ClinVar Genomic variation as it relates to human health

The p.T378I variant (also known as c.1133C>T), located in coding exon 10 of the CHEK2 gene, results from a C to T substitution at nucleotide position 1133. The threonine at codon 378 is replaced by isoleucine, an amino acid with similar properties. This alteration behaved as functional in an in vivo, yeast-based growth rate assay (Delimitsou A et al. Hum Mutat, 2019 05;40:631-648). This amino acid position is well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.

This sequence change replaces threonine, which is neutral and polar, with isoleucine, which is neutral and non-polar, at codon 378 of the CHEK2 protein (p.Thr378Ile). This variant is present in population databases (rs587780167, gnomAD 0.01%). This missense change has been observed in individual(s) with breast cancer (PMID: 34282249). ClinVar contains an entry for this variant (Variation ID: 128046). 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 does not substantially affect CHEK2 function (PMID: 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 missense variant replaces threonine with isoleucine at codon 378 in the kinase domain of the CHEK2 protein. The reference threonine residue has been shown to be phosphorylated in response to DNA damage stimuli (PMID: 17698850, 20713355) and to play a role in regulating ionizing radiation-induced kinase activity (PMID: 20713355). Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). A functional study has shown the mutant protein to be functional in DNA damage repair assay in yeast (PMID: 30851065). In a large breast cancer case-control study, this variant was observed in 9/60466 cases and 7/53461 unaffected controls (OR=1.137, 95%CI 0.423 to 3.053, p-value=1) (PMID: 33471991, Leiden Open Variation Database DB-ID CHEK2_000355). This variant has been identified in 13/250714 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.

Variant summary: CHEK2 c.1133C>T (p.Thr378Ile) results in a non-conservative amino acid change located in the Protein kinase domain (IPR000719) of the encoded protein sequence. Three of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 5.2e-05 in 250714 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in CHEK2 causing Hereditary Breast And Ovarian Cancer Syndrome (5.2e-05 vs 0.00031), allowing no conclusion about variant significance. c.1133C>T has been reported in the literature in unaffected controls and individuals with breast cancer (example, Weitzel_2019, Dorling_2021/LOVD database, Stolarova_2023). These report(s) do not provide unequivocal conclusions about association of the variant with Hereditary Breast And Ovarian Cancer Syndrome. A co-occurrence with a pathogenic variant has been reported (BRIP1 c.2108delinsTCC, p.Lys703Ilefs*3 in Helgadottir_2021), providing supporting evidence for a benign role. In a functional study, the variant results in reduced KAP1 phosphorylation and CHK2 autophosphorylation activity (35.8% and 47.5% versus wild type, respectively) in vitro in a human CHEK2-knockout background (example, Stolarova_2023). A further study showed no damaging effect of this variant in an in vivo yeast based assay comparing growth rates of each strain with positive and negative controls, however the relevance of this assay with respect to CHEK2 function in humans is unclear (example, Delimitsou_2019). The following publications have been ascertained in the context of this evaluation (PMID: 30851065, 33471991, 34282249, 37449874, 31206626). ClinVar contains an entry for this variant (Variation ID: 128046). Based on the evidence outlined above, the variant was classified as uncertain significance.

This variant is classified as a variant of uncertain significance as there is insufficient evidence to determine its impact on protein function and/or cancer risk.

The frequency of this variant in the general population, 0.00012 (4/34568 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. This variant has been reported in individuals affected with breast cancer and unaffected controls (PMIDs: 30851065 (2019) and 33471991 (2021), see also LOVD (http://databases.lovd.nl/shared/genes/CHEK2)). A yeast-based functional study has indicated that this variant maintains CHEK2 protein function (PMID: 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 conflicting predictions that this variant is deleterious or benign. Based on the available information, we are unable to determine the clinical significance of this variant.

Published functional studies demonstrate DNA damage response and cell growth comparable to wild type in yeast-based assays but impaired kinase activity and auto-phosphorylation in human-cell based studies (PMID: 30851065, 37449874); Observed in individuals with breast cancer and in unaffected controls (PMID: 31206626, 33471991, 34282249, 37449874); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 33471991, 30851065, 31206626, 34282249, 37449874, 22419737, 19782031, 20713355, 17698850)

The CHEK2 c.1133C>T variant is predicted to result in the amino acid substitution p.Thr378Ile. This variant was reported in an individual with breast cancer in the presence of a pathogenic variant in a different gene (Helgadottir et al. 2021. PubMed ID: 34282249). However, this variant has also been documented in controls (Table S3, Weitzel et al. 2019. PubMed ID: 31206626). An in vivo, yeast-based growth rate assay showed this variant behaved similar to wild type CHEK2 (Delimitsou et al. 2019. PubMed ID: 30851065). This variant is reported in 0.012% of alleles in individuals of Latino descent in gnomAD and has conflicting interpretations in ClinVar of likely benign and uncertain (https://www.ncbi.nlm.nih.gov/clinvar/variation/128046/). Although we suspect that this variant may be benign, at this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence.