B.J Hum Jochimsen Genet et(2002) al.: Stetteria 47:147–171 hydrogenophila © Jpn Soc Hum Genet and Springer-Verlag4600/147 2002 ORIGINAL ARTICLE Susumu Saito · Aritoshi Iida · Akihiro Sekine Yukie Miura · Chie Ogawa · Saori Kawauchi Shoko Higuchi · Yusuke Nakamura Identification of 779 genetic variations in eight genes encoding members of the ATP-binding cassette, subfamily C (ABCC/MRP/CFTR) Received: January 8, 2002 / Accepted: January 9, 2002 Abstract We screened DNAs from 48 Japanese individuals ac.uk/nomenclature/genefamily/abc.html]). Members of the for single-nucleotide polymorphisms (SNPs) in eight genes MRP/CFTR family have been recognized as export pumps encoding the ATP-binding cassette, subfamily C (ABCC/ for amphiphilic anions, particularly for conjugates of lipo- MRP/CFTR), by direct sequencing of their entire genomic philic compounds with glutathione or several other anionic regions, except repetitive sequence elements. This ap- residues (Keppler et al. 1998, 1999; König et al. 1999). proach identified 688 SNPs and 91 insertion/deletion ABCC1 (MRP1) has been found to be overexpressed in polymorphisms among the eight genes. Of the 688 SNPs, many drug-resistant cell lines and tumor tissues (Cole et al. 81 were identified in the ABCC1 gene, 41 in ABCC2, 30 in 1992; Loe et al. 1996). When being overexpressed in tumor ABCC3, 230 in ABCC4, 76 in ABCC5, 58 in CFTR, 102 in cells, ABCC1 confers multidrug resistance by reducing ABCC8, and 70 in ABCC9. Six SNPs were located in the intracellular drug concentrations in an ATP-dependent 5Ј flanking regions, 617 in introns, 46 in exons, and 19 in the manner (Cole et al. 1994; Zaman et al. 1994). ABCC1 is 3Ј flanking regions. These variants should contribute to ubiquitously expressed, but at a very low expression level in studies that investigate possible correlations of genotypes the liver (Stride et al. 1996). This protein serves as an efflux with disease-susceptibility phenotypes and responsiveness pump for xenobiotics, drugs, and cellular metabolites or adverse effects to drugs. (König et al. 1999). In the brain, ABCC1 together with ABCB1 (MDR1) forms part of the blood-brain barrier Key words Single-nucleotide polymorphism (SNP) · ATP- (Rao et al. 1999). Although various polymorphisms have binding cassette, subfamily C (ABCC) · Multidrug resis- been reported in the ABCC1 gene, their phenotypic conse- tance protein (MRP) · Cystic fibrosis transmembrane quences have not been observed (Perdu and Germain 2000; conductance regulator (CFTR) · Sulfonylurea receptor Conrad et al. 2001; Ito et al. 2001). (SUR) ABCC2 (MRP2) is mainly expressed in the canalicular membrane of hepatocytes (Kool et al. 1997). Its major physiological function is the secretion of bilirubin glucu- Introduction ronides and organic anions into bile (Jedlitschky et al. 1997). ABCC2 also confers resistance to multiple anti- cancer chemotherapeutic agents when it is overexpressed in Multidrug resistance protein/cystic fibrosis transmembrane tumor cells (Cui et al. 1999). Mutations in the ABCC2 conductance regulator (MRP/CFTR) constitutes a subfam- gene were reported to cause Dubin-Johnson syndrome ily of ATP-binding cassette (ABC) proteins (Human ABC (Kartenbeck et al. 1996; Paulusma et al. 1997; Toh et al. Gene Nomenclature Committee [http://www.gene.ucl. 1999). By screening an expressed sequence tag (EST) database, ABCC3 (MRP3), ABCC4 (MRP4), and ABCC5 (MRP5) were identified (Kool et al. 1997). ABCC3 is mainly ex- S. Saito · A. Iida · A. Sekine · Y. Miura · C. Ogawa · S. Kawauchi · S. Higuchi · Y. Nakamura pressed in liver, colon, intestine, and adrenal gland (Kool Laboratory for Genotyping, SNP Research Center, Institute of et al. 1997) and plays an important role in the removal of Physical and Chemical Research, Tokyo, Japan toxic organic anions from the liver under cholestatic condi- Y. Nakamura (*) tions (Kool et al. 1999). ABCC4 is widely expressed in Laboratory of Molecular Medicine, Human Genome Center, human tissues and predominantly in the prostate (Lee et al. Institute of Medical Science, The University of Tokyo, 4-6-1 1998), and its overexpression was shown to be correlated Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Tel. ϩ81-3-5449-5372; Fax ϩ81-3-5449-5433 with ATP-dependent efflux of acyclic nucleoside mono- e-mail: [email protected] phosphates such as 9-(2-phosphonylmethoxyethyl) guanine 148 and the anti-human immunodeficiency virus (HIV) drug 9- in cardiac and skeletal muscle, whereas the SUR2B tran- (2-phosphonylmethoxyethyl)adenine (PMEA) (Schuetz et script was detected mainly in vascular smooth muscle al. 1999). Consequently, ABCC4 confers resistance to these (Isomoto et al. 1996; Davis-Taber et al. 2000). drugs. ABCC5 is mainly expressed at high transcript levels To investigate the nature of apparent genotype/ in skeletal muscle, brain, and heart, and at a very low level phenotype correlations for some ABC-transporters in more in liver (McAleer et al. 1999). This protein acts as the cellu- detail, we began by searching for additional single- lar export of cyclic nucleotides and introduces resistance nucleotide polymorphisms (SNPs) in the eight ABCC genes to thiopurine anticancer drugs such as 6-mercaptopurine described earlier, including their promoter regions and in- and thioguanine, and the anti-HIV drug PMEA (Wijnholds trons, except repetitive elements. We report here a total of et al. 2000). 779 genetic variations, of which 454 have not been reported CFTR (cystic fibrosis transmembrane conductance regu- before. lator) acts as a chloride channel (Bear et al. 1992) and also regulates other ion transport pathways including the amiloride-sensitive epithelial sodium channel (Schreiber et al. 1999). Mutations in the CFTR gene were found in Subjects and methods patients with cystic fibrosis (Cystic Fibrosis Mutation Data Base [http://www.genet.sickkids.on.ca/cftr/]) and congenital Total genomic DNAs were isolated from peripheral leuko- bilateral absence of the vas deferens (Costes et al. 1995; cytes by the standard phenol/chloroform extraction method Zielenski et al. 1995). CFTR mutations were found at sig- in 48 unrelated Japanese individuals. Informed consent was nificantly higher frequencies in patients with disseminated obtained from each participant. On the basis of sequence bronchiectasis or allergic bronchopulmonary aspergillosis information from GenBank, we designed polymerase than expected (Miller et al. 1996; Pignatti et al. 1996). chain reaction (PCR) primers to amplify DNA fragments The sulfonylurea receptor (SUR) contains ATP-sensi- from all eight genes, excluding repetitive elements, by tive potassium (K ) channels (Aguilar-Bryan et al. 1995) ATP invoking the REPEAT MASKER computer program that are involved in many diverse functions such as insulin (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). secretion from pancreatic cells, regulation of skeletal PCR experiments and DNA sequencing were performed muscle excitability, neurotransmitter release, and smooth according to methods described previously (Iida et al. 2001; muscle relaxation (Quayle et al. 1997). The SUR subunit Sekine et al. 2001; Saito et al. 2001). All SNPs detected by consists of two members, ABCC8 (SUR1) and ABCC9 the PolyPhred Computer Program (Nickerson et al. 1997) (SUR2). Channels containing isoform ABCC8 (SUR1) were confirmed by sequencing both strands of each PCR found in neuronal cells and pancreatic -cells are closed in product. response to the sulfonylurea hypoglycemic drugs (Gribble et al. 1998) and can be activated by diazoxide (D’hahan et al. 1999). Mutations and deficiencies in this protein were detected in patients with hyperinsulinemic hypoglyce- mia of infancy, an autosomal recessive disorder of unregu- Results lated and high insulin secretion (Thomas et al. 1995; Nestorowicz et al. 1996, 1998; Glaser et al. 1999; Verkarre Exon–intron boundaries of the eight genes were defined by et al. 1998). An arginine residue at codon 1273 was reported comparison of genomic sequences with cDNA sequences. to be associated with hyperinsulinemia in Mexican Accession numbers of the genomic and cDNA sequences American nondiabetic individuals (Goksel et al. 1998) and used for this study are listed in Table 1. We screened 96 with type 2 diabetes mellitus in French Caucasians (Reis et Japanese chromosomes for SNPs in the eight ABCC genes al. 2000). ABCC9 (SUR2) is the primary regulatory subunit by means of direct DNA sequencing. Figure 1 (a–h) illus- expressed in muscle cells and generates two splice variants, trates the location of each variation in the respective genes; SUR2A (exon 38a) and SUR2B (exon 38b) (Isomoto et al. detailed information for nucleotide positions and substitu- 1996). The SUR2A transcript was predominantly expressed tions is summarized in Table 2 (a–h). Table 1. Accession numbers for the genomic and cDNA sequences used in this study Accession number Gene name Chromosomal localization Genomic sequence cDNA sequence ABCC1 16p13.1 AC026452.5 AC025778.4 NM_004996.2 ABCC2 10q24 AL392107.4 U49248.1 ABCC3 17q22 AC004590.1 AC005921.3 NM_003786.2 ABCC4 13q32 AL356257.11 AL157818.12 AL139381.12 NM_005845.1 ABCC5 3q27 AC068644.5 NM_005688.1 CFTR 7q31.2 AC000111.1 AC000061.1 XM_004980.1 ABCC8 11p15.1 AC000406.1 NM000352.2 ABCC9 12p12.1 AC084806.9 AC008250.23 NM_005691.1 149 Fig. 1a–h. Locations of single-nucleotide polymorphisms (SNPs) in of repetitive elements. ATG, and TGA or TAA, initiation and stop ABCC1 (a), ABCC2 (b), ABCC3 (c), ABCC4 (d), ABCC5 (e), CFTR codons, respectively; ABCC, ATP-binding