Report of New Haplotype for ABCC2 Gene

The ATP-binding cassette, subfamily C [CFTR/MRP], member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across cellular membranes. Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine. Because of the pharmacogenomics implications, we developed a clinical laboratory–developed assay to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F, rs56220353), c.-24C>T (5′UTR, rs717620), and c.4544G>A (p.C1515Y, rs8187710). During the validation process, we noted several DNA samples, obtained from the Coriell Cell Repository, that contained both c.3563T>A, c.4544G>A, and a third variant, suggesting that c.3563T>A and c.4544G>A are in cis on the chromosome in some individuals. We obtained DNA samples from a trio (father, mother, and child), tested their ABCC2 variants, and confirmed that c.3563T>A and c.4544G>A were in cis on the same chromosome. Here, we report a new haplotype in ABCC2. The ATP-binding cassette, subfamily C [CFTR/MRP], member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across cellular membranes. Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine. Because of the pharmacogenomics implications, we developed a clinical laboratory–developed assay to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F, rs56220353), c.-24C>T (5′UTR, rs717620), and c.4544G>A (p.C1515Y, rs8187710). During the validation process, we noted several DNA samples, obtained from the Coriell Cell Repository, that contained both c.3563T>A, c.4544G>A, and a third variant, suggesting that c.3563T>A and c.4544G>A are in cis on the chromosome in some individuals. We obtained DNA samples from a trio (father, mother, and child), tested their ABCC2 variants, and confirmed that c.3563T>A and c.4544G>A were in cis on the same chromosome. Here, we report a new haplotype in ABCC2. Interindividual variation in drug response among patients is well known and poses a serious challenge in medicine. At present, there are a limited number of biomarkers that can predict which group of patients will respond positively, which patients are nonresponders, and which will experience adverse reactions for the same medication and dose. Physicians often optimize dosage regimens for an individual patient by a trial-and-error method, leading to adverse drug reactions in some patients. In fact, adverse reactions are found to occur in more than two million cases annually in the United States and account for 100,000 deaths.1Lazarou J. Pomeranz B.H. Corey P.N. Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies.JAMA. 1998; 279: 1200-1205Crossref PubMed Scopus (4100) Google Scholar Similarly, in a large prospective German study, about 6% of new hospital admissions were due to adverse drug reactions.2Dormann H. Neubert A. Criegee-Rieck M. Egger T. Radespiel-Troger M. Azaz-Livshits T. Levy M. Brune K. Hahn E.G. Readmissions and adverse drug reactions in internal medicine: the economic impact.J Intern Med. 2004; 255: 653-663Crossref PubMed Scopus (118) Google Scholar This interindividual variability in drug response could be due to multiple factors such as disease determinants, genetic and environmental factors, variability in drug target response (pharmacodynamic response) or idiosyncratic response. These factors affect drug absorption, distribution, metabolism, and excretion.3Zheng C.J. Sun L.Z. Han L.Y. Ji Z.L. Chen X. Chen Y.Z. Drug ADME associated protein data base as a resource for facilitating pharmacogenomics research.Drug Dev Res. 2004; 62: 134-142Crossref Scopus (14) Google Scholar Thus, an understanding of the variability of drug response, especially genetic variability, in efficacy and toxicity of medications may provide safer and more effective drug therapy. The ATP-binding cassette, subfamily C (CFTR/MRP), member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters. ABCC2 is located on the apical membrane and is important in detoxification and chemoprotection. It transports a wide range of compounds, especially conjugates of lipophilic substances with glutathione, glucuronate, and sulfate, which are phase II products of biotransformation. ABCC2 can also transport uncharged compounds in cotransport with glutathione, and thus modulates the pharmacokinetics of many drugs.4Jedlitschky G. Hoffman U. Kroemer H.K. Structure and function of MRP2 (ABCC2) protein and its role in drug disposition.Expert Opin Metab Toxicol. 2006; 2: 351-366Crossref PubMed Scopus (177) Google Scholar The ABCC subfamily contains 12 full transporters that perform functions in ion transport, toxin secretion, and signal transduction. This protein is a member of the MRP subfamily, which is involved in multidrug resistance, and is expressed in the canalicular (apical) part of the hepatocyte and functions in biliary transport. It is additionally present in the kidneys, intestines, and placenta (National Cancer Biotechnology Institute, , last accessed October 17, 2014). Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine (the Pharmacogenomics Knowledgebase, , last accessed March 13, 2014). An example of deleterious effects of DNA variants within the ABCC subfamily is cystic fibrosis, which is an inherited multisystem disorder characterized by abnormalities in exocrine gland function consequent to loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) transporter. Similar to cystic fibrosis, defects in the ABCC2 gene have been shown to cause Dubin-Johnson syndrome.5Zimniak P. Dubin-Johnson and Rotor syndromes: molecular basis and pathogenesis.Semin Liver Dis. 1993; 13: 248-260Crossref PubMed Scopus (34) Google Scholar, 6Wada M. Toh S. Taniguchi K. Nakamura T. Uchiumi T. Kohno K. Yoshida I. Kimura A. Sakisaka S. Adachi Y. Kuwano M. Mutations in the canalicular multispecific organic anion transporter (cMOAT) gene, a novel ABC transporter, in patients with hyperbilirubinemia II/Dubin-Johnson syndrome.Hum Mol Genet. 1998; 7: 203-207Crossref PubMed Scopus (238) Google Scholar, 7Toh S. Wada M. Uchiumi T. Inokuchi A. Makino Y. Horie Y. Adachi Y. Sakisaka S. Kuwano M. Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome.Am J Hum Genet. 1999; 64: 739-746Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 8Mor-Cohen R. Zivelin A. Rosenberg N. Shani M. Muallem S. Seligsohn U. Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome.J Biol Chem. 2001; 276: 36923-36930Crossref PubMed Scopus (105) Google Scholar, 9Pacifico L. Carducci C. Poggiogalle E. Caravona F. Antonozzi I. Chiesa C. Maggiore G. Mutational analysis of ABCC2 gene in two siblings with neonatal-onset Dubin syndrome.Clin Genet. 2010; 78: 598-600Crossref PubMed Scopus (15) Google Scholar Dubin-Johnson syndrome is an autosomal recessive disorder characterized by jaundice. In addition to hyperbilirubinemia, affected individuals have an increase in the urinary excretion of coproporphyrin isomer I, deposition of melanin-like pigment in hepatocytes, and prolonged retention of sulfobromophthalein, but otherwise normal liver function (Online Mendelian Inheritance in Man, , last accessed October 31, 2014). Because of the pharmacogenomic implications, we developed a clinical laboratory assay to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F, rs56220353), c.-24C>T (5′UTR, rs717620), and c.4544G>A (p.C1515Y, rs8187710). During the analytical validation process, we noted several DNA samples obtained from the Coriell Cell Repository (Camden, NJ) that contained both c.3563T>A (p.V1188E, rs17222723), c.4544G>A (p.C1515Y, rs8187710), and a third variant, suggesting that c.3563T>A and c.4544G>A are in cis in some of these individuals. Fifty-one reference DNA samples were selected and obtained from the Coriell Cell Repository, based on partial genotypic information.10Pratt V.M. Zehnbauer B. Wilson J.A. Baak R. Babic N. Bettinotti M. Buller A. Butz K. Campbell M. Civalier C. El-Badry A. Farkas D.H. Lyon E. Mandal S. McKinney J. Muralidharan K. Noll L. Sander T. Shabbeer J. Smith C. Telatar M. Toji L. Vairavan A. Vance C. Weck K.E. Wu A.H.B. Yeo K.T.J. Zeller M. Kalman L. Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1 and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project.J Mol Diagn. 2010; 12: 835-846Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar An additional 50 anonymous DNA samples were obtained from the Indiana BioBank (Indianapolis, IN). Three anonymized whole-blood and matched saliva samples from unrelated individuals were also obtained. DNA was extracted from the whole blood and saliva (DNA Genotek, Toronto, ON, Canada) using the Qiagen QiAMP reagents (Valencia, CA) according to the manufacturer's instructions. Approximately 1 μg of DNA was extracted from the blood and saliva samples, with an average concentration of approximately 30 ng/μL. Seven variants were chosen for the genotyping assay based on commercial reagents and positive reference material availability: c.1249G>A (p.V417I; rs2273697), c.3972C>T (p.I1324I; rs3740066), c.-24C>T (5′UTR; rs717620), c.3563T>A (p.V1188E; rs17222723), c.4544G>A (p.C1515Y; rs8187710), c.2302C>T (p.R768W; rs56199535), and c.2366C>T (p.S789F; rs56220353). DNA was amplified in singlicate by real-time PCR on the LifeTech QuantStudio 12K Flex software version 1.2.2 (Grand Island, NY) and subjected to TaqMan allele discrimination using commercially available LifeTech reagents in a custom-designed open array (Table 1).Table 1Allelic Sequences for TaqMan ReagentsdbSNP#Sequencers37400665′-TCCTCAGAGGGATCACTTGTGACAT[C/T]GGTAGCATGGAGAAGGTAGGTGGAG-3′rs81877105′-CTAGACAACGGGAAGATTATAGAGT[A/G]CGGCAGCCCTGAAGAACTGCTACAA-3′rs561995355′-AAATCTTAGTGGGGGTCAGAAGCAG[C/T]GGATCAGCCTGGCCAGAGCTACCTA-3′rs562203535′-ATCTATCTTCTAGATGACCCCCTGT[C/T]TGCAGTGGATGCTCATGTAGGAAAA-3′rs22736975′-CAACTTGGCCAGGAAGGAGTACACC[A/G]TTGGAGAAACAGTGAACCTGATGTC-3′rs172227235′-CAGCGATTTCTGAAACACAATGAGG[A/T]GAGGATTGACACCAACCAGAAATGT-3′rs7176205′-ACAATCATATTAATAGAAGAGTCTT[C/T]GTTCCAGACGCAGTCCAGGAATCAT-3′ Open table in a new tab Pairwise linkage disequilibrium coefficients, D′ and r2, were computed from the observed genotype data for the variants of interest. Calculations were performed using the Haploview software version 4.2 (Broad Institute, Cambridge, MA).11Barrett J.C. Fry B. Maller J. Daly M.J. Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11960) Google Scholar Minor allele frequency and the Hardy-Weinberg test statistic were also computed for each variant using Haploview from the same genotype data. One hundred four DNA samples were genotyped for seven variants in the ABCC2 gene. We observed five samples in which c.3563T>A and c.4544G>A occurred with a third variant [ie, c.3972C>T (p.I1324I; rs3740066), c.2302C>T (p.R768W; rs56199535), c.1249G>A (p.V417I; rs2273697)]. We noted that c.3563T>A and c.4544G>A could also occur independently. One sample (1%) was heterozygous for c.3563T>A, 6 samples (6%) were heterozygous for c.4544G>A, and 11 samples (11%) were compound heterozygous (Table 2). No deviation from Hardy-Weinberg equilibrium was observed for either c.3563T>A or c.4544G>A among the individuals genotyped (P > 0.5). On the basis of the genotypes observed in our samples, the c.3563T>A and c.4544G>A loci are in high linkage disequilibrium (D′ = 0.91; r2 = 0.58), which is consistent with their close physical proximity within ABCC2 (15.3 kb apart) and their similar minor allele frequencies (6.1% and 8.5%, respectively).Table 2New Haplotypes for ABCC2ABCC2 Haplotypesrs1885301rs2804402rs717620rs2273697Intron26-34T>Crs17222723rs3740066rs8187710c.-1549G>Ac.-1019A>Gc.-24C>T (5′UTR)c.1249G>A (p.V417I)c.3563T>A (p.V1188E)c.3972C>T (p.I1324I)c.4544G>A (p.C1515Y)H1GACGTTCGH2AGTGTTH3GACATCH4AGCGTTH5AGCGTCH6AGCGCCH7GACGTTH8GACGCCH9AGTGTCH10AACGTCH11GACATTH12AACGCCH13CATH14TACH15TATH16CGACH17CAACH18CGTCAH19CGACAHaplotypes in bold are the new haplotypes described within this paper. The minor allele is denoted in italics. Open table in a new tab Haplotypes in bold are the new haplotypes described within this paper. The minor allele is denoted in italics. A trio of samples were obtained (mother, father, offspring) and genotyped for ABCC2 to resolve phasing of c.3563T>A and c.4544G>A. The father was a compound homozygote for c.3563T>A and c.4544G>A, whereas the mother and offspring were compound heterozygotes for both variants. These results confirmed that c.3563T>A and c.4544G>A are in cis within this region of high linkage disequilibrium (Figure 1) in this trio, and that the haplotype containing both variant alleles is indeed present in the population. ABCC2 facilitates ion transport, toxin secretion, and signal transduction. As such, we developed a laboratory test for clinically relevant ABCC2 variants for pharmacogenomic testing. For well-established tests, there is consensus as to which variants should be present in a clinical assay. For pharmacogenetic assays where the field is continuing to evolve, there is currently lack of consensus or standardization as to which variants should be assayed for many, if not most genes. The Clinical Pharmacogenetics Implementation Consortium (CPIC) evaluates the evidence of each variant, but does not provide a genotyping minimum standard as that is outside the purview of their charge. At this time, there are no CPIC guidelines that support ABCC2 testing. Thus, we chose the variants in our clinical assay based on information available for each variant, the availability of commercial reagents, and reference materials. We reviewed the ABCC2 haplotypes that had been reported in the literature.12Nguyen T.D. Markova S. Liu W. Gow J.M. Baldwin R.M. Habashian M. Relling M.V. Ratain M.J. Kroetz D.L. Functional characterization of ABCC2 promoter polymorphisms and allele-specific expression.Pharmacogenomics J. 2013; 13: 396-402Crossref PubMed Scopus (25) Google Scholar, 13Hilger E. Reinthaler E.M. Stogmann E. Hotzy C. Pataraia E. Baumgartner C. Zimprich A. Zimprich F. Lack of association between ABCC2 gene variants and treatment response in epilepsy.Pharmacogenomics. 2012; 13: 185-190Crossref PubMed Scopus (30) Google Scholar, 14Hassane I. Hulot J.-S. Villard E. Goyenvalle C. Dominguez S. Ghosn J. Valantin M.A. Lechat P. Deray A.G. Association between ABCC2 gene haplotypes and tenofovir-induced proximal tubulopathy.J Infect Dis. 2006; 194: 1481-1491Crossref PubMed Scopus (215) Google Scholar The current curated database of haplotypes for ABCC2 in The Pharmacogenomics Knowledgebase (, last accessed May 1, 2014) demonstrates redundancy and lack of consensus mentioned above. For example, H11 and H13 are positive for the same two single nucleotide polymorphisms, c 1249G>A and c.3972C>T. Because the two articles referenced for these haplotypes do not completely overlap as to the variants genotyped (Table 2), the curators have chosen to keep them separate with regard to the annotated associations reported in the two prior papers. Often, the haplotypes are inferred from variant genotyping and not always confirmed in family studies. The two common variants in ABCC2 on which we focus in this report (c.3563T>A and c.4544G>A) are reported to be associated with impaired drug transport and adverse drug reactions.15Sookoian S. Castaño G. Gianotti T.F. Gemma C. Pirola C.J. Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease.J Nutr Biochem. 2009; 20: 765-770Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 16Sookoian S. Castaño G. Burgueño A. Gianotti T.F. Pirola C.J. Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancy.J Hepatol. 2008; 48: 125-132Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Although not specifically noted nor confirmed, both studies appear to have inferred a haplotype containing c.3563T>A and c.4544G>A in cis in a small number of subjects. We directly confirmed the cis phasing of these variants in a trio informative for phase at both loci. Through our studies, we also noted that c.3563T>A and c.4544G>A could occur independently as well. Interestingly, two other ABCC2 variants, rs717620 (c.-24C>T) and rs3740066 (c.3972C>T), have been reported to be within the same haplotype (ie, H2 and H15; , last accessed May 1, 2014). Sookoian et al16Sookoian S. Castaño G. Burgueño A. Gianotti T.F. Pirola C.J. Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancy.J Hepatol. 2008; 48: 125-132Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar reported or inferred several haplotypes not in agreement with pharmgkb.org (, accessed May 1, 2014), including an H7 that has both the c.3563T>A and c.4544G>A variants. In addition, the authors had the reference haplotype as H3, where standard convention would have the reference sequence as H1. Pharmacogenetic sequence variants are often designated by a nomenclature of star (*) alleles, where *1 is designated as normal (commonly referred to as wild type), and subsequent variant alleles are numbered in the order that they are described. Within each star allele designation, variants that are in cis with the defining single nucleotide polymorphism create subfamilies that are designated alphabetically in the order that they are described (eg, *2A, *2B, *2C). The use of the star allele nomenclature is considered by some to be confusing and outdated; but because pharmacogenomic testing often involves multiple variants in a haplotype, rather than a single mutation (similar to the histocompatibility leukocyte antigen, HLA, nomenclature system), its use persists today. Because of the lack of clarity in the current haplotypes, we propose that ABCC2 also be described using the star allele nomenclature. We have made an attempt to translate the haplotype information to ABCC2 star alleles (Table 3). The functionality of many of the variants is not known in vitro or in vivo. Also, where functionality has been studied, the data have been conflicting.17Anderson P.L. Lamba J. Aquilante C.L. Schuetz E. Fletcher C.V. Pharmacogenetic characteristics of indinavir, zidovudine, and lamivudine therapy in HIV-infected adults: a pilot study.J Acquir Immune Defic Syndr. 2006; 42: 441-449Crossref PubMed Scopus (142) Google Scholar, 18Haenisch S. May K. Wegner D. Caliebe A. Cascorbi I. Siegmund W. Influence of genetic polymorphisms on intestinal expression and rifampicin-type induction of ABCC2 and on bioavailability of 2008; PubMed Scopus Google Scholar, S. U. E. C.J. P. Siegmund W. Kroemer H.K. Cascorbi I. Influence of polymorphisms of and ABCC2 on and protein expression in normal and J. 7: PubMed Scopus Google Scholar, M. H. M. S. J. I. K. Y. Characterization of the cellular expression and function of variants of Res. 2004; 21: PubMed Scopus Google Scholar, Y. C. K. E. N. M. B. Interindividual variability of canalicular ATP-binding-cassette expression in 2006; PubMed Scopus Google for ABCC2 variant defining are in Open table in a new tab The defining are in In the star allele nomenclature that we are alleles were where there were haplotypes were in single star alleles this this is a has an as does of for are considered and are not to protein It was more to in the promoter as well as DNA variants that not in in alleles were to each haplotype (Table 3). Functional studies will be to the haplotypes should be as testing molecular of genetic variants to predict variability in drug response. are a number of polymorphisms in many of the associated with pharmacokinetics or of Here, we report and a new haplotype in ABCC2 and propose a new star allele nomenclature. This was made by the Indiana of and

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