DISCUSSION
We have made an effort to classify variants in the low-moderate
penetrance CHEK2 gene. For that, we analyzed the whole coding
region of CHEK2 in a large HC cohort, performed in-depth
literature review and have defined specific cut-offs for ACMG criteria
to allow classification of variants with low effect. Furthermore, we
applied different combinatorial rules that enabled us to compare
classification rates. Concluding that the Bayesian model is the most
optimal framework to classify variants to a greater extent.
From our experience in variant classification and after a comprehensive
literature review, we propose two adaptations of the ACMG criteria.
Regarding PS4 we propose to score PS4_moderate for low-moderate
penetrant genes if an OR is given between 1.5 and 5, with a p value of
<0.01, when the phenotype is in accordance with the previously
described. In relation to PM2 evidence, in our laboratory we use an
extremely conservative approach and assign PM2 only if the variant is
absent or present in less than 1 out of 100,000 alleles in gnomAD
(0.001% of maximum frequency) for high penetrant genes. However, we
propose to assign PM2_supporting when the variant is ≤1 out of 20,000
alleles.
Variants meeting PVS1 criterion tend to be easier to classify as LP/P.
For instance, the founder mutation c.1100delC is the most studiedCHEK2 mutation and it has a prevalence of 0.26% in NFE
population. CHEK2 c.1100delC has a moderate penetrance
(Meijers-Heijboer et al., 2002; Oldenburg et al., 2003), conferring an
increased BC risk for overall population (OR= 2.89, 95% CI, 2.63–3.16)
(Liang et al., 2018) and for carriers with familial BC (OR= 3.21, 95%
CI, 2.41-4.29) (Liang et al., 2018). It has been reported absent in
Spanish population (Bellosillo et al., 2005), or with frequencies of
0.93% in Basque population, 0.36% in Galician population and 0.3% in
a study of BRCA -negative HBC Basque and Catalan families (Fachal,
Santamariña, Blanco, Carracedo, & Vega, 2013; Gutiérrez-Enríquez,
Balmaña, Baiget, & Díez, 2008; Martínez-Bouzas et al., 2007). In our
larger cohort, only one case was identified (0.08%, 1 out of 1,251 BC
affected cases), confirming its low prevalence in our population.
Moreover, in a recent study analyzing 15 truncating CHEK2variants in 213 patients and 29 control carriers, the BC risk OR was
3.11 (95% CI, 2.15-4.69) (Decker et al., 2017). Here we identified 10
proband carriers of truncating variants, 8 of which developed the first
tumor before the age of 50, consistent with previous findings of early
cancer development in carriers of truncated variants (Decker et al.,
2017; Han et al., 2013). Nonetheless, the median age at first cancer
diagnosis in our study was not very different amongst carriers of
truncating and missense LP/P variants, being 42 (range 25-65) and 40
(range 22-51) years, respectively. Bilateral BC has been mainly reported
in c.1100delC carriers (M Kriege & J M Collée, 2014), and truncating
variants in this gene have been associated to other non-breast second
primary tumor diagnosis in a study using multigene panel testing
(Fostira et al., 2020). In our cohort, 4 cases with two or multiple
cancers were carriers of truncating variants and only one was carrier of
a LP missense, confirming a higher aggressiveness of truncating variants
over missense variants.
Conflicting results are common for missense hypomorphic variants and
represent one of the biggest challenges we faced for CHEK2variant classification due to the lack of more sensitive functional
assays and the use of different controls, complicating replication and
therefore bypassing PS3 application. The c.470T>C founder
mutation conveys a moderate susceptibility for overall cancer (OR= 1.39;
p<0.00001) and for BC only (OR= 1.58; p<0.00001) in
a large meta-analysis (Han et al., 2013). Its pathogenicity has been
established for ovary cystadenomas in young Polish carriers (OR = 2.6;
p=0.006) (Szymanska-Pasternak et al., 2006) and is associated to a
2-fold risk of non-Hodgkin lymphoma, colon, kidney, thyroid and prostate
cancers (Cybulski et al., 2004). We found it in a male patient diagnosed
of testicular cancer at 25 years. Interestingly, in a recent study of
448 Croatian testicular cancer patients it was found in 5.1% of them,
resulting in an OR of 3.93 (95% CI, 1.53-9.95) even when its population
frequency is of 1-2% (AlDubayan et al., 2019). Of note, when applying
ACMG-AMP guidelines, c.470T>C remains as VUS even applying
PS4_moderate. To our knowledge, c.470T>C is the most
studied CHEK2 missense variant, but as shown in Table S3, it has
conflictive interpretations of pathogenicity at almost all functional
studies, therefore PS3 was ruled out, remaining as VUS in the ACMG
context. However, we were able to classify it as ERA according to the
risk allele-based classification (Senol-Cosar et al., 2019). Of note,
this variant is classified as LP by GeneDx, and as P by Ambry, Color and
Invitae diagnostic laboratories (Table S3), which could convey errors in
clinical interpretation. PS3 was also not possible to apply for 2 other
missense variants: c.190G>A and c.1427C>T.CHEK2 c.190G>A is a fairly frequent variant found in
0.03% of NFE by gnomAD, with partial reduction of Thr68
phosphorylation, auto-phosphorylation and Cdc25C phosphorylation, but
DNA repair assays in yeast are discordant (Table S3). Variant
c.1427C>T is another relatively frequent variant present in
0.05% of NFE (gnomAD). It has been reported to affect DNA damage
response in yeast at intermediate-high level. In addition, it shows
reduced SOX phosphorylation almost equally to c.1100delC. However,in vivo and in vitro studies of KAP1 phosphorylation from
the same group showed discordant results of pathogenicity (Table S3). As
noted in Table S2, lack of robust association studies and meta-analysis
of these variants hampered the possibility of applying risk allele-based
classification. Both remained as VUS in any classification framework,
although are classified as LP by at least 2 different reputable sources
(Table S3).
To summarize, we describe here a comprehensive CHEK2 mutational
analysis in a large Spanish cohort of HC patients, providing full data
of the actual prevalence of CHEK2 pathogenic variants in our
population. The frequency of LP/P variants in the HBC suspected cases in
the whole gene analysis was 1.3% (9 out of 689), similar to the
reported by Couch et al (Couch et al., 2017) in a study of 58,798 BC
patients, in which they found 1.41% of truncating variants and 2.22%
of LP/P CHEK2 missense variants. Interestingly, 3 young CRC cases
carried an LP/P CHEK2 variant and none of them had any additional
pathogenic variant in our NGS panel analysis. By this means,CHEK2 represents the most frequently mutated gene after MMR genes
in our hereditary non-polyposis CRC (HNPCC) cohort. CHEK2c.1100delC was reported in 6 out of 234 HNPCC families from Poland
(Meijers-Heijboer et al., 2003). In their study, 3 of them also carried
germline MMR P variants. In addition, c.470T>C has been
found in familial CRC. To our knowledge this is the largest Spanish
dataset presenting the sequencing of the whole CHEK2 coding
region together with the first attempt to apply ACMG-AMP guidelines for
this gene. We detailed different strategies that can be helpful to
classify VUS using different frameworks with the aim of being of help
not only for the curation of CHEK2 variants but also for other
genes. We hope our work serves as a starting point to better tune ACMG
criteria in the case of low-penetrance and low effect size variants
associated with disease risk.