Identification and Allelic Frequencies of Novel Single-Nucleotide
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J Hum Genet (2001) 46:155–157 © Jpn Soc Hum Genet and Springer-Verlag 2001 SHORT COMMUNICATION Chie Suzuki · Motoko Unoki · Yusuke Nakamura Identification and allelic frequencies of novel single-nucleotide polymorphisms in the DUSP1 and BTG1 genes Received: December 20, 2000 / Accepted: December 22, 2000 Abstract Defects in the activity of the PTEN gene, a tumor defective endometrial cancer cell lines, and we found that suppressor, are implicated in many types of cancer in hu- exogenous PTEN induced expression of the DUSP1 and mans. However, not all mediators of PTEN signaling path- BTG1 genes (unpublished data). ways have been clarified, and, during efforts to identify such DUSP1/MKP1/CL100/PTPN10, a dual-specificity phos- molecules, we previously induced expression of the DUSP1 phatase for tyrosine and threonine, specifically inactivates and BTG1 genes by introducing exogenous PTEN into mitogen-activated protein kinase (MAPK) and suppresses endometrial cancer cell lines. In the course of analyzing its activation by ras (Alessi et al. 1993). BTG1 encodes a these two genes for mutations in ovarian carcinomas, we member of the anti-proliferative Tob/BTG1 family of mol- identified a novel single-nucleotide polymorphism (SNP) in ecules; transfection experiments have indicated that BTG1 the DUSP1 gene, and three novel SNPs in the BTG1 gene, negatively regulates cell proliferation (Rouault et al. 1992). and we have established their allelic frequencies in a Japa- Therefore, in view of our results from the microarray nese population sample. These polymorphic sites will be experiments, we considered these two genes to be candi- useful for detecting losses of heterozygosity (LOH) in dates for mediating PTEN signaling pathways. We screened tumors and for examining latent associations between both genes for mutations in ovarian cancers and found specific alleles and disease susceptibility. none, but we did identify four single-nucleotide polymor- phisms (SNPs) in non-coding regions. Here we describe Key words Single-nucleotide polymorphism · DUSP1 · these novel SNPs and report their allelic frequencies in a BTG1 · PTEN · Tumor suppressor Japanese population sample. Introduction Subjects and methods The PTEN tumor suppressor gene encodes a multifunc- DNA samples. Materials used in this study were obtained, tional phosphatase that plays an important role in inhibiting with informed consent, from 132 Japanese individuals. the phosphatidylinositol-3-kinase pathway, and in down- Genomic DNAs were extracted according to standard stream functions that include the activation of Akt/protein protocols. kinase B, cell survival, and cell proliferation (Maehama and Dixon 1998; Stambolic et al. 1998). Enforced expression of Screening of SNPs. Amplification of genomic DNA frag- PTEN in various cancer cell lines decreases cell prolifera- ments by polymerase chain reactions (PCRs) and DNA tion through arrest of the cell cycle, accompanied, in some sequencing of the amplified fragments were performed as instances, by the induction of apoptosis (Furnari et al. 1998; described previously (Lin et al. 2000). Minaguchi et al. 1999). We used cDNA microarrays con- taining 4009 cDNAs to examine changes in gene expression SNP typing. SNPs were genotyped by allele-specific oligo- profiles when exogenous PTEN was introduced into PTEN- nucleotide (ASO) hybridization as described previously (Unoki et al. 2000). Allele-specific probes (32P-labeled 13- C. Suzuki · M. Unoki · Y. Nakamura ( ) to 17-mers) of 59-TACGCCCTCGGGG-39 or 59-TACGCC Laboratory of Molecular Medicine, Human Genome Center, GTCGGGG-39 for C(IVS 1 8)G of DUSP1, and 59- Institute of Medical Science, University of Tokyo, 4-6-1 GGGTGGCTGCTCC-39 or 59-GGGTGGTTGCTCC-39 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Tel. 181-3-5449-5372; Fax 181-3-5449-5433 for C(-67T) of BTG1, were hybridized at 37°C to the poly- e-mail: [email protected] merase chain reaction (PCR) products on the membranes 156 N. Matsuda et al.: EGF receptor and osteoblastic differentiation Table 1. Primer sequences for amplifying the coding regions and splice sites of the DUSP1 and the BTG1 genes Name of Product Gene primer Nucleotide sequences size (bp) DUSP1 DUSP1-F1 59-CGTCACGTGATCACCATTCA-39 499 DUSP1-R1 59-TGAAGCGCACGTTGACAGAG-39 DUSP1-F2 59-CCAAAAGCGGCTTTTGGTTC-39 695 DUSP1-R2 59-AGGGCTGGAAGTTTACATCG-39 DUSP1-F3 59-CGATGTAAACTTCCAGCCCT-39 420 DUSP1-R3 59-AGCCTACAATTGGAGACTCG-39 DUSP1-F4 59-CCTCCAGCAACAGAACTGAG-39 492 DUSP1-R4 59-ACAGGAATGTTGCCCACACC-39 DUSP1-F5 59-CATGTGATGGCATGTGGTGA-39 609 DUSP1-R5 59-AATAAGGACCAGCCCTCTCG-39 DUSP1-F6 59-CACCACCACCGTGTTCAACT-39 607 DUSP1-R6 59-CAGTGCTGAAAACAAACCTGC-39 DUSP1-F7 59-CCCGACGACACATATACATAT-39 535 DUSP1-R7 59-AGAGTTCAGCTGTAGCGTCC-39 BTG1 BTG1-F1 59-GGTGCGATATTCGGATTGGC-39 616 BTG1-R1 59-GACTCTGACCCAGGGATGTG-39 BTG1-F2 59-AACAACGGATGCAATCCTGG-39 932 BTG1-R2 59-CTTTCCTATTAAAAGCTGCCG-39 Table 2. Allelic frequencies of SNPs of the DUSP1 and the BTG1 genes in a Japanese population Gene Region SNPs and their flanking sequence Allelic frequenciesa DUSP1 Intron1 AAGGTACGCC (C/G) TCGGGGAAGC C G IVS1 1 8 0.68 0.32 BTG1 59UTR (exon1) AGCTATTGAG (A/G) TCTTCGAATG A G (2278) 0.64 0.36 59UTR (exon1) CCCGGGGTGG (C/T) TGCTCCGCCG C T (267) 0.65 0.35 59UTR (exon1) GCCGAGCCCC (G/A) GCCGCCCCGG G A (233) 0.67 0.33 SNP, Single-nucleotide polymorphism; UTR, untranslated region a Estimated from the genotyping of 264 chromosomes and washed in 6 3 standard saline citrate (SSC) at 42°C of translation or transcription. Nevertheless, these polymor- (DUSP1) or at 46°C (BTG1) according to standard proto- phisms should be useful for detecting losses of heterozygos- cols. Genomic DNAs (50-ng each) were amplified by PCR, ity (LOH) in tumors and for examining potential and each PCR product was digested with BglII for associations between specific alleles and disease A(-278)G of BTG1 or with Eco52I for G(-33)A of BTG1, susceptibility. according to the manufacturer’s protocol (TaKaRa). References Results and discussion Alessi DR, Smythe C, Keyse SM (1993) The human CL100 gene en- By screening all coding exons and their neighboring introns codes a tyr/thr-protein phosphatase which potently and specifically in the DUSP1 and BTG1 genes, using the PCR primers inactivates MAP kinase and suppresses its activation by oncogenic listed in Table 1, we detected four novel SNPs, one in the ras in Xenopus oocyte extracts. Oncogene 8:2015–2020 DUSP1 gene and three in the BTG1 gene. To estimate the Furnari FB, Huang HJ, Cavenee WK (1998) The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth allelic frequency of each SNP in the Japanese population, arrest in glioma cells. Cancer Res 58:5002–5008 we genotyped a panel of 132 subjects, with the results shown Lin YM, Kato T, Satoh S, Nakamura Y, Furukawa Y (2000) Identifica- in Table 2. tion of novel polymorphisms in the AXIN1 and CDX-2 genes. J Hum Genet 45:254–256 The SNP in the DUSP1 gene is located in intron 1 and all Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1, three SNPs in the BTG1 gene are located in the 59 dephosphorylates the lipid second messenger, phosphatidylinositol untranslated region. Because none of these polymorphic 3,4,5-trisphosphate. J Biol Chem 273:13375–13378 sites is within a coding region, none is likely to influence the Minaguchi T, Mori T, Kanamori Y, Matsushima M, Yoshikawa H, Taketani Y, Nakamura Y (1999) Growth suppression of human function of the gene product, although we cannot exclude ovarian cancer cells by adenovirus-mediated transfer of the PTEN the possibility that certain alleles might alter the efficiency gene. Cancer Res 54:6063–6067 B. Jochimsen et al.: Stetteria hydrogenophila 157 Rouault JP, Rimokh R, Tessa C, Paranhos G, Ffrench M, Duret L, Unoki M, Furuta S, Onouchi Y, Watanabe O, Doi S, Fujiwara H, Garoccio M, Germain D, Samarut J, Magaud JP (1992) BTG1, a Miyatake A, Fujita K, Tamari M, Nakamura Y (2000) Association member of a new family of antiproliferative genes. 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