Detection of APC mutations by a yeast‐based protein truncation test (YPTT)

Suzuki, Takao; Ishioka, Chikashi; Kato, Satoshi; Mitachi, Yasushi; Shimodaira, Hideki; Sakayori, Masato; Shimada, Atsushi; Asamura, M; Kanamaru, Ryunosuke

doi:10.1002/(sici)1098-2264(199804)21:4<290::aid-gcc2>3.3.co;2-l

Cited by 5 publications

(5 citation statements)

References 12 publications

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“…In general, the mutational spectrum of the NF1 gene seems to be very similar to that of other tumor-suppressor and mutator genes, which all seem to be characterized by a large proportion of truncating mutations-for example, nearly 100% for APC (Suzuki et al 1998) and TSC1 (Jones et al 1999), 180% for RB1 (Lohmann et al 1996;, ∼80% for BRCA1 (Miki et al 1994) and ATM (Sandoval et al 1999), and ∼60% for TSC2 (Jones et al 1999), and all six NBS1 gene mutations identified to date (Varon et al 1998). Similarly, 5%-20% missense mutations, as well as wholegene deletions, have been found in some of these genes (RB1, ATM, TSC2).…”

Section: Figurementioning

confidence: 87%

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain

Fahsold

Hoffmeyer²,

Mischung

et al. 2000

The American Journal of Human Genetics

296

246

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More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.

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Section: Figurementioning

confidence: 87%

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain

Fahsold

Hoffmeyer²,

Mischung

et al. 2000

The American Journal of Human Genetics

296

246

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“…The systematic functional analysis in yeast of PTEN missense mutations may help in the unequivocal definition of pathogenic mutations. Similar approaches have been exploited in yeast to monitor the pathogenicity of mutations in some other tumor suppressor genes, such as p53, hMLH1, BRCA1 and APC (Ishioka et al, 1993;Humphrey et al, 1997;Shimodaira et al, 1998;Suzuki et al, 1998). The fact that the PI3K/PTEN yeast reconstitution system allows the measurement of the reaction products of PI3K and PTEN in vivo makes such system highly sensitive and reliable.…”

Section: Pten Inhibitory Drugsmentioning

confidence: 99%

Assessment of PTEN tumor suppressor activity in nonmammalian models: the year of the yeast

et al. 2008

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Model organisms have emerged as suitable and reliable biological tools to study the properties of proteins whose function is altered in human disease. In the case of the PI3K and PTEN human cancer-related proteins, several vertebrate and invertebrate models, including mouse, fly, worm and amoeba, have been exploited to obtain relevant functional information that has been conserved from these organisms to humans along evolution. The yeast Saccharomyces cerevisiae is an eukaryotic unicellular organism that lacks a canonical mammalian-like PI3K/PTEN pathway and PIP3 as a physiological second messenger, PIP2 being essential for its life. The mammalian PI3K/PTEN pathway can be reconstituted in S. cerevisiae, generating growth alteration phenotypes that can be easily monitored to perform in vivo functional analysis of the molecular constituents of this pathway. Here, we review the current nonmammalian model systems to study PTEN function, summarize our knowledge of PTEN orthologs in yeast species and propose the yeast S. cerevisiae as a sensitive biological sensor of PI3K oncogenicity and PTEN tumor suppressor activity.

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“…9 Hence, the widespread distribution of many different mutations within the very large APC gene poses problems in molecular genetic diagnosis, and detection of the APC mutations is labor-intensive when nucleic acidbased approaches are used. An alternative method is the protein truncation test which detects the protein truncating mutations in vitro 10,11 or in yeast, 12 because the vast majority (Ͼ95%) of the APC mutations have truncation of the APC protein because of frameshift, nonsense, or splice-site mutations. 13 We present here a new, rapid, and sensitive yeastbased screening method for detection of the truncating mutation in the APC.…”

mentioning

confidence: 99%

Somatic Mutations of the APC Gene in Primary Breast Cancers

Furuuchi

Tada

Yamada

et al. 2000

The American Journal of Pathology

104

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APC gene mutations play an important role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) patients and non-FAP patients. Although the APC gene is expressed in most tissues, including the lung, liver, kidney, and mammary gland, its somatic mutations have rarely been found in primary tumors affecting these organs. We have developed a sensitive yeast-based assay for screening almost the entire coding region of the APC gene. By this method, we have been able to detect somatic mutations of the APC gene in 57% of colorectal cancers and none in non-small cell lung cancers. Interestingly, the assay detected somatic APC gene mutations in 18% of breast cancers, in which APC gene mutation was previously considered rare. In the breast cancers, most of the APC mutations were distributed outside the mutation cluster region that has been advocated for colorectal cancers. We also noted a difference in the mutation pattern of the APC between colorectal and breast cancers. In colorectal cancers, all base substitutions were observed at C residues (5 of 5), whereas in breast cancers the majority of them were found at G residues (4 of 5). Furthermore, APC mutations were observed at a significantly high frequency in advanced stages of primary breast cancers (TNM classification, P < 0.05; T category, P < 0.01). Our data suggest that the etiology of the APC mutations and their biological role in carcinogenesis may differ between colorectal and breast cancers.

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Detection of APC mutations by a yeast‐based protein truncation test (YPTT)

Cited by 5 publications

References 12 publications

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain

Assessment of PTEN tumor suppressor activity in nonmammalian models: the year of the yeast

Somatic Mutations of the APC Gene in Primary Breast Cancers

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