Variants not meeting PVS1
We found 6 missense variants with discordant classifications of
pathogenicity in ClinVar (Table 1) in 13 unrelated patients. In
addition, one of the healthy (non-cancer) controls carried theCHEK2 c.349A>G, p.(Arg117Gly) variant. To better
interpret missense variants, a comprehensive review of previous
functional studies was done, main results are summarized in Table S3 and
Table S4. In a further effort to improve variant classification, after
classical ACMG we also followed the allele risk criteria reported
recently (Senol-Cosar et al., 2019). For this, we searched for
association studies of our CHEK2 variants (Table S2).
CHEK2c.190G>A, p.(Glu64Lys) is located in a weakly conserved
amino acid in the SQ/TQ cluster domain (SCD). It is predicted
deleterious by in silico analysis. It shows a partially reduced
phosphorylation by ATM at the Thr68 residue, as well as partially
reduced auto-phosphorylation and Cdc25C phosphorylation. It affects KAP1
phosphorylation and has discrepant results about DNA damage response
(Table S3). Furthermore, there are no high-quality case-control studies.
Therefore, this variant only meets PP3 criterion, remaining as VUS
(Table 1). Variant c.349A>G, p.(Arg117Gly) affects a highly
conserved amino acid (class C65 according to GVGD) in the FHA domain. It
is predicted deleterious by in silico analysis. It does not
affect phosphorylation by ATM nor oligomerization, but affects all the
rest of the studied protein
functions (Table S3). This variant
accomplished PS3, PS4_moderate, PM1 and PP3 criteria, being classified
as LP by all frameworks. It has been studied in a large high-quality
case-control study, reporting a BC OR of 2.26 (95% CI, 1.29-3.95)
(Table 1), therefore it could be considered as LRA within the
Senol-Cosar framework (Table S2). Variant c.433C>T,
p.(Arg145Trp) is located in a moderately conserved amino acid of the FHA
domain. It is predicted deleterious by in silico . It reduces CHK2
expression and stability. In functional assays, it has been consistently
reported to impair kinase and DNA repair
activity. Evidences for
classification includes PS3, PM1 and PP3, being classified as LP by all
frameworks. Variant c.470T>C, p.(Ile157Thr) lies in a
weakly conserved amino acid of the FHA domain. It is predicted
deleterious by in silico analysis. It has been widely studied,
nevertheless the functional assays reported to date show discordant
results (Table S3). The reported OR in the biggest CHEK2meta-analysis was 1.58 (95% CI, 1.42 - 1.75), therefore PS4_moderate
was applied, application of PP3 was not enough to classify this variant
as LP/P. However, following recommendations from Senol-Cosar et al, it
would be an ERA due to the existence of multiple case-control studies
(Senol-Cosar et al., 2019). Variant c.499G>A, p.(Gly167Arg)
is located in a highly conserved amino acid of the FHA domain. It is
predicted deleterious by in silico analysis. Although there are
only 2 functional studies, they both reported an impaired DNA repair
activity in yeast assays (Table S3). PS3, PM1, PM2_supporting and PP3
were assigned, being classified as LP by all frameworks. Variant
c.1427C>T, p.(Thr476Met) lies in a moderately conserved
amino acid of the kinase domain. It is predicted deleterious by in
silico analysis. Functional assessment of KAP1 phosphorylation results
deleterious in vitro and good enough in vivo . Furthermore,
SOX phosphorylation was reported equal to that of the pathogenic
c.1100delC variant. Assays on DNA repair activity have found it damaging
or with intermediate activity (Table S3). Due to these discordant
functional assay results PS3 was not weighted. Classification remained
as VUS since c.1427C>T only accomplished PP3.