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.