract. 2022; 20: 1-19Crossref PubMed Scopus (11) Google Scholar). In this work, we used a commercial panel to analyze 94 genes associated with cancer predisposition, in two Portuguese families with FNMTC. This approach allowed the identification of two likely pathogenic variants in the CHEK2 gene. CHEK2 is a tumor suppressor gene that encodes the serine/threonine protein kinase CHK2, which is a key mediator of the DNA damage checkpoint that responds to DNA double-strand breaks, playing a crucial role in the maintenance of genomic integrity (28Zannini L. Delia D. Buscemi G. CHK2 kinase in the DNA damage response and beyond.J. Mol. Cell. Biol. 2014; 6: 442-457Crossref PubMed Scopus (281) Google Scholar). Mutations in CHEK2 not only exist in subsets of sporadic cancers, but they also predispose patients to several types of familial cancers (29Cybulski C. Gorski B. Huzarski T. Masojc B. Mierzejewski M. Debniak T. et al.CHEK2 is a multiorgan cancer susceptibility gene.Am. J. Hum. Genet. 2004; 75: 1131-1135Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), with increasing evidence emerging in the context of FNMTC (21Wang Y. Liyanarachchi S. Miller K.E. Nieminen T.T. Comiskey Jr., D.F. Li W. et al.Identification of rare variants predisposing to thyroid cancer.Thyroid. 2019; 29: 946-955Crossref PubMed Scopus (37) Google Scholar, 22Zhao Y. Yu T. Chen L. Xie D. Wang F. Fu L. et al.A germline CHEK2 mutation in a family with papillary thyroid cancer.Thyroid. 2020; 30: 924-930Crossref PubMed Scopus (17) Google Scholar, 23Pires C. Marques I.J. Dias D. Saramago A. Leite V. Cavaco B.M. A pathogenic variant in CHEK2 shows a founder effect in Portuguese Roma patients with thyroid cancer.Endocrine. 2021; 73: 588-597Crossref PubMed Scopus (3) Google Scholar, 24Srivastava A. Giangiobbe S. Skopelitou D. Miao B. Paramasivam N. Diquigiovanni C. et al.Whole genome sequencing prioritizes CHEK2, EWSR1, and TIAM1 as possible predisposition genes for familial non-medullary thyroid cancer.Front. Endocrinol. 2021; 12600682Crossref PubMed Scopus (13) Google Scholar, 25Kamihara J. Zhou J. LaDuca H. Wassner A.J. Dalton E. Garber J.E. et al.Germline pathogenic variants in cancer risk genes among patients with thyroid cancer and suspected predisposition.Cancer Med. 2022; 11: 1745-1752Crossref PubMed Scopus (3) Google Scholar). In this study, the identified CHK2 variants were structurally and functionally characterized, using biophysical methods and molecular dynamics (MD) simulations. In the present study, a screening for germline variants in 94 cancer predisposing genes was conducted in the DNA of the probands from two FNMTC families [Family 24 (F24), individual II.1; Family 83 (F83), individual II.2] (Fig. 1), through NGS analysis, using the TruSight Cancer Kit. Following NGS, bioinformatics analysis was carried out. A total of 805 variants were detected in the two samples. Thus, in order to select the potentially pathogenic variants, specific criteria and filters were applied, revealing two germline checkpoint kinase 2 (CHEK2) (NM_007194.4) missense variants: c.470T > C, p.I157T (exon 4) detected in F24, and c.962A > C, p.E321A (exon 9) in F83 (NGS data bioinformatics analyses detailed in the Supporting information). Both variants were confirmed by Sanger sequencing. Segregation analysis in Family F83 showed that the CHEK2 variant segregated with TC, thyroid FND, and no otherwise specified thyroid nodule(s) (NOS-TN) in the family. The variant was detected in heterozygosity in the proband (II.2; PTC), her two sisters [one with PTC (II.4) and one with NOS-TN (II.5)], and sons (III.1 and III.2; FND and NOS-TN, respectively). It was not possible to investigate the presence of the variant in the proband's mother (I.2), who presented FND and ovarian cancer, since she was already deceased. In family F24, the CHEK2 variant p.I157T segregated with TC but not with the benign/NOS thyroid lesions. The variant was found in heterozygosity in the proband (II.1) and his sister (II.6), both with PTC, but not in the siblings with FND (II.8) and a NOS-TN (II.4). Further segregation analysis was not possible due to families' small size. Both CHEK2 variants were absent in 100 Portuguese healthy controls. Loss of heterozygosity (LOH) is a common genetic event in cancer development and is known to be involved in the somatic inactivation of WT alleles from tumor suppressor genes, in many inherited cancer syndromes. To investigate the second hit, the presence of LOH involving CHEK2 was evaluated in available benign and malignant thyroid lesions from CHEK2 variant carriers (F24: II.1 and II.6; F83: II.2 and II.4). However, no pattern suggestive of LOH was detected, as the germline variants were found in heterozygosity in the patients' tumors (data not shown). In addition, NGS analysis of the thyroid cancer (PTC) from patient II.4 from family F83 showed that there were no somatic CHEK2 variants. This indicates that other mechanisms may be underlying tumor development in these cases (e.g., loss-of-function possibly leading to haploinsufficiency or dominant-negative effects). The expression of the CHEK2 gene, both at protein and mRNA level in normal thyroid gland tissues, are defined as "medium" in The Human Protein Atlas database (Table 1). The potential functional consequences of CHEK2 candidate variants identified in this study were further investigated, adding MetaLR, Mutation Assessor and REVEL to the common set of in silico prediction tools (SIFT, PolyPhen, MutationTaster). Prediction of the substitutions' effects on protein function ranged from low effect to deleterious, for the two CHEK2 variants (Table 1). The variants are located in relevant CHK2 regions: the I157T substitution is located in the forkhead-associated (FHA) functional domain and the E321A in the kinase domain, where most pathogenic CHEK2 variants are located (mutational hotspots) (30Sutcliffe E.G. Stettner A.R. Miller S.A. Solomon S.R. Marshall M.L. Roberts M.E. et al.Differences in cancer prevalence among CHEK2 carriers identified via multi-gene panel testing.Cancer Genet. 2020; 246: 12-17Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) (Fig. S1A). Both variants are present in population databases, however, p.E321A (rs374395284) is extremely rare (Table 1), whereas p.I157T (rs17879961) has a higher minor allele frequency, being reported in 0.4% of the European population. Comparative sequence analysis of CHK2 showed that amino acid E321 is highly conserved among the selected species (Fig. S1B), whereas the I157 position is conserved in mammals, but not in more distantly related organisms (e.g., Danio rerio), which may suggest that a change in this amino acid may have a less relevant biological role. The tolerance of amino acid substitutions at and around positions 157 and 321 of CHK2 was predicted by SNAP2 (31Hecht M. Bromberg Y. Rost B. Better prediction of functional effects for sequence variants.BMC Genomics. 2015; 16: 1-12Crossref PubMed Google Scholar) and presented as a heat-map. The isoleucine to threonine substitution (I157T), a hydrophobic to polar uncharged change, is predicted to have a neutral effect (score −23; Fig. S1C), while the change from a glutamic acid to alanine (E321A), a large acidic residue to a small hydrophobic one, is predicted to alter the native protein function (score +56; Fig. S1C). Both p.E321A and p.I157T variants are described in the ClinVar database, being respectively classified as variant of uncertain significance (VUS) (ClinVar ID: 232111) and variant with conflicting interpretations of pathogenicity (ClinVar ID: 5591), whereas the data aggregator VarSome assigned the same classification to p.E321A and classified p.I157T as likely pathogenic (Table 1). The p.I157T variant has been extensively described in the literature, in different cancer types, including breast, colon, prostate, gastric, renal, and TCs (32Stolarova L. Kleiblova P. Janatova M. Soukupova J. Zemankova P. Macurek L. et al.CHEK2 germline variants in cancer predisposition: stalemate rather than checkmate.Cells. 2020; 9: 2675Crossref PubMed Scopus (93) Google Scholar). However, the interpretation of these findings is conflicting, as they depend on the population (33Kilpivaara O. Vahteristo P. Falck J. Syrjäkoski K. Eerola H. Easton D. et al.CHEK2 variant I157T may be associated with increased breast cancer risk.Int. J. Cancer. 2004; 111: 543-547Crossref PubMed Scopus (137) Google Scholar, 34Kilpivaara O. Alhopuro P. Vahteristo P. Aaltonen L.A. Nevanlinna H. CHEK2 I157T associates with familial and sporadic colorectal cancer.J. Med. Genet. 2006; 43: e34Crossref PubMed Scopus (67) Google Scholar, 35Leedom T.P. LaDuca H. McFarland R. Li S. Dolinsky J.S. Chao E.C. Breast cancer risk is similar for CHEK2 founder and non-founder mutation carriers.Cancer Genet. 2016; 209: 403-407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 36Konstantinova D.V. Kadiyska T.K. Kaneva R.P. Tosheva E.G. Guseva V.T. Dimitrov B.H. et al.CHEK2 I157T and endometrial cancer.DNA Cell Biol. 2009; 28: 9-12Crossref PubMed Scopus (7) Google Scholar, 37Kleibl Z. Havranek O. Hlavata I. Novotny J. Sevcik J. Pohlreich P. et al.The CHEK2 gene I157T mutation and other alterations in its proximity increase the risk of sporadic colorectal cancer in the Czech population.Eur. J. Cancer. 2009; 45: 618-624Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 38Lener M.R. Kashyap A. Kluźniak W. Cybulski C. Soluch A. Pietrzak S. et al.The prevalence of founder mutations among individuals from families with familial pancreatic cancer syndrome.Cancer Res. Treat. 2017; 49: 430-436Crossref PubMed Scopus (17) Google Scholar), also, the variant cosegregation with the disease in the families is variable (39Roeb W. Higgins J. King M.C. Response to DNA damage of CHEK2 missense mutations in familial breast cancer.Hum. Mol. Genet. 2012; 21: 2738-2744Crossref PubMed Scopus (64) Google Scholar), and the previous functional studies insights are not consensual (39Roeb W. Higgins J. King M.C. Response to DNA damage of CHEK2 missense mutations in familial breast cancer.Hum. Mol. Genet. 2012; 21: 2738-2744Crossref PubMed Scopus (64) Google Scholar, 40Lee S.B. Kim S.H. Bell D.W. Wahrer D.C. Schiripo T.A. Jorczak M.M. et al.Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome.Cancer Res. 2001; 61: 8062-8067PubMed Google Scholar, 41Falck J. Lukas C. Protopopova M. Lukas J. Selivanova G. Bartek J. Functional impact of concomitant versus alternative defects in the Chk2-p53 tumour suppressor pathway.Oncogene. 2001; 20: 5503-5510Crossref PubMed Scopus (97) Google Scholar, 42Schwarz J.K. Lovly C.M. Piwnica-Worms H. Regulation of the Chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation.Mol. Cancer Res. 2003; 1: 598-609PubMed Google Scholar, 43Delimitsou A. Fostira F. Kalfakakou D. Apostolou P. Konstantopoulou I. Kroupis C. et al.Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system.Hum. Mutat. 2019; 40: 631-648Crossref PubMed Scopus (29) Google Scholar, 44Kleiblova P. Stolarova L. Krizova K. Lhota F. Hojny J. Zemankova P. et al.Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.Int. J. Cancer. 2019; 145: 1782-1797Crossref PubMed Scopus (57) Google Scholar). On the other hand, the p.E321A variant has not been published in the literature, in spite of being already described in the context of hereditary breast cancer in databases (ClinVar ID: 232111) (Table 1). Altogether, compared to p.E321A, the p.I157T variant is more frequent in the population, less conserved in evolution, and already has variable and contradictory descriptions regarding its pathogenicity.Table 1In silico characterization of the two candidate CHEK2 variants identified by NGS in families F24 and F83FamilyGene(RefSeq Transcript)Chromosomal positon (GRCh38)Nucleotide (CDS)Amino acid (protein)dbSNP IDMAF (%)In silico prediction of variant effect in protein functionAnnotations in databasesExpression in normal thyroidACMG classificationFinalbFinal ACMG classification supported by the results obtained in the present study.GnomAD/ALFA(NFE/European)SIFTPolyPhenMutation TasterMetaLRMutation AssessorREVELClinVarVarsomemRNA/ProteinaThe Human Protein Atlas database.F24CHEK2(NM_007194.4)22: 28725099c.470T>Cp.I157Trs178799610.2/0.4UncertainDeleteriousDeleteriousDeleteriousLowUncertainClinVar ID: 5591Conflicting interpretations of pathogenicity/risk factor:Pathogenic(5); Likely pathogenic(13); Pathogenic, low penetrance(1); Established risk allele(1); Uncertain significance(9)Likely pathogenicMedium/MediumLikely pathogenicF83CHEK2(NM_007194.4)22: 28699884c.962A>Cp.E321Ars3743952840.0007/0UncertainDeleteriousDeleteriousBenignMediumUncertainClinVar ID: 232111 Uncertain significance(9)Uncertain significanceMedium/MediumLikely pathogenicAbbreviations: ACMG, American College of Medical Genetics and Genomics; CDS, coding DNA sequence; MAF, minor allele frequency; NFE, Non-Finish European.Gene, transcript and chromosomal positions were taken from Ensembl build 38.a The Human Protein Atlas database.b Final ACMG classification supported by the results obtained in the present study. Open table in a new tab Abbreviations: ACMG, American College of Medical Genetics and Genomics; CDS, coding DNA sequence; MAF, minor allele frequency; NFE, Non-Finish European. Gene, transcript and chromosomal positions were taken from Ensembl build 38. In this study, in order to further clarify the role of the two missense CHEK2 variants in the development of TC, and to extend the present knowledge beyond that already available from in silico, databases and literature data, we used biophysical and MD approaches for functional and structural characterization of the encoded proteins. All recombinant truncated CHK2 proteins (according to (45Cai Z. Chehab N.H. Pavletich N.P. Structure and activation mechanism of the CHK2 DNA damage checkpoint kinase.Mol. Cell. 2009; 35: 818-829Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar); represented in Fig. S1A) were successfully purified, with yields ranging from 1.2 to 26 mg of purified monomeric protein per liter of culture. The CHK2 p.E321A variant exhibited a higher tendency to precipitate during the purification process and resulted in a significant amount of higher oligomeric forms or soluble aggregates in the preparative size exclusion chromatography (SEC), as compared to the expected functional oligomeric form (data not shown). Oligomeric profiles, monitored by analytical SEC, were similar for all proteins, exhibiting one major peak (shown in Fig. S2 for WT CHK2) corresponding to a 65 kDa-protein, ∼30% higher than the expected molecular mass for a truncated monomer. Further analyses were performed using exclusively this oligomeric form. The ADP-Glo Kinase assay was performed to measure CHK2 proteins (WT, p.E321A, and p.I157T) kinase activity by quantifying the amount of ADP produced during the kinase reaction, using a peptide derived from CDC25 as the client phosphorylation target. Using equal enzyme and peptide substrate concentrations, but varying the ATP concentration, we observed that the WT protein presented a higher enzymatic activity when compared to both variants, regardless of the ATP concentration (Fig. S3). At saturating ATP, the CHK2 p.I157T and p.E321A variants presented 40 to 50% of the WT CHK2 enzymatic activity (Fig. 2). CHK2 WT, p.I157T, and p.E321A exhibited similar far-UV circular dichroism (CD) spectra (Fig. 3, A–C), indicating no significant changes in the content of secondary structure elements. The obtained spectra displaying a local minimum centered at ∼210 nm and a broader band at higher λ indicated a predominantly α-helical structure with contribution from β sheets, consistent with the reported 3D structures for truncated CHK2 (45Cai Z. Chehab N.H. Pavletich N.P. Structure and activation mechanism of the CHK2 DNA damage checkpoint kinase.Mol. Cell. 2009; 35: 818-829Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). To evaluate the effect of the substitutions on protein stability, we obtained thermal denaturation profiles by monitoring the CD at 222 nm (Fig. 3D). A decrease in CD at 222 nm signal to more negative values was observed, unlike what was expected for thermal unfolding of the α-helices. By taking the spectrum of fully denatured CHK2 at 90 °C, we could observe that the predominantly α-helical spectrum in native CHK2 was converted into a broad band centered at 218 nm (Fig. 3, A–C), indicative of parallel β sheets, such as those occurring in amyloid fibrils, insoluble cytotoxic protein aggregates. Upon cooling the denatured protein back to 20 °C, the spectrum remained nearly identical to the 90 °C spectrum, indicating irreversible thermal unfolding (Fig. 3, A–C). Irrespective of the final thermally denatured structure, the melting curves (presented as ascending curves for simplicity) yielded a single transition for all proteins fitted with a monophasic sigmoidal curve (Fig. 3D). The corresponding Tm values are presented in Table 2, with the WT protein exhibiting a slightly higher Tm value (by +1.1 to 1.7 °C) when compared to the two variants. Resistance to thermal denaturation was also evaluated by differential scanning fluorimetry (DSF) (indirect method). Thermal denaturation profiles of the three studied proteins presented a single appa

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