TY - JOUR
AU - O’Leary, Timothy, J
AB - To the Editor: Deletion mutations of the juxtamembrane domain of the KIT protooncogene, which encodes a tyrosine kinase receptor, have been associated with malignant behavior of gastrointestinal stromal/smooth muscle tumors (GISTs) (1)(2)(3)(4)(5). Identification of KIT mutations in GISTs is clinically useful because metastasis and death may occur in tumors that are histologically and clinically benign (6). Direct sequence analysis is the method of choice for identifying these mutations. To facilitate routine identification of the most common KIT gene mutations associated with malignant behavior, we have developed a fluorescence-based PCR method in which amplified DNA fragments are analyzed by capillary electrophoresis (CE). The method can be used to detect mutations of KIT exon 11 in the clinical diagnostic laboratory. DNA from 25 cases (26 samples) of paraffin-embedded GISTs originating in the small bowel or colon was isolated by previously published methods (5)(7). Briefly, specimens were first deparaffinized with xylene, and then dehydrated with ethanol. Following dehydration, they were digested in 100 μL of extraction buffer containing, per liter, 0.15 mol of NaCl, 0.1 mol of EDTA, 20 mmol of Tris (pH 8), 10 g of sodium dodecyl sulfate, and 0.1 g proteinase K overnight at 55 °C. Specimens were then heated at 95 °C for 5 min to inactivate proteinase K. To prepare specimens for CE, DNA extracts were amplified using exon 11 primer sequences (forward, 5′-TTTCCCTTTCTCCCCACAGA-3′; reverse, 5′-FAM-CTCAGCCTGTTTCTGGGAAAC, where FAM is 6-carboxyfluorescein). PCR was carried out in a 50-μL reaction mixture containing 2 μL of the DNA extraction solution; 10 mM Tris (pH 8.3); 50 mM KCl; 3 mM MgCl2 0.2 mM each of dATP, dGTP, dCTP, and dTTP (Pharmacia Biotech); 0.4 μM each primer; and 2.5 U of AmpliTaq (Perkin-Elmer). After incubation for 5 min at 94 °C, amplification was carried out for 40 cycles of 94 °C (1 min), 55 °C (1 min), and 72 °C (1 min). After cycling, the reaction mixture was incubated at 72 °C for 10 min. We combined 1 μL of PCR product diluted 1:10, 12 μL of deionized formamide, and 0.5 μL of 6-carboxytetramethylrhodamine (TAMRA)-labeled size marker. The mixture was heated for 5 min at 95 °C and then cooled on ice for 2 min. Samples were loaded into an ABI 310 genetic analyzer for CE analysis of the PCR fragments. Amplification of wild-type DNA produced a 141-bp fragment. For direct sequencing, DNA was amplified by PCR using different primers (5)(7). The sequence for the “forward” primer was 5′-CCAGAGTGCTCTAATGACTGAGAC-3′; the sequence for the “reverse” primer was 5′-AGCCCCTGTTTCATACTGACC-3′. Reactions were carried out in a 50-μL volume containing 2 μL of the nucleic acid extract; 0.5 μM each primer; 0.5 μM each of dATP, dGTP, dCTP, and dTTP; and 0.5 U of AmpliTaq Gold (Perkin-Elmer) in a 2.5 μM MgCl2 buffer. Amplification consisted of 1 cycle at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s, and concluded with 1 cycle at 72 °C for 10 min. The DNA was gel purified in an 2% agarose gel and extracted; direct DNA sequencing was performed on the ABI Prism 377 DNA Sequencer, using the same primers that were used for amplification. Sequencing reactions were conducted with the Big Dye Terminator Sequencing Ready Reaction reagent set (Perkin-Elmer) according to the manufacturer’s instructions. Frameshift mutations within KIT exon 11 produced PCR products with altered sizes, which were readily distinguished from wild-type KIT by CE (Fig. 1 ). In each of the eight cases in which CE demonstrated altered product sizes, the presence of a frameshift mutation was confirmed by direct sequencing [shown in the data supplement available at Clinical Chemistry Online (http://www.clinchem.org/content/vol47/issue7)]. For example, the presence of a single peak of 141 bp for case 94 (Fig. 1 , GIST 94) demonstrates the presence of homozygous wild-type KIT sequences. In contrast, identification of two equal-intensity peaks of 141 and 144 bp, respectively, in case 102a (Fig. 1 , GIST 102a) demonstrates the presence of an insertion mutation and strongly suggests monoallelic deletion. Similarly, the identification of peaks at 126 and 141 bp for case 96 (Fig. 1 , GIST 96) suggests monoallelic or heterogeneous deletion. Figure 1. Open in new tabDownload slide Electrophoretic traces of three GISTs. A trace for the molecular weight markers appears below each tumor trace. Y axes have been enhanced for clarity. Figure 1. Open in new tabDownload slide Electrophoretic traces of three GISTs. A trace for the molecular weight markers appears below each tumor trace. Y axes have been enhanced for clarity. Two specimens demonstrated point mutations on direct sequencing. Point mutations are observed in ∼20% of those GISTs that harbor an exon 11 mutation, but their significance is unknown. The inability to identify point mutations is the major disadvantage of the CE approach. Nevertheless, PCR fragment length analysis by CE is a fast and simple method to detect KIT gene mutations when sequencing is not available. References 1 Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998 ; 279 : 577 -580. Crossref Search ADS PubMed 2 Nakahara M, Isozaki K, Hirota S, Miyagawa J, Hase-Sawada N, Taniguchi M, et al. A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors. Gastroenterology 1998 ; 115 : 1090 -1095. Crossref Search ADS PubMed 3 Taniguchi M, Nishida T, Hirota S, Isozaki I, Ito T, Nomura T, et al. Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors. Cancer Res 1999 ; 59 : 4297 -4300. PubMed 4 Lasota J, Jasinski M, Sarlomo-Rikala M, Miettinen M. Mutations in exon 11 of c-Kit occur preferentially in malignant versus benign gastrointestinal stromal tumors and do not occur in leiomyomas or leiomyosarcomas. Am J Pathol 1999 ; 154 : 53 -60. Crossref Search ADS PubMed 5 Ernst SI, Hubbs AE, Przygodzki RM, Emory TS, Sobin LH, O’Leary TJ. KIT mutation portends poor prognosis in gastrointestinal stromal/smooth muscle tumors. Lab Invest 1998 ; 78 : 1633 -1636. PubMed 6 Cunningham RE, Federspiel BH, McCarthy WF, Sobin LH, O’Leary TJ. Predicting prognosis of gastrointestinal smooth muscle tumors. Role of clinical and histologic evaluation, flow cytometry, and image cytometry. Am J Surg Pathol 1993 ; 17 : 588 -594. Crossref Search ADS PubMed 7 Li SQ, O’Leary TJ, Sobin LH, Erozan YS, Rosenthal DL, Przygodzki RM. Analysis of KIT mutation and protein expression in fine needle aspirates of gastrointestinal stromal/smooth muscle tumors. Acta Cytol 2000 ; 44 : 981 -986. Crossref Search ADS PubMed © 2001 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Rapid Screening for KIT Mutations by Capillary Electrophoresis
JF - Clinical Chemistry
DO - 10.1093/clinchem/47.7.1325
DA - 2001-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/rapid-screening-for-kit-mutations-by-capillary-electrophoresis-1Q3ifdBHOg
SP - 1325
VL - 47
IS - 7
DP - DeepDyve
ER -