Study population
The whole exome was studied in 13 FCCTX families recruited at the Genetic Counseling Unit of Hospital Clínico San Carlos (Madrid, Spain). All of the families fulfilled the Amsterdam I or II clinical criteria for HNPCC (Vasen et al., 1991, 1999) and presented MMR-proficient tumors with neither MSI nor lack of expression of MMR proteins. In addition, none of them carried germline mutations in the MMR genes. Other family members, whether healthy or affected, were also recruited for segregation studies. Formalin-fixed paraffin-embedded (FFPE) tumor blocks from the probands and/or their cancer-affected relatives were obtained whenever available. Information on personal and family cancer history was obtained, and cancer diagnoses were confirmed by medical and pathology records. The study was approved by the Institutional Review Board of Hospital Clínico San Carlos, and a written informed consent was signed by each participant.
An independent series of 473 genetically unexplained MMR-proficient familial and/or early-onset non-polyposis CRC unrelated patients recruited at the Institut Català d’Oncologia (IDIBELL, Barcelona, Spain), was included for validation purposes (Belhadj et al., 2019). While 443 fulfilled the Amsterdam or Bethesda (Umar et al., 2004) criteria at the time of referral to the genetic counseling, 30 did not but were included in the study based on a clinical referral for non-polyposis CRC and absence of MMR deficiency. Healthy individuals with no cancer family history were recruited from the Blood Bank of Hospital Clínico San Carlos (Madrid) and used as controls. FFPE tumor blocks obtained from sporadic CRC patients were used as CRC controls, while FFPE blocks containing non-tumor colon tissue were used as healthy colon controls. All of these control subjects had previously signed an informed consent.