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Germline mutations in the β-catenin gene are not associated with the FAP phenotype without an APC mutation
  1. Research Group of Human Genetics, Department of Research, University Hospital, CH-4031 Basel, Switzerland, and Department of Medical Genetics, University Children’s Hospital, Basel, Switzerland

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    Editor—Familial adenomatous polyposis (FAP) is generally considered a typical monogenic disease caused by germline mutations within the adenomatous polyposis coli (APC) gene. Despite applying several screening techniques, however, mutational studies world wide have failed to identify a germline mutation within the APC gene in up to 50% of all FAP cases (called APC negative below).1 2 Intronic alterations, mutations within the regulatory regions causing altered APC gene expression, or large scale rearrangements of the APC gene could account for the failure to identify an APC mutation in some FAP families. Assuming no hot spots, however, such mutations are not likely to cause a high percentage of APC negative cases.

    Fifty families which were referred to our department with the clinical diagnosis of FAP were tested for APC germline mutations. In 14 of them (28%), no APC mutation could be identified after screening the entire coding region of the gene (APC negative group) using the protein truncation test (PTT, the whole coding region), single strand conformation polymorphism analysis (SSCP, the affected PTT segment), and direct DNA sequencing (the affected SSCP exon).3 As these families were either too small or only a limited number of family members were available, linkage analysis with respect to the APC locus could not be performed. They all fulfil the major criteria for the clinical diagnosis of FAP (table 1). We have shown previously that these families differ phenotypically from those with an APC mutation, suggesting that they might represent a distinct genetic entity.4 In summary, the APC negative group tended to have less severe disease characteristics with significantly increased age at diagnosis, fewer colonic polyps, and fewer extracolonic manifestations, so they are similar to those with attenuated adenomatous polyposis coli (AAPC). However, all three regions of the APC gene which have been reported to correlate with AAPC5 were analysed without detecting DNA abnormalities.

    Table 1

    Phenotypes of FAP patients without an identified APC mutation

    A candidate which might be involved in the locus heterogeneity in FAP is the β-catenin gene, a member of the same cellular pathway as APC. Free β-catenin is targeted for degradation by the glycogen-synthase kinase (GSK) 3β and the APC protein. An increase in the stability of free β-catenin is triggered by the Wnt signal as well as by the absence of APC protein or the presence of mutated APC protein.6 Finally, if APC is intact, mutations within the β-catenin gene itself could also result in an increased level of free β-catenin.7 In recent studies, somatic mutations in the β-catenin gene were found in colorectal and other types of tumours.7-12 However, none of the studies observed a germline mutation within the β-catenin gene in tumour matched constitutional DNA.

    Based on the observation that somatic mutations in the β-catenin gene can mimic mutations in the APC gene in colonic tumours, the possibility arises that, similarly, germline β-catenin mutations could mimic germline APC mutations giving rise to an FAP-like phenotype. In the present study this hypothesis was tested.

    In 14 APC negative FAP families, the entire coding region (16 exons) of the β-catenin gene (CTNNB1) was screened in DNA isolated from peripheral blood. SSCP analysis with PCR primers allowing investigation of intron/exon boundaries (table 2) was applied. All but one exon were analysed in a single PCR-SSCP reaction. Owing to its length exon 9 was screened in two separate reactions. No new conformers in any of the exons were detected, suggesting that DNA alterations in the β-catenin gene did not occur. We are aware of the possibility that some mutations might have been missed; however, SSCP is one of the most reproducible techniques for mutation analysis with the reported sensitivity under optimised conditions reaching 90%.13

    Table 2

    PCR primers for SSCP analysis of 16 exons of the β catenin gene

    Interestingly, in previous studies, β-catenin mutations were found almost exclusively in the NH2-terminus of the gene and altered either the phosphorylation sites or a domain responsible for binding to the APC protein.7-12 Most frequently affected codons were localised in exon 3 (33, 44, and 45). Therefore, we additionally analysed exon 3 by direct DNA sequencing using the same primers as for SSCP, the Thermosequenase labelled primer cycle sequencing kit (Amersham), and an automated DNA sequencer (LI-COR) without identifying any changes in the nucleotide sequence.

    As there were no differences detected in the coding region, β-catenin gene expression in patients’ lymphocytes was examined using reverse transcription PCR. PCR amplification of cDNA was performed in two separate PCR reactions using PCR primer pairs 3.1/6.2 and 6.1/13.2, as listed in table 2. This showed that in all persons tested the expected β-catenin transcript was present and of expected size, suggesting that differential splicing did not occur. However, as our experiments regarding β-catenin expression were performed on lymphocytes, differential expression in colonic or other tissues affected in FAP, which were not available for our study, cannot be excluded.

    Taken together, using a combination of several techniques for the mutational analysis, in our group of 14 FAP APC negative families, no hereditary alterations were identified in the β-catenin coding sequence or gene expression, suggesting that β-catenin germline mutations do not account for APC negative FAP cases. Even though these results need confirmation in a larger sample of APC negative FAP families, they indicate that β-catenin might play a different role in the pathogenesis of hereditary colon carcinoma compared to sporadic colorectal cancer.


    We thank Dr Karl Heinimann for critical review of manuscript. This work was supported by a grant from the Swiss National Foundation 3200-049310.96. All experiments comply with the current laws of Switzerland.