Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole-genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25-1 G > T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48-12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which, in a human colonic cancer cell line, controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

Bioinformatics and Omics Data Analytics German Cancer Research Center D 69120 Heidelberg Germany

Computational Oncology Molecular Diagnostics Program National Center for Tumor Diseases D 69120 Heidelberg Germany

Department of Genetics and Pathology Hereditary Cancer Center Pomeranian Medical University Unii Lubelskiej 1 71 252 Szczecin Poland

Department of Medical Oncology The 2nd Affiliated Hospital of Zhejiang University School of Medicine Hangzhou 310009 China

Division of Cancer Epidemiology German Cancer Research Center D 69120 Heidelberg Germany

Division of Molecular Genetic Epidemiology German Cancer Research Center Im Neuenheimer Feld 580 D 69120 Heidelberg Germany

Division of Pediatric Neurooncology German Cancer Research Center D 69120 Heidelberg Germany

Faculty of Medicine and Biomedical Center in Pilsen Charles University Prague 30605 Pilsen Czech Republic

Hopp Children's Cancer Center D 69120 Heidelberg Germany

Institute of Bioinformatics International Technology Park Bengaluru 560066 India

Manipal Academy of Higher Education Manipal 576104 India

Medical Faculty Heidelberg Heidelberg University D 69120 Heidelberg Germany

Splicing and Genetic Susceptibility to Cancer Instituto de Biología y Genética Molecular 47003 Valladolid Spain

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Search for germline gene variants in colorectal cancer families presenting with multiple primary colorectal cancers