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RET Oligonucleotide Microarray for the Detection of RET Mutations in Multiple Endocrine Neoplasia Type 2 Syndromes1

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RET Oligonucleotide Microarray for the Detection of RET Mutations in Multiple Endocrine Neoplasia Type 2 Syndromes1 Vol. 8, 457–463, February 2002 Clinical Cancer Research 457 RET Oligonucleotide Microarray for the Detection of RET Mutations in Multiple Endocrine Neoplasia Type 2 Syndromes1 Il-Jin Kim, Hio Chung Kang, Jae-Hyun Park, tide microarray can detect RET missense mutations at these Ja-Lok Ku, Jong-Soo Lee, Hyuk-Joon Kwon, nine codons. Theoretically, a total of 55 missense mutation Kyong-Ah Yoon, Seung Chul Heo, types can occur at eight codons (codons 609, 611, 618, 620, 630, 634, 768, and 804). RET oligonucleotide microarray is Hee-Young Yang, Bo Youn Cho, designed to detect all of these 55 missense mutation types at Seong Yeon Kim, Seung Keun Oh, these eight codons and one predominant type at codon 918. Yeo-Kyu Youn, Do-Jun Park, Myung-Shik Lee, Fifty-six oligonucleotides were designed for the 56 mutation Kwang-Woo Lee, and Jae-Gahb Park2 types at nine codons, and 11 oligonucleotides were designed Familial Cancer Clinic, National Cancer Center, Gyeonggi 411-764, for the wild types and positive controls. We found RET Korea [J-G. P., J-S. L.]; Korean Hereditary Tumor Registry, mutations in all eight of the Korean MEN2A families (a total Laboratory of Cell Biology, Cancer Research Center and Cancer of 75 members; 27 affected members, 19 gene carriers, and Research Institute, Seoul National University College of Medicine, 29 unaffected members) using the developed RET oligonu- Seoul, Korea 110-744 [I-J. K., H. C. K., J-H. P., J-L. K., H-J. K., K- cleotide microarray and an automatic sequencing. Because A. Y., H-Y. Y., J-G. P.]; Departments of Surgery [S. C. H., S. K. O., Y-K. Y.] and Internal Medicine [B. Y. C., S. Y. K., D-J. P.], Seoul we found only five mutation types from eight MEN2A fam- National University College of Medicine, Seoul, Korea 110-744; ilies, the international collaborations are required to see Department of Medicine, Samsung Medical Center, Sungkyunkwan whether the RET oligonucleotide microarray may be used as University School of Medicine, Seoul, Korea 135-230 [M-S. L.]; and a genetic diagnostic tool. Taken together, the RET oligonu- Department of Internal Medicine, The Catholic University College of Medicine, Seoul, Korea 137-070 [K-W. L.] cleotide microarray can function as a fast and reliable ge- netic diagnostic device, which simplifies the process of de- tecting RET mutations. ABSTRACT Multiple endocrine neoplasia type 2 (MEN2) syndromes INTRODUCTION are inherited in an autosomal dominant fashion with high The human RET gene encodes a transmembrane receptor of penetrance. There are three subtypes, namely, MEN2A the protein tyrosine kinase family, which is implicated in neural (multiple endocrine neoplasia type 2A), MEN2B (multiple crest tissue development and differentiation (1, 2). The RET endocrine neoplasia type 2B), and familial medullary thy- gene, located on chromosome 10q11.2, is composed of 21 exons roid carcinoma. The variations in the RET gene play an and is ϳ55 kb in size (3). The RET protein is composed of an important role in the MEN2 syndromes. In this work, we extracellular domain, a transmembrane domain, and intracellu- have developed a RET oligonucleotide microarray of 67 lar tyrosine kinase domains (3, 4). The RET gene is responsible oligonucleotides to quickly detect RET mutations in MEN2 for MEN23 syndromes, which are inherited in an autosomal syndromes. The predominant RET mutations are missense dominant fashion with high penetrance and diverse clinical mutations and are restricted to nine codons (codons 609, manifestations. The syndromes have three subtypes: MEN2A, 611, 618, 620, 630, 634, 768, 804, and 918) in MEN2 syn- MEN2B, and FMTC (2, 5). MEN2A, the most frequent subtype dromes. Missense mutations at codons 609, 611, 618, 620, of the MEN2 syndromes, is characterized by MTC (or C-cell and 634 have been identified in 98% of MEN2A families and hyperplasia), pheochomocytoma, and hyperparathyroidism. in 85% of familial medullary thyroid carcinoma families. RET mutations in MEN2 syndromes have usually been detected More than 95% of MEN2B patients also had a predominant by single-strand conformational polymorphism or by direct se- mutation type at codon 918 (Met 3 Thr). RET oligonucleo- quencing. A method for the rapid mutation analysis of a few gene sequences has been developed using an oligonucleotide microarray, which can be effectively used for sequence analysis, diagnostics for genetic diseases, and gene polymorphism studies Received 7/13/01; revised 10/29/01; accepted 11/2/01. (6). A typical DNA microarray-based method is less time con- The costs of publication of this article were defrayed in part by the suming and is cheaper than conventional sequencing, and plays 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 Supported by a 2001 research grant from National Cancer Center, Korea. I-J. K., H. C. K., and J-H. P. were supported by the 2001 BK Brain Korea 21 project for Medicine, Dentistry, and Pharmacy. 3 The abbreviations used are: MEN2, multiple endocrine neoplasia type 2 To whom requests for reprints should be addressed, at National Cancer 2; MEN2A, multiple endocrine neoplasia type 2A; MEN2B, multiple Center, 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi, 411-764, Korea. endocrine neoplasia type 2B; FMTC, familial medullary thyroid carci- Phone: 82-31-920-1501; Fax: 82-31-920-1511; E-mail: jgpark@plaza. noma; MTC, medullary thyroid carcinoma; PHEO, pheochromocytoma; snu.ac.kr. UDG, uracil-DNA glycosylase; dNTP, deoxynucleotide triphosphate. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. 458 Development of RET Oligonucleotide Microarray Table 1 Germ-line mutations of the RET proto-oncogene in SNU-MEN2A families No. of affected Family No. of affected members with No. of gene Cloning RET (SNU-MEN2A) members PHEO carriers Mutation Direct seq.a and seq. microarray I 5 2 4 C634Wb ϩϩϩ II 2 2 3 C634R ϩϩϩ III 2 2 1 C634R ϩϩϩ IV 11 1 9 C618S ϩϩϩ V 1 1 – C634Y n/d ϩϩ a seq., sequencing; ϩ, detected; n/d, not detected. b C634W, mutation at codon 634 (TGC3TGG, Cys3Trp). a valuable role in high throughput sequence analysis (7, 8). cycler (Gene Amp PCR System 9700; Applied Biosystems Inc., Oligonucleotide microarrays show high sensitivity in terms of Foster City, CA). PCR conditions consisted of 35 cycles of 94°C point mutation detection. Thus, it would be very useful to use an for 30 s, 60°C for 30 s, and 72°C for 1 min, with a final oligonucleotide microarray for analyzing genes with more fre- elongation of 7 min at 72°C. Each exon was amplified sepa- quent point mutations, such as the RET gene in MEN2 syn- rately. dromes. The predominant mutations in MEN2 syndromes are Cloning and Sequencing. Fresh PCR products were li- missense mutations, and these are restricted to some codons of gated into PCR-TOPO vectors, and subcloned using the TA the RET gene. In MEN2A and FMTC, most RET mutations are cloning system (Invitrogen, Carlsbad, CA). Bidirectional se- found in exons 10 and 11, and are located in the extracellular quencing was performed using a Taq dideoxy terminator cycle cysteine-rich region, which is a part of the putative ligand- sequencing kit and an ABI 377 DNA sequencer (Perkin-Elmer, binding domain (codons 609, 611, 618, 620, 630, and 634). Foster City, CA). Infrequently, noncysteine codon mutations have been reported Segregation Analysis by PCR-restriction Enzyme Di- in FMTC (at codons 768 and 804; Ref. 9). Missense mutations gestion. Each PCR product of exon 11 in SNU-MEN2A-I-III at codons 609, 611, 618, 620, and 634 have been identified in was digested with HinP1I restriction enzyme (NEB Inc., Bev- 98% of MEN2A families and in 85% of FMTC families. Mis- sense mutations at codons 768 and 804 have been known to be erly, MA), and the fragments obtained were separated by elec- responsible for 5–10% of the FMTC families (10). We devel- trophoresis on 2% agarose gels. oped an oligonucleotide microarray for rapid and simplified RET Oligonucleotide Microarray Manufacturing. All RET mutation detection. In our microarray system, a glass slide 67 of the oligonucleotides with 12 carbon spacers were synthe- Ј spotted with 67 oligonucleotides and fluorescence-labeled PCR sized by MWG-Biotech (Ebersberg, Germany). They were 5 products were used to detect RET mutations. The RET oligo- modified with amino residues for Schiff’s base reaction with an ␮ nucleotide microarray can find RET missense mutations at nine aldehyde group on a glass slide. Ten pmol/ l of oligonucleotide codons in MEN2 syndromes (MEN2A, MEN2B, and FMTC). In in micro spotting solution (TeleChem International Inc., Sunny- this study, the RET proto-oncogene was analyzed in eight Ko- vale, CA) was printed on the aldehyde-coated glass slide (26 ϫ rean MEN2A families using automatic bidirectional sequencing 76 ϫ 1 mm; CEL Associates Inc., Houston, TX) using a pin and the RET oligonucleotide microarray. microarrayer (Cartesian Microsys 5100; Cartesian Technologies Inc., Irvine, CA). A total of 67 oligonucleotides were printed on ϫ MATERIALS AND METHODS each glass slide in a 3.7 mm 7.6 mm. Spot spacing was 300 ␮ ␮ Families and DNA Samples. Blood samples of five Ko- m, and spot size was 130 m. After printing, the RET mi- rean MEN2A families (SNU-MEN2A-I, -II, -III, -IV, and -V) croarray was dried at room temperature, at least overnight, and from Seoul National University Hospital and three MEN2A then stored at 4°C. All 67 of the oligonucleotide sequences are families (SMC-MEN2A-I, -II, and-III) from Samsung Medical shown in Table 2.
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