Genetic characterization of nucleoside analogue transporters ABCC4 and ABCC5 gene loci 1

Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci TABLE OF CONTENTS SUMMARY ………………………………………………………………………….v LIST OF PUBLICATIONS DURING PHD TENURE …………………………….vii LIST OF ABBREVIATIONS ……………………………………………………….viii LIST OF TABLES ………………………………………………………………… xi LIST OF FIGURES ……………………………………………………………… xiii I GENERAL INTRODUCTION Chapter Background …………………………………………1 …………………………………………………………1 1.1 Overall objectives of thesis …………………………………………1 1.2 SNP genotyping strategies …………………………………………3 1.3 Population-based association studies …………………………………8 1.4 SNPs and haplotypes …………………………………………………9 1.5 Haplotypes and linkage disequilibrium 1.6 Gene-based versus genome-based approaches 1.7 Gene-based haplotypes ……………………………………… 13 1.8 Haplotype reconstruction ……………………………………… 13 1.9 Haplotype tagging 1.10 International Hapmap Project ……………………………………… 19 1.11 Signatures of positive selection 1.12 New tests of selection ……………………………………………… 24 Chapter …………………………9 ……………… 12 ……………………………………………… 17 ……………………………… 21 The nucleotide analogue transporters ……………………………… 29 2.1 The ATP binding cassette (ABC) transporter superfamily ……… 29 2.2 ABCC4 and ABCC5 in antiretroviral and anticancer therapy ……… 34 2.3 Tissue distribution of ABCC4 and ABCC5 proteins ……………… 41 i Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci 2.4 Polymorphisms in coding regions of ABCC4 and ABCC5 gene loci ……………………………………………………………………… 46 2.5 Chapter II Background and specific aims of studies ……………………… 60 3.1 Workflow of studies ……………………………………………… 60 3.2 Genotyping assay development 3.3 Genetic characterization of ABCC4 and ABCC5 gene loci……… …63 MATERIALS AND METHODS Chapter ……………………………… 61 ……………………………………… 67 Genotyping assay development ……………………………… 67 4.1 Multiplex PCR amplification ……………………………………… 67 4.2 Multiplex minisequencing 4.3 Capillary electrophoresis and data analysis ……………………… 68 Chapter III Clinical implications on ABCC4 and ABCC5 in drug efflux ……… 53 ……………………………………… 67 Genetic structure characterization ……………………………… 73 5.1 DNA samples ……………………………………………………… 73 5.2 SNP selection ……………………………………………………… 74 5.3 Nomenclature of SNPs 5.4 Genotyping strategy ……………………………………………… 78 5.5 Data analysis ……………………………………………………… 86 5.6 Identification of tagging SNPs 5.7 Scanning for evidence of positive selection ……………………… 87 5.8 Coalescent simulations 5.9 Prediction of transcription factor binding sites RESULTS ……………………………………… 78 ……………………………… 87 ……………………………………… 88 ……………… 90 ……………………………………………………………… 92 Chapter Genotyping assay development ……………………………… 92 Chapter Genetic characterization of ABCC4 gene locus ……………… 95 ii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci 7.1 Large variability in SNP frequencies amongst populations ……… 95 7.2 Rapidly declining LD profile across ABCC4 gene locus 7.3 High variability in profiles of common haplotypes amongst populations ………103 ………………………………………………………104 7.4 Population-specific tagging SNP sets ………………………………107 7.5 Scanning for evidence of positive selection by Fst and Pexcess 7.6 Revelation of recent positive selection at 5’ flanking region by longrange haplotype method Chapter 8.1 109 ………………………………………112 Genetic characterization of ABCC5 gene locus ………………122 Diversity of ABCC5 SNP allele frequencies amongst different populations ………………………………………………………122 8.2 Strong LD at the ABCC5 gene locus in all five populations ………127 8.3 Low but varied ABCC5 haplotype diversity among the five populations ………………………………………………………………………131 8.4 Tagging SNPs at the ABCC5 gene locus ………………………134 8.5 Evidence of positive selection not apparent at the ABCC5 gene locus ………………………………………………………………………136 IV INFERENCES Chapter ………………………………………………………138 Simultaneous Genotyping of Seven Single Nucleotide Polymorphisms (SNPs) of the MDR1 gene by Single-Tube Multiplex Minisequencing ………………………………………………………………………138 Chapter 10 Inference of Positive Selection on a Polymorphism in the 5’ Flanking Region of the Nucleotide Analogue Transporter ABCC4/MRP4 10.1 141 Variability in allele frequencies in ABCC4 SNPs amongst populations ………………………………………………………………………141 iii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci 10.2 A rapidly declining LD profile of ABCC4 142 10.3 Population-specific tSNPs are required ………………………143 10.4 LRH test identifies signature of positive selection in a 5’ flanking SNP of ABCC4 Chapter 11 Strong ………………………………………………………144 Linkage Disequilibrium Transporter ABCC5 Gene Locus 11.1 at the Nucleotide Analogue … .………………………….147 Similarities and differences in SNP, haplotype and LD profiles between populations ………………………………………………147 V 11.2 Haplotype diversity and LD at the ABCC5 gene locus 11.3 Tagging SNPs at the ABCC5 gene locus ………………………150 GENERAL DISCUSSION 152 Chapter 12 VI ………148 Concluding remarks and future perspectives 152 12.1 ABCC4 and ABCC5: partners in crime or chalk and cheese 152 12.2 So, will the real functional variant please stand up? 160 12.3 Sources of complexity to LD and haplotype analysis 171 12.4 Future perspectives 12.5 Conclusions REFERENCES …………………………………………… 173 176 178 iv Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci SUMMARY More than million Single Nucleotide Polymorphisms (SNPs) and other sequence variations have been identified following the Human Genome Project. The construction of a dense SNP map has been aided by accumulation of large numbers of human samples for analysis, advances in genotyping techniques, and improvement in computational power for statistical analysis. However, the interrogation of which of these allelic variations in the genome are responsible for common diseases and other human traits is still in its infancy. Testing individual polymorphisms to provide evidence for genotype-phenotype correlation is an arduous and time-consuming task, not to mention the low power of detection. The approach of this dissertation focuses on using haplotypes or multilocus genotypes for the identification of candidate causal variants in a gene-based association study. The information gained from this work would then provide the direction for future functional studies that will lead to insights into the causal mechanisms of human diseases or drug response. The candidate genes of interest explored in this dissertation are two closely related nucleotide analogue transporters, MRP4 (ABCC4) and MRP5 (ABCC5). As nucleotide analogue drugs are used in antiretroviral and antitumor therapy, polymorphisms in ABCC4 and ABCC5 may play a role in determining interindividual variation in drug responses. Potential functional SNPs amongst other markers were chosen to provide a wellspaced SNP set across the candidate gene. A robust and cost-efficient genotyping technique was developed for the simultaneous analysis of seven or more SNPs in a single reaction. This enabled a small laboratory to adopt a quick mid-throughput assay for genotyping multiple SNPs in candidate genes. The non-random association of SNPs or Linkage Disequilibrium (LD) was utilized to provide greater power in v Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci detecting the effects of functional variants that may or may not be in the analyzed SNP set. From the detailed LD and haplotype analyses, subsets of tagging SNPs (tSNPs) were then constructed. This provided further a reduction in the number of SNPs to be used for gene-based association studies without sacrificing resolution or coverage. As the LD structure varied even in the same genomic region amongst different populations, comparative analysis of LD profiles in ethnically unique populations was performed. It was deduced that despite their similarity in drug transport profile, ABCC5 was in a region of strong linkage disequilibrium whereas the LD profile in ABCC4 decayed rather rapidly. The larger sets of tSNPs in ABCC4 as compared to ABCC5 also attested to the apparent strong LD in the latter. A complementary strategy was also introduced to infer genetic variants determining drug response variability by detecting signatures of natural selection in the ABCC4 gene locus. Evidence of positive selection was observed in the A allele of SNP i-1 1015G>A residing within the 5’ flanking region in which the putative promoter of ABCC4 might be found. In summary, the strategies employed in this dissertation may be useful for studies associating disease / drug response and these genes. This thesis also highlights the potential usefulness of utilizing positive selection to elucidate putative functional variants responsible for drug response and other human traits. vi Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci LIST OF PUBLICATIONS DURING PHD TENURE Publications resulting from research carried out during the period of postgraduate programme are listed below. Gwee PC, Tang K, Sew PH, Lee EJ, Chong SS, Lee CG (2005) Strong linkage disequilibrium at the nucleotide analogue transporter ABCC5 gene locus. Pharmacogenet Genomics 15:91-104 [ISI Impact Factor 6.406 as of Year 2003] Gwee PC, Tang K, Chua JM, Lee EJ, Chong SS, Lee CG (2003) Simultaneous genotyping of seven single-nucleotide polymorphisms in the MDR1 gene by single-tube multiplex minisequencing. Clin Chem 49:672-676. [ISI Impact Factor 6.501 as of Year 2003] Tang K, Ngoi SM, Gwee PC, Chua JM, Lee EJ, Chong SS, Lee CG (2002) Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations. Pharmacogenetics 12:437-450. [ISI Impact Factor 6.406 as of Year 2004] vii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci LIST OF ABBREVIATIONS (S)HPMPC 3TC 5-Br-dUrd 5-FU 5-HP AA ABC ADME ADR ALL AML ASE AS-PCR AZT AZT-MP BBB BCEC BCSFB bis-POM-PMEA cAMP CAU CEPH CEU CFTR cGMP CH CNS cPr-PMEDAP CSF DCM ddNTP dHPLC DNP-SG dNTP E217βG EHH EM Exo I FDA GST HAART HBD HCB HIV HKA HWE 1-(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine 2'-deoxy-3'-thiacytidine, lamivudine 5-bromo-2'deoxyuridine 5-flurouracil. 5-hydroxypyridine-2-carboxaldehyde thiosemicarbazone African American ATP-Binding Cassette Absorption, Distribution, Metabolism, and Excretion adverse drug reactions acute lymphoblastic leukemia acute myeloid leukemia allele-specific extension allele-specific polymerase chain reaction zidovudine, azidothymidine, 2’-azido-2’,3’-dideoxythymidine azidothymidine-monophosphate blood-brain barrier brain capillary endothelial cells blood-cerebrospinal fluid barrier bis(pivaloyloxymethyl)-9-(2-phosphonomethoxyethyl)adenine adenosine 3’,5’-cyclic monophosphate European American Centre d’Etude du Polymorphisme Humain - U.S. Utah residents with ancestry from northern and western Europe) in the International Hapmap Project CEPH/UTAH cystic fibrosis transmembrane conductance guanosine 3’,5’-cyclic monophosphate Chinese Central Nervous System cyclopropyl-PMEDAP cerebrospinal fluid dilated cardiomyopathy dideoxyribonucleoside triphosphate Denaturing High-Performance Liquid Chromatography S-(2,4-dinitrophenyl)-glutathione deoxynucleotide triphosphate estradiol 17-β-D-glucuronide extended haplotype homozygosity expectation-maximization E.coli exonuclease I U.S. Food and Drug Administration glutathione S-transferase highly active antiretroviral therapy Haplotype Branching Diagram Han Chinese in Beijing human immunodeficiency virus McDonald-Kreitman-Aguade Hardy-Weinberg Equilibrium viii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci ICM IN JPY LD LD0.5 LRH LTRA MCMC MDM MDR1 MDS mesDA MH MK571 ML MRP MSD NAc-DNP-Cys NAT NBD NIGMS NRTI NSAID NSCLC OLA PBL PCR PDE5 PFIC PG P-gp PI PL PMEA PMEDAP PMEG Preference PTC Ptest r2 R2 rEHH RFLP RT SAP SBE SCLC SNP SSCP ischemic cardiomyopathy Indian Japanese in Tokyo linkage disequilibrium half-length LD Long-Range Haplotype Leukotrienes Receptor Antagonist Markov chain–Monte Carlo monocyte-derived macrophages multidrug resistance myelodysplastic syndromes subtypes mesencephalic dopaminergic major haplotype 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid Malay multidrug resistance related protein membrane spanning domain N-acetyl (2,4,-dinitrophenyl)-cysteine N-acetyltransferase nucleotide binding domains National Institute of General Medical Sciences nucleoside reverse transcriptase inhibitor non-steroidal anti-inflammatory drug non-small cell lung cancers Oligonucleotide Ligation Assay peripheral blood lymphocytes Polymerase Chain Reaction phosphodiesterase progressive familial intrahepatic cholestasis prostaglandin P-glycoprotein protease inhibitor partition-ligation 9-(2-phosphonomethoxyethyl)adenine 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine 9-(2-phosphonomethoxyethyl)guanine allele frequency in ancestral (reference) population premature termination codon allele frequency in population under study (test) Pearson correlation correlation coefficient relative extended haplotype homozygosity Restriction Fragment Length Polymorphism reverse transcriptase shrimp alkaline phosphatase Single-Base Extension small cell lung cancers Single Nucleotide Polymorphism single strand conformation polymorphism ix Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci SSD STR tSNPs VCR VP-16 YRI Stephens-Smith-Donnelly Short-Tandem-Repeat Tagging SNPs vincristine etoposide Yoruba in Ibadan x Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci LIST OF TABLES I GENERAL INTRODUCTION Table 1. Summary of the major classes of tests for natural selection. ………………………………………………………………… …23 Table 2. Overall amino acid identity amongst selected ABCC subfamily members ………………………………………………………………… …31 Table 3. Substrates of ABCC4 and ABCC5 proteins. …………………………………………………………………… …38 Table 4. Substrates of ABCC4 and ABCC5 proteins with inhibitory actions. …………………………………………………………………… …40 Table 5. Coding polymorphisms/mutations in ABCC4 and ABCC5 as reported from published reports. …………………………………………………………………… …48 Table 6. Coding SNPs found from public polymorphism/mutation databases. …………………………………………………………………… …51 II MATERIALS AND METHODS Table 7. Multiplex PCR and minisequencing of the MDR1 gene. …………………………………………………………………… …71 Table 8. Primers, PCR and minisequencing conditions for 28 SNPs at the ABCC4 gene locus. …………………………………………………………………… …82 Table 9. Primers, PCR and minisequencing conditions for 20 SNPs at the ABCC5 gene locus. ……………………………………………………………………… 84 III RESULTS Table 10. Genotype and allele frequency analysis of 28 SNPs at the ABCC4 gene locus. .…………………………………………………………………… .97 Table 11. Combinations of tagging SNPs (tSNPs) at the ABCC4 gene locus. …………………………………………………………………… 108 Table 12. P-values derived from comparing relative EHH values of SNPs -1 -1015G>A, i-1 -527C>G and i31 1454C>T against simulated data points against 12 proposed population models. ……………………………………………………………………116 xi Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci Table 13. Allele frequencies of the different SNPs in the different ethnic populations. ………………………………………………………………………124 Table 14. Tagging SNPs at the ABCC5 gene locus in ethnic populations. …………………………………………………………………… 135 Table 15. ABCC5 SNPs and their respective rEHH values. …………………………………………………………………… 137 IV INFERENCES V GENERAL DISCUSSION Table 16. List of therapeutic drugs that are effluxed by ABCC4 or ABCC5 and their clinical applications. …………………………………………………………………… 157 Table 17. Summary of LD and haplotype analyses around ABCC4 and ABCC5 gene loci. …………………………………………………………………… 159 VI REFERENCES xii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci LIST OF FIGURES I GENERAL INTRODUCTION Figure 1. Linkage Disequilibrium and Linkage Equilibrium. ……………………………………………………………………… 10 Figure 2. Long-range haplotype test. ……………………………………………………………………… 27 Figure 3. Structural illustration of selected members of ABC transporter superfamily. ……………………………………………………………………… 32 Figure 4. Predicted secondary structures of ABCC4/MRP4 and ABCC5/MRP5 proteins. ……………………………………………………………………… 33 II MATERIALS AND METHODS Figure 5. Overview of studies. ……………………………………………………………………… 60 Figure 6. Schematic illustration of multiplex minisequencing protocol. ……………………………………………………………………… 70 Figure 7. Multiplex PCR for the seven MDR1 SNPs. ……………………………………………………………………… 72 Figure 8. Distribution of SNPs across the ABCC4 gene. ……………………………………………………………………… 76 Figure 9. Distribution of SNPs across the ABCC5 gene. ……………………………………………………………………… 77 III RESULTS Figure 10. Genotyping results for the seven MDR1 SNPs. ……………………………………………………………………… 94 Figure 11. Pair-wise linkage disequilibrium profiles of ABCC4 gene locus. …………………………………………………………………… 101 Figure 12. Haplotype profiles of SNPs at the ABCC4 gene locus. ………………………………………………………………………105 Figure 13. Local profiles of Fst and Pexcess values around ABCC4 gene locus. ………………………………………………………………………111 xiii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci Figure 14. Haplotype Branching Diagrams at selected SNP loci ………………………………………………………………………113 Figure 15. Extended haplotype homozygosity (EHH) and relative EHH (rEHH) plots of SNPs i-1 -1015G>A and i31 1454C>T. ………………………………………………………………………114 Figure 16. Pair-wise linkage disequilibrium profile of CEPH population in International Hapmap Project. ………………………………………………………………………118 Figure 17. Long range haplotype test in CEPH data from International Hapmap Project ………………………………………………………………………121 Figure 18. Pair-wise linkage disequilibrium profiles of ABCC5 gene locus. ………………………………………………………………………128 Figure 19. LD decay profile at the ABCC5 locus. ………………………………………………………………………130 Figure 20. Haplotype profiles of SNPs at the ABCC5 gene locus. ………………………………………………………………………132 IV INFERENCES V GENERAL DISCUSSION VI REFERENCES xiv [...]... …………………………………………………………………… 10 1 Figure 12 Haplotype profiles of SNPs at the ABCC4 gene locus …………………………………………………………………… 10 5 Figure 13 Local profiles of Fst and Pexcess values around ABCC4 gene locus …………………………………………………………………… 11 1 xiii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci Figure 14 Haplotype Branching Diagrams at 4 selected SNP loci …………………………………………………………………… 11 3 Figure 15 ... (tSNPs) at the ABCC4 gene locus …………………………………………………………………… 10 8 Table 12 P-values derived from comparing relative EHH values of 3 SNPs -1 -10 15G>A, i -1 -527C>G and i 31 1454C>T against simulated data points against 12 proposed population models ………………………………………………………………… 11 6 xi Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci Table 13 Allele frequencies of the different... around ABCC4 and ABCC5 gene loci …………………………………………………………………… 15 9 VI REFERENCES xii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci LIST OF FIGURES I GENERAL INTRODUCTION Figure 1 Linkage Disequilibrium and Linkage Equilibrium ……………………………………………………………………… 10 Figure 2 Long-range haplotype test ……………………………………………………………………… 27 Figure 3 Structural illustration of selected.. .Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci LIST OF TABLES I GENERAL INTRODUCTION Table 1 Summary of the major classes of tests for natural selection ………………………………………………………………… …23 Table 2 Overall amino acid identity amongst selected ABCC subfamily members ………………………………………………………………… … 31 Table 3 Substrates of ABCC4 and ABCC5 proteins ……………………………………………………………………... (EHH) and relative EHH (rEHH) plots of SNPs i -1 -10 15G>A and i 31 1454C>T …………………………………………………………………… 11 4 Figure 16 Pair-wise linkage disequilibrium profile of CEPH population in International Hapmap Project …………………………………………………………………… 11 8 Figure 17 Long range haplotype test in CEPH data from International Hapmap Project …………………………………………………………………… 12 1 Figure 18 Pair-wise linkage disequilibrium profiles... …………………………………………………………………… 12 4 Table 14 Tagging SNPs at the ABCC5 gene locus in 5 ethnic populations …………………………………………………………………… 13 5 Table 15 ABCC5 SNPs and their respective rEHH values …………………………………………………………………… 13 7 IV INFERENCES V GENERAL DISCUSSION Table 16 List of therapeutic drugs that are effluxed by ABCC4 or ABCC5 and their clinical applications …………………………………………………………………… 15 7 Table 17 Summary of LD and haplotype... …………………………………………………………………… … 71 Table 8 Primers, PCR and minisequencing conditions for 28 SNPs at the ABCC4 gene locus …………………………………………………………………… …82 Table 9 Primers, PCR and minisequencing conditions for 20 SNPs at the ABCC5 gene locus ……………………………………………………………………… 84 III RESULTS Table 10 Genotype and allele frequency analysis of 28 SNPs at the ABCC4 gene locus …………………………………………………………………… 97 Table 11 Combinations of tagging... for the seven MDR1 SNPs ……………………………………………………………………… 72 Figure 8 Distribution of SNPs across the ABCC4 gene ……………………………………………………………………… 76 Figure 9 Distribution of SNPs across the ABCC5 gene ……………………………………………………………………… 77 III RESULTS Figure 10 Genotyping results for the seven MDR1 SNPs ……………………………………………………………………… 94 Figure 11 Pair-wise linkage disequilibrium profiles of ABCC4 gene locus ……………………………………………………………………... …………………………………………………………………… 12 1 Figure 18 Pair-wise linkage disequilibrium profiles of ABCC5 gene locus …………………………………………………………………… 12 8 Figure 19 LD decay profile at the ABCC5 locus …………………………………………………………………… 13 0 Figure 20 Haplotype profiles of SNPs at the ABCC5 gene locus …………………………………………………………………… 13 2 IV INFERENCES V GENERAL DISCUSSION VI REFERENCES xiv ... Substrates of ABCC4 and ABCC5 proteins with inhibitory actions …………………………………………………………………… …40 Table 5 Coding polymorphisms/mutations in ABCC4 and ABCC5 as reported from published reports …………………………………………………………………… …48 Table 6 Coding SNPs found from public polymorphism/mutation databases …………………………………………………………………… … 51 II MATERIALS AND METHODS Table 7 Multiplex PCR and minisequencing of the MDR1 gene . distribution of ABCC4 and ABCC5 proteins ……………… 41 i Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci 2.4 Polymorphisms in coding regions of ABCC4 and ABCC5 gene. ……………………………… 92 Chapter 7 Genetic characterization of ABCC4 gene locus ……………… 95 ii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci 7 .1 Large variability. …………………………………………………………………… 11 1 xiii Genetic Characterization of Nucleotide Analogue Transporters ABCC4 and ABCC5 Gene Loci Figure 14 . Haplotype Branching Diagrams at 4 selected SNP loci …………………………………………………………………… 11 3