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

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 genetic 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.

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. Center were collected. SNU-MEN2A-I and three SMC-MEN2A Sample Preparation for the Generation of Single- ␭ families have been reported previously (11, 12). Sixty-two stranded DNA ( Exonuclease- and Asymmetric PCR-based ␭ SNU-MEN2A family members (21 affected members, 17 gene Approach). In the exonuclease-based method, PCR was carriers, and 24 unaffected members) were studied using the carried out using 5Ј-phosphate modified forward primer and the clinical approach, and 46 members of the 62 were analyzed original reverse primer in a volume of 25 ␮l containing the genetically (Table 1). following: 100 ng of genomic DNA, 10 pmol of each primer, DNA Extraction. Total genomic DNA was extracted dNTP at 40 ␮M each, 20 ␮M Cy5-dCTP (Amersham Pharmacia- using Ficoll-Paque (Amersham Pharmacia-biotech Ltd., Upp- biotech Ltd., Buckinghamshire, United Kingdom), and 0.5 units sala, Sweden) and TRI reagent (Molecular Research Center, of Taq polymerase (Intron-biotechnology, Seoul, Korea). After Cincinnati, OH) following the manufacturer’s instructions. purification, the PCR product was digested with 5 units of ␭ PCR Amplification. The PCR primers for exons 10 and exonuclease (Amersham Pharmacia-biotech Ltd.) to obtain sin- 11 were used as described in Ref. 13, and primers for exons 13, gle-stranded DNA. Asymmetric PCR, which contains a reverse 14, and 16 were as described in Ref. 2. Reactions were initiated primer and 20 ␮M of Cy5-dCTP, was performed using ethanol- by denaturation for 5 min at 94°C in a programmable thermal precipitated PCR product without fluorescence-labeled dNTP.

Table 2 Oligonucleotides sequences of the RET oligonucleotide microarray No. Probe name Exon Codon Sequence 1 10-PCa 10 5Ј-GGGGATTAAAGCTGGCTATGG-3Ј 2 609M-(R)b 10 609 5Ј-CTATGGCACCCGCAACTGCTT-3Ј 3 609M-(S) 5Ј-CTATGGCACCAGCAACTGCTT-3Ј 4 609M-(G) 5Ј-TGGCACCGGCAACTGC-3Ј 5 609M-(F) 5Ј-CTATGGCACCTTCAACTGCTTC-3Ј 6 609M-(Y) 5Ј-CTATGGCACCTACAACTGCTTC-3Ј 7 609M-(S) 5Ј-TATGGCACCTCCAACTGCTTC-3Ј 8 609M-(W) 5Ј-TATGGCACCTGGAACTGCTTC-3Ј 9 609W-(C) 5Ј-TATGGCACCTGCAACTGCTTC-3Ј 10 611M-(R) 10 611 5Ј-ACCTGCAACCGCTTCCCTGA-3Ј 11 611M-(S) 5Ј-CACCTGCAACAGCTTCCCTGA-3Ј 12 611M-(G) 5Ј-ACCTGCAACGGCTTCCCTGA-3Ј 13 611M-(F) 5Ј-ACCTGCAACTTCTTCCCTGAG-3Ј 14 611M-(Y) 5Ј-ACCTGCAACTACTTCCCTGAG-3Ј 15 611M-(S) 5Ј-ACCTGCAACTCCTTCCCTGAG-3Ј 16 611M-(W) 5Ј-ACCTGCAACTGGTTCCCTGAG-3Ј 17 611W-(C) 5Ј-ACCTGCAACTGCTTCCCTGAG-3Ј 18 618M-(R) 10 618 5Ј-AGGAGAAGCGCTTCTGCGAG-3Ј 19 618M-(S) 5Ј-GAGGAGAAGAGCTTCTGCGAG-3Ј 20 618M-(G) 5Ј-GAGAAGGGCTTCTGCG-3Ј 21 618M-(F) 5Ј-AGGAGAAGTTCTTCTGCGAG-3Ј 22 618M-(Y) 5Ј-AGGAGAAGTACTTCTGCGAG-3Ј 23 618M-(S) 5Ј-AGGAGAAGTCCTTCTGCGAG-3Ј 24 618M-(W) 5Ј-AGGAGAAGTGGTTCTGCGAG-3Ј 25 618W-(C) 5Ј-GAGGAGAAGTGCTTCTGCGAG-3Ј 26 620M-(R) 10 620 5Ј-AAGTGCTTCCGCGAGCCCGA-3Ј 27 620M-(S) 5Ј-AAGTGCTTCAGCGAGCCCGA-3Ј 28 620M-(G) 5Ј-AAGTGCTTCGGCGAGCCCGA-3Ј 29 620M-(F) 5Ј-AAGTGCTTCTTCGAGCCCGA-3Ј 30 620M-(Y) 5Ј-AAGTGCTTCTACGAGCCCGA-3Ј 31 620M-(S) 5Ј-AAGTGCTTCTCCGAGCCCGA-3Ј 32 620M-(W) 5Ј-AAGTGCTTCTGGGAGCCCGA-3Ј 33 620W-(C) 5Ј-AAGTGCTTCTGCGAGCCCGA-3Ј 34 630M-(R) 11 630 5Ј-GATCCACTGCGCGACGAGCT-3Ј 35 630M-(S) 5Ј-GATCCACTGAGCGACGAGCT-3Ј 36 630M-(G) 5Ј-GATCCACTGGGCGACGAGCT-3Ј 37 630M-(F) 5Ј-GATCCACTGTTCGACGAGCT-3Ј 38 630M-(Y) 5Ј-GATCCACTGTACGACGAGCT-3Ј 39 630M-(S) 5Ј-GATCCACTGTCCGACGAGCT-3Ј 40 630M-(W) 5Ј-GATCCACTGTGGGACGAGCT-3Ј 41 630W-(C) 5Ј-GATCCACTGTGCGACGAGCT-3Ј 42 634M-(R) 11 634 5Ј-GACGAGCTGCGCCGCACGGT-3Ј 43 634M-(S) 5Ј-GACGAGCTGAGCCGCACGGT-3Ј 44 634M-(G) 5Ј-CGAGCTGGGCCGCACG-3Ј 45 634M-(F) 5Ј-GACGAGCTGTTCCGCACGGT-3Ј 46 634M-(Y) 5Ј-GACGAGCTGTACCGCACGGT-3Ј 47 634M-(S) 5Ј-GACGAGCTGTCCCGCACGGT-3Ј 48 634M-(W) 5Ј-GACGAGCTGTGGCGCACGGT-3Ј 49 634W-(C) 5Ј-GACGAGCTGTGCCGCACGGT-3Ј 50 11-PCa 11 5Ј-CCGCTGTCCTCTTCTCCTTC-3Ј 51 768M-(Q) 13 768 5Ј-TCCCCGAGTCAGCTTCGAGA-3Ј 52 768M-(K) 5Ј-CTCCCCGAGTAAGCTTCGAGA-3Ј 53 768M-(A) 5Ј-TCCCCGAGTGCGCTTCGAGA-3Ј 54 768M-(G) 5Ј-TCCCCGAGTGGGCTTCGAGA-3Ј 55 768M-(V) 5Ј-TCCCCGAGTGTGCTTCGAGA-3Ј 56 768M-(D) 5Ј-TCCCCGAGTGACCTTCGAGA-3Ј 57 768M-(D) 5Ј-TCCCCGAGTGATCTTCGAGAC-3Ј 58 768W-(E) 5Ј-TCCCCGAGTGAGCTTCGAGA-3Ј 59 804M-(L) 14 804 5Ј-CTCCTCATCCTGGAGTACGC-3Ј 60 804M-(M) 5Ј-CCTCCTCATCATGGAGTACGC-3Ј 61 804M-(L) 5Ј-CCTCCTCATCTTGGAGTACGC-3Ј 62 804M-(E) 5Ј-CTCCTCATCGAGGAGTACGC-3Ј 63 804M-(A) 5Ј-CTCCTCATCGCGGAGTACGC-3Ј 64 804M-(G) 5Ј-CTCCTCATCGGGGAGTACGC-3Ј 65 804W-(V) 5Ј-CTCCTCATCGTGGAGTACGC-3Ј 66 918M-(T) 16 918 5Ј-CAGTTAAATGGACGGCAATTGAAT-3Ј 67 918W-(M) 5Ј-CAGTTAAATGGATGGCAATTGAAT-3Ј a PC, positive control; M, mutant type; W, wild type. b (R), amino acid was indicated in the parenthesis, i.e. (R)-Arg.

Sample Preparation for the Fragmentation of DNA AGC, Cys 3 Ser) in exon 10. In the case of three SNU-MEN2A (UDG and DNase I-based Approach). In the UDG-based families (SNU-MEN2A-I-III), each PCR product was digested approach, PCR reactions were carried out in a volume of 25 ␮l with Hin Pl I to determine the cosegregation of these mutations. containing 100 ng of genomic DNA, 10 pmol of each primer, All of the samples produced a new Hin Pl I restriction site at dNTP at 40 ␮M each, 16 ␮M of dUTP, 20 ␮M of Cy5-dCTP, and codon 634. The SNU-MEN2A-V family appeared to be a wild 0.5 units of Taq polymerase. After the PCR amplification, the type by direct sequencing. After the RET oligonucleotide mi- Cy5-labeled PCR product was purified using a purification kit croarray analysis, cloning and sequencing were performed, and (Qiagen Inc., Valencia, CA) and fragmented by adding 2 units the C634Y mutation (codon 634, TGC 3 TAC, Cys 3 Tyr) of UDG (MBI Fermentas Inc., Hanover, MD) at 37°Cfor4h. was confirmed (see “Discussion”; Table 1). The SMC- Enzyme inactivation was performed at 95°C for 5 min in 50 mM MEN2A-I, -II, and -III families had a C634R (codon 634, TGC 3 3 3 of NaOH and 5 mM EDTA (14). For the DNase I-based system, CGC, Cys Arg), C618R (codon 618, TGC CGC, Cys 3 3 3 PCR amplification containing dNTP at 40 ␮M each and 20 ␮M Arg), and C634Y (codon 634, TGC TAC, Cys Tyr), of Cy5-dCTP was performed. The PCR products were then respectively. purified and digested with 0.25 units of DNase I (Takara, Shiga, RET Mutations Identified Using the Oligonucleotide Japan) at 25°C for 10 min. Enzyme was inactivated at 95°C for Microarray. Twenty-three of 62 members from five SNU- 10 min. MEN2A families and 13 members from three SMC-MEN2A Hybridization and Washing. The RET microarray was families were available for the RET oligonucleotide microarray rinsed with 0.2% SDS three times and denatured at 95°C for 3 analysis. Twenty-two of 23 SNU-MEN2A family members min. It was then rinsed with sodium borohydride (Sigma Chem- had been automatically sequenced previously. One (SNU- ical Co., St. Louis, MO) and 0.2% SDS. Finally, it was washed MEN2A-V) sample was analyzed using the RET oligonucleo- in distilled water. Prepared PCR products of each exon were tide microarray, and confirmed by cloning and sequencing. mixed, resuspended in prewarmed 1 ϫ UniHyb Solution DNase I- based hybridization results of 22 previously sequenced (TeleChem International Inc., Sunnyvale, CA) at 3␮ᐉ, and hy- samples were consistent with the sequencing data. SNU- bridized in a saturated vapor tube at 60°C for 3 h. The hybrid- MEN2A-I showed specific signals in all eight of the positions ized microarray was rinsed at room temperature in a buffer of (positions 9, 17, 25, 33, 41, 49, 58, and 65) exhibiting wild type, 2 ϫ SSC and 0.2% SDS. Both the hybridization and the washing two positive controls (positions 1 and 50), and an additional steps were performed in the dark. signal at position 48. This signal at position 48 indicated the Analysis of Signals. The RET oligonucleotide microar- presence of a C634W mutation in codon 634 (Fig. 1A). SNU- ray was scanned using a ScanArray Lite (Packard Instrument MEN2A-II and -III families showed an additional signal at Co., Meriden, CT) and analyzed using Quantitative Microarray position 42 of C634R (Fig. 1B). SNU-MEN2A-IV had an ad- analysis software (QuantArray, version 2.0). To establish a ditional signal at position 19 indicating the C618S mutation. In cutoff, all of the data analysis was carried out using a SigmaPlot SNU-MEN2A-V, we found an additional signal at position 46, (SPSS Inc., San Rafael, CA), and means and SDs were calcu- indicating the C634Y mutation. SNU-MEN2A family members lated. with no RET mutation showed only signals at wild type and positive control positions. SMC-MEN2A-I, -II, and -III had an additional signal at positions 42, 18, and 46, which indicate RESULTS C634R, C618R, and C634Y, respectively. RET Oligonucleotide Microarray. Each oligonucleo- Data Analysis. The signal intensities of the oligonucleotide was printed four times horizontally, in an upward direction tides that were spotted in quadruplicate were averaged, and starting from position 1, and the printing was divided into three these averages values at each position were regarded as the real groups. Positions 1–25 were printed first, then positions 26–50, signal values. All of the signals from the wild types and the and finally, positions 51–67. Positions 1 and 50 were positive positive controls were then excluded. In the case for the pres- controls for exons 10 and 11, respectively. The other oligonu- ence of a mutation, a signal showing the strongest intensity cleotides were as follows: 2–33 for exon 10 (codons 609, 611, among the remainder was also excluded, and the remaining 618, and 620), 34–49 for exon 11 (codons 630 and 634), 51–58 values were defined as background signals. The mean (BA) and for exon 13 (codon 768), 59–65 for exon 14 (codon 804), and SD (BSD) of the background signals were calculated, and BA ϩ 66 and 67 for exon 16 (codon 918). 2.58 BSD was established as the cutoff level. We regarded any RET Mutations Identified by DNA Sequencing and signals over the cutoff level to be significant signals. Data are Enzyme Segregation Analysis. We screened for the presence presented graphically in Fig. 2, which also shows a horizontal of RET mutations in all five of the SNU-MEN2A families and solid line showing BA ϩ 2.58 ϫ BSD (cutoff value) in SNU- three SMC-MEN2A families. Forty-six of 62 members of five MEN2A-I. SNU-MEN2A families were sequenced. Screenings of the RET RET Mutations and Phenotype. PHEO is more com- gene were performed by bidirectional sequencing in exons 10, mon in patients with C634 mutations than in those with C618 11, 13, and 14. RET mutations were found in five SNU-MEN2A mutations (9). Korean MEN2A patients with MTC and C634 families and three SMC-MEN2A families. SNU-MEN2A-I had mutations (SNU-MEN2A-I, -II, -III, -V, SMC-MEN2A-I, and a C634W mutation in exon 11 (codon 634, TGC 3 TGG, Cys -III) had ϳ80% (12 of 15) frequency of PHEO. However, 3 Trp). SNU-MEN2A-II and SNU-MEN2A-I had a C634R SNU-MEN2A-IV and SMC-MEN2A-II with the MTC and the mutation (codon 634, TGC 3 CGC, Cys 3 Arg) in exon 11. C618 mutation showed a PHEO occurrence of 15% (2 of 13). SNU-MEN2A-II had a C618S mutation (codon 618, TGC 3 It was reported that PHEO is shown in 50% of MEN2A (9).

Fig. 1 RET oligonucleotide microarray result using DNase I in SNU-MEN2A-I and SNU- MEN2A-III. A, SNU-MEN2A-I had a signal at 634 M-(W), in addition to the signals of the positive controls and wild types. This meant that SNU-MEN2A-I had a C634W mutation in exon 11. Oligonucleotides were spotted four times horizontally. The numbers refer to oligonucleotide order [e.g., 48 3 634 M-(W)]. Photomultiplier tube gain: 87%, laser power: 87%, and resolution: 5 ␮m. B, SNU-MEN2A-III showed an additional signal at 634 M-(R). The signal at 634 M-(R) indicates a C634R mutation in exon 11 (TGC 3 CGC, Cys 3 Arg). The numbers refer to oligonucleotide order [e.g., 42 3 634 M-(R)]. PMT gain: 76%, laser power: 76%, and the resolution: 5 ␮m.

nucleotide chip can efficiently detect point mutations, it is not easily applied to some genes with multi-bp frameshift mutations. Thus, the oligonucleotide microarray would be very effective as a mutation detection tool in those genes dominated by point mutations. (Because the predominant RET mutations are missense mutations of some restricted codons in MEN2 syndromes, the oligonucleotide microarray method can efficiently detect mutations in this case.) We developed the RET oligonucleotide microarray covering nine codons in which predominant RET missense mutations have been reported in MEN2 syndromes. Seven or eight oligonucleotides per codon were designed for MEN2A and FMTC. Because Ͼ95% of MEN2B patients had a predominant mutation Fig. 2 Vertical histogram of data analysis in SNU-MEN2A-I. OOO 3 indicates the cutoff value (BA ϩ 2.58 BSD), and the OOOindicates type in codon 918 (Met Thr), we designed only two oligo- the mean value of the background. The cutoff value of SNU-MEN2A-I nucleotides of the wild and the mutant one (2). All of the was 6897 (BA, 4461, BSD, 944; n ϭ 50). All signals over the cutoff oligonucleotides were printed four times to obtain the accurate were recognized as true signals, which were at positions 1, 9, 17, 25, 33, signal value. 41, 48, 49, and 50. After preliminary experiments, we found that some G-A mismatches often exhibited nonspecific signals (positions 4, 20, and 44). It was reported that G-T and G-A mismatches slightly The frequency of PHEO in Korean MEN2A patients was also destabilize a duplex, whereas A-A, T-T, C-T, and C-A mis- 50% (14 of 28). SNU-MEN2A-IV had 1 of 11 PHEO patients, matches cause significant destabilization (15). Whereas it was but SNU-MEN2A-I, SNU-MEN2A-II, SNU-MEN2A-III, and also reported that a high washing temperature enables a clear SNU-MEN2A-V had 2 of 5, 2 of 2, 2 of 2, and 1 of 1 patients distinction to be made between the perfectly matched duplex with PHEO, respectively. SMC-MEN2A-I, -II, and -III had 3 of and the mismatched duplex (15), the signal intensities in our 3, 1 of 2, and 2 of 2 PHEO incidence. None of eight Korean work were reduced without sufficient distinction between spe- MEN2A families had hyperparathyroidism. cific signals and nonspecific signals throughout room temperature and 60°C. Thus, we reduced the length of the oligonucleo- DISCUSSION tides from 20 mer to 16 mer in three cases (positions 4, 20, and Many cancer-causing genes show different mutation types 44) exhibiting a G-A mismatch to increase the destabilizing such as frameshift, nonsense, missense, and an alternative splic- effect. These 16 mer oligonucleotides were analyzed with per- ing. To identify mutation types in cancer patients, many tools fectly matched oligonucleotides. In another report, the length of such as single-strand conformational polymorphism, protein oligonucleotides was reduced to 14 bases to increase specificity truncation test, and gel-based sequencing have been used. Be- (16). However, an excessively short oligonucleotide could result cause these tools are relatively time-consuming and labor inten- in hybridization with unrelated DNA (17). sive, a rapid and high throughput system, such as that using the In sample preparations for the hybridization, we incorpo- oligonucleotide chip, has been developed. Although the oligo- rated Cy5-labeled dCTP directly to the synthesizing strand

during PCR without any extra step. We attempted a multiplex dure, we could reduce false positive and false negative. Non- PCR for the sake of simplicity, but found that individual PCRs specific signals might be caused by some problems in PCR produced the better result. Four approaches were used to prepare amplification or sample preparations for hybridization such as samples for hybridization to determine which provided the best enzyme digestion. In a false positive case, one sample with hybridization result, namely, asymmetrical PCR-based, ␭ exo- C634R showed a nonspecific signal at position 27 along with a nuclease-based, UDG-based, and DNase I-based preparations. specific signal at position 42 (codon 620). In this case, signals Asymmetric PCR-based and ␭ exonuclease-based preparations were generally strong, and wild-type oligonucleotide at codon were investigated to obtain single-stranded DNA for effective 620 showed signals twice as strong as other wild-type signals. hybridization. UDG and DNase I digestion were introduced to This might be affected by the DNase I enzyme digestion. We fragment PCR products, which decrease the interference of spotted 67 type oligonucletides covering nine codons (codons hairpin structures on hybridization with oligonucleotides (6). 609, 611, 618, 620, 630, 634, 768, 804, and 918) of five exons Unpredictable signals were shown in the asymmetric PCR, (exons 10, 11 13, 14, and 16). Because codons 609 and 618 are although all of the specific signals were present. The ␭ exonu- contiguous to codons 611 and 620, respectively, these codons clease-based preparation generally showed reinforced signals at share some parts of oligonucleotides each other. DNA fragments positions 2–17 (codon 609 and 611). When localizing positions randomly digested by DNase I may affect the specificity of 2–17, specific signals were shown with more than three times hybridization at nearby codon and cause either a false-positive the intensity of the background signals. However, the mean or false-negative result. value of the background signals in these areas (positions 2–17) When analyzing the data, we averaged the signal values of was similar to the specific signal value of the other codons in four spots and accepted the average as the real signal value, and terms of the entire area of the RET oligonucleotide microarray. when fluorescent debris was shown in a spot, we excluded it and Therefore, this approach required a more detailed analysis, as calculated the signal mean value from the remaining three. The such comparing each signal within an individual codon. In the signal recognition cutoff level was set as (BA ϩ 2.58 ϫ BSD). UDG-based hybridization approach, SNU-MEN2A-I, -II, -III, (BA ϩ 2.58 ϫ BSD) indicates the upper limit of the 99% and -V families with a mutation in exon 11 showed a specific confidence interval, and signals more than this value were signal in exon 11 area. However, in the case of the exon 10 identified as specific signals. hybridization, the UDG-based method often showed nonspecific Genotype-phenotype correlations in RET proto-oncogene signals at positions 26–28, regardless of the ratio of dUTP: and MEN2 syndromes have been well characterized, and it has dTTP. The DNase I-based approach showed relatively low been suggested that the codon 634 mutations are highly predic- incidence of the nonspecific signals. Specific signals in DNase tive of the presence of PHEO. We found that the C634 muta- I-based method were usually twice as strong as those obtained tions (6 of 8) are prevalent in Korean MEN2A families and the using the other methods and showed relatively similar signal correlation between C634 mutations, and that the occurrence of intensity in the entire region of the RET microarray. The DNase PHEO (14 of 28) in Korean MEN2A patients is in agree- I-based preparation was straightforward enough to allow the ment with much of the data published previously. Six of eight whole experiment (from PCR amplification to the scanning of Korean MEN2A families (SNU-MEN2A-I, -II, -III, -V, SMC- the hybridized microarray) to be finished within a day. There- MEN2A-I, and SMC-MEN2A-I) showed mutations at codon fore, we concluded that DNase I-based sample preparation was 634. C634R mutation was the most common in MEN2A patients the most efficient and preferable approach for the RET oligo- followed by C634Y in MEN2A patients (9). Three of eight nucleotide microarray. The hybridization buffer was considered Korean MEN2A families showed C634R, and two showed to minimize the difference between the G:C and A:T bp (18). C634Y. Five of eight Korean MEN2A families showed C634R SNU-MEN2A-V was first analyzed by RET oligonucleotide or C634Y, which is consistent with the data reported previously. microarray and then reexamined by direct sequencing. To con- Although there were some discrepancies, it has been suggested firm the signal at position 46 (C634Y type) of the RET oligo- that the C634R mutation is consistent with a higher frequency of nucleotide microarray in SNU-MEN2A-V, direct sequencing hyperparathyroidism (2, 3, 9). An age-related and mutation- was performed. As a result, codon 634 seemed to be of the wild specific hyperparathyroidism penetrance was reported in a se- type. We could not confirm the presence of the signal at position ries of patients carrying codon 634 mutations; e.g., the pen- 46 (C634Y mutation, TGC 3 TAC, exon 11) until we analyzed etrance was 14% by age 30 and rose to 81% by age 70 (2, 3, 10). exon 11 by cloning and sequencing. If we had relied on the We could not find any clinical symptom of hyperparathyroidism direct sequencing approach, we could have missed the C634Y in eight Korean MEN2A families. The age of MEN2A family mutation in the SNU-MEN2A-V patient. We examined the members with the codon 634 mutations ranged from 6 to 67. incidence of false positive and false negative three times using Thus, we could not exclude the possibility that Korean MEN2A 13 samples from three SMC-MEN2A families under a blind families with C634 mutations will be at risk for hyperparathy- status. One of 39 showed false negative (codon 618, position 18) roidism in the future. It is also possible that the thyroidectomy and another case of 39 showed false positive (codon 620, performed in some patients with the removal of parathyroid position 27). One SMC-MEN2A-II member with C618R glands had influence on the occurrence of hyperparathyroidism showed no distinctive signal at position 18, and the wild-type (2). SNU-MEN2A-IV and SMC-MEN2A-II had mutations at signal at codon 618 was also weak in this case. We spotted codon 618, and the incidences of PHEO were 1 of 11 and 1 of wild-type oligonucelotides in each codon. When we could not 2, respectively. Usually 5Ј cysteine codon mutations (e.g., codon see the specific signals over the cutoff level even at wild-type 609, 611, and 618) are associated with FMTC, and these muta- oligonucleotides, our experiment was repeated. By this proce- tions might result in a weaker activation than those nearer the

transmembrane domain. Codon 634 mutations appear to have ance of a p53 sequencing microarray chip using 140 previously se- the penetrance high enough for MTC, PHEO, and hyperpara- quenced bladder tumor samples. Clin. Chem., 46: 1555–1561, 2000. thyroidism. However, 5Ј cysteine codon mutations seemed to 8. Hacia, J. G., Sun, B., Hunt, N., Edgemon, K., Mosbrook, D., Rob- cause a reduced proportion of receptor molecules on the cell bins, C., Fodor, S. P., Tagle, D. A., and Collins, F. S. Strategies for surfaces, thus resulting in only MTC (4). The low PHEO fre- mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays. Genome Res., 8: 1245–1258, 1998. quency in families with C618 mutations might be caused by this Ј 9. Eng, C., Clayton, D., Schuffenecker, I., Lenoir, G., Cote, G., Gagel, 5 cysteine codon mutation. R. F., van Amstel, H. K., Lips, C., Nashisho, I., Takai, S. I., Marsh, D. J., In summary, we have developed a RET oligonucleotide Robinson, B. G., Frank-Raue, K., Raue, F., Xue, F., Noll, W. W., microarray for the detection of RET mutations, and found RET Romei, C., Pacini, F., Fink, M., Niederle, B., Zedenius, J., Norden- mutations in all five SNU-MEN2A and three SMC-MEN2A skjold, M., Komminoth, P., Hendy, G. N., Gharib, H., Thibodeau, S. N., families by gel-based sequencing and the RET oligonucleotide Lacroix, A., Frilling, A., Ponder, B. A. J., and Mulligan, L. M. The relationship between specific RET proto-oncogene mutations and dis- microarray. RET mutations were reliably detected by the RET ease phenotype in multiple endocrine neoplasia type 2. J. Am. Med. microarray from the DNA of MEN2A families within a day. Assoc., 276: 1575–1579, 1996. Because the MEN2 syndromes are inherited in an autosomal 10. Schuffenecker, I., Virally-Monod, M., Brohet, R., Goldgar, D., dominant fashion and show high penetrance, it is important that Conte-Devolx, C., Leclerc, L., Chabre, O., Boneu, A., Caron, J., we test for the presence of RET mutations in MEN2A families Houdent, C., Modigliani, E., Rohmer, V., Schlumberger, M., Eng, C., in the early stages. Twenty-two mutation carriers in eight Ko- Guillausseau, P. J., and Lenoir, G. M. Risk and penetrance of primary hyperparathyroidism in multiple endocrine neoplasia type 2A families rean MEN2A families were found using RET oligonucleotide with mutation at codon 634 of the RET proto-oncogene. Groupe D’etude microarray and direct sequencing. A close monitor is required des Tumeurs a Calcitonine. J. Clin. Endocrinol. Metab., 83: 487–491, for these mutation carriers. Because we found only five muta- 1998. tion types in eight MEN2A families, the international collabo- 11. Yang, H. Y., Park, Y. J., Kwon, H. J., Cho, K. J., and Park, J. 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Il-Jin Kim, Hio Chung Kang, Jae-Hyun Park, et al.

Clin Cancer Res 2002;8:457-463.

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