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.