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[CANCER RESEARCH 53, 5581-5584, December 1, 1993] Advances in Brief A Rapid Screening Method to Detect Nonsense and Frameshift Mutations: Identification of Disease-causing A P C Alleles Liliana Varesco, Joanna Groden, 2 Lisa Spirio, Margaret Robertson, Robert Weiss, Viviana Gismondi, G. B. Ferrara, and Ray White 3 lstituto Nazionale per la Ricerca sul Cancro, Genoa, Italy [L. V, V G., G. B. F..], and Howard Hughes Medical Institute [J. G., M. R., R. Wh.] and Department of Human Genetics [L. S., R. W., R. Wh.], Eccles Institute of Human Genetics, The University of Utah, Salt Lake City, Utah 84112 Abstract Materials and M e t h o d s A functional screen for nonsense and frameshift mutations has been devised that allows genes of interest to be scanned in segments. This assay is based on the cloning of these segments in-frame with a colorimetric marker gene (lacZ) followed by screening for the level of functional activity from the marker polypeptide (fl-galactosidase). Individuals at risk for any one of a number of genetic diseases, in particular familial adenomatous polyposis coil (APC), can be quickly screened for chain-terminating mutations introduced by stops and frameshifts. At present, scanning of the APC gene for mutation requires significant effort because it is a large gene and most APC mutations are unique. Therefore, this assay offers a powerful option for the diagnosis of this and other genetic diseases, as well as great potential for the development of a similar rapid screen to detect APC mutations in colorectai adenomas and carcinomas. Plasmid Construct and Preparation. The pSTDTAC-IKS plasmid is a 7.3-kilobase plasmid derived from pBR322 that contains the entire lacZ coding sequence (14). A 300-base pair insert containing multiple in-frame stops is flanked by ApaI and HindIII cleavage sites. This insertion is located between expression control sequences that include a Tac promoter, lac operator and translational start sequences and the/3-galactosidase coding sequence; in-frame replacement of this out-of-frame insert is required for high levels of expression of the lacz gene. The plasmid also carries an ampicillin resistance gene. Plasmid DNA was used to transform host bacterial strain XL1-Blue (Stratagene), which was grown in Terrific Broth and was isolated by means of a Qiagen plasmid preparation column. The vector was cut with HindlII and ApaI restriction endonucleases (Boehringer Mannheim) and was gel purified using Geneclean. Amplification of Test DNA and Cloning. Template DNA was obtained from APC patients, normal individuals, and colorectal carcinoma cell line SW480 (available from the American Type Culture Collection) as described previously (2, 8). APC mutations in DNAs had already been identified by other methods (2, 3, 8). Insert DNA was prepared using PCR with the following pair of oligonucleotide primers: BWL2-GGCTGCAGGAATI'CGATATCAAGCTITG CTGCCCATACACATI'CAAACAC and BWR4-GGCTGCGGAATTCGATATGGG CCCATGAGTGGGGTCTCCTGAAC. These primers amplify 1370 base pairs of APC sequence from nucleotide 2779 to 4151 in exon 15 of the APC gene and include appropriate restriction sites for directional cloning into pSTDTAQ-IKS as well as nucleotides within BWL2 to adjust the frame of the DNA sequence produced. (There are no known ApaI or HindlII restriction sites in this segment of the APC gene.) DNA samples were amplified by the PCR: 5 min at 95~ once; 1 min at 95~ 1 min at 60~ 1 rain at 72~ 30 cycles. Reaction mixtures contained the following: 200-400 ng of DNA template; 10 ~M each primer; 75 p~Meach deoxynucleotide triphosphate; 10 mM Tris, pH 8.3; 50 mM KCI; 1.5 mM MgCI2; 0.01% gelatin; 2.5 units of Taq polymerase. For each DNA sample analyzed, six PCR reactions were performed. Aliquots of each of these six PCR reactions were electrophoresed through a 2% ME agarose gel to check for amplification. The PCR products were pooled and split into two samples, which were each phenol/chloroform extracted and ethanol precipitated. Each pellet was washed once with 70% ethanol and resuspended in 20 /xl of water. PCR products were digested sequentially with ApaI and HindlII. Enzymes were heat inactivated at 65~ for 20 rain. Digested products were purified with Centricon 100 columns (Amicon) and electrophoresed to estimate DNA concentration. Products were mixed at a molar ratio of 1:2 or 1:3 (vector to insert) in a 20-/xl ligation reaction using a Stratagene ligation kit. Transformation and Plating. Cloned DNAs (1 /xl of each of the 20 ~1 ligation reactions, or about 30 ng of vector DNA) were introduced into Sure Electrocompetent Cells (40 ixl) (Stratagene) by electroporation in Bio-Rad Gene Pulser cuvets. The transformation efficiency of these cells was approximately 5 • 109, as determined by the use of pUC18 as a control. The electroporated cells were resuspended in 960/xl of S.O.C. medium (Bethesda Research Laboratories). Aliquots of 250 txl were then plated on each of two LB agar plates (180 cm) with 100/xg/ml ampicillin, 15/.Lg/ml tetracycline, and 80 /.zg/ml X-gal. IPTG was not included in the plating as the levels of constitutive expression were adequate for colorimetric determination and high expression levels appeared to inhibit colony formation. Plates were incubated overnight at 37~ and stored at 4~ to enhance the color of the colonies. Colony color and numbers were then scored, 2 days and again 4 days after incubation. Introduction T h e accurate detection o f mutation in h u m a n t u m o r suppressor genes is important for the clinical diagnosis of inherited diseases that predispose to neoplasia. Linkage analysis can follow the inheritance of these mutant genes within kindreds with a calculated likelihood for carrier status, but only a direct analysis of the g e n e will determine the g e n o t y p e of an at-risk individual in the absence of familial analysis. Therefore, rapid and reliable m e t h o d s for detecting mutation m u s t be developed. A P C 4 represents an excellent candidate disease for d e v e l o p i n g such approaches. Disruption o f the A P C gene leads to a highly penetrant, inherited f o r m of colon cancer (1-3). Mutational analyses o f the o p e n reading frame of APC, undertaken by a n u m b e r o f groups, have s h o w n that virtually all germline mutations are frameshift or point mutations that result in premature stop c o d o n s (2-11). T h e majority of these m u t a t i o n s are unique within a given family, with the exception of two deletions that together represent about 10% o f k n o w n germline m u tations (4, 7, 8, 10, 11). Because the A P C c o d i n g sequence is greater than 8.5 kilobases, it is extremely difficult to scan or sequence this gene rapidly (12, 13). Therefore, a functional test has been designed that allows direct detection of mutations in individuals. This assay is based on the level of expression of a fl-galactosidase coding sequence present at the 3' end of a P C R - a m p l i f i e d and cloned D N A s e g m e n t in this expression vector system. Received 9/29/93; accepted 10/20/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Howard Hughes Medical Institute, NIH Grant 5P30-HG00199-03, the Associazionc ltaliana Ricerca sul Cancro, C.N.R. Target Project "Biotechnology and Bioinstrumentation" Contract N. 93.0110PF70, and C.N.R. Progetto Finalizzato Ingegneria Genetica Contract N. 93.00050.PF99. 2 Present address: Department of Molecular Genetics, Biochemistry and Microbiology, The University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524. To whom requests for reprints should be addressed. 3 Investigator of the Howard Hughes Medical Institute. 4 The abbreviations used are: APC, adenomatous polyposis coli; PCR, polymerase chain reaction; IPTG, isopropyl-13-D-thiogalactopyranoside;X-gal, 5-bromo-4-chloro-3indolyl-t3-o-galactopyranoside. 5581 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer Research. SCREEN FOR APC MUTATIONS o W O O g~ e, W 4J gee Oe O 9 eee 9 eb 99 9 o~ O~ # 0 e 9 9 9 fl~8 o 9 O O O:I 0 ~* OO00 9 9 9 o 9 9 9 o Q O. . e 9 o 9 e O 9 O 0 e tD 9 . 9 e 9 9 0 9 9 o "l 9 |. ~~I 9 0 0 . e tills, ~e : O 0 eO Fig. 1. Three types of colonies that result from cloning an A P C segment amplified from the DNA of a normal individual. Clear arrow, blue colony (in-frame cloning); short black arrow , white colony (vector background); long black arrow, light blue colony (translational restart of in-frame cloning or PCR error that changed the frame). g 9 o / / , / o 9 9 9 .//; 9 ee 9 ..../.. ,e , ~ ./ /~ / / Mutation Detection and DNA Sequencing. Colonies to be checked for the sequence of insert DNA were picked from the plates into 100/xl of water and boiled for 10 min. An aliquot was then PCR amplified with custom APC primers (also carrying M13 universal or reverse sequencing primers) flanking the known mutation or with primers that were allele specific for either of the two common APC deletions (8). Samples for sequencing were purified with Centricon 100 columns (Amicon) and finally sequenced with an Applied Biosystems model 373A DNA sequencer, as described previously (2, 8, 9). , ,. jj in-frame cloning and maintenance of the reading frame in DNA that has a normal APC sequence; this results from the production of high levels of functional /3-galactosidase protein. A white colony is produced by plasmid not carrying a cloned DNA insert, resulting in almost no production of/3-galactosidase. The in-frame cloning of an insert that contains a sequence with a frameshift or a premature stop codon, however, produces a colony of intermediate blue color the intensity of which varies with the specific mutation. This result may be due to reinitiation at downstream in-frame ATGs in the cloned APC segment (positions 2845, 2935, 3631, and 4150) or to factors specific to the plasmid and bacterial strains used in this assay (see "Discussion"). The intermediate blue colonies derive from two types of inserts: mutations present in the original template DNA and errors generated by PCR amplification of the insert. This conclusion was supported by sequencing of dark and intermediate blue colonies derived from the testing of APC patient 3400. Each of three intermediate blue colonies sequenced contained the stop mutation found in this patient and each of three dark blue colonies contained normal APC sequence (data not Results A functional assay has been designed to detect mutations that disrupt translation of the A P C reading frame. The assay is composed of four steps: step 1, PCR amplification of APC sequence from genomic DNA or cDNA; step 2, cloning of these sequences into a plasmid, upstream and in-frame, with a/3-galactosidase coding sequence; step 3, transformation of bacteria; and step 4, plating onto X-gal-containing medium and differential counting of blue and white colonies to indicate the presence or absence of a mutation in the insert. In practice, three types of bacterial colonies are seen with this protocol, as shown in Fig. 1. An intense blue colony is produced from B 9 ~ Q 4.9 ~ 4 Q 9 9 e e o i O o * IP '- Q O Q ~ t 9 p . ~Q * 9 9 ~O I ~ ~ ql, z . , 9 0 i Fig. 2. Result of three clonings and platings; each DNA sample was prepared from an individual or cell line with a different A P C genotype. Colors and distribution of colonies containing amplified DNA from (A) an individual with a normal genotype at the A P C locus; (B) premature stop codon in A P C at position 4015; (C) a colorectal carcinoma cell line (SW480) that is homo- or hemizygous for an A P C stop mutation at position 4017. All three samples are represented numerically in Table 1. 5582 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer Research. SCREEN FOR APC MUTATIONS Table 1 Colony colors and numbers obtained by cloning APC segments from different templates into the reporter plasmid Scoring for most samples was done at 2 and 4 days; both numbers are included below with the 2-day count to the left of the shillingand the 4-day number shownon the right. Sample name 97a' 2379 3400 a 3400 12046 3493 3794 SW480 a SW480 APC genotype apc+/apc+ apc+/apc+ apc~/apc+ apcm/apc+ apc~/apc+ apc'/apc+ apc~/apc+ apcm/apcm or apcm/apcm/apcm or apcm/- Position of APC mutation No. of blue colonies No. of white colonies No. of light blue colonies 4015 4015 3183 3926 2805 4017 4017 388 91/91 188 74/74 33/33 64/64 76 4 3/3 18 10/3 25 23/10 20/16 16/7 2 14 19/7 16 3/10 257 108/121 44/48 73/82 94 332 224/236 a DNA ligation was set up in a 1:2 vector:insert ratio. shown). However, 4% of colonies derived from DNA of a normal subject also were intermediate blue, implying that mutations were also present in these inserts, most likely generated by PCR. The five DNAs used for this assay had been characterized previously using other methods of mutation detection. Each DNA sample was taken from an individual or tumor cell line with a different APC genotype: apc+/apc +, apcm/apc § and apcm/apcm (or apcm/apc-). All five carried unique APC mutations within the region of the gene amplified by the primers. Each of these five different germline or tumor cell line APC mutations was detected with the PCR/cloning strategy presented here. Table 1 summarizes the results. The colony colors and their ratios for three samples are shown in Fig. 2. DNA sequencing of colonies derived from several of these assays gave the expected sequences in every case (data not shown). Discussion or stability of the chimeric protein could be important. Indeed, the omission of IPTG from the plating steps of this assay was indicated by initial experiments suggesting that overexpression of the cloned gene product, when induced by IPTG, resulted in small, slow-growing colonies. This observation implies toxicity either from the high expression of the chimeric protein or from the vector in the Sure strain of bacteria. Heterogeneity in colony color is observed with the samples tested here. In some platings, the intensity of the blue color obtained from normal DNA inserts varied, especially in comparison to the lighter blue color obtained from the mutant inserts. Although it was not difficult to differentiate colors among colonies on a single plate, comparisons from one plate to another were difficult to interpret. Colors obtained from samples with frameshifts in APC showed clear differences between the blue and the intermediate blue colonies. Colors obtained from samples with premature stop codons, however, gave a less obvious, although still distinguishable, difference in color. The type of stop codon present also slightly affected the colony colors obtained: ochres (UAG) gave a clearer difference in the two colony colors than did amber (UAA) codons. We believe that this observation reflects the ability of bacteria in general to read through some stop codons more efficiently than others. In general, ochre codons can be read through more efficiently than amber codons, although in this instance, this ability is tempered by the supE44 genotype of the Sure strain. This gene is a nonsense suppressor that allows for about 1020% of ambers to be read as glutamines. Finally, UGA is the least efficient of the termination codons (18). Work is in progress to test other bacterial strains with different genotypes to optimize color differences. Surprisingly, other strains tested (su1675 and HB101) that do not have the supE44 mutation do not generate results that as are clear as those using Sure. The genomic structure of the APC gene is unusual, in that one of its exons is 6.5 kilobases. This size permits the majority of the open reading frame to be screened using genomic DNA. Moreover, the majority of germline mutations identified to date have occurred in this exon (2-11). The remainder of the gene is composed of at least 19 exons that are alternatively spliced (12, 17). 5 Therefore, the remainder of the open reading frame will be most effectively screened through complementary DNA. This can be accomplished with one or two sets of primers, depending on the splice forms that are targeted for analysis. 6 Most human disease genes that are candidates screening with the method reported here, i.e., those known to carry a significant proportion of nonsense or frameshift mutations, will likewise be screened most efficiently by using complementary DNA. Mutational analyses of colorectal adenomas and carcinomas at the APC locus have shown that most, if not all, tumors carry at least one nonsense or frameshift APC mutation (3, 19-21). Many of the tumors analyzed carry two APC mutations or show a loss of heterozygosity affecting the APC region of chromosome 5 (19-21). 7 These observations underscore the potential effectiveness of the mutation screening method presented here for the detection of APC mutations in colorectal adenomas and carcinomas, as well as in APC patients. These same mutational analyses of tumors also have suggested that over 70% of APC mutations occur within a 3-kilobase region of exon 15. The fact that this region is contained within the 6.5-kilobase contiguous genomic sequence of open reading frame suggests that genomic DNA can be used as a PCR template in the assay reported here. The experiments presented here have shown that a specific segment of the APC gene can be scanned efficiently and reliably for nonsense and frameshift mutations with a colorimetric assay. Work is under way This mutational screening technique detects germline mutations in the APC gene in patients and their family members who may be at risk for the inheritance of the same mutation. Detection of the mutations is accomplished by a rapid colorimetric assay. If the result suggests that a nonsense or frameshift mutation is present, repetition of the same assay with shorter stretches of the gene would localize the mutation more specifically. Direct sequencing of the PCR product would identify the exact mutation. The efficiency of this assay is determined by the size of DNA segments that can be screened. More conventional methods of mutation screening, such as single strand conformation polymorphism analysis, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, direct sequencing, or RNase protection assays, are all labor-intensive, gel-based assays that are normally reliable for DNA segments no longer than about 500 nucleotides. Other methods that are not gel based, such as allele-specific PCR and the oligoligation assay, are directed only toward the identification of known mutations and are not applicable for detection of unknown mutations (8, 15). In APC screening, for example, these methods would be inefficient, as none of the mutations identified to date has been found at a frequency of more than 1 in 10. A functional assay for the detection of p53 mutations in Li-Fraumeni patients has been described; however, in this case, it is the function of the gene product itself that is assayed and thus not generally applicable (16). Although the DNA segment length chosen for this assay is under 2 kilobases, the size of a fragment that can be screened depends upon a combination of the ability of PCR to generate larger fragments of DNA and the ability of/3-galactosidase to be expressed and to function appropriately with a second, uncharacterized protein domain attached. Although the function of/3-galactosidase is not usually affected by the gene product attached, factors such as solubility, toxicity, 5583 5 R. White, unpublished data. 6 L. Spirio, unpublished data. 7 L. Varesco, unpublished observations. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer Research. SCREEN FOR APC MUTATIONS to i m p r o v e the c o l o r i m e t r i c d e t e r m i n a t i o n o f the assay and to e x t e n d this m e t h o d to include the r e m a i n d e r o f the A P C gene, b y u s i n g other p r i m e r pairs and b y m u l t i p l e x i n g the P C R and c l o n i n g reactions. W e h o p e that the m e t h o d can b e applied in clinical situations for the a c c u r a t e and rapid d i a g n o s i s o f A P C . Acknowledgments We would like to thank Richard Cawthon, Tim Galitski, and Thrr~se Touhy for helpful discussions and Ruth Foltz for critical reading of the manuscript. References 1. Burr, R. W. Polyposis syndromes. In: T. Yamada, D. H. Alpers, C. Owyang, D. W. Powell, and E E. Silverstein (eds.), Textbook of Gastroenterology, Vol. 2, pp. 16741696. Philadelphia: J. B. Lippincott Company, 1991. 2. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burr, R. W., Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., Le Paslier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. Identification and characterization of the familial adenomatous polyposis coli gene. Cell, 66: 589-600, 1991. 3. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., Hedge, P., Markham, A., Krush, A. J., Peterson, G., Hamilton, S. R., Nilbert, M. C., Levy, D. B., Bryan, T. M., Preisinger, A. M., Smith, K. J., Su, L-K., Kinzler, K. W., and Vogelstein, B. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science (Washington DC), 253: 665-669, 1991. 4. Miyoshi, Y., Ando, H., Nagase, H., Nishisho, I., Horii, A., Miki, Y., Mori, T., Utsunomiya, J., Baba, S., Petersen, G., Hamilton, S. R., Kinzler, K. W., Vogelstein, B., and Nakamura, Y. Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients. Proc. Natl. Acad. Sci. USA, 89: 4452-4456, 1992. 5. Nagase, H., Miyoshi, Y., Horii, A., Aoki, T., Ogawa, M., Utsunomiya, J., Baba, S., Sasazuki, T., and Nakamura, Y. Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients. Cancer Res., 52: 4055-4057, 1992. 6. Fodde, R., van der Luijt, R., Wijnen, J., Tops, C., van der Klift, H., van LeeuwenCornelisse, I., Griffioen, G., Vasen, H., and Khan, P. M. Eight novel inactivating germline mutations at the APC gene identified by denaturing gradient gel electrophoresis (DGGE). Genomics, 13: 1162-1168, 1992. 7. Cottrell, S., Bicknell, D., Kaklamanis, L., and Bodmer, W. E Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas. Lancet, 340: 626--630, 1992. 8. Groden, J., Gelbert, L., Thliveris, A., Nelson, L., Robertson, M., Joslyn, G., Samowitz, W., Spirio, L., Carlson, M., Burt, R., Leppert, M., and White, R. Mutational analysis of patients with adenomatous polyposis: identical inactivating mutations in unrelated individuals. Am. J. Hum. Genet., 52: 263-272, 1993. 9. Olschwang, S., Laurent-Puig, P., Groden, J., White, R., and Thomas, G. Germline mutations in the first fourteen exons of the APC gene. Am. J. Hum. Genet., 52: 273-279, 1993. 10. 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Identification of deletion mutations and three new genes at the familial polyposis locus. Cell, 66: 589-600, 1991. 13. Kinzler, K. W., Nilbert, M. C., Su, L-K., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. M., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M S., Altschul, S. E, Horii, A., Ando, H., Miyoshi, Y., Miki, Y., Nishisho, I., and Nakamura, Y. Identification of FAP locus genes from chromosome 5q21. Science (Washington DC), 253: 661-665, 1991. 14. Weiss, R. B., Dunn, D. M., Atkins, J. E, and Gesteland, R. E Slippery runs, shifty stops, backward steps and forward hops:'-2, -1, +1, +2, +5 and +6 ribosomal frameshifting. Cold Spring Harbor Symposia on Quantitative Biology: Proceedings, 52: 687--693, 1987. 15. Nickerson, D., Kaiser, R., Lappin, S., Stewart, J., Hood, L., and Landegren, U. Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay. Proc. Natl. Acad. Sci. USA, 87: 8923--8927, 1990. 16. Frebourg, T., Barbier, N., Kassel, J., Ng, Y-S., Romero, P., and Friend, S. H. A functional screen for germ line p53 mutations.based on transcriptional activation. Cancer Res., 52: 6976--6978, 1992. 17. Hori, A., Nakatsuru, S., Ichii, S., Nagase, H., and Nakamura, Y. Multiple forms of the APC gene transcripts and their tissue-specific expression. Hum. Mol. Genet., 2: 283-287, 1993. 18. Lewin, B. Genes IV. New York: Oxford University Press, 1990. 19. Powell, S. M., Zilz, N., Beazer-Barclay, Y., Bryan, T. M., Hamilton, S. R., Thibodeau, S. N., Vogelstein, B., and Kinzler, K. W. APC mutations occur early during colorectal tumorigenesis. Nature (Lond.), 359: 235-237, 1992. 20. Miyoshi, Y., Nagase, H., Ando, H., Horii, A., Ichii, S., Nakatsuru, S., Aoki, T., Miki, Y., Mori, T., and Nakamura, Y. Somatic mutations of the APC gene in colorectal tumors: mutation cluster region in the APC gene. Hum. Mol. Genet., 1: 229-233, 1992. 21. Ichii, S., Horii, A., Nakatsuru. S., Furuyama, J., Utsunomiya, J., and Nakamura, Y. Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis (FAP). Hum. Mol. Genet., 1: 387-390, 1992. 5584 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer Research. A Rapid Screening Method to Detect Nonsense and Frameshift Mutations: Identification of Disease-causing APC Alleles Liliana Varesco, Joanna Groden, Lisa Spirio, et al. Cancer Res 1993;53:5581-5584. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/53/23/5581 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer Research.