Germline Mutations in FAF1 Are Associated With Hereditary Colorectal Cancer Q7 Laia Bonjoch, 1 Sebastià Franch-Expósito, 1 Pilar Garre, 2 Sami Belhadj, 3 Jenifer Muñoz, 1 Coral Arnau-Collell, 1 Marcos Díaz-Gay, 1 Anna Gratacós-Mulleras, 1 Giulia Raimondi, 4 Clara Esteban-Jurado, 1 Yasmin Soares de Lima, 1 Cristina Herrera-Pariente, 1 Miriam Cuatrecasas, 5 Teresa Ocaña, 1 Antoni Castells, 1 Cristina Fillat, 4 Gabriel Capellá, 3 Francesc Balaguer, 1 Trinidad Caldés, 2 Laura Valle, 3 and Sergi Castellví-Bel 1 1 Gastroenterology Department, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Universitat de Barcelona, Barcelona, Spain; 2 Molecular Oncology Laboratory, Centro Investigación Biomédica en Red de Cáncer (CIBERONC). Hospital Clínico San Carlos. Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain; 3 Hereditary Cancer Program, Catalan Institute of Oncology, Oncobell, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Barcelona, Spain; 4 Gene Therapy and Cancer, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Universitat de Barcelona, Barcelona, Spain; and 5 Pathology Department, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD) and Tumor Bank-Biobank, Hospital Clínic, Barcelona, Spain Familial CRC Functional characterization by CRISPR/Cas9 modeling c.1111G>A Next-generation sequencing CAACCTGCCCTC ATCCTGGCCCG CGACTGTAAGAC CGGACTCCACAT CCAGACCAATAT (p.Asp371Asn) c.254G>C (p.Arg85Pro) FAF1 predicted pathogenic variants p.Asp371Asn p.Arg85Pro Apoptosis NF-κB signaling β-catenin Proliferation Predisposition to CRC BACKGROUND & AIMS: A significant proportion of colorectal cancer (CRC) cases have familial aggregation but little is known about the genetic factors that contribute to these cases. We per- formed an exhaustive functional characterization of genetic vari- ants associated with familial CRC. METHODS: We performed whole-exome sequencing analyses of 75 patients from 40 families with a history of CRC (including early-onset cases) of an unknown germline basis (discovery cohort). We also sequenced speci fic genes in DNA from an external replication cohort of 473 families, including 488 patients with colorectal tumors that had normal expression of mismatch repair proteins (validation cohort). We disrupted the Fas-associated factor 1 gene (FAF1) in DLD-1 CRC cells using CRISPR/Cas9 gene editing; some cells were transfected with plasmids that express FAF1 missense variants. Cells were analyzed by immunoblots, quantitative real-time polymerase chain reaction, and functional assays monitoring apoptosis, proliferation, and assays for Wnt signaling or nuclear factor (NF)-kB activity. RESULTS: We identified predicted pathogenic variant in the FAF1 gene (c.1111G>A; p.Asp371Asn) in the discovery cohort; it was present in 4 patients of the same family. We identified a second variant in FAF1 in the validation cohort (c.254G>C; p.Arg85Pro). Both variants encoded unstable FAF1 proteins. Expression of these variants in CRC cells caused them to become resistant to apoptosis, accumulate b-catenin in the cytoplasm, and translocate NF-kB to the nucleus. CONCLUSIONS: In whole-exome sequencing analyses of patients from families with a history of CRC, we identified variants in FAF1 that associate with development of CRC. These variants encode unstable forms of FAF1 that increase resistance of CRC cells to apoptosis and increase activity of b- catenin and NF-kB. Keywords: Wnt Signaling; Programmed Cell Death; Gene Edit- ing; Functional Genomics. FLA 5.6.0 DTD  YGAST63273_proof  1 June 2020  4:37 pm  ce Gastroenterology 2020;-:1–14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 BASIC AND TRANSLATIONAL AT