Functional characterization of the novel APC N1026S variant associated with attenuated familial adenomatous polyposis.

Background & Aims: We identified the APC N1026S variant of unknown malignant potential in the adenomatous polyposis coli (APC) gene in a Spanish attenuated familial adenomatous polyposis (AFAP) family. The variant was located in the first of the 4 highly conserved 15-amino acid (AA) repeats within the β-catenin union domain. Our aim was to determine its functional relevance to establish its pathogenicity. Methods: N1026S variant was analyzed in 22 members of the AFAP family studied, in 236 sporadic colorectal cancer cases, 203 matched controls, and 205 unrelated familial colorectal cancer cases. To assess its effects on β-catenin binding, β-catenin/Tcf-4–mediated transcription and β-catenin subcellular distribution we performed affinity chromatography experiments, BIAcore 1000 (BIAcore AB, Uppsala, Sweden) assays, luciferase reporter assays, assessment of c-myc messenger RNA levels, and cell fractionation. Results: N1026S variant cosegregated with the disease in the AFAP family studied. None of the sporadic or familial cases as well as the controls analyzed was positive for the variant. N1026S variant completely precluded β-catenin binding to the first 15-AA repeat and diminished it when all four 15-AA repeats were present. Expression of APC N1026S in SW480 and DLD-1 cells did not diminish β-catenin/Tcf-4–mediated transcription as effectively as APC wild-type. N1026S did not decrease c-myc transcription in DLD1 cells and nuclear β-catenin in SW480 cells as effectively as WT. Conclusions: These findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype, reinforcing the importance of functional characterization of APC variants for genetic counseling.

[1]  M. Nieuwenhuis,et al.  Correlations between mutation site in APC and phenotype of familial adenomatous polyposis (FAP): a review of the literature. , 2007, Critical reviews in oncology/hematology.

[2]  A. de la Chapelle,et al.  Pathogenicity of MSH2 missense mutations is typically associated with impaired repair capability of the mutated protein. , 2006, Gastroenterology.

[3]  Stephen B Gruber,et al.  The Genetics of Colorectal Cancer , 2006, Annals of Internal Medicine.

[4]  O. Sieber,et al.  Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly but depend on the site of the germline mutation , 2006, Gut.

[5]  Gerrit Groenhof,et al.  GROMACS: Fast, flexible, and free , 2005, J. Comput. Chem..

[6]  F. Barany,et al.  Classification of BRCA1 missense variants of unknown clinical significance , 2005, Journal of Medical Genetics.

[7]  M. Peris,et al.  Colorectal cancer risk and the APC D1822V variant , 2004, International journal of cancer.

[8]  Stefano Landi,et al.  Association of common polymorphisms in inflammatory genes interleukin (IL)6, IL8, tumor necrosis factor alpha, NFKB1, and peroxisome proliferator-activated receptor gamma with colorectal cancer. , 2003, Cancer research.

[9]  Mariann Bienz,et al.  Nuclear export of the APC tumour suppressor controls β‐catenin function in transcription , 2003, The EMBO journal.

[10]  S. Woolf,et al.  Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. , 2003, Gastroenterology.

[11]  Alison L. Livingston,et al.  Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors , 2002, Nature Genetics.

[12]  W. Weis,et al.  Molecular mechanisms of β‐catenin recognition by adenomatous polyposis coli revealed by the structure of an APC–β‐catenin complex , 2001, The EMBO journal.

[13]  G. Marra,et al.  Nontruncating APC germ-line mutations and mismatch repair deficiency play a minor role in APC mutation-negative polyposis. , 2001, Cancer research.

[14]  M. Leppert,et al.  A molecular variant of the APC gene at codon 1822: its association with diet, lifestyle, and risk of colon cancer. , 2001, Cancer research.

[15]  K. Kinzler,et al.  The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression. , 2000, Cancer research.

[16]  D. Morton,et al.  Molecular analysis of the APC gene in 205 families: extended genotype-phenotype correlations in FAP and evidence for the role of APC amino acid changes in colorectal cancer predisposition , 1999, Journal of medical genetics.

[17]  David S. Goodsell,et al.  Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function , 1998, J. Comput. Chem..

[18]  A. Sparks,et al.  Identification of c-MYC as a target of the APC pathway. , 1998, Science.

[19]  P. Hartge,et al.  The APC I1307K allele and cancer risk in a community-based study of Ashkenazi Jews , 1998, Nature Genetics.

[20]  W F Bodmer,et al.  The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  W. Birchmeier,et al.  Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. , 1998, Science.

[22]  Hans Clevers,et al.  Activation of β-Catenin-Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC , 1997, Science.

[23]  Michael Kühl,et al.  Functional interaction of β-catenin with the transcription factor LEF-1 , 1996, Nature.

[24]  K. Kinzler,et al.  Apoptosis and APC in colorectal tumorigenesis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Polakis,et al.  Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  K. Kinzler,et al.  Association of the APC tumor suppressor protein with catenins. , 1993, Science.

[27]  G. Thomas,et al.  Alleles of the APC gene: An attenuated form of familial polyposis , 1993, Cell.

[28]  L. Lipton,et al.  The genetics of FAP and FAP-like syndromes , 2005, Familial Cancer.

[29]  J. Jiricny,et al.  Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system. , 2002, Gastroenterology.