FIGURE LEGENDS
FIGURE 1. (a) Family pedigree showing a miscellany of
neurological phenotypes in the last generation of the family. +/+
homozygous wild-type; +/− heterozygous carrier; −/− homozygous mutant
for the TDP2 c.636+3_636+6del variant. (b) Clinical features of
the probands (III-38 and III-40), showing highly overlapping facial
dysmorphisms, large hands, clinodactyly of the 5thfinger, and flatfoot.
FIGURE 2 . Functional analysis on the probands’ fibroblasts. (a)
RT-PCR shows exon 5 skipping at the RNA level in the probands compared
to control pools; NTC: no template control. (b) Sequencing analysis of
the exon 5 skipped band in the RT-PCR. The junction between exon 4 and
exon 6 creates a frame shift leading to a premature termination codon
within six amino acid residues. (c) TDP2 immunoblotting showing no
detectable TDP2 expression in either Italian (novel mutation) or US
patients (previous mutations); 1BR: control human fibroblast cell line;
850BR: TDP2-mutated patient primary human fibroblasts; III-38, III-40:
probands’ fibroblasts; YJR1504: primary fibroblast control cell line.
(d) Repair kinetics of yH2AX foci after 25µM etoposide treatment showing
decreased repair of TOP2-induced DSBs in affected cells (probands and
850BR) compared to control cell line (1BR). Data are the mean (±s.e.m.)
of four independent experiments.
FIGURE S1. WES analysis and segregation in the family. WES
results visualized on the Integrative Genomics Viewer (IGV) in the
probands and parents (top left panel). NGS findings were confirmed by
Sanger sequencing (top right panel), which was also used for segregation
analysis in other family members (bottom panel).