Rapid Detection of Translation-Terminating Mutations at the Adenomatous Polyposis Coli (APC) Gene by Direct Protein Truncation Test

Luijt, Rob van der; Khan, P. Meera; Vasen, Hans F. A.; Leeuwen, C. van; Tops, Carli M.J.; Roest, Pauline A.M.; Dunnen, Johan T. den; Fodde, Riccardo

doi:10.1006/geno.1994.1119

Cited by 191 publications

(109 citation statements)

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“…For the investigation of the APC gene, exon 15 mutations were performed using the protein truncation test (PTT) on genomic DNA as described previously (Van der Luijt, 1994). Exons 1 -14 were screened by SSCP using published oligonucleotides and PCR conditions (Van der Luijt, 1994;Li et al, 1999).…”

Section: Apc and B-catenin Gene Mutational Analysismentioning

confidence: 99%

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Nuclear β-catenin expression is closely related to ulcerative growth of colorectal carcinoma

et al. 2002

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Although most colorectal cancer develops based on the adenoma -adenocarcinoma sequence, morphologically, colorectal cancer is not a homogeneous disease entity. Generally, there are two distinct morphological types: polypoid and ulcerative colorectal tumours. Previous studies have demonstrated that K-ras codon 12 mutations are preferentially associated with polypoid growth of colorectal cancer; however, little is known about the molecular mechanism that determines ulcerative growth of colorectal cancer. b-catenin complex plays a critical role both in tumorigenesis and morphogenesis. We examined the differential expression of b-catenin and its related factors among different types of colorectal cancer in order to determine any relationship with gross tumour morphology. Immunohistochemical staining of b-catenin, E-cadherin and MMP-7 was performed on 51 tumours, including 26 polypoid tumours and 25 ulcerative tumours. Protein truncation tests and single-strand conformational polymorphism for mutation of the adenomatous polyposis coli tumour suppressor gene, as well as singlestrand conformational polymorphism for the mutation of b-catenin exon 3 were also done. Nuclear expression of b-catenin was observed in 18 out of 25 (72%) cases of ulcerative colorectal cancer and seven out of 26 (26.9%) cases of polypoid colorectal cancer. A significant relationship of nuclear b-catenin expression with ulcerative colorectal cancer was found (P50.001). However, this finding was independent of adenomatous polyposis coli tumour suppressor gene mutation and Ecadherin expression. Together with previous data, we propose that different combinations of genetic alterations may underlie different morphological types of colorectal cancer. These findings should be taken into consideration whenever developing a new genetic diagnosis or therapy for colorectal cancer.

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Section: Apc and B-catenin Gene Mutational Analysismentioning

confidence: 99%

“…Exons 1 -14 were screened by SSCP using published oligonucleotides and PCR conditions (Van der Luijt, 1994;Li et al, 1999). After PCR amplification, products were loaded onto 12.5% polyacrylamide Sweden) and underwent electrophoresis at 208C.…”

Section: Apc and B-catenin Gene Mutational Analysismentioning

confidence: 99%

Nuclear β-catenin expression is closely related to ulcerative growth of colorectal carcinoma

et al. 2002

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“…Currently the protein truncation test (PTT) is the technique most widely used to screen for mutations in the APC gene. 10,11 However, PTT only allows the detection of truncating mutations. While over 90% of pathogenic mutations detected in classic FAP are truncating mutations, missense mutations, polymorphisms and variants of unknown significance that have been reported are spread over the whole gene.…”

Section: Introductionmentioning

confidence: 99%

Pathogenic mutations and rare variants of the APC gene identified in 75 Belgian patients with familial adenomatous polyposis by fluorescent enzymatic mutation detection (EMD)

et al. 2002

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Familial adenomatous polyposis (FAP) is a dominant inherited colorectal cancer syndrome which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Enzymatic mutation detection (EMD) has potential advantages over the standard protein truncation test (PTT) that is currently used in screening the APC gene for mutations. First we wanted to validate the EMD technique in comparison to PTT. Secondly, we wanted to develop an efficient working protocol for EMD screening of APC. Seventy-five unrelated patients were screened for mutations. All mutations that had previously been detected by PTT were also identified by EMD; the sizes of the cleavage fragments were as expected according to the position of the mutations within the amplicons. A new screening strategy based on EMD allows the analysis of the APC gene in 31 overlapping PCR fragments. In total, EMD efficiently detected the 26 truncating mutations in this series. In addition, two rare variants were also detected: the first is the typical Ashkenazi missense mutation I1307K while the second variant, E1317Q, has been identifed in Belgian patients and controls, and should no longer be considered as a pathogenic mutation, but rather classified as a polymorphism.

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“…For example, 95% of the APC gene mutations associated with familial adenomatous polyposis 3 and 71% of the mutations in the mismatch repair genes predisposing to hereditary nonpolyposis colorectal cancer 4 are translation terminating mutations. The breast cancer susceptibility genes BRCA1 and BRCA2 follow the same pattern.…”

Section: Introductionmentioning

confidence: 99%

A complete protein truncation test for BRCA1 and BRCA2

Garvin

1998

Eur J Hum Genet

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The protein truncation test (PTT) is currently the fastest method in general use for detecting previously unidentified mutations in tumor suppressor genes. Greater than three kilobases of coding sequence can be screened by one PCR reaction, one coupled in vitro transcription/translation reaction, and one lane on an SDS-PAGE gel. The 16 kb of BRCA1/2 coding sequence can be screened with nine overlapping segments. Since 90% of BRCA1/2 mutations result in a truncated protein product, the theoretical false negative rate for a BRCA1/2 PTT screen should be 10%. In practice the false negative rate is much higher, especially when cDNA is used as template. However, the actual false negative rate for a given screen will depend on the details of how the test is performed. Design of the overlapping segments, gel parameters, and nonsense mediated mRNA decay can all influence the effectiveness of the screen. BRCA1/2 screening by PTT can be optimised by considering these variables. Furthermore, nonsense-mediated mRNA decay can be inhibited by blocking protein synthesis with cycloheximide.

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Rapid Detection of Translation-Terminating Mutations at the Adenomatous Polyposis Coli (APC) Gene by Direct Protein Truncation Test

Cited by 191 publications

References 0 publications

Nuclear β-catenin expression is closely related to ulcerative growth of colorectal carcinoma

Nuclear β-catenin expression is closely related to ulcerative growth of colorectal carcinoma

Pathogenic mutations and rare variants of the APC gene identified in 75 Belgian patients with familial adenomatous polyposis by fluorescent enzymatic mutation detection (EMD)

A complete protein truncation test for BRCA1 and BRCA2

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