Genetic analysis of SNPs in and around the MDRI gene locus

Acknowledgement I would like to acknowledge all who have helped and inspired me during my years at the National University of Singapore. I am very grateful to my supervisor, Assistant Professor Lee, Caroline G.L., and her husband, Associate Professor Chong, Samuel S. for their invaluable inspiration, guidance and encouragement throughout the course of my Ph.D study. I want to thank Miss Wong Li Peng for her diligent and excellent work. I would also like to thank all the lab members for their kind help. They made my four years in this “family” fun and exciting. In addition, I would like to specially thank my friends Mr. Wang, Baoshuang, Mr. Ren, Jianwei, Mr. Wang, Zihua, Mr. Zhang, Dongwei, Mr. Gwee, PaiChung and Dr. Lee, Alvin T.C. for their constant support and friendship. I acknowledge the National University of Singapore, for honoring me with studentship and financial assistance in the form of scholarship. i Table of Contents Acknowledgements ……………………………………………………………… i Table of Contents………………… ……………………………………………… . ii Summary …………………………………………………………………. vi Publications and Awards arising during PhD tenure………………………….… . viii List of Tables …………………………………………………………………… . xi List of Figures ……………………………………………………………………. xii PART I INTRODUCTION Chapter 1: General Introduction …………………………………….……………. 1.1 SNP profiling in candidate genes and genomic regions – the practical pharmacogenetics approach ……………………………………… .…….…2 1.1.1 Introduction of SNP profiling and several concepts related to it.……. 1.1.2 Association studies ………………………………………………… . 1.1.3 Detection of signature of natural selection …………………………. 11 1.2 A drug-response related genomic region around chromosome 7q21.1: the importance of MDR1, MDR3 and the CYP3A clusters …………………….16 1.2.1 Drug response related genes. The ATP binding cassette (ABC) transporter super-family and the P450 cytochrome enzyme superfamily ……………………………………………………………… .16 1.2.2 ABCB1 (MDR1) gene and its related functional and genetic studies 21 1.2.3 ABCB4 (MDR3) gene and its related functional and genetic studies 26 1.2.4 An important CYP3A cluster: the CYP3A4, CYP3A5, CYP3A7 and CYP3A43 genes ……………………………………………………. 27 1.3 Objectives and Significance ……………………………………………… 30 ii 1.4 References …………………………………………………………………. 31 PART II GENOTYPING TECHNOLOGY Chapter 2: Simultaneous Genotyping of Multiple Single-Nucleotide Polymorphisms in Candidate Gene by Single-Tube Multiplex Minisequencing 2.1 Introduction ……………………………………………………………… 43 2.2 Material and methods ………………………………………………………43 2.3 Examples and Discussion …………………………………………………. 47 2.4 References ………………………………………………………………….48 PART III GENETIC CHARACTERIZATION OF ABCB1/4 AND CYP3A Chapter 3: Distinct Haplotype Profiles and Strong Linkage Disequilibrium at the MDR1 Multidrug Transporter Gene Locus in Three Ethnic Asian Populations …………………………………………………………… 49 3.1 Introduction …………………………………………………………… 50 3.2 Materials and Methods ………………………………………… .……… 54 3.3 Results ………………………………………………………… .……… . 56 3.3.1 SNPs in the promoter and 59 UTR of the MDR1 gene .…….……. 58 3.3.2 SNPs in the coding region of the MDR1 gene ………… .………… 60 3.3.3 Haplotype profile and linkage disequilibrium of SNPs in the MDR1 gene ………………………………………………………………… .……. 63 3.4 Discussions ….………………………………………………………… 67 3.5 References ……………………………………………………… .………. 74 Chapter 4: Genomic Evidence for Recent Positive Selection at the Human MDR1 Gene Locus …………………………………………………………… 78 4.1 Introduction ……………………………………… .…………………… . 79 iii 4.2 Materials and Methods ……………………………………………………. 82 4.3 Results …………………………………………… .…………………… . 88 4.3.1 MDR1 SNP allele frequencies differ among populations ……… … 88 4.3.2 MDR1 haplotype diversity differs among populations …………… 89 4.3.3 Highly variable LD between SNP loci ……………………… .…… 93 4.3.4 SNPs 10 and 11 of the MDR1 gene are positively selected .……… 96 4.4 Discussion ……………………………………………… .…………… 101 4.4.1 Varied haplotype diversity and long-range LD in the MDR1 gene 101 4.4.2 Evidence of recent positive selection at the MDR1gene locus …. 103 4.4.3 Implication of recent positive selection with respect to functional disease association studies ……………………….……………… . 104 4.5 Reference …………………………………………… .……………… . 107 Chapter 5: An Extended Genetic Study of a Drug-response Related Region: Differential Selections Detected in Both MDR1 and MDR3 genes .112 5.1 Introduction ………………………………………… ………………… 113 5.2 Materials and methods …………………………………………………… 115 5.3 Results ………………………………………… ……………………… 119 5.3.1 Genotyping results and allele frequencies ……… .………………. 119 5.3.2 Linkage Disequilibrium profiles ………………… .…………… . 122 5.3.3 Haplotype frequency profiles ……………………… .…………… 123 5.3.4 Detection of positive selection ……………………………… .… 126 5.4 Discussion ………………………………………… .………………… . 133 5.5 Reference ………………………………………………………………… 137 Chapter 6: The CYP3A Gene Shows Strong Evidences of Positive Selection in Caucasians ……………………………………………………………. 140 iv 6.1 Introduction ………………………………………… .…………………. 141 6.2 Materials and methods …………………………………………………… 143 6.3 Results …………………………………………………………… .……. 149 6.3.1 Allele frequency and the Fst, Pexcess tests ………………… ……… 149 6.3.2 Haplotype and Linkage Disequilibrium profiles …… ………… . 151 6.3.3 LRH test of positive selection ……………………………… .… . 156 6.4 Discussions …………………………………………………… ……… 160 6.5 References ……………………………………………………………… 162 PART IV ASSOCIATION STUDY Chapter 7: MDR1, the Blood–brain Barrier Transporter, Is Associated with Parkinson’s Disease in Ethnic Chinese ………………… .……… . 165 7.1 Introduction ……………………………………………… …………… 165 7.2 Methods ………………………………… …………………………… 168 7.3 Results …………………………………………………………… .……. 172 7.3.1 Association of MDR1 SNPs and their haplotypes with Parkinson’s disease.…………………………………………………………… 172 7.3.2 Sex differences in risk determination …………………………… . 173 7.3.3 Role of SNPs/haplotypes in the MDR1 gene in later onset of Parkinson’s disease …………………………………………….…. .175 7.4 Discussions………………………… .……………………….………… 177 7.5 Conclusions ……………………………………… .…………………. 181 7.6 References ……………………………………………………………… 182 SUMMARY AND CONCLUSIONS ……………………………………… . 186 v Summary The ABCB1/MDR1 multidrug transporter is the prototype of drug transporters and one of the major determinants of drug/xenobiotics response. The MDR1 gene, together with several other important drug-response genes, namely the ABCB4/MDR3 gene and the CYP3A gene cluster, maps to a 12 Mb region around Chromosome 7q21.1. A large number of studies here reported associations between MDR1/CYP3A genetic polymorphisms and a diversity of functional traits including gene expression, pharmacokinetic properties as well as susceptibilities to various diseases. Functional polymorphisms were also identified at the CYP3A4 and CYP3A5 gene loci. As polymorphisms in genes controlling drug response may influence an individual’s response to medication, it is necessary to understand this important drug-response locus, localizing the causative variants and clarifying how its genetic variants affect function. This thesis describes a series of studies aimed at addressing the above questions, using the MDR1 gene and several nearby drug-response genes as models. Specifically, comprehensive SNP profiling was carried out in and around these genes in major world populations: Chinese, Malay, Indian, Caucasian and African American. The relationships between individual markers were described in terms of linkage disequilibrium (LD) profiles; and the haplotype frequencies were estimated using Expectation Maximization (EM) approaches and compared amongst the different populations. We detected substantial, but highly variable and complex LD at this 12Mb region of Chromosome 7q21.1. Haplotype frequencies vary amongst populations, with the African population being the most different from the nonAfrican populations. We further investigated the impact of natural selection at these gene loci through several tests including a modified Long Range Haplotype (LRH) vi test and Fst / Pexcess based tests. The MDR1 and MDR3 genes demonstrated significant evidence of positive selection for several variants residing on a common extended haplotype, in the non-African groups. Tests of positive selection, including the LRH approach and Fst / Pexcess tests together revealed strong signatures of selection at the CYP3A gene cluster in Caucasians. We further examined the association between SNPs / haplotypes of SNPs within the MDR1 gene with Parkinson’s disease. Several MDR1 polymorphisms were found to significantly affect one’s susceptibility to Parkinson’s disease. The studies described in this thesis are amongst the first efforts to clarify the genetic profiles at this important drug-response region at Chromosome 7q21.1. We presented the genetic relationships of several functionally associated drug-response genes at this chromosome locus. Our studies would provide a basis for future studies directed at single locus in different populations to be compared systematically. It should also facilitate the inference of the genomic location of causative variants. The evidences of natural selection demonstrated in our studies are among the first to be reported for genes important for drug response. These evidences strongly support the notion that genes controlling drug/xenobotics responses were under substantial selection pressures during recent human migrations. Additionally, the approaches for detecting signatures of natural selection and functional association, applied and evaluated in our studies could contribute to the identification of other functional variants in the genome. vii Publications and Awards arising during PhD tenure: Peer-Reviewed Publications: 1. Kun Tang, Soo-Mun Ngoi, Pai-Chung Gwee, John MZ Chua, Edmund JD Lee, Samuel S. Chong and Caroline G. Lee*. “Distinct Haplotype Profiles and Strong Linkage Disequilibrium in the MDR1 multidrug transporter gene locus in three Asian populations. Pharmacogenetics 12(6):437-450 (2002). In focus comments on our article: Kim RB. MDR1 single nucleotide polymorphisms: multiplicity of haplotypes and functional consequences. Pharmacogenetics 12(6): 425 (2002) (2003 Impact Factor: 5.851) 2. Pai-Chung Gwee, Kun Tang, John MZ Chua, Edmund JD Lee, Samuel S Chong, Caroline G. Lee*. Simultaneous Genotyping of Seven Single Nucleotide Polymorphisms (SNPs) of the MDR1 gene by Single Tube Multiplex Minisequencing. Clinical Chemistry 49(4):672-676 (2003). (2003 Impact Factor: 5.538) 3. Caroline GL Lee*, Kun Tang, Yin Bun Cheung, Li Peng Wong, Chris Tan, Hui Shen, Yi Zhao, R. Pavanni, Meng-Cheong Wong, Samuel S Chong and Eng King Tan. MDR1, the blood-brain barrier transporter, is associated with Parkinson’s Disease in Ethnic Chinese. Journal of Medical Genetics 41:e60 (2004). (2003 Impact Factor: 6.368) 4. Kun Tang, Li Peng Wong, Edmund JD Lee, Samuel S. Chong, Caroline G.L. Lee*. Genomic Evidence for Positive Selection at the MDR1 Gene Locus. Human Molecular Genetics 13(8): 783-797 (2004). (2003 Impact Factor: 8.597) 5. Eng-King Tan, Marek Drozdzik, Monika Bialecka, Krystyna Honczarenko, Gabriela Klodowska-Duda, YY Teo, Kun Tang, Li-Peng Wong, Samuel S viii Chong, Chris Tan, Kenneth Yew, Yi Zhao, Caroline GL Lee. Analysis of MDR1 Haplotypes in Parkinson’s Disease in a White Population. Neuroscience Letts 372: 240-244(2004). 6. Eng-King Tan, Daniel Kam-Yin Chan, Ping-Wing Ng, Jean Woo, Y Y Teo, Kun Tang, Li-Peng Wong, Samuel S Chong, Chris Tan, Hui Shen, Yi Zhao, Caroline GL Lee. MDR1 Haplotype (e21/2677T and e26/3435T) Modulates Risk of Parkinson’s Disease. Archives of Neurology (in press) (2003 Impact Factor: 4.684) 7. Pai Chung Gwee, Kun Tang, Pui Hoon Sew, Edmund J.D. Lee, Samuel S. Chong, and Caroline G.L. Lee*. Strong Linkage Disequilibrium at the Nucleotide Analogue Transporter ABCC5 Gene Locus. Pharmacogenetics (in press). (2003 Impact Factor: 5.851) ix Awards: 1. 2003 American Association for Cancer Research (AACR) Pfizer Scholarin-Training Award to present at the AACR Special Meeting: “SNPs, Haplotypes, and Cancer: Applications in Molecular Epidemiology” September 13-17 (2003) at the Sonesta Beach Resort Key Biscayne, in Key Biscayne, Florida (did not go because of visa problems). Details of presentation: Kun Tang, Li Peng Wong, Edmund JD Lee, Samuel S Chong, and Caroline GL Lee. e21/26777T and e26/3435T alleles in the MDR1 gene showed evidence of recent positive selection. 2. 2004 AACR-ITO EN Ltd Scholar-in-Training Award to present at the AACR 95th Annual Meeting in Orlando, Florida, March 27-31, 2004. Abstract # 2923. Details of presentation: Kun Tang, Li Peng Wong, Edmund JD Lee, Samuel S Chong, and Caroline GL Lee. Recent positive selection of SNPs e21/2677 and e26/3435 in the MDR1 gene. x Only SNP e12/1236C, and haplotypes i1/-41A-e12/1236C, e12/1236Ce21/2677G-e26/3435C-e28/4036A, and i1/-41A-e12/1236C-e21/2677G-e26/3435Ce28/4036A were not significantly associated with Parkinson’s disease in men, although their association with the disease in the overall population was significant. In addition, haplotypes e21/2677G-e26/3435Ce28/4036G (OR 3.644 (1.652 to 7.269), e12/1236T-e21/2677Ge26/3435C-e28/4036G (OR 2.715 (1.182 to 6.888), e12/1236C-e21/2677G-e26/3435C-e28/4036G (OR 5.778 (1.286 to 93.813), and i1/41A-e12/1236T-e21/2677G-e26/3435C-e28/4036G (OR 2.804 (1.090 to 7.126) were significantly associated with Parkinson’s disease in men but not overall (Table 7.3). 7.3.3 Role of SNPs/haplotypes in the MDR1 gene in later onset of Parkinson’s disease Interesting observations were made when we examined the age of onset specific association of SNPs/haplotypes in the MDR1 gene with Parkinson’s disease. While the promoter SNP i1/-41(A/G) was found not to be associated with Parkinson’s disease in our overall or sex specific analyses, the low frequency G allele of this SNP was found to be significantly associated (p=0.01), with a decreased risk of developing Parkinson’s disease at or before the age of 55 years (OR 0.307 (95% CI, 0.125 to 0.758) (Table 7.4). Conversely, SNPs e21/2677(G/T/A) (p=0.0102), e26/3435(C/T) (p=0.0061), and SNP combinations e26/3435(C/T)-e28/4036(A/G) (p=0.0423) and e21/2677(G/T/A)-e26/3435(C/T)-e28/4036(A/G) (p=0.0225) were associated with increased risk of developing Parkinson’s disease at or after age 60, with SNPs e21/2677G (OR 1.748 (1.209 to 2.534)) and e26/3435C (OR 1.642 (1.148 to 2.354)), and haplotypes e26/3435C-e28/4036A (OR 1.657 (1.081 to 2.583)) and e21/2677Ge26/3435C-e28/4036A (OR 1.963 (1.250 to 3.106)) being associated with the 174 increased risk (Table 7.4). Some haplotypes that include either or both of the SNPs e21/2677(G/T/A) and e26/3435(C/T) were also associated with an increased risk of developing Parkinson’s disease (Table 7.4). Curiously, although SNPs i1/-41(A/G) 175 0.97412124 0.12988741 0.93508326 0.6112244 0.25033874 0.04886644 0.15750045 0.84732688 0.09546228 0.26934543 0.5064368 0.64957189 0.6580088 0.99342381 T C T A G T C A G A-T A-C T-T T-G C-A C-G T-T G-C T-A C-A C-G A-T-T A-T-G A-C-A A-C-G T-T-T T-G-C C-G-C T-T-A G-C-A G-C-G A-T-T-T A-T-G-C A-C-A-C A-C-G-C T-T-T-A T-G-C-A T-G-C-G C-G-C-A C-G-C-G A-T-T-T-A A-T-G-C-A A-T-G-C-G A-C-A-C-G A-C-G-C-A A-C-G-C-G i-1/-41(A/G) e12/1236(C/T) e21/2677(G/T/A) e26/3435(C/T) e28/4036(A/G) i1/-41(A/G)-e12/1236(C/T) e12/1236(C/T)-e21/2677(G/T/A) e21/2677(G/T/A)-e26/3435(C/T) e26/3435(C/T)-e28/4036(A/G) i1/-41(A/G)-e12/1236(C/T)e21/2677(G/T/A) e12/1236(C/T)-e21/2677(G/T/A)e26/3435(C/T) e21/2677(G/T/A)-e26/3435(C/T)e28/4036(A/G) i1/-41(A/G)-e12/1236(C/T)e21/2677(G/T/A)-e26/3435(C/T) e12/1236(C/T)-e21/2677(G/T/A)e26/3435(C/T)-e28/4036(A/G) i1/-41(A/G)-e12/1236(C/T)e21/2677(G/T/A)-e26/3435(C/T)e28/4036(A/G) 108.000 8.000 69 47.000 40 16 60.000 38 78.000 83 33.000 66.3 41.700 37.2 31.800 16.000 28.200 37 60.000 30.7 52.300 25.700 36.1 29.700 12.200 28.600 34.3 31.600 28.400 30.6 46.000 14.000 33.1 29.600 11.105 28.7 29.3 24.500 7.400 21.300 6.800 28.5 24.300 5.700 7.6 20.000 7.900 58.000 14.000 43 29.000 27 17.000 28.002 24 48.000 49.0013 23.000 36.2 21.800 27 12.200 13.200 15.800 21.8 26.910 16.59 32.400 15.600 23.1 9.900 6.000 15.500 21.8 12.500 14.400 14.7 22.400 4.500 20.7 9.800 6.200 14 15 8.500 4.500 13.900 0.000 15.9 6.500 3.700 4.1 14.100 0.000 2.092 0.864 1.0229 0.783 1.481 0.830 0.784 2.017 1.185 1.4227 0.988 1.504 1.611 1.248 1.916 1.299 1.184 0.871 0.891 1.314 1.892 0.880 1.294 1.045 0.847 1.0261 0.635 1.446 0.992 0.307 =60 years old Odds Freq PD ratio 1.060 0.385 0.8583 1.017 1.034 0.462 1.159 1.045 1.013 1.1659 1.250 0.639 1.069 1.177 1.100 1.019 1.127 1.081 0.910 1.241 1.095 0.943 1.134 1.005 0.676 1.1482 0.898 1.209 0.950 0.521 CI 3.570 2.490 3.9307 3.527 3.377 2.393 3.540 2.976 3.873 3.369 3.106 2.279 2.820 3.078 2.843 3.793 2.849 2.583 2.352 2.732 2.720 2.945 2.742 2.143 1.453 2.3541 2.693 2.534 1.896 2.074 Data for the alleles of the SNPs are shown. Only relevant haplotypes that have significant CI values in either Tables 2, or are shown Data for the alleles of the SNP are shown. Only relevant haplotypes that have significant CI values in either Tables 2, or are shown. 0.010 A G P-value allele/ haplotype SNP/Haplotype Table 4. Effect of Age-of-Onset in the association of SNPs / Haplotypes in the MDR1 gene with Parkinsons’ Disease Table 7.4 Effect of Age-of-Onset in the association of SNPs / Haplotypes in the MDR1 gene with Parkinson’s Disease. and e12/1236(C/T) were not individual risk factors, the haplotype i1/-41A-e12/1236C (OR 1.470 (1.005 to 2.143)) was significantly associated with increased risk of late onset Parkinson’s disease (Table 7.4). Overall, the results from Table 7.4 suggest that SNP i1/-41(A/G) may be associated with decreased risk for developing Parkinson’s disease at or before the age of 55, while SNPs e21/2677(G/T/A) and e26/3435(C/T) and haplotypes containing these SNPs are associated with later onset disease (>60 years). 7.4 Discussion Environmental xenobiotics have been implicated in the development of Parkinson’s disease, a complex genetically heterogeneous disorder (1, 22, 23). The blood–brain barrier plays an important role in regulating the traffic of environmental xenobiotics in the brain, and individual differences in the ‘‘quality’’ of this barrier may influence the susceptibility to Parkinson’s disease. The MDR1 multidrug transporter represents an important component of the blood–brain barrier and has been shown to regulate the uptake of drugs and xenobiotics into this sensitive organ (25, 26, 44). It is conceivable that polymorphisms which alter the expression levels or transport ability of this transporter could result in altered susceptibility to neurotoxic substances and thus alter the genetic threshold for the development of Parkinson’s disease. Two recent case–control studies have examined the role of MDR1 gene polymorphisms (SNPs e1/-129(T/C), e21/2677(G/T/A), and e26/3435(C/T)) in Parkinson’s disease development. The studies involved approximately 100 white Italian and Polish patients and 100 controls from the same geographical regions (34, 35). No significant associations between these SNPs and Parkinson’s disease were 176 detected. However, our present study of 206 Chinese patients and 224 controls showed that three SNPs—e12/1236(C/T) (p=0.0367), e21/2677(G/T/A) (p=0.00067), and e26/3435(C/T) (p=0.00074), all in tight linkage disequilibrium with each other— are significantly associated with an altered risk of developing Parkinson’s disease (Table 7.2). The odds ratios of the haplotypes that were associated with Parkinson’s disease were not very high. These observations are, however, consistent with the widely held view that Parkinson’s disease is a complex disorder involving the interaction of multiple genes with different environmental factors, whereby the individual contribution of each causative gene may not be large. We recently found strong evidence of positive selection for the e21/2677T and e26/3435T alleles in the Chinese, but only marginal evidence for this in white Americans (45). The Chinese samples in that study were from anonymized umbilical cord blood from Chinese neonates, and allele frequencies of the seven SNPs were found to be very similar to those in the present study. When we used DNA samples from cord blood as controls and compared it against DNA samples from individuals of Parkinson’s, we obtained a similar, statistically significant association between Parkinson’s disease and these two SNPs (data not shown). The strong evidence of a recent positive selection for the T alleles of these two SNPs supports our current observation that these alleles are significantly underrepresented in patients with Parkinson’s disease compared with unaffected controls, suggesting that the T alleles of these SNPs may confer better protection for the brain against xenobiotic insults in the Chinese population. It is possible that the earlier Italian and Polish association studies did not detect a significant statistical association because of their limited sample size. There may be another reason why neither study was able to detect a significant association 177 between any MDR1 SNPs and Parkinson’s disease. If we assume that the Italian and Polish subjects (34, 35) were genetically similar to white Americans, their MDR1 haplotype and LD profiles may not favor the detection of associations. Our observation of only marginal evidence of recent positive selection in white Americans compared with the Chinese supports this hypothesis. Nonetheless, it remains to be determined whether the white Italians and Poles are in fact similar to white Americans in their underlying genetic architecture at this locus. It is possible that either SNP e21/2677(G/T/A) or e26/3435(C/T) could be potential causal SNPs as they had much lower p values than SNP e12/1236(C/T). Consistent with our observation that individuals carrying the G allele at the nonsynonymous SNP e21/2677(G/T/A) have a higher risk of developing Parkinson’s disease, the MDR1 transporter carrying the e21/2677G allele—coding for Ala at amino acid position 893—has been shown to be a less effective transporter than one carrying the T allele (Ser 893) (27). The synonymous SNP e26/3435(C/T) appears to be associated with altered MDR1 transporter expression and function. While several reports found that the T allele is associated with lower MDR1 expression (29, 30, 33, 46), resulting in lower efflux or higher plasma levels of drugs and xenobiotics (29, 30), others have reported lower drug plasma concentration in individuals carrying the T allele (27, 31, 33). Most of these studies examined only SNP e26/3435(C/T) without taking into account the underlying haplotype and linkage disequilibrium architecture of the study population. Detailed characterization of the genetic and evolutionary history of the entire MDR1 gene in each study population, and the influence of recent events in the history of each population on linkage disequilibrium and the likelihood of detecting an association, could resolve these conflicting reports. Our data showing an association between e26/3435T and a lower risk of developing Parkinson’s disease 178 support observations that the T allele alters MDR1 function, resulting in a greater efflux of drugs or xenobiotics. Although SNP e26/3435(C/T) is a synonymous SNP and does not result in an amino acid change, there are several possible explanations for this observation. The observed correlation with e26/3435T could reflect either differential codon usage of the C or T allele at the wobble position of the isoleucine codon, or allele specific differences in RNA folding (47), sometimes influencing RNA processing (48) or splicing (49, 50), or differences in translation control (51) and regulation (52). It is also possible that neither SNP e21/2677(G/T/A) nor e26/3435(C/T) represents the causal SNP, but that they are merely in strong linkage disequilibrium with an unobserved causal SNP. A strong association of these two SNPs with Parkinson’s disease could suggest that the unknown linked causal variant resides within a region defined by strong LD. An interesting observation was made when male and female patients with Parkinson’s disease were investigated independently—the MDR1 gene appears to play a more important role in determining risk of developing the disease in men than in women (Table 7.3). This is consistent with the view that the MDR1 transporter regulates the accumulation of neurotoxic xenobiotics in the brain to modulate the risk of developing Parkinson’s disease. As older women in urban Singapore are primarily home makers while men often work out of doors, it is conceivable that the observed greater risk for Parkinson’s disease in men compared with women is related to increased exposure to environmental susceptibility factors among men, given the same genetic risk factors in the two sexes. When patients with Parkinson’s disease were compared on the basis of their age at disease onset, we found that several polymorphisms in the MDR1 gene seemed to play a greater role in later onset disease (>60 years) (Table 7.4). One hypothesis is 179 that, in individuals with particular MDR1 genotypes (for example, e12/1236C, e21/2677G, e26/3435C) and haplotypes, the blood–brain barrier allows neurotoxic xenobiotics easier access and gradual accumulation in the brain, eventually leading to Parkinson’s disease. Conversely, individuals with the alternative alleles (that is, e12/1236T, e21/2677T and e26/3435T) are better protected from xenobiotic insults and hence from Parkinson’s disease. In contrast, early onset Parkinson’s disease is probably a result of other genetic factors and hence is less dependent on genetic variation at the MDR1 locus. The promoter SNP i1/-41(A/G), which resides in a putative CCAAT box, was found to influence the risk of Parkinson’s disease in patients with a younger age of onset (p=0.01) (Table 7.4). The G allele of this SNP appeared to protect individuals from Parkinson’s disease (OR 0.307 (95% CI, 0.125 to 0.758)). This observation, however, should be interpreted cautiously, given the low frequency (60 years). The wide variations in allele frequencies of the MDR1 SNPs (especially SNP e12/1236(C/T), e21/2677(G/T/A), and e26/3435(C/T)) among different ethnic populations may account for the differences in the ability to detect an association between MDR1 and Parkinson’s disease in other ethnic groups, especially if the increase in relative risk is small. 180 7.6 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Langston, J.W. (1998) Epidemiology versus genetics in Parkinson's disease: progress in resolving an age-old debate. Ann Neurol, 44, S45-52. Mouradian, M.M. (2002) Recent advances in the genetics and pathogenesis of Parkinson disease. Neurology, 58, 179-85. Bonifati, V., Rizzu, P., van Baren, M.J., Schaap, O., Breedveld, G.J., Krieger, E., Dekker, M.C., Squitieri, F., Ibanez, P., Joosse, M. et al. 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To establish a comprehensive view of how genetic variability affects drug response, genes of important drug response modifiers should be identified, and detailed genetic profiles of candidate genes should be investigated and compared between populations. This thesis describes a series of studies in candidate drug response genes (or regions) directed towards these goals. Important observations from this study includes the comprehensive genetic profiling of several drug-response related genes around the locus Chr. 7q21.1, as well as the demonstration of several recent computational approaches that were successfully applied to identify important loci. Linkage disequilibrium and haplotype analyses were initially conducted at the ABCB1 gene locus in three major Asian populations: Chinese, Indian and Malay (Chapter 3). Previous studies reported significant associations of several coding SNPs, especially C3435T and G2677A/T, to the ABCB1 functions, but controversial observations were made. In this study, we reported strong pairwise LD between SNPs for all the three populations in this gene. Expectation Maximization approach was used to estimate the haplotype frequencies on three coding SNPs, and substantial differences in haplotype frequency were observed between different populations. The 185 strong LD suggests the real causative locus might lie further away from C3435T and G2677A/T. Given the genes that determine one’s response to various environmental agents, i.e. toxins/xenobiotics may have experienced selective pressures during the early human migration, we propose that signatures of natural selection might be evident in these important drug response genes. We tested this hypothesis in the ABCB1 gene and extended the genetic characterization of this gene in search of further candidate functional loci (Chapter 4). We first conducted genetic profiling on 12 SNPs in the ABCB1 gene in global populations, namely Chinese, Malay, Indian, Caucasian and African American. SNP allele frequencies as well as EM estimated haplotype frequencies revealed much higher differences in genetic diversity between the African group and the non-African groups, than within the four non-African populations. Using a modified Long Range Haplotype (LRH) test, we detected significant evidence of positive selection on the T alleles of the two SNPs ABCB1C3435T and G2677A/T in Chinese and Malays. The Indians and Caucasians demonstrated similar patterns of positive selection; however, they failed to show statistical significance in the LRH test. The African American population however showed evidence of positive selection of the alternative 3435C at the ABCB1 gene locus. The evidences of natural selection found on C3435T and G2677A/T are consistent with the strong associations of these two SNPs with differences in function reported. We extended the study in Chapter 3, where we examined both the ABCB1 gene and a neighboring drug transporter gene ABCB4 with greater number of high frequency SNP markers, which together span ~300 kb, to achieve better power of the LRH test (Chapter 5). We detected significant evidence of positive selection of 186 various SNPs (including C3435T and G2677A/T) in the non-African groups which reside in the same high frequency haplotype that extend for over 150 kb as well as the African-American population of the alternative allele residing in a different haplotype. This suggests that the four non-African populations may have been subjected to common selective pressures. Strikingly, a SNP within the ABCB4 gene (e6/160) exhibited stronger and more consistent evidence of natural selection, raising the possibility that the causative SNP may reside at the ABCB4 rather than the ABCB1 gene. The CYP3A cluster consists of several important CYP3A drug metabolizing genes, located 12 Mb downstream from ABCB1. Several SNPs in these CYP3A genes have been strongly associated with altered CYP3A gene functions. In particular, one intronic variant of CYP3A5 (CYP3A5*3 G), was reported to cause total interruption of CYP3A5 expression through alternative splicing. The much higher population frequency of this variant in Caucasians than in Africans explained the lower prevalence of CYP3A5 expression in Caucasians than in Africans. However, the genetic profiles of these CYP genes have not been fully investigated. As described in Chapter 5, we characterized the genetic profile of this 300kb genome region based on genotyping of 24 SNPs in populations, namely Chinese, Indian, Malay, Caucasian and African American. Three recently developed tests of positive selection, namely the Fst and Pexcess based tests and Long Range Haplotype (LRH) approach, were also applied. These tests revealed strong evidence of natural selection in Caucasians, consistent with the previous report that the functional variant (CYP3A5*3 G) is highly prevalent in Caucasians than in Africans. Our data suggest that other regions of the CYP3A cluster, in addition to the CYP3A5 gene were also under selection pressures. 187 We further explored the relationship between the ABCB1 gene and Parkinson’s disease. Parkinson’s disease has been linked to exposure to neurotoxic xenobiotics. The susceptibility of this disease may therefore be modified by polymorphisms of important drug-response determinants such as ABCB1. In Chapter 7, we tested the associations between the susceptibility of Parkinson’s disease and polymorphisms in the ABCB1 gene in ethnic Chinese. We detected significant association between the three coding SNPs C1236T, G2677A/T and C3435T, as well as several haplotypes defined by them and Parkinson’s disease. The T alleles of the three SNPs seem to predict lower risks of Parkinson’s disease in Chinese. Factors such as gender and age of onset were found to be important modifiers, with gender or later age of onset contributing more to the correlations between differential disease risks and genetic polymorphisms. The studies described in this thesis are among the first efforts to comprehensively clarify the profiles of genetic variations in drug-response related genes and regions in different populations. Before this, polymorphisms in drug response related genes and their relationships to functions were mainly studied in isolated ways, and the complex relationship between each other were not taken into consideration. Our studies established a workflow for the comprehensive genetic survey of candidate drug response genes, taking into account the interacting relationship of polymorphisms by the analyses of LD and haplotype. Significantly, our genetic profiling of the genes in this study has confirmed the strong association between the SNPs and functional differences and demonstrated that functional association doesn’t necessarily indicate a causative role. We also reported substantial differences in the genetic profiles of these drug response related genes between different ethnic groups. This highlights the importance of taking into consideration the 188 genetic backgrounds in association studies. Furthermore, we detected significant evidences of positive selection in several important drug transport genes, with differential patterns in different populations. Our findings are among the first to demonstrate that the genes controlling drug responses and xenobiotics defense could have had important impacts on human evolution process. The approaches for detecting signatures of natural selection, evaluated and improved in our studies should also benefit the search of candidate genes harboring important functional genetic variants. 189 [...]... examined in this thesis, the MDR1, MDR3 genes and the CYP3A cluster are introduced Previous genetic and pharmacogenetics studies on these candidate genes are also reviewed in this section Finally, the objectives and significance of this study will be given in the last section 1.1 SNP profiling for candidate genes (regions) – the practical Pharmacogenetics approach 1.1.1 Introduction to SNP profiling and. .. (MDR3) gene and the CYP3A gene cluster, based mainly on the approach of Single Nucleotide Polymorphisms (SNP) profiling The introduction is given in three sections The first section is a brief review of the importance and the problems of SNP profiling approaches in the current Pharmacogenetics studies Thereafter, the two major drug-response related members of the gene super-families, ABC and CYP, and their... medicines/dosages tested on one population do not directly apply to other populations Fortunately, the practice of medicine is seeing a great change at the dawn of the new century, as a result of the recent surges of genetic studies With the accomplishment of the Human Genome Project and the rapid development of genetic assays and technologies, geneticists are now seeing the possibility of identifying the. .. Reviews in more details about functional and genetic studies on ABCB1 are presented in later sections However, before the studies presented in this thesis, there were few systematic genetic profiling studies in this gene, which are critical in directing the identification of the actual causative variants Furthermore, there is a lack of thorough comparisons of the genetic profiles among 20 the global... determine the variability in drug responses Association study is the most common strategy for mapping the candidate functional variants The principle of genetic association study is to detect the statistical association between genetic variations/mutations and the variances in the traits -of- interest, such as the differences in individual’s predisposition to certain diseases and the response to certain... Pgp limited the bioavailability of various drugs by secreting them back to the gastrointestinal tracts, after either oral or intravenous administration (103) Several inhibitors of Pgp on the other hand, increased the bioavailability of oral drugs (104, 105) Recently much attention has been focused on genetic polymorphisms at the MDR1 locus, since any functional genetic variants in this gene may act... limited in identifying the causal variants as well as assessing the validity of associations with the candidate loci As the importance of LD and haplotype profiling becomes more apparent, there is an increasing number of candidate gene studies that carried out detailed LD and haplotype analyses (33, 34, 42, 57, 58) Besides the tests based on single SNPs, novel association tests using multiple -SNPs and. .. obtain optimal treatment for each individual; and target the specific gene that causes adverse responses or diseases This thesis aims to shed some light on the understanding of the genetics of drug-response genes In this thesis, I will present several genetics studies carried out on the Multidrug Resistant 1 (MDR1, ABCB1) gene and a few others closely located 1 drug-response-related genes, namely the. .. play crucial roles in determining drug responses, and are attracting increasing attentions in the past few years Most of the identified drug transporters so far belong to the ATP binding 18 cassette transporters (ABC) super-family, the largest transmembrane (TM) protein family in the genome The name of this gene superfamily comes from these transporters’ characteristic ATP binding domain(s) A typical... prevalence of 4 SNPs among all forms of polymorphisms also suggests it may be responsible for the majority of the phenotype variance (13) SNP is of special importance also to the study of Linkage Disequilibrium (LD), or the non-random association between the genetic variations, which plays pivotal role in many genetic studies including Pharmacogenetics Linkage Disequilibrium rises and decays by mutation and . 3.3.1 SNPs in the promoter and 59 UTR of the MDR1 gene …….……. 58 3.3.2 SNPs in the coding region of the MDR1 gene ………… ………… 60 3.3.3 Haplotype profile and linkage disequilibrium of SNPs in the. on the understanding of the genetics of drug-response genes. In this thesis, I will present several genetics studies carried out on the Multidrug Resistant 1 (MDR1, ABCB1) gene and a few others. members of the gene super-families, ABC and CYP, and their specific members that are examined in this thesis, the MDR1, MDR3 genes and the CYP3A cluster are introduced. Previous genetic and pharmacogenetics