Variants of unknown significance
Thirty-five unique VUS (with less than 2 LP/P interpretations in
ClinVar) were encountered in our cases (Table S1). We aimed to perform
RNA analysis in 3 of these, due to in silico prediction results
(c.320-5T>A and c.1376-8T>C) or to the nature
of the variant (duplication of exons 3 and 4). Lymphocytes for RNA
analysis were available from one carrier of the duplication of exons 3
and 4, for several samples with c.320-5T>A and were
unattainable from c.1376-8T>C carriers. RNA analysis showed
that the duplication of exons 3 and 4 occurs in tandem and produces
∼30% of aberrant transcript containing an in-frame insertion of 273 bp
(Figure 3). This affects the region that codifies for the FHA domain,
unfortunately there were no polymorphisms in the region to perform
quantitative analysis. This variant remains as VUS following all
guidelines. Regarding c.320-5T>A variant, in silicoprograms predicted a reduction in the recognition of the splicing
acceptor site of exon 3. cDNA analysis in two carriers showed the
generation of an aberrant transcript, consisting in an in-frame deletion
of exons 3 and 4 (Figure 4), as previously reported (Kraus et al.,
2017). The amount of abnormal transcript seemed greater than 20%,
although the absence of exonic polymorphisms prevented an accurate
quantification. Of note, the frequency of c.320-5T>A is
0.12% in gnomAD (NFE) and of 1.35% (25 out of 1,848) in our HC cohort.
In order to understand the differences in frequency in our population
with relation to international databases, we screened 1,501 control
samples. CHEK2 c.320-5T>A had a frequency of 0.8%
(12 out of 1,501) in our controls, not a statistically significant
difference, preventing it to be considered as risk allele.