Research Article High-throughput Loss-of-Heterozygosity Study of Chromosome 3p in Lung Cancer Using Single-Nucleotide Polymorphism Markers Amy L.S. Tai,1 William Mak,4 Phoebe K.M. Ng,4 Daniel T.T. Chua,1 Mandy Y.M. Ng,4 Li Fu,1 Kevin K.W. Chu,1 Yan Fang,5 You Qiang Song,2,4 Muhan Chen,1 Minyue Zhang,1 Pak C. Sham,3,4 and Xin-Yuan Guan1,5 Departments of 1Clinical Oncology, 2Biochemistry, and 3Psychiatry, 4Genome Research Center, The University of Hong Kong, Hong Kong, China and 5State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou, China Abstract candidate TSGs, such as FHIT (7), RASSF1A (8), CACNA2D2 (9), and Loss of DNA copy number at the short arm of chromosome 3 is DLC1 (10), have been widely studied. However, comparative one of the most common genetic changes in human lung genomic hybridization results showed that the loss of 3p was often involved in the whole short arm in NSCLCs (5, 11). Therefore, cancer, suggesting the existence of one or more tumor suppressor genes (TSG) at 3p. To identify most frequently the existence of some unknown TSGs at loci other than 3p21.3 and deleted regions and candidate TSGs within these regions, 3p14.2 regions is likely. a recently developed single-nucleotide polymorphism (SNP)- Conventional LOH uses limited microsatellite polymorphism mass spectrometry-genotyping (SMSG) technology was ap- markers (12), which is a time-consuming process with low- pliedto investigate the loss of heterozygosity (LOH) in 30 resolution genotyping. In addition, it is difficult to automate this primary non–small-cell lung cancers. A total of 386 SNP method in large-scale LOH analysis. Because of these limitations, it markers that spanneda region of 70 Mb at 3p, from 3pter to is very hard to perform high-resolution LOH analysis using 3p14.1, were selectedfor LOH analysis. The average inter- conventional LOH method. The progress of the human genome marker distance in the present study is f180 kb. Several project makes it possible to apply single-nucleotide polymorphism (SNP) markers in LOH analysis. SNPs represent the most common frequently deleted regions, including 3p26.3, 3p25.3, 3p24.1, f 3p23, and 3p21.1, were found. Several candidate TSGs within form of sequence variation in the human genome, occurring 1 these frequently detected LOH regions have been found, every 1,200 bp (13). SNPs can be used as high-density polymorphic markers for studying genetic variations, including LOH (14), including APG7L at 3p25.3, CLASP2 at 3p23, and CACNA2D3 at 3p21.1. This study also showed that SMSG technology is a although their heterozygosity is relatively low compared with very useful approach to rapidly define the minimal deleted microsatellites. In this study, a high-throughput and high- resolution LOH study of chromosome 3p in 30 NSCLCs was done region andto identifytarget TSGs in a given cancer. (Cancer using SNP-mass spectrometry-genotyping (SMSG) technology. Res 2006; 66(8): 4133-8) Several high-LOH loci have been identified and three candidate TSGs were further studied. Introduction Lung cancer is one of the most common malignancies in the world and one of the leading causes of cancer death in the United Materials and Methods States, which accounts for f28% of all cancer death during 2001 Tumor samples. Thirty NSCLC samples were collected from the time (1). The overall 5-year survival rate of this prevalent cancer is <15% of surgical resection at the Cancer Institute, Sun Yat-Sen University (2). Lung cancer can be classified into two major types: small-cell (Guangzhou, China), during the period of 1998 to 2002. The specimens À j lung cancer and non–small-cell lung cancer (NSCLC), which is were snap frozen and kept at 80 C until DNA extraction. All the f25% and 75% of all lung cancers, respectively. The epidemiology samples were examined and macrodissected under a microscope by a pathologist to ensure the purity of tumor samples with <10% normal cell of lung cancer is multifactorial with cigarette smoking as the major contamination. cause (3). Deletion of chromosome 3p is one of the most frequent SNP marker selection andprimer design. A total of 386 SNP markers allelic imbalances in various human tumors, including NSCLC spanning over 0 to 70 Mb of 3p with minor allele frequency over 0.3 were (4, 5). Loss of heterozygosity (LOH) analysis is one of the most selected, including 326 SNPs with minor allele frequency over 0.45, 41 SNPs efficient methods to study chromosomal deletion and to narrow between 0.4 and 0.44, 16 SNPs with between 0.35 and 0.43, and 4 SNPs down the deleted region. High rates of LOH loci are commonly between 0.3 and 0.34 (see Supplementary Table S1). All minor allele found to harbor putative tumor suppressor genes (TSG). Previously frequencies were calculated from Caucasian populations. published LOH studies showed that 3p21.3 and 3p14.2 are the two MassARRAY AssayDesign software (Sequenom, San Diego, CA) was used most frequently deleted regions in lung cancer (4, 6). Several to design amplification and allele-specific extension primers for uniplexed or multiplexed assays. In the design, PCR primers have an additional 10-base tag (5V-ACGTTGGATG-3V) to prevent their interference in the resulting mass spectra. The designed assays were constrained to produce products of optimized size (90-120 bp) to maximize the PCR successful Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). rate. The extension primer was designed to hybridize to the amplicon near Requests for reprints: Xin-Yuan Guan, Department of Clinical Oncology, The the SNP locus for the extension of a single base or a few bases depending University of Hong Kong Medical Center, Room 109, Estate Building, 10 Sassoon Road, on the genotype of the allele (Supplementary Table S2). Pokfulam, Hong Kong, China. Phone: 852-25890458; Fax: 852-28169126; E-mail: PCR amplification anddephosphorylation. PCR reactions were done [email protected]. A I2006 American Association for Cancer Research. in a 384-well-plate format in a total volume of 6 L per reaction with 5 ng doi:10.1158/0008-5472.CAN-05-2775 genomic DNA, 0.3 pmol each of the specific forward and reverse primers, www.aacrjournals.org 4133 Cancer Res 2006; 66: (8). April 15, 2006 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research expected molecular weights of all relevant peaks were calculated by the Table 1. Summary of the frequently detected LOH in 30 MassARRAY AssayDesign Software before analysis and identified from the NSCLC cases mass spectrum. In every assayed plate, one well for blank control and five wells for duplicate check on five samples for quality control were included. SNP ID Location No. LOH Mapped genes Twenty-four SpectroChips for 8-plexed reactions, in which each chip is composed of 3,072 genotypes, can be analyzed within 1 day. A high- rs3772313 3p26.3 10 CNTN6 throughput genotyping consisting of 73,728 genotypes is routinely done rs6794523 3p26.2 10 IL5RA, CNTN4 within 24 hours. rs6766718 3p26.1 11 To test the effect of the tumor purity in LOH detection using SMSG, rs7638724 3p25.3 9 APG7L DNA from one tumor sample that contained <5% normal cells was used rs3856803 3p25.3 11 VGLL4 to determine the accuracy of genotyping. DNA samples from tumor, rs6550072 3p24.3 10 matched normal tissue, and series of mixtures (containing 5%, 10%, 20%, rs3849542 3p24.3 9 FLJ22419 and 50% normal DNA, respectively) have been assessed with five SNP rs3856621 3p24.2 11 markers. rs2102801 3p24.1 10 Comparative genomic hybridization. Comparative genomic hybrid- rs987693 3p24.1 12 RBMS3 ization was done as previously described (15). Briefly, 1 Ag tumor test and rs6550226 3p23 13 CLASP2 normal reference DNA were labeled with Spectrum Green-dUTP and rs4389435 3p22.3 10 DLC1 Spectrum Red-dUTP (Vysis, Downers Grove, IL), respectively, by nick rs3816779 3p21.31 9 FLJ20211 translation at 15jC for 2 hours. The slide containing normal metaphase rs6797113 3p21.1 9 CACNA2D3* spreads was denatured and then hybridized with 200 ng of each probe at rs1449325 3p21.1 12 CACNA2D3 37jC for 2 days. After hybridization, the slide was washed and counter- rs1113042 3p21.1 8 CACNA2D3 stained with 1 Ag/mL 4V,6-diamidino-2-phenylindole in an antifade solution. rs589281 3p21.1 10 CACNA2D3 c The image of hybridized metaphases was captured using a Zeiss Axioplan 2 rs722070 3p14.2 9 FHIT microscope equipped with a Sensys cooled-charged device camera (Photo- rs2044613 3p14.2 9 FHIT metrics, Ltd., Tucson, AZ), analyzed using Quips comparative genomic rs832187 3p14.3 9 LOC132200 hybridization program (Vysis), and interpreted according to the fluores- rs264083 3p14.1 9 BAIAP1 cence intensity profile under program guidance. The threshold value for rs6548999 3p14.1 9 TAFA1 DNA copy number gain and loss was defined as intensity ratio of tumor/ normal >1.2 and <0.8, respectively. *CACNA2D3 covers four SNPs in this study and overall LOH involving CACNA2D3 was detected in 19 NSCLC cases. Results c FHIT covers two SNPs and overall LOH was detected in 15 NSCLCs. SMSG technology. In the SMSG system, a 90- to 120-bp DNA fragment containing the target SNP site was amplified by PCR with a pair of specific primers. The PCR product was then extended with another primer near the SNP site and extended sequence was A 200 mol/L of each of the deoxynucleotide triphosphates, 3.25 mmol/L automatically detected by MALDI-TOF.