Variant validation, segregation and loss of heterozygosity (LOH)
studies
Candidate variants were validated by PCR followed by Sanger sequencing
of the corresponding region of each gene. The segregation and LOH
studies for the PTPRT variant were also assessed by Sanger
sequencing of the selected area of the PTPRT gene (exon 30,
ENST00000373198). The segregation study was carried out in germline DNA
from the available members of the family. For the LOH analyses, tumor
and germline DNA were assessed by Sanger sequencing and the
corresponding electropherograms were compared in order to detect
possible variations in the peak height of any of the alleles. All the
primers used are described in Supplementary Table S2.