Clinical omics analysis of colorectal cancer incorporating copy number aberrations and gene expression data.

BACKGROUND: Colorectal cancer (CRC) is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC) and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an "omics" study by integrating many kinds of analysis into a single comprehensive investigation. In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers. Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC. This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC. MATERIALS AND METHODS: Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language. RESULT: We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene expression analysis, together with the clinical information, suggested UGT2B28, LOC440995, CXCL6, SULT1B1, RALBP1, TYMS, RAB12, RNMT, ARHGDIB, S1000A2, ABHD2, OIT3 and ABHD12 as genes that are possibly associated with CRC. Some of these genes have already been reported as being related to CRC. TYMS has been reported as being associated with resistance to the anti-cancer drug 5-fluorouracil, and we observed a copy number increase for this gene. RALBP1, ARHGDIB and S100A2 have been reported as oncogenes, and we observed copy number increases in each. ARHGDIB has been reported as a metastasis-related gene, and our data also showed copy number increases of this gene in cases with metastasis. CONCLUSION: The combination of CNA analysis and gene expression analysis was a more effective method for finding genes associated with the clinicopathological classification of CRC than either analysis alone. Using this combination of methods, we were able to detect genes that have already been associated with CRC. We also identified additional candidate genes that may be new markers or targets for this form of cancer.

Genetic variations of the ABC transporter gene ABCB11 encoding the human bile salt export pump (BSEP) in a Japanese population.

The bile salt export pump (BSEP) encoded by ABCB11 is located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus. In this study, 28 ABCB11 exons (including non-coding exon 1) and their flanking introns were comprehensively screened for genetic variations in 120 Japanese subjects. Fifty-nine genetic variations, including 19 novel ones, were found: 14 in the coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3'-UTR, and 41 in the introns. Three novel nonsynonymous variations, 361C>A (Gln121Lys), 667C>T (Arg223Cys), and 1460G>T (Arg487Leu), were found as heterozygotes and at 0.004 allele frequencies. These data provide fundamental and useful information for genotyping ABCB11 in the Japanese and probably other Asian populations.

Genetic variations and haplotypes of ABCC2 encoding MRP2 in a Japanese population.

The multidrug resistance-associated protein 2 (MRP2) encoded by the ABCC2 gene is expressed in the liver, intestine and kidneys and preferentially exports organic anions or conjugates with glucuronide or glutathione. In this study, all 32 exons and the 5'-flanking region of ABCC2 in 236 Japanese were resequenced, and 61 genetic variations including 5 novel nonsynonymous ones were detected. A total of 64 haplotypes were determined/inferred and classified into five *1 haplotype groups (*1A, *1B, *1C, *1G, and *1H) without nonsynonymous substitutions and *2 to *9 groups with nonsynonymous variations. Frequencies of the major 4 haplotype groups *1A (-1774delG), *1B (no common SNP), *1C (-24C>T and 3972C>T), and *2 [1249G>A (Val417Ile)] were 0.331, 0.292, 0.172, and 0.093, respectively. This study revealed that haplotype *1A, which has lowered activity, is quite common in Japanese, and that the frequency of *1C, another functional haplotype, was comparable to frequencies in Asians and Caucasians. In contrast, the haplotypes harboring 3972C>T but not -24C>T (*1G group), which are reportedly common in Caucasians, were minor in Japanese. Moreover, the allele 1446C>T (Thr482Thr), which has increased activity, was not detected in our Japanese population. These findings imply possible differences in MRP2-mediated drug responses between Asians and Caucasians.

Genetic variation and haplotype structure of the ABC transporter gene ABCG2 in a Japanese population.

The ATP-binding cassette transporter, ABCG2, which is expressed at high levels in the intestine and liver, functions as an efflux transporter for many drugs, including clinically used anticancer agents such as topotecan and the active metabolite of irinotecan (SN-38). In this study, to elucidate the linkage disequilibrium (LD) profiles and haplotype structures of ABCG2, we have comprehensively searched for genetic variations in the putative promoter region, all the exons, and their flanking introns of ABCG2 from 177 Japanese cancer patients treated with irinotecan. Forty-three genetic variations, including 11 novel ones, were found: 5 in the 5'-flanking region, 13 in the coding exons, and 25 in the introns. In addition to 9 previously reported nonsynonymous single nucleotide polymorphisms (SNPs), 2 novel nonsynonymous SNPs, 38C>T (Ser13Leu) and 1060G>A (Gly354Arg), were found with minor allele frequencies of 0.3%. Based on the LD profiles between the SNPs and the estimated past recombination events, the region analyzed was divided into three blocks (Block -1, 1, and 2), each of which spans at least 0.2 kb, 46 kb, and 13 kb and contains 2, 24, and 17 variations, respectively. The two, eight, and five common haplotypes detected in 10 or more patients accounted for most (>90%) of the haplotypes inferred in Block -1, Block 1, and Block 2, respectively. The SNP and haplotype distributions in Japanese were different from those reported previously in Caucasians. This study provides fundamental information for the pharmacogenetic studies investigating the relationship between the genetic variations in ABCG2 and pharmacokinetic/pharmacodynamic parameters.

Functional analysis of SNPs variants of BCRP/ABCG2.

PURPOSE: The aim of the current study was to identify the effect of single nucleotide polymorphisms (SNPs) in breast cancer resistance protein (BCRP/ABCG2) on its localization, expression level, and transport activity. METHODS: The cellular localization was identified using the wild type and seven different SNP variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP) after transfection of their cDNAs in plasmid vector to LLC-PK1 cells. Their expression levels and transport activities were determined using the membrane vesicles from HEK293 cells infected with the recombinant adenoviruses containing these kinds of BCRP cDNAs. RESULTS: Wild type and six different SNP variants of BCRP other than S441N BCRP were expressed on the apical membrane, whereas S441N BCRP showed intracellular localization. The expression levels of Q141K and S441N BCRP proteins were significantly lower compared with the wild type and the other five variants. Furthermore, the transport activity of E1S, DHEAS, MTX, and PAH normalized by the expression level of BCRP protein was almost the same for the wild type, V12M, Q141K, A149P, R163K, Q166E, and P269S BCRP. CONCLUSIONS: These results suggest that Q141K SNPs may associate with a lower expression level, and S441N SNPs may affect both the expression level and cellular localization. It is possible that subjects with these polymorphisms may have lower expression level of BCRP protein and, consequently, a reduced ability to export these substrates.

Ethnic differences in genetic polymorphisms of CYP2D6, CYP2C19, CYP3As and MDR1/ABCB1.

Metabolic capacities for debrisoquin, sparteine, mephenytoin, nifedipine, and midazolam, which are substrates of polymorphic CYP2D6, CYP2C19, and CYP3A, have been reported to exhibit, in many cases, remarkable interindividual and ethnic differences. These ethnic differences are partly associated with genetic differences. In the case of the drug transporter ABCB1/MDR1, interindividual differences in its transporter activities toward various clinical drugs are also attributed to several ABCB1/MDR1 genetic polymorphisms. In this review, the existence and frequency of various low-activity alleles of drug metabolizing enzymes as well as populational drug metabolic capacities are compared among several different races or ethnicities. Distribution of nonsynonymous ABCB1/MDR1 SNPs and haplotype frequency in various races are summarized, with the association of nonsynonymous SNPs with large functional alterations as a rare event.

Seven novel single nucleotide polymorphisms in the human SLC22A1 gene encoding organic cation transporter 1 (OCT1).

Twenty genetic variations, including seven novel ones, were found in the human SLC22A1 gene, which encodes organic cation transporter 1, from 116 Japanese individuals. The novel variations were as follows: -94C>A in the 5'-untranslated region (A of the translation start codon is numbered +1 in the cDNA sequence; MPJ6_OC1001), 350C>T (MPJ6_OC1004), IVS1-35T>C (MPJ6_OC1006), 561G>A (MPJ6_OC1010), IVS6+75C>G (MPJ6_OC1014), IVS8+108A>G (MPJ6_OC1017), and 1671_1673delATG (MPJ6_OC1020). The frequencies were 0.082 for IVS1-35T>C, 0.022 for IVS6+75C>G, 0.009 for 561G>A, and 0.004 for the other 4 variations. Among them, 350C>T resulted in the amino acid substitution Pro117Leu, which is located in the large extracellular loop between transmembrane domains 1 and 2. Also, we detected the four previously reported nonsynonymous variations, 123C>G (Phe41Leu), 480C>G (Phe160Leu), 1022C>T (Pro341Leu), and 1222A>G (Met408Val) with frequencies of 0.004, 0.086, 0.168, and 0.810, respectively.

Characterization of the cellular localization, expression level, and function of SNP variants of MRP2/ABCC2.

PURPOSE: The presence of single nucleotide polymorphisms (SNPs) has been reported for multidrug resistance-associated protein 2 (MRP2/ABCC2). The purpose of the current study was to characterize the localization, expression level, and function of MRP2 variants. METHODS: The expression and cellular localization of the wild-type and three kinds of reported SNP variants of MRP2 molecules were analyzed in LLC-PK1 cells after infection with the recombinant Tet-off adenoviruses. Their function was determined by using the isolated membrane vesicles from the infected LLC-PK1 cells. RESULTS: The transport activity for E217betaG, LTC4, and DNP-SG, normalized by the expression level of MRP2, was similar between the wild-type, V417I, and A1450T MRP2s. The transport activity of S789F MRP2 was slightly higher than that of wild-type MRP2. However, the expression level of S789F and A1450T MRP2 proteins was significantly lower compared with the wild-type and V417I MRP2. In addition, although the wild-type and V417I MRP2 were exclusively localized in the apical membrane, S789F and A1450T MRP2 were located in the apical membrane and also in the intracellular compartment. CONCLUSIONS: These results suggest that the most frequently observed V417I substitution may not affect the in vivo function of MRP2, whereas the much less frequently observed S789F and A1450T may be associated with the reduced in vivo function.

Identification of novel alternative splice variants of human constitutive androstane receptor and characterization of their expression in the liver.

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.

Haplotype analysis of ABCB1/MDR1 blocks in a Japanese population reveals genotype-dependent renal clearance of irinotecan.

We performed comprehensive haplotyping of ABCB1/MDR1 gene blocks using 49 genetic polymorphisms, including seven novel ones, obtained from 145 Japanese subjects. The ABCB1/MDR1 gene was divided into four blocks (Blocks -1, 1, 2, and 3) based on linkage disequilibrium analysis of polymorphisms. Using an expectation-maximization based program, 1, 2, 8, and 3 haplotype groups (3, 12, 32, and 18 haplotypes) were identified in Blocks -1, 1, 2, and 3, respectively. Within Block 2, haplotype groups *1, *2, *4, *6, and *8 reported by Kim and colleagues (Clin Pharmacol Ther 2001; 70:189-199) were found, and additional three groups (*9 to *11) were newly defined. We analyzed the association of haplotypes with the renal clearance of irinotecan and its metabolites in 49 Japanese cancer patients given irinotecan intravenously. There was a significant association of the *2 haplotype in Block 2, which includes 1236C>T, 2677G>T and 3435C>T, with a reduced renal clearance of those compounds. Moreover, tendencies of reduced and increased renal clearance were also observed with *1f in Block 2 and *1b in Block 3, respectively. These findings suggest that the P-glycoprotein encoded by ABCB1/MDR1 in the proximal tubules plays a substantial role in renal exclusion of drugs and, moreover, that block-haplotyping is valuable for pharmacogenetic studies.

Five novel single nucleotide polymorphisms in the EPHX1 gene encoding microsomal epoxide hydrolase.

Five novel single nucleotide polymorphisms (SNPs) were found in the EPHX1 gene from 96 Japanese epileptic patients. The detected SNPs were as follows: 1) SNP, MPJ6_EX1009; GENE NAME, EPHX1 ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-CCTCACTTCAGTG/ACTGGGCTTTGCC-3'. 2) SNP, MPJ6_EX1013; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TCCGCAGCCAGGG/CAGGACGACAGCA-3'. 3) SNP, MPJ6_EX1026; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-GTTCTCCCTGGAC/TGACCTGCTGACC-3'. 4) SNP, MPJ6_EX1028; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-AGGCAGGGGGACG/AGCCAGTCTTGGG-3'. 5) SNP, MPJ6_EX1030; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TGAAAAGTGGGTG/AAGGTTCAAGTAC-3'. The frequencies were 0.016 for MPJ6_EX1028 (IVS8+54G>A) and 0.005 for the other SNPs. The SNP MPJ6_EX1013 (130G>C) results in an amino acid alteration (E44Q). The other three SNPs in the coding region, MPJ6_EX1009 (30G>A), MPJ6_EX1026 (1056C>T), and MPJ6_EX1030 (1239G>A) result in synonymous changes (V10V, D352D, and V413V, respectively).

Eight novel single nucleotide polymorphisms in ABCG2/BCRP in Japanese cancer patients administered irinotacan.

Eight novel single nucleotide polymorphisms (SNPs) were found in the gene encoding the ATP-binding cassette transporter, ABCG2/BCRP, from 60 Japanese individuals administered the anti-cancer drug irinotecan. The detected SNPs were as follows: 1) SNP, MPJ6_AG2005 (IVS2-93T>C); Gene Name, ABCG2; Accession Number, NT_006204; 2) SNP, MPJ6_AG2007 (IVS3+71_72 insT); Gene Name, ABCG2; Accession Number, NT_006204; 3) SNP, MPJ6_AG2012 (IVS6-204C>T); Gene Name, ABCG2; Accession Number, NT_006204; 4) SNP, MPJ6_AG2015 (at nucleotide 1098G>A (exon 9) from the A of the translation initiation codon); Gene Name, ABCG2; Accession Number, NT_006204; 5) SNP, MPJ6_AG2017 (1291T>C (exon 11)); Gene Name, ABCG2; Accession Number, NT_006204; 6) SNP, MPJ6_AG2019 (IVS11-135G>A); Gene Name, ABCG2; Accession Number, NT_006204; 7) SNP, MPJ6_AG2020 (1465T>C (exon 12)); Gene Name, ABCG2; Accession Number, NT_006204; 8) SNP, MPJ6_AG2023 (IVS13+65T>G); Gene Name, ABCG2; Accession Number, NT_006204.MPJ6_AG2015 was a synonymous SNP (E366E). MPJ6_AG2017 and MPJ6_AG2020 resulted in amino acid alterations, F431L and F489L, respectively.

Inhibition of 3 alpha/beta,20 beta-hydroxysteroid dehydrogenase by dexamethasone, glycyrrhetinic acid and spironolactone is attenuated by deletion of 12 carboxyl-terminal residues.

We constructed a pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase (3alpha/beta,20beta-HSD) mutant, which lacks 12 carboxyl-terminal amino acids residues. Enzyme activity studies indicated that the deleted amino acids have a role in steroid metabolism and may assist in substrate binding in wild-type 3alpha/beta,20beta-HSD. Furthermore, substrate binding likely induces a conformational change allowing the 12 carboxyl-terminal amino acids interact with the steroid substrate [Nakajin S. et al., Biochim. Biophys. Acta, 1550, 175-182 (2001)]. In this paper, we clarified that although pig 3alpha/beta,20beta-HSD is potently inhibited by dexamethasone, glycyrrhetinic acid and spironolactone, this inhibition is remarkably attenuated by deleting the 12 carboxyl-terminal residues. The inhibition constant (Ki) of pig 3alpha/beta,20beta-HSD for dexamethasone increased 115-fold. These observations also indicate that these amino acid residues interact with steroid substrates or steroid inhibitors and have an important role in substrate or inhibitor binding to the active site.

Polymorphisms in the ABCC2 (cMOAT/MRP2) gene found in 72 established cell lines derived from Japanese individuals: an association between single nucleotide polymorphisms in the 5'-untranslated region and exon 28.

We found nucleotide variability in the 5'-upstream region and exonic sequences of a gene-encoding canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 (cMOAT/MRP2) by polymerase chain reaction-based sequencing using genomic DNA from 72 established cell lines derived from 72 Japanese individuals. Four single nucleotide polymorphisms (SNPs) were found in the 5'-untranslational region and 21 in the exonic regions. Of them, 14 were nonsynonymous SNPs. One deletion of seven consecutive adenines resulting in a frameshift variant was also found. Four SNPs, c-24t, g1249a (V417I), c2366t (S789F), and c3972t (I1324I), were the same as those recently reported. A strong association was found between c-24t (5'-untranslated region) and c3972t (exon 28), with the promoter activity of the former worth being compared.

CYP3A4 gene polymorphisms influence testosterone 6beta-hydroxylation.

Three non-synonymous single nucleotide polymorphisms (SNPs) in the CYP3A4 gene were found in 34 cell lines derived from Japanese individuals. These three SNPs (T185S, L293P, and T363M)(1) have been previously reported, but little is known about the effect that these polymorphisms, especially T185S, have on catalytic activity. We measured testosterone hydroxylation in wild-type CYP3A4 and these three variants using a mammalian expression system. Testosterone 6beta-, 2beta-, and 15beta-hydroxylations by the variant CYP3A4 forms T363M (<40%) and T185S (<60%) were reduced as compared with the wild-type in transient expression assays. L293P was similar to the wild-type in testosterone 6beta- and 2beta-hydroxylase activities. Western blot analysis confirmed lower amounts of CYP3A4 protein in the T363M and T185S variants than in the wild-type. Interestingly, Northern blot analysis showed no significant difference among mRNA levels between the wild-type and variants. These results suggest that the T363M and T185S substitutions in CYP3A4 affect either protein expression or stability. These established cell lines provided useful CYP3A4 SNP information in the Japanese.

Twelve novel single nucleotide polymorphisms in ABCB1/MDR1 among Japanese patients with ventricular tachycardia who were administered amiodarone.

Twelve novel single nucleotide polymorphisms (SNPs) were found in the gene encoding the ATP-binding cassette transporter, P-glycoprotein, from 60 Japanese individuals who were administered the anti-antiarrythmic drug, amiodarone. The detected SNPs were as follows: 1) SNP, MPJ6_AB1017 (IVS6-109); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 2) SNP, MPJ6_AB1018 (IVS7+14); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 3) SNP, MPJ6_AB1021 (IVS9-44); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 4) SNP, MPJ6_AB1052 (IVS12+17); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 5) SNP, MPJ6_AB1029 (IVS15-69); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 6) SNP, MPJ6_AB1040 (IVS24+16); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 7) SNP, MPJ6_AB1053 (IVS27-189); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 8) SNP, MPJ6_AB1054 (IVS27-172); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 9) SNP, MPJ6_AB1048 (IVS27-167); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 10) SNP, MPJ6_AB1055 (IVS27-152); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 11) SNP, MPJ6_AB1049 (IVS27-119); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168; 12) SNP, MPJ6_AB1051 (at nucleotide 3751 (exon 28) from the A of the translation initiation codon); GENE NAME, ABCB1; ACCESSION NUMBER, NT_017168. Among these SNPs, only MPJ6_AB1051 resulted in an amino acid alteration, V1251I.