Genetic analysis and functional validation in the patients
Whole-exome sequencing (WES; SureSelect Human All Exome V7) identified
in both siblings a homozygous variant (NM_016614.3:c.636+3_636+6del)
within the exon-intron boundary of TDP2 exon 5, removing 4
nucleotides near the splicing donor site (Figure S1). The variant,
unreported in gnomAD and found in only one carrier individual in the
Bravo/TOPMed dataset (1/125,568, 0.000796%), was at the heterozygous
state in the healthy parents. Homozygosity mapping detected a large
region of homozygosity (ROH) of ∼30 Mb (chr6:10,398,474-40,431,707;
GRCh38/hg38), shared by the affected siblings and spanning TDP2(chr6:24,649,977-24,667,033).
Different bioinformatic tools predicted donor splice-site loss due to
the c.636+3_636+6del variant. In agreement with the in-silicopredictions, fibroblasts’ analysis revealed that the variant caused the
skipping of TDP2 exon 5, leading to the insertion of a premature
stop codon within six amino acid residues (p.His174GlufsTer6) and
consequent nonsense-mediated mRNA
decay (Figure 2a and Figure 2b). Accordingly, immunoblotting failed to
detect TDP2 protein in the
fibroblasts of both siblings,
totally overlapping the expression pattern previously observed in the
fibroblasts of the 850BR patient (Zagnoli-Vieira et al., 2018)
harbouring the c.425+1G>A variant in TDP2 (Figure
2c). Furthermore, cells from affected individuals carrying either the
c.636+3_636+6del or the c.425+1G>A variants revealed
defective repair of TOP2-induced DSBs relative to control cells, as
measured by the persistence of γH2AX immunostaining, an indirect marker
of DSBs, after treatment with the TOP2 poison etoposide (Figure 2d).
According to the ACMG criteria (Richards et al., 2015), the variant was
classified as pathogenic.