ClinVar Genomic variation as it relates to human health

Variant summary: CHEK2 c.1420C>T (p.Arg474Cys) results in a non-conservative amino acid change located in the protein kinase-like domain of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. This variant reportedly alters the tertiary structure of CHEK2 by disrupting the salt bridge between p.R474 and p.E394 suggesting that protein stability of both inactive and active states might be disturbed (Kukita_2016, Delimitsou_2019). The variant allele was found at a frequency of 1.7e-05 in 233784 control chromosomes (gnomAD). It has also been reported in the FLOSSIES database of women over the age of 70 with no history of cancer. c.1420C>T has been reported in the literature in individuals affected with a a variety of cancer types such as colorectal cancer, prostate cancer, breast cancer and lung cancer (e.g. Kukita_2016, Wu_2016, Schubert_2019), including two homozygous siblings who experienced multiple primary lung cancers as well as cancers in other organs (Kukita_2016). These data indicate that the variant may be associated with disease. The variant was determined to be damaging in an in vivo yeast functional assay (Delimitsou_2019). Kukita_2016 also found the variant expression protein did not increase nor activate in response to UV damage. Nine ClinVar submitters have assessed this variant since 2014: eight classified the variant as of uncertain significance and one as likely pathogenic. Some submitters cite overlapping evidence utilized in the context of this evaluation. Based on the evidence outlined above, the variant was classified as VUS-possibly pathogenic.

The p.R474C variant (also known as c.1420C>T), located in coding exon 12 of the CHEK2 gene, results from a C to T substitution at nucleotide position 1420. The arginine at codon 474 is replaced by cysteine, an amino acid with highly dissimilar properties. This alteration has been detected in the homozygous state in siblings with multiple primary lung cancers; the sister was also affected with breast cancer and uterine myoma and the brother was also affected with prostate and colon cancers (Kukita Y et al Cold Spring Harb. Mol. Case Stud. 2016 Nov;2(6):a001032). In addition, this alteration has been reported in the heterozygous state in multiple cohorts of affected patients with various tumor types (Wu Y et al. Prostate. 2018 Jun;78(8):607-615; Momozawa Y et al. Nat Commun, 2018 Oct;9:4083; Schubert S et al. Int. J. Cancer 2019 06;144(11):2683-2694; Xie Y et al. Clin. Genet. 2018 Jan;93(1):41-51; Tsaousis GN et al. BMC Cancer 2019 Jun;19(1):535; Dorling et al. N Engl J Med. 2021 02;384:428-439; Aksoy F et al. Hum Hered, 2022 Jan;:). This alteration behaved as non-functional in an in vivo, yeast-based growth rate assay (Delimitsou A et al. Hum. Mutat. 2019 05;40(5):631-648). In addition, this alteration was reported as functionally impaired in a study assessing CHEK2-complementation through quantification of KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells (Stolarova L et al. Clin Cancer Res. 2023 Aug;29(16):3037-3050). The p.R474C variant has been shown to eliminate a salt bridge between the core kinase domain and the highly conserved activation loop, and is critical for stability and function of Ser/Thr kinases (Ambry internal analysis; Cai Z et al. Mol. Cell. 2009 Sep;35:818-29; Yang J et al. J. Mol. Biol. 2012 Jan;415:666-79). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.

This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 474 of the CHEK2 protein (p.Arg474Cys). This variant is present in population databases (rs540635787, gnomAD 0.003%). This missense change has been observed in individual(s) with breast cancer, lung cancer, Lynch syndrome-associated cancer(s), and/or prostate cancer (PMID: 25980754, 27900359, 28580595, 29520813, 30287823, 30426508, 33309985). This variant is also known as c.1549C>T (p.Arg517Cys). ClinVar contains an entry for this variant (Variation ID: 128059). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Experimental studies have shown that this missense change affects CHEK2 function (PMID: 27900359, 30851065). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 27900359]. This variant is strongly associated with more severe personal and family histories of cancer, typical for individuals with pathogenic variants in this gene [PMID: 25085752].

The frequency of this variant in the general population, 0.000028 (3/108758 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in individuals with breast cancer (PMIDs: 30426508 (2018), 30287823 (2018), 28580595 (2018), and 33471991 (2021)), prostate cancer (PMID: 29520813 (2018)), colorectal cancer (PMID: 33309985 (2020)), as well as in healthy individuals (PMIDs: 32980694 (2020), 33309985 (2020), and 33471991 (2021)). Additionally, the variant was reported in an individual with suspected Lynch Syndrome (PMID: 25980754 (2015)). Yeast-based and mammalian studies have reported that this variant may have a deleterious effect on CHEK2 protein function. Further studies are needed to validate these findings (PMIDs: 27900359 (2016) and 30851065 (2019)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, we are unable to determine the clinical significance of this variant.

This missense variant replaces arginine with cysteine at codon 474 of the CHEK2 protein. This variant impacts a highly conserved arginine in the kinase domain of the protein (PMID: 15060014, 19782031). Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have reported that this variant may impact CHEK2 protein stability and DNA damage response (PMID: 27900359, 30851065). This variant has been reported in at least six individuals affected with breast cancer (PMID: 28580595, 29522266, 30287823, 30426508, 34991090) and in a breast cancer case-control meta-analysis in 6/60466 cases and 2/53461 unaffected individuals (PMID: 33471991; Leiden Open Variation Database DB-ID CHEK2_000093). This variant also has been reported in individuals affected with colorectal and prostate cancer (PMID: 25980754, 29520813, 33309985) and in an unaffected individual in a pancreatic cancer case-control study (PMID: 32980694). This variant has been identified in 4/233784 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

CHEK2: PS3, PM1, PM5:Supporting

Published functional studies demonstrate a damaging effect: decreased CHEK2 protein expression and impaired kinase activity (PMID: 27900359, 30851065, 37449874); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 25980754, 26506619, 22138346, 26094658, 19782031, 27900359, 29522266, 29520813, 28580595, 30426508, 29731985, 30851065, 30287823, 30858171, 31398194, 31159747, 33309985, 36061833, 34991090, 35661486, 32980694, 33471991, 36243179, 22419737, 37449874)

Two siblings, an elder sister and a younger brother, were found to have multiple primary lung cancers at the age of 60. The former subsequently developed breast cancer, and had a history of myoma uteri. The latter had initially developed prostate cancer at the age of 59, and had a history of colon cancer. This variant was homozygous in both patients.

Variant summary: CHEK2 c.1420C>T results in the replacement of Arg474 by a Cys residue (p.Arg474Cys). The variant was identified in 1 out of 396 patients analysed (freq: 0.0025, doi: 10.3389/fgene.2023.1274108). The patient was a woman that developed breast cancer at the age of 69 years (luminal-A tumor phenotype). Two second-degree relatives were also diagnosed with BC, although a co-segregation study could not be performed. Arg474 is located in the kinase domain and appears well conserved in vertebrate species. The replacement of Arg474 by a cysteine could affect the interaction with some residues at the dimerization interface, particularly with Ala392. It thus may affect the protein integrity and therefore its function. In addition, Arg474, forms a cation/π interaction with Trp411 (at the same monomer) and a salt bridge with Glu394 (at the partner monomer). Replacement of Arg474 with a cysteine residue will therefore remove the indicated stabilizing interactions of Arg474. The variant is reported in gnomAD v4 in 32 cases out of 1595950 alleles analysed (freq=2.0x10-3 %). In ClinVar a dozen of reports appear classifying the variant as of Uncertain Significance, whereas in 3 reports the submitters have suggested the classification of Likely Pathogenic. Other replacements found at this position (R474L, R474G, R474S and R474H) are classified by ClinVar as of either Uncertain Significance or Conflicting Interpretation. The metapredictor PirePred (relies on verdicts from other 15 predictor or metapredictor tools) and the AI-based predictor AlphaMissense classify Arg474Cys as Pathogenic, whereas the ACMG classification tool Franklin suggests this variant as Likely Pathogenic. Our in-silico study on the protein stability based on relaxation molecular dynamics simulations (doi: 10.3389/fgene.2023.1274108) indicates that Arg474Cys induces conformational unstability on CHEK2 protein. Moreover, Stolarova L. et al’s functional study on KAP1 phosphorylation and CHEK2 autophosphorylation protein capability reports this variant as functionally impaired (PMID: 37449874). In summary, we believe this variant have more chances of being Pathogenic.