No mutations were observed in either patient, strongly suggesting that both alleles were normal in these two patients. there may be at least one additional gene, besides gene cause approximately 80% of all cases of FAP (3C5). Before the advent of genetic testing, it was necessary that all members of FAP kindreds be screened for the presence of disease by using colonoscopy or related clinical methods. Genetic testing can spare a significant number of first- and second-degree relatives the need for frequent colonoscopies and also can alleviate anxiety associated with the uncertainty of their genetic state. Though the only definitive treatment for FAP historically has been colectomy, chemopreventive agents currently are showing promise, and genetic testing should allow such agents to be used before disease onset (6, Rabbit Polyclonal to ARHGAP11A 7). Experience with FAP also illustrates the technical problems associated with genetic testing. is a large gene, encoding a protein of 2,843 aa contained within 15 exons (8, 9). Sequencing the entire gene, including introns, untranslated, and promoter regions, is impractical. Fortunately, all confirmed FAP-causing mutations detected to date result in truncations of Lenalidomide-C5-NH2 the protein (4, 5). One major consequence of these truncating mutations is the disruption of APCs ability to inhibit the function of -catenin (10, 11). It is believed that APC normally binds to -catenin (12, 13) and promotes its degradation (14), thereby preventing activation of growth-promoting genes, such as (15), by a -catenin/Tcf-4 transcription complex (16, 17). This mutation spectrum has stimulated the development of testing approaches that can reveal truncated APC proteins. In particular, the most commonly used test [called synthesized protein (IVSP) or protein truncation test (PTT)] involves transcription and translation of APC PCR products. Gel electrophoretic analysis of the translated polypeptides Lenalidomide-C5-NH2 reveals truncated proteins indicative of mutations (3, 18). Extensive analyses of FAP kindreds with IVSP and direct or indirect DNA sequencing methods have been used to identify more than 200 different mutations (4, 5). The frequency of mutations detected among FAP kindreds varies with the technique used, but in no case has it been more than 80%. The basis for the inability to identify mutations in a substantial proportion of such kindreds is unclear. One possibility involves the existence of mutations that are difficult to detect by standard mutational analyses. Indeed, the patient whose analysis originally led to the chromosome 5 localization of had a large deletion that would have been impossible to detect with any standard sequencing or IVSP assay (19). Furthermore, some FAP patients without truncating mutations appeared to express significantly reduced levels of transcript from one allele (3). A second possibility is that some cases of FAP are caused by mutations in genes Lenalidomide-C5-NH2 other than mutations could be detected with standard methods. The results show that more than 95% of FAP patients have inactivating mutations in and that a combination of MAMA and standard genetic testing can identify abnormalities in the vast majority of FAP patients. Additionally, the results suggest that there may be at least one other gene besides that can give rise to FAP. MATERIALS AND METHODS Cell Culture. Lymphoblastoid lines were established by EpsteinCBarr virus infection of peripheral blood leukocytes from patients diagnosed with FAP who had no evidence of mutation upon IVSP analysis. These lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and grown at 37C and 5% CO2. The UCW-56 hamster cell line (22) was grown Lenalidomide-C5-NH2 in DMEM supplemented with 10% fetal bovine serum and 60 g/ml l-proline and grown at 32C Lenalidomide-C5-NH2 and 5% CO2. Cell Fusions. Fusions initially were performed with polyethylene glycol as described (20). In later experiments, electrofusion was employed because a greater yield of viable fusion clones could be obtained. UCW-56 cells were combined with lymphoblastoid cells in FS (0.3 M mannitol/0.1 mM MgCl2/0.1 mM CaCl2) at a ratio of 3:1. The cells were washed and centrifuged three times in FS before resuspending in FS at a final concentration of 5 107 cells/ml. Thirty microliters of this solution then was mixed and pipetted into a 0.5-mm gap microfusion plate (BTX Microslide 450; BTX, San Diego). Fusions were performed by using a BTX Electrocell Manipulator, ECM 200. The settings that yielded the greatest number of fusion clones were 15 V (AC) for 10 sec followed by two 100-V (DC) pulses of 30 sec each. The cells from one 30-l fusion were plated into four wells of a 48-well plate (Costar) in DMEM supplemented with 10% fetal bovine serum and 60.

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