Volume 146, Issue 2, Pages 520-529.e6 (February 2014) Germline Mutations in Oncogene-Induced Senescence Pathways Are Associated With Multiple Sessile Serrated Adenomas  Manish K. Gala, Yusuke Mizukami, Long P. Le, Kentaro Moriichi, Thomas Austin, Masayoshi Yamamoto, Gregory Y. Lauwers, Nabeel Bardeesy, Daniel C. Chung  Gastroenterology  Volume 146, Issue 2, Pages 520-529.e6 (February 2014) DOI: 10.1053/j.gastro.2013.10.045 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Strong LoF mutations among study participants. (A) Gene structure diagrams for all official isoforms of mutated genes. Vertical structures along the line show exons. Down arrows indicate the site of mutations. Isoforms are labeled with RefSeq IDs. (B) Pedigrees of study participants with identifiable mutations in the gene set and family histories of colon cancer. Ages are current as of the time of publication. Study participants 5 and 13 did not have family histories of colon or extracolonic cancers and are not displayed. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Strong LoF mutations among controls. (A) Direct protein-protein interaction network of gene set mutations discovered in controls. Red halos designate genes also found mutated in cases. (B) Cumulative minor allele frequencies (MAFs) of each mutated gene in order of decreasing frequency. Arrows designate genes found in cases. The skewness was 8.8, and kurtosis was 92.1. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 RNF43 modulates the ATM-ATR–mediated DDR pathway. (A) Gene structure diagram of the germline RNF43 mutation identified from the study participants. The arrowhead shows the site of nonsense mutation. (B) Pedigrees of study participants with germline RNF43 mutations. (C) Western blots of protein isolated from KRAS mutated pancreatic duct cells. Cells were stably transfected with short hairpin constructs targeting either GFP or RNF43 and exposed to UV irradiation as indicated. (D) Efficacy of RNF43 silencing in stably transfected pancreatic duct cells. Error bars designate the SEM. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 1 Pedigrees of study participants with strong family histories of cancers and no identifiable mutations. Study participants are highlighted with an arrow. Ages are current as of the time of publication. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 2 Sanger tracings of mutations of interest. Genomic coordinates are listed in reference to hg19. Sequence tracings displayed are in the 5′ orientation from left to right in relation to the coding domain sequence. Red arrows display site of mutation. Tracings may be inverted from the opposite strand for uniform display. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 3 Venn diagram of literature-derived gene set and genes from cancer GWAS. Assessment of the overlap between the gene predefined set and genes identified from GWAS of all malignancies. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 4 Relative RNF43 expression among serrated adenomas and carcinomas. Microarray comparison of RNF43 expression among various studies and data sets. A P value of <.05 and a fold change greater than 1.2 in either direction was considered significant. N.S., not significant; PMID, PubMed ID number; GSE, National Center for Biotechnology Information Gene Expression Omnibus (GEO) accession number. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 5 Effect of RNF43 silencing on increasing UV doses. Protein was extracted 4 hours after exposure. Western blot of phosphorylated H2Ax after exposure to different doses of UV radiation. β-actin serves as a loading control. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 6 Cell proliferation assay. Absorbance is directionally proportional to cell number. No significant differences between control and RNF43 knockdown cell lines were noted. Error bars represent SD. Gastroenterology 2014 146, 520-529.e6DOI: (10.1053/j.gastro.2013.10.045) Copyright © 2014 AGA Institute Terms and Conditions