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Genetic Variations of NR1I3 and NR2B1 in Asian Populations
Drug Metab. Pharmacokinet. 28 (2): 169–176 (2013).
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
SNP Communication Genetic Variations of NR1I3 and NR2B1 in Asian Populations Sin Chi C HEW 1, 2, Joanne Siok Liu L IM 1, 2, Onkar S INGH 1 , Mabel W ONG 3 , Edmund JD L EE 2 and Balram C HOWBAY 1,4, * 1
Laboratory of Clinical Pharmacology, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore 2 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore 3 Department of Medical Oncology, National Cancer Centre, Singapore, Singapore 4 Office of Clinical Sciences, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Several nuclear receptors are being increasingly recognized for their role as master xenosensors. Among them, CAR-RXR¡ heterodimer, as encoded by NR1I3 and NR2B1, responds to the presence of drug compounds and regulates the transcription of a wide array of genes involved in their disposition. To investigate the frequency distribution and linkage disequilibrium patterns of NR1I3 and NR2B1 genetic variations, these genes were screened in 168 healthy local Asian subjects, namely Chinese, Malays, and Indians (n = 56 subjects each). A total of 38 and 88 SNPs were identified in NR1I3 and NR2B1, respectively. Among them, there were 13 and 43 novel SNPs present at low allelic frequencies (<10%) in NR1I3 and NR2B1, respectively. Notably, the genetic variations in the NR1I3 and NR2B1 genes were mainly confined to the introns whilst the exons were highly conserved across the ethnic populations. Indians harboured distinct frequency distributions from Chinese and Malays in both genes. Based on the linkage disequilibrium patterns of both genes, a number of tag-SNPs were selected for each population (n = 813 for NR1I3; n = 1218 for NR2B1). In-silico prediction analyses revealed a number of possible functional SNPs. Our data would be valuable for future pharmacogenetic studies on the drug substrates of CAR-RXR¡ target genes. Keywords: NR1I3; NR2B1; CAR; RXR¡; pharmacogenetics; Asians; polymorphisms; tag-SNPs; in-silico prediction
one and spans across 8.5 kb. It contains one non-coding exon and eight coding exons. NR1I3 mRNA has been found to be highly expressed in the liver and kidney.2) However, a wide range of transcript sizes was identified, indicating the presence of alternatively spliced transcripts.3) There are approximately 26 splice variants of NR1I3 identified so far, and most of them demonstrate tissue-specific expression.4,5) CAR is activated by a range of endogenous ligands such as androstane and bilirubin, and exogenous ligands such as phenobarbital and phenytoin. Similar to other nuclear receptors, there is a carboxyl-terminal ligand binding domain and an amino-terminal DNA-binding domain in CAR. The ligand binding domain also mediates dimerization with RXR¡.6) NR2B1, the gene encoding RXR¡, is located in chromosome nine spanning 114 kb and contains 10 exons. Similar to CAR, it is expressed abundantly in the liver and kidney.2) The hetero-
Introduction Drug disposition is highly affected by the intrinsic activities of drug metabolism enzymes and transporters in the body. The activities of these enzymes and transporters are vital in governing the drug level and ultimately the effectiveness of the therapy. The expression of genes encoding drug metabolism enzymes and transporters is under regulatory control by nuclear receptors which act as master xenosensors.1) These receptors respond to the presence of endogenous or exogenous compounds and regulate the transcription of a wide array of genes involved in their disposition.1) Among them, constitutive androstane receptor (CAR) transactivates a wide array of disposition genes following heterodimerization with retinoid X receptor alpha (RXR¡).1) NR1I3, the gene encoding CAR, is located in chromosome
Received June 11, 2012; Accepted July 10, 2012 J-STAGE Advance Published Date: July 24, 2012, doi:10.2133/dmpk.DMPK-12-SC-060 *To whom correspondence should be addressed: Balram CHOWBAY, Ph.D., Clinical Pharmacology Lab, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore. Tel. ©65-6436-8321, Fax. ©656372-0161, E-mail: [email protected] This study was supported by grants from National Medical Research Council Singapore (NMRC/1159/2008, NMRCB1011, NMCCG10122). 169
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dimerization of these two receptors is required for the transactivation of various Phase I to III genes involved in drug disposition, such as CYP1A1/2,7) CYP2B6,8) CYP3A4,9) UGT1A110) and MRP211) as well as maintaining other housekeeping functions such as bile acids and endocrine homeostasis.12) Moreover, RXR¡ also heterodimerizes with other partners such as vitamin D receptor (VDR) to promote cell differentiation through transcription13) or peroxisome proliferator-activated receptors (PPAR), bile acid/ farnesoid receptor (FXR) and the oxysterol receptors (LXRs) to control metabolic regulation.14) A striking 240-fold inter-individual difference in the expression level of NR1I3 mRNA in humans was previously reported.15) Interestingly, this observation strongly correlated with a 278-fold variability observed in the mRNA level of CYP2B6, a CAR target gene.15) Additionally, a transcriptome study following the treatment with a CAR inducer, phenobarbital, revealed a variable gene expression pattern between Hispanics and Whites.16) These findings suggest that the existence of differential CAR expression can contribute to altered target gene induction leading to interethnic or inter-individual variability in drug disposition. In view of the role of CAR as a master regulator of xenobiotic metabolism and that of RXR¡ as its heterodimeric partner, knowledge of the pattern of genetic variability and ethnic diversity of the genes encoding CAR and RXR¡ is necessary to discern the variations in drug disposition. A total of 26 single nucleotide polymorphisms (SNPs) in NR1I3 were previously reported in the Japanese population,17) while the data for NR2B1 is available only in the NCBI dbSNP public database. Pharmacogenetic data for both genes are lacking in the local Asian population. Therefore, this study was carried out to characterize (1) the presence of NR1I3/NR2B1 SNPs and their frequencies, (2) the linkage disequilibrium (LD) pattern and (3) population-specific tag-SNPs in three local Asian populations, namely Chinese, Malays and Indians. Methods Subjects: A total of 168 healthy Asian subjects [Singapore Chinese (n = 56), Singapore Malays (n = 56), Singapore Indians (n = 56)] were screened for NR1I3 and NR2B1 SNPs. The subjects were recruited in the National University of Singapore and the National Cancer Centre Singapore. The ethnicities of all subjects were checked by verbal questioning and verified against their National Registry Identification Cards. The study was approved previously by the Institutional Review Board of the National University of Singapore and the National Cancer Centre Singapore. Informed consent was obtained from all subjects for participation in the study. Pharmacogenetic analysis of NR1I3 and NR2B1 genetic polymorphisms: The Puregene DNA isolation kit (Qiagen, Valencia, CA, USA) was used to extract genomic DNA from the peripheral blood of healthy subjects. Pharmacogenetic analysis was carried out in the National Cancer Centre Singapore. The 5A-flanking region (up to 2.5 kb upstream from the 5A untranslated region), all exons and introns, and the 3A untranslated region as well as the 3A-flanking region (up to 2 kb downstream from the 3A untranslated region) of the NR1I3 gene (NM_005122) were amplified by PCR. Similar regions were covered in NR2B1 (NM_002957), except that only the exon-intron junctions were screened due to the presence of large introns. Purification of the PCR products was carried out with Exonuclease 1 and Shrimp Alkaline Phosphatase followed
by direct sequencing using an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, CA, USA). LD and tag-SNP selection: The chi-square and Fisher’s exact tests were performed to evaluate the conformity of genotype distribution to Hardy-Weinberg equilibrium (HWE). The chisquare test was used to determine the interethnic variability of the genotype distribution. Pairwise LD among the polymorphisms was quantified by «DA« and rho square (R2) values (Haploview software, v.3.32, Daly Lab, Broad Institute, MA, USA).18) Tag-SNPs were selected by Tagster based on the following criteria: minor allelic frequency (MAF) ² 0.05 and R2 ² 0.8 (Tagster software, v.1.0, NIEHS, NC, USA).19) In-silico prediction of SNP function: FastSNP was used to evaluate the possible regulatory effects (e.g., through affecting splicing and transcription factor binding sites) of SNPs located in the 5A flanking region, 5A UTR, exons and introns.20) PolymiRTS Database 2.0 was employed to identify 3A UTR SNPs that may affect the base-pairing between miRNA and its target site.21) As 5A UTRs and exons were previously shown to harbour miRNA binding sites,22,23) these regions were further examined by miRBase.24) Results and Discussion Population genetic analysis of NR1I3 in healthy Asian populations: A total of 38 SNPs, including 13 novel ones (NCBI dbSNP database; information correct as of 15/2/2012) were detected from the complete screening of the NR1I3 genomic region in the Asian populations (Supplementary Table S1). Among all the variants, thirteen mapped to the 5A flanking region, twenty-four were intronic SNPs and one was a synonymous exonic SNP. All SNPs conformed to HWE. About one-third of the SNPs (n = 13) had genotype distributions that were significantly different between at least two populations, with Indians harbouring markedly distinct allelic and genotype distributions from Chinese and Malays (Supplementary Table S1). Five variants in this study overlapped with those reported in the Japanese population previously [IVS2¹131C>A (rs6686001), IVS3¹99C>T (rs2502815), 540C>T (Pro180Pro; rs2307424), IVS8+17A>C (rs2307418), IVS8+18G>A].17) Caucasian data were obtained from the HapMap database for comparison purposes.25) The allelic frequencies of IVS2¹131C>A (rs6686001) and 540C>T (Pro180Pro; rs2307424) were comparable among the local Asian populations, Japanese and Caucasians. The frequencies of IVS8+17A>C (rs2307418) in the local Chinese (0.020) and Japanese (0.069) populations were lower than the Malays, Indians and Caucasians (0.12–0.16), whilst the frequency of IVS8+18G>A in the Japanese (0.004) was lower compared to the local populations (0.01–0.04; Supplementary Table S1). The allelic frequency of IVS3¹99C>T (rs2502815) in Indians (0.23) was significantly lower compared with Chinese and Malays (0.40– 0.46; p ¯ 0.02), while Caucasians had the same allelic frequency as Indians. This variant was previously reported to be present at the allelic frequency of 0.40 in 548 healthy Japanese postmenopausal women. In the same study, this SNP was statistically associated with bone mineral density; however the functional basis is not known.26) The exonic polymorphism 540C>T (Pro180Pro; rs2307424)], which maps to the ligand binding domain,27) was previously screened in a cohort of local Asian breast cancer patients.28) The allelic frequency of the polymorphism in the patient population was similar to those of the local healthy populations
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
NR1I3 and NR2B1 Pharmacogenetics in Asians
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Fig. 1. (Color online) Linkage disequilibrium plot of NR1I3 polymorphisms in healthy (A) Chinese, (B) Malay and (C) Indian populations LD values are denoted as «DA« = 100. Flags represent tag-SNPs that were selected based on MAF ² 0.05 and R2 ² 0.8.
(0.51 versus 0.45–0.55). While this SNP was shown to correlate with worse docetaxel-induced neutropenia in interaction with NR2A1 variant Met49Val, the effects of the SNPs located in
other regions were not investigated as the study limited the SNP screening to exons and splice-site junctions.28) The NR1I3 gene is known to have several alternatively spliced isoforms.4,5) Notice-
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Sin Chi CHEW, et al. Table 1. In-silico functional predictions of NR1I3 polymorphisms Possible functional effects SNP
rsID
FastSNPa
Region
¹3640_¹3639insG
NA
5A flanking
¹3492A>G
NA
5A flanking
¹3486G>A
rs2502805
5A flanking
¹2809C>T
rs79769623
5A flanking
¹2808G>A
NA
5A flanking
¹1184_¹1183insT
NA
5A flanking
¹1022T>C
rs75114882
5A flanking
¹350T>A
rs75090438
5A flanking
¹166_¹165ins
rs71701871
5A flanking
IVS2¹131C>A
rs6686001
Intron 2
Function Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Intronic enhancer
IVS3+457C>T IVS3+1099G>A IVS3+1211T>C
rs62001415 rs2501873 rs2501874
Intron 3 Intron 3 Intron 3
Intronic enhancer Intronic enhancer Intronic enhancer
IVS3¹1104_¹1086del
NA
Intron 3
Intronic enhancer
IVS3¹1089T>C
rs3003596
Intron 3
Intronic enhancer
540C>T (P180P; ligand binding domain) IVS5+41A>G
rs2307424
Exon 5
rs2307420
Intron 5
Sense/synonymous; Splicing regulation Intronic enhancer
IVS5+573G>C
rs112168438
Intron 5
Intronic enhancer
IVS8+223_+225del
rs71730831
Intron 8
Intronic with no known function
IVS8+327C>T
NA
Intron 8
Intronic enhancer
miRBaseb
PolymiRTSc
—
—
Disrupt CdxA and C/EBPb binding sites Disrupt C/EBP binding site
—
—
—
—
Create C/EBPb binding site
—
—
Disrupt GATA-1 binding site
—
—
Disrupt HFH-2 binding site
—
—
Disrupt USF binding site
—
—
Disrupt Pbx-1 and HNF-3b binding sites Disrupt SRY binding site
—
—
—
—
—
—
— — —
— — —
—
—
—
—
No function
—
—
—
—
—
—
Disrupt hsa-miR-30a-3p binding site and create hsa-miR-506-5p binding site in transcript variant 10 (NM_001077479) —
Description Disrupt TATA binding site
Create deltaE and C/EBP binding sites Create Lyf-1 binding site Create c-Ets binding site Create AML-1a, CRE-BP and CREB binding sites Disrupt CP2 and SRY binding sites Disrupt CP2 and SRY binding sites Affect SRp55 splicing factor binding Disrupt Nkx-2 and CdxA binding sites Disrupt deltaE binding site —
Create CdxA binding site
—
a
FastSNP prediction was restricted to SNPs located in 5A flanking region, 5A UTR, exons and introns. miRBase blast was restricted to SNPs located in exon, 5A UTR and 3A UTR. PolymiRTS database contains only information on 3A UTR SNPs in selected transcripts. — indicates that the SNP was not tested using the respective bioinformatics tool as described in footnotes a, b and c. No function indicates that no known function of the SNP was predicted by the bioinformatics tool. b c
ably, the intronic regions of CAR are rather polymorphic, in comparison to the well-conserved exonic regions, possibly suggesting the susceptibility of these genes to differential splicing effects. Among the four rare non-synonymous exonic variants [398T>G (Val133Gly), 737A>G (His246Arg), 923T>C (Leu308Pro) and 968A>G (Asn323Ser)] that mapped to ligand-binding domain of CAR, 737A>G (His246Arg) and 923T>C (Leu308Pro) led to significantly diminished transactivation activities of CAR.29) These variants were absent in our populations and present at extremely low frequencies in the Japanese (¯0.0030).29) A total of 19, 21 and 20 SNPs with MAF²0.05 were included in the LD analysis in Chinese, Malays and Indians, respectively, of which the following pairs of SNPs were in perfect linkage («DA« and R2 = 1): IVS3+1099G>A (rs2501873) with IVS3+1211T>C (rs2501874) in Chinese and Malays; and IVS3+1211T>C (rs2501874) with IVS3¹1089T>C (rs3003596) in Indians (Fig. 1). There were 8, 13 and 11 tag-SNPs selected in Chinese,
Malays and Indians, respectively and seven tag-SNPs [¹350T>A (rs75090438), ¹1022T>C (rs75114882), IVS3+587A>G (rs7414014), IVS3+1099G>A (rs2501873), IVS3¹499T>C (rs28738963), IVS5¹310C>T (rs9725457) and IVS8+116G>T (rs4073054)] were common across all three populations (Fig. 1). In-silico analysis (Table 1) revealed that the exonic SNP [540C>T (Pro180Pro; rs2307424)] may affect splicing regulation by altering the binding of splicing factor SRp55. Additionally, nine SNPs mapped to the 5A flanking region were predicted to alter promoter regulatory activity, and nine intronic SNPs may potentially affect intronic enhancer regions. The SNP IVS8+223_+225del (rs71730831) was predicted to alter the miRNA binding site in certain NR1I3 transcript variants. Population genetic analysis of NR2B1 in healthy Asian populations: Eighty-eight polymorphisms were identified in NR2B1. The majority of them were intronic SNPs (n = 45), nine were located in the 5A flanking region, and thirty-two were in
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Fig. 2. (Color online) Linkage disequilibrium plot of NR2B1 polymorphisms in healthy (A) Chinese, (B) Malay and (C) Indian populations LD values are denoted as «DA« = 100. Flags represent tag-SNPs that were selected based on MAF ² 0.05 and R2 ² 0.8.
3A UTR and flanking region (Supplementary Table S2). Similar to NR1I3, the exonic region of NR2B1 was well-conserved with only two exonic SNPs identified: 105G>A (Pro35Pro) which possibly
maps to the AF-1 region and 782C>T (Pro261Leu; rs2234960) located in the ligand binding domain.30) Only one heterozygous carrier was identified in the Indian population for each of
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Sin Chi CHEW, et al. Table 2. In-silico functional predictions of NR2B1 polymorphisms Possible functional effects SNP
rsID
FastSNPa
Region
miRBaseb
PolymiRTSc
—
—
Disrupt NF-E2 binding site
—
—
Disrupt Sox-5 binding site
—
—
— — Disrupt hsa-miR4763-3p binding site and create hsa-miR5088 binding site — — —
— — —
Function Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Intronic enhancer Intronic enhancer Exonic enhancer region
Description Disrupt Ik-2, Lyf-1 and Ik-1 binding sites Disrupt Oct-1 and USF binding sites Disrupt MZF1 binding site
Disrupt MZF-1 binding site Create v-Myb binding site Disrupt Ik-2 and c-Rel binding sites — Create GATA-2, YY1 and GATA-1 binding sites Create AML-1a binding site — Affect the binding of No miRNA SF2/ASF splicing factor binding site Create SRY binding site — Disrupt CP2 binding site — Disrupt p300 binding site — Create GR binding site — Disrupt Th1/E4 binding site — Create GATA-2 binding site — — Disrupt Tax/CR and MZF1 binding sites Create AML-1a binding site — Disrupt GATA-2 binding site — — No miRNA binding site
¹2650G>A
rs11185647
5A flanking
¹2637T>A
NA
5A flanking
¹2613C>T
NA
5A flanking
¹2147C>G
NA
5A flanking
¹1741G>A
rs62576288
5A flanking
IVS1+171C>T IVS1¹169C>T 105G>A (Pro35Pro; AF-1)
NA NA NA
Intron 1 Intron 1 Exon 2
IVS2+33G>A IVS2+57T>C IVS2¹46C>A
rs2234753 NA rs1805352
Intron 2 Intron 2 Intron 2
Intronic enhancer Intronic enhancer Intronic enhancer
IVS5+288C>T
rs7861779
Intron 5
Intronic enhancer
Create CDP binding site Create p300 binding site Create GATA-1 and GATA-2 binding sites
IVS5¹115G>T rs2234754 rs2234960 782C>T (P261; ligand binding domain) IVS6¹257T>G rs34502422 IVS7¹126G>A NA IVS8+106A>G rs35443779 IVS8+278T>C rs3132294 IVS8+308C>T NA IVS8¹126T>C rs3132293 IVS9+288C>G rs34703525
Intron 5 Exon 6
Intronic enhancer splicing site
Intron Intron Intron Intron Intron Intron Intron
Intronic Intronic Intronic Intronic Intronic Intronic Intronic
IVS9¹97G>A IVS9¹92C>G *294C>G
rs1805347 NA rs35438206
Intron 9 Intron 9 3A UTR
*846G>A
rs4240711
3A UTR
—
—
No miRNA binding site
*1848C>T
rs180869938 3A UTR
—
—
No miRNA binding site
*2100C>T
rs4842194
3A UTR
—
—
No miRNA binding site
*3649C>T
rs34109509
3A UTR
—
—
Disrupt hsa-miR4745-5p binding site
6 7 8 8 8 8 9
enhancer enhancer enhancer enhancer enhancer enhancer enhancer
Intronic enhancer Intronic enhancer —
— — — — — — — — — — — — — — — Disrupt hsa-miR-3425p, hsa-miR-4455, hsa-miR4651, hsa-miR-4664-5p and hsa-miR-608 binding sites and create hsa-miR4746-3p binding site Disrupt hsa-miR-3620 binding site and create hsamiR-3184-3p binding site Disrupt hsa-miR-4706 and hsa-miR-4749-5p binding sites Disrupt hsa-miR-298 and hsa-miR-3158-5p binding sites and create hsa-miR-548 binding site —
a
FastSNP prediction was restricted to SNPs located in 5A flanking region, 5A UTR, exons and introns. miRBase blast was restricted to SNPs located in exon, 5A UTR and 3A UTR. c PolymiRTS database contains only information on 3A UTR SNPs in selected transcripts. — indicates that the SNP was not tested using the respective bioinformatics tool as described in footnotes a, b and c. b
them. A total of 43 novel polymorphisms were identified (NCBI dbSNP database; information correct as of 22/2/2012) with allelic frequencies ranging from 1–4% (Supplementary Table S2). All SNPs conformed to HWE. Significant interethnic variations in the genotype distributions were detected in 25% of all identified polymorphisms. Similar to NR1I3, the pattern of genetic variability in Indians differed distinctly from those in Chinese and Malays
(Supplementary Table S2). Twenty-five SNPs with MAF ² 0.05 were included in the LD analysis in Chinese and Indians, while there were only twenty-three for Malays (Fig. 2). The following pairs of SNPs were in perfect linkage in Malays («DA« and R2 = 1): IVS9¹138T>C (rs6413514) with IVS9¹97G>A (rs1805347); IVS9¹27G>A (rs1805343) with *846G>A (rs4240711) and *+4768C>A (rs4842196) with
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
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NR1I3 and NR2B1 Pharmacogenetics in Asians
*+4988A>G (rs4842198), but not in Chinese or Indians (Fig. 2). The number of tag-SNPs required to represent the genetic diversity of NR2B1 in Chinese, Malays and Indians was 12, 17, and 18, respectively, of which 5 tag-SNPs [¹2650G>A (rs11185647), ¹1741G>A (rs62576288), IVS2+33G>A (rs2234753), IVS2¹46C>A (rs1805352) and IVS8¹126T>C (rs3132293)] were common across all three populations. By virtue of its role as a heterodimerization partner of other regulatory receptors involved in cell proliferation, cholesterol metabolism or insulin sensitivity as mentioned above, the effects of NR2B1 polymorphisms have mostly been investigated in relation to disease susceptibilities such as Alzheimer’s disease, diabetes and various cancers to date.31–34) For instance, IVS2¹46C>A (rs1805352) was previously identified as one of the markers within the 6-SNP haplotype significantly associated with colon cancer.33) The remaining 5 SNPs were deep intronic SNPs which were not covered in the present study. Additionally, IVS8¹126T>C (rs3132293), when being in haplotypic linkage with IVS6¹99G>T (rs3118570) and IVS7+70A>G (rs1536475), was previously shown to correlate with Alzheimer’s disease.32) These three SNPs were also moderately linked in our three local populations, with pairwise «DA« values ranging from 0.62–0.96. In-silico analysis revealed the exonic polymorphism 782C>T (Pro261Leu; rs2234960) to be located at an exonic splicing site, while the novel polymorphism 105G>A (Pro35Pro) may affect transcription factor and/or miRNA binding sites. Five SNPs located in the 5A flanking region were predicted to exert altered regulatory function and sixteen intronic SNPs may be located within an intronic enhancer. Additionally, five 3A UTR SNPs may disrupt or create miRNA binding sites (Table 2). CAR/RXR¡ heterodimer is a master regulator of xenobiotic metabolism, in addition to governing various physiological processes. The collinearity between the mRNA levels of CAR and its target genes suggests that the variability in the expression level of the regulatory genes can possibly account for the altered induction and activity of the target genes.35) Nonetheless, the potential of the genetic variations in these regulatory genes to affect target gene expression and drug disposition has not been investigated extensively. It is also to be noted that the genetic polymorphisms in the NR1I3 and NR2B1 genes were confined to mostly the introns whilst the exons were highly conserved across the ethnic populations. Thus far, the studies on the effects of SNPs in these genes were mostly limited to disease susceptibility. The common cis-acting genetic variations in the Phase I to III drug disposition genes have often failed to account for the interindividual differences in drug pharmacokinetics and pharmacodynamics.36,37) Hence, exploration of the genes encoding orphan nuclear receptors that regulate the expression of downstream genes involved in drug disposition may provide useful insights into the mechanistic basis of inter-individual variability in drug pharmacokinetics and pharmacodynamics.38) Although several SNPs in our study were predicted to be functional, their biological relevance requires further experimental evaluation. In conclusion, our study provides a comprehensive overview of genetic variations present in NR1I3 and NR2B1 in the local Asian populations. The selection of tag-SNPs based on the LD pattern provides an economical means of representing the genetic variability of the genes without the loss of power for future studies. As these receptors are the focal components in xenobiotic detoxification, as well as in many developmental and metabolic pathways,
our data would be valuable for future pharmacogenetic or disease association studies. References 1) 2) 3)
4) 5)
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9) 10)
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12) 13)
14) 15)
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Xu, C., Li, C. and Kong, A.: Induction of phase I, II and III drug metabolism/transport by xenobiotics. Arch. Pharm. Res., 28: 249–268 (2005). Nishimura, M., Naito, S. and Yokoi, T.: Tissue-specific mRNA expression profiles of human nuclear receptor subfamilies. Drug Metab. Pharmacokinet., 19: 135–149 (2004). Baes, M., Gulick, T., Choi, H. S., Martinoli, M. G., Simha, D. and Moore, D. D.: A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Mol. Cell. Biol., 14: 1544–1552 (1994). Arnold, K. A., Eichelbaum, M. and Burk, O.: Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor. Nucl. Recept., 2: 1–16 (2004). Lamba, J. K., Lamba, V., Yasuda, K., Lin, Y. S., Assem, M., Thompson, E., Strom, S. and Schuetz, E.: Expression of constitutive androstane receptor splice variants in human tissues and their functional consequences. J. Pharmacol. Exp. Ther., 311: 811–821 (2004). Suino, K., Peng, L., Reynolds, R., Li, Y., Cha, J. Y., Repa, J. J., Kliewer, S. A. and Xu, H.: The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization. Mol. Cell, 16: 893–905 (2004). Yoshinari, K., Yoda, N., Toriyabe, T. and Yamazoe, Y.: Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5A-flanking region. Biochem. Pharmacol., 79: 261–269 (2010). Sueyoshi, T., Kawamoto, T., Zelko, I., Honkakoski, P. and Negishi, M.: The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J. Biol. Chem., 274: 6043–6046 (1999). Goodwin, B., Hodgson, E., D’Costa, D. J., Robertson, G. R. and Liddle, C.: Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor. Mol. Pharmacol., 62: 359–365 (2002). Sugatani, J., Kojima, H., Ueda, A., Kakizaki, S., Yoshinari, K., Gong, Q., Owens, I., Negishi, M. and Sueyoshi, T.: The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology, 33: 1232–1238 (2001). Kast, H. R., Goodwin, B., Tarr, P. T., Jones, S. A., Anisfeld, A. M., Stoltz, C. M., Tontonoz, P., Kliewer, S., Willson, T. M. and Edwards, P. A.: Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem., 277: 2908–2915 (2002). Swales, K. and Negishi, M.: CAR, driving into the future. Mol. Endocrinol., 18: 1589–1598 (2004). Haussler, M. R., Haussler, C. A., Bartik, L., Whitfield, G. K., Hsieh, J. C., Slater, S. and Jurutka, P. W.: Vitamin D receptor: molecular signaling and actions of nutritional ligands in disease prevention. Nutr. Rev., 66: S98– S112 (2008). Lefebvre, P., Benomar, Y. and Staels, B.: Retinoid X receptors: common heterodimerization partners with distinct functions. Trends Endocrinol. Metab., 21: 676–683 (2010). Chang, T. K., Bandiera, S. M. and Chen, J.: Constitutive androstane receptor and pregnane X receptor gene expression in human liver: interindividual variability and correlation with CYP2B6 mRNA levels. Drug Metab. Dispos., 31: 7–10 (2003). Finkelstein, D., Lamba, V., Assem, M., Rengelshausen, J., Yasuda, K., Strom, S. and Schuetz, E.: ADME transcriptome in Hispanic versus White donor livers: evidence of a globally enhanced NR1I3 (CAR, constitutive androstane receptor) gene signature in Hispanics. Xenobiotica, 36: 989–1012 (2006). Ikeda, S., Kurose, K., Ozawa, S., Sai, K., Hasegawa, R., Komamura, K., Ueno, K., Kamakura, S., Kitakaze, M., Tomoike, H., Nakajima, T., Matsumoto, K., Saito, H., Goto, Y., Kimura, H., Katoh, M., Sugai, K., Minami, N., Shirao, K., Tamura, T., Yamamoto, N., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., Saito, Y. and Sawada, J.: Twenty-six novel single nucleotide polymorphisms and their frequencies of the NR1I3 (CAR) gene in a Japanese population. Drug Metab. Pharmacokinet., 18: 413–418 (2003). Barrett, J. C., Fry, B., Maller, J. and Daly, M. J.: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics, 21: 263–265 (2005).
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Sin Chi CHEW, et al. Xu, Z., Kaplan, N. L. and Taylor, J. A.: TAGster: efficient selection of LD tag SNPs in single or multiple populations. Bioinformatics, 23: 3254– 3255 (2007). Yuan, H. Y., Chiou, J. J., Tseng, W. H., Liu, C. H., Liu, C. K., Lin, Y. J., Wang, H. H., Yao, A., Chen, Y. T. and Hsu, C. N.: FASTSNP: an always up-to-date and extendable service for SNP function analysis and prioritization. Nucleic Acids Res., 34: W635–W641 (2006). Ziebarth, J. D., Bhattacharya, A., Chen, A. and Cui, Y.: PolymiRTS Database 2.0: linking polymorphisms in microRNA target sites with human diseases and complex traits. Nucleic Acids Res., 40: D216–D221 (2012). Lytle, J. R., Yario, T. A. and Steitz, J. A.: Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5A UTR as in the 3A UTR. Proc. Natl. Acad. Sci. USA, 104: 9667–9672 (2007). Fang, Z. and Rajewsky, N.: The Impact of miRNA Target Sites in Coding Sequence and in 3A UTR. PLoS ONE, 6: e18067 (2011). Griffiths-Jones, S., Saini, H. K., van Dongen, S. and Enright, A. J.: miRBase: tools for microRNA genomics. Nucleic Acids Res., 36: D154– D158 (2008). International HapMap Consortium: The International HapMap Project. Nature, 426: 789–796 (2003). Urano, T., Usui, T., Shiraki, M., Ouchi, Y. and Inoue, S.: Association of a single nucleotide polymorphism in the constitutive androstane receptor gene with bone mineral density. Geriatr. Gerontol. Int., 9: 235–241 (2009). di Masi, A., Marinis, E. D., Ascenzi, P. and Marino, M.: Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects. Mol. Aspects Med., 30: 297–343 (2009). Hor, S. Y., Lee, S. C., Wong, C. I., Lim, Y. W., Lim, R. C., Wang, L. Z., Fan, L., Guo, J. Y., Lee, H. S., Goh, B. C. and Tan, T.: PXR, CAR and HNF4alpha genotypes and their association with pharmacokinetics and pharmacodynamics of docetaxel and doxorubicin in Asian patients. Pharmacogenomics J., 8: 139–146 (2008). Ikeda, S., Kurose, K., Jinno, H., Sai, K., Ozawa, S., Hasegawa, R., Komamura, K., Kotake, T., Morishita, H., Kamakura, S., Kitakaze, M., Tomoike, H., Tamura, T., Yamamoto, N., Kunitoh, H., Yamada, Y., Ohe, Y., Shimada, Y., Shirao, K., Kubota, K., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., Saito, Y. and Sawada, J. I.: Functional analysis of four naturally occurring variants of human constitutive androstane receptor. Mol. Genet. Metab., 86: 314–319 (2005). Mangelsdorf, D. J., Ong, E. S., Dyck, J. A. and Evans, R. M.: Nuclear receptor that identifies a novel retinoic acid response pathway. Nature,
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SNP Communication Genetic Variations of NR1I3 and NR2B1 in Asian Populations Sin Chi C HEW 1, 2, Joanne Siok Liu L IM 1, 2, Onkar S INGH 1 , Mabel W ONG 3 , Edmund JD L EE 2 and Balram C HOWBAY 1,4, * 1
Laboratory of Clinical Pharmacology, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore 2 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore 3 Department of Medical Oncology, National Cancer Centre, Singapore, Singapore 4 Office of Clinical Sciences, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Several nuclear receptors are being increasingly recognized for their role as master xenosensors. Among them, CAR-RXR¡ heterodimer, as encoded by NR1I3 and NR2B1, responds to the presence of drug compounds and regulates the transcription of a wide array of genes involved in their disposition. To investigate the frequency distribution and linkage disequilibrium patterns of NR1I3 and NR2B1 genetic variations, these genes were screened in 168 healthy local Asian subjects, namely Chinese, Malays, and Indians (n = 56 subjects each). A total of 38 and 88 SNPs were identified in NR1I3 and NR2B1, respectively. Among them, there were 13 and 43 novel SNPs present at low allelic frequencies (<10%) in NR1I3 and NR2B1, respectively. Notably, the genetic variations in the NR1I3 and NR2B1 genes were mainly confined to the introns whilst the exons were highly conserved across the ethnic populations. Indians harboured distinct frequency distributions from Chinese and Malays in both genes. Based on the linkage disequilibrium patterns of both genes, a number of tag-SNPs were selected for each population (n = 813 for NR1I3; n = 1218 for NR2B1). In-silico prediction analyses revealed a number of possible functional SNPs. Our data would be valuable for future pharmacogenetic studies on the drug substrates of CAR-RXR¡ target genes. Keywords: NR1I3; NR2B1; CAR; RXR¡; pharmacogenetics; Asians; polymorphisms; tag-SNPs; in-silico prediction
one and spans across 8.5 kb. It contains one non-coding exon and eight coding exons. NR1I3 mRNA has been found to be highly expressed in the liver and kidney.2) However, a wide range of transcript sizes was identified, indicating the presence of alternatively spliced transcripts.3) There are approximately 26 splice variants of NR1I3 identified so far, and most of them demonstrate tissue-specific expression.4,5) CAR is activated by a range of endogenous ligands such as androstane and bilirubin, and exogenous ligands such as phenobarbital and phenytoin. Similar to other nuclear receptors, there is a carboxyl-terminal ligand binding domain and an amino-terminal DNA-binding domain in CAR. The ligand binding domain also mediates dimerization with RXR¡.6) NR2B1, the gene encoding RXR¡, is located in chromosome nine spanning 114 kb and contains 10 exons. Similar to CAR, it is expressed abundantly in the liver and kidney.2) The hetero-
Introduction Drug disposition is highly affected by the intrinsic activities of drug metabolism enzymes and transporters in the body. The activities of these enzymes and transporters are vital in governing the drug level and ultimately the effectiveness of the therapy. The expression of genes encoding drug metabolism enzymes and transporters is under regulatory control by nuclear receptors which act as master xenosensors.1) These receptors respond to the presence of endogenous or exogenous compounds and regulate the transcription of a wide array of genes involved in their disposition.1) Among them, constitutive androstane receptor (CAR) transactivates a wide array of disposition genes following heterodimerization with retinoid X receptor alpha (RXR¡).1) NR1I3, the gene encoding CAR, is located in chromosome
Received June 11, 2012; Accepted July 10, 2012 J-STAGE Advance Published Date: July 24, 2012, doi:10.2133/dmpk.DMPK-12-SC-060 *To whom correspondence should be addressed: Balram CHOWBAY, Ph.D., Clinical Pharmacology Lab, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore. Tel. ©65-6436-8321, Fax. ©656372-0161, E-mail: [email protected] This study was supported by grants from National Medical Research Council Singapore (NMRC/1159/2008, NMRCB1011, NMCCG10122). 169
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dimerization of these two receptors is required for the transactivation of various Phase I to III genes involved in drug disposition, such as CYP1A1/2,7) CYP2B6,8) CYP3A4,9) UGT1A110) and MRP211) as well as maintaining other housekeeping functions such as bile acids and endocrine homeostasis.12) Moreover, RXR¡ also heterodimerizes with other partners such as vitamin D receptor (VDR) to promote cell differentiation through transcription13) or peroxisome proliferator-activated receptors (PPAR), bile acid/ farnesoid receptor (FXR) and the oxysterol receptors (LXRs) to control metabolic regulation.14) A striking 240-fold inter-individual difference in the expression level of NR1I3 mRNA in humans was previously reported.15) Interestingly, this observation strongly correlated with a 278-fold variability observed in the mRNA level of CYP2B6, a CAR target gene.15) Additionally, a transcriptome study following the treatment with a CAR inducer, phenobarbital, revealed a variable gene expression pattern between Hispanics and Whites.16) These findings suggest that the existence of differential CAR expression can contribute to altered target gene induction leading to interethnic or inter-individual variability in drug disposition. In view of the role of CAR as a master regulator of xenobiotic metabolism and that of RXR¡ as its heterodimeric partner, knowledge of the pattern of genetic variability and ethnic diversity of the genes encoding CAR and RXR¡ is necessary to discern the variations in drug disposition. A total of 26 single nucleotide polymorphisms (SNPs) in NR1I3 were previously reported in the Japanese population,17) while the data for NR2B1 is available only in the NCBI dbSNP public database. Pharmacogenetic data for both genes are lacking in the local Asian population. Therefore, this study was carried out to characterize (1) the presence of NR1I3/NR2B1 SNPs and their frequencies, (2) the linkage disequilibrium (LD) pattern and (3) population-specific tag-SNPs in three local Asian populations, namely Chinese, Malays and Indians. Methods Subjects: A total of 168 healthy Asian subjects [Singapore Chinese (n = 56), Singapore Malays (n = 56), Singapore Indians (n = 56)] were screened for NR1I3 and NR2B1 SNPs. The subjects were recruited in the National University of Singapore and the National Cancer Centre Singapore. The ethnicities of all subjects were checked by verbal questioning and verified against their National Registry Identification Cards. The study was approved previously by the Institutional Review Board of the National University of Singapore and the National Cancer Centre Singapore. Informed consent was obtained from all subjects for participation in the study. Pharmacogenetic analysis of NR1I3 and NR2B1 genetic polymorphisms: The Puregene DNA isolation kit (Qiagen, Valencia, CA, USA) was used to extract genomic DNA from the peripheral blood of healthy subjects. Pharmacogenetic analysis was carried out in the National Cancer Centre Singapore. The 5A-flanking region (up to 2.5 kb upstream from the 5A untranslated region), all exons and introns, and the 3A untranslated region as well as the 3A-flanking region (up to 2 kb downstream from the 3A untranslated region) of the NR1I3 gene (NM_005122) were amplified by PCR. Similar regions were covered in NR2B1 (NM_002957), except that only the exon-intron junctions were screened due to the presence of large introns. Purification of the PCR products was carried out with Exonuclease 1 and Shrimp Alkaline Phosphatase followed
by direct sequencing using an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, CA, USA). LD and tag-SNP selection: The chi-square and Fisher’s exact tests were performed to evaluate the conformity of genotype distribution to Hardy-Weinberg equilibrium (HWE). The chisquare test was used to determine the interethnic variability of the genotype distribution. Pairwise LD among the polymorphisms was quantified by «DA« and rho square (R2) values (Haploview software, v.3.32, Daly Lab, Broad Institute, MA, USA).18) Tag-SNPs were selected by Tagster based on the following criteria: minor allelic frequency (MAF) ² 0.05 and R2 ² 0.8 (Tagster software, v.1.0, NIEHS, NC, USA).19) In-silico prediction of SNP function: FastSNP was used to evaluate the possible regulatory effects (e.g., through affecting splicing and transcription factor binding sites) of SNPs located in the 5A flanking region, 5A UTR, exons and introns.20) PolymiRTS Database 2.0 was employed to identify 3A UTR SNPs that may affect the base-pairing between miRNA and its target site.21) As 5A UTRs and exons were previously shown to harbour miRNA binding sites,22,23) these regions were further examined by miRBase.24) Results and Discussion Population genetic analysis of NR1I3 in healthy Asian populations: A total of 38 SNPs, including 13 novel ones (NCBI dbSNP database; information correct as of 15/2/2012) were detected from the complete screening of the NR1I3 genomic region in the Asian populations (Supplementary Table S1). Among all the variants, thirteen mapped to the 5A flanking region, twenty-four were intronic SNPs and one was a synonymous exonic SNP. All SNPs conformed to HWE. About one-third of the SNPs (n = 13) had genotype distributions that were significantly different between at least two populations, with Indians harbouring markedly distinct allelic and genotype distributions from Chinese and Malays (Supplementary Table S1). Five variants in this study overlapped with those reported in the Japanese population previously [IVS2¹131C>A (rs6686001), IVS3¹99C>T (rs2502815), 540C>T (Pro180Pro; rs2307424), IVS8+17A>C (rs2307418), IVS8+18G>A].17) Caucasian data were obtained from the HapMap database for comparison purposes.25) The allelic frequencies of IVS2¹131C>A (rs6686001) and 540C>T (Pro180Pro; rs2307424) were comparable among the local Asian populations, Japanese and Caucasians. The frequencies of IVS8+17A>C (rs2307418) in the local Chinese (0.020) and Japanese (0.069) populations were lower than the Malays, Indians and Caucasians (0.12–0.16), whilst the frequency of IVS8+18G>A in the Japanese (0.004) was lower compared to the local populations (0.01–0.04; Supplementary Table S1). The allelic frequency of IVS3¹99C>T (rs2502815) in Indians (0.23) was significantly lower compared with Chinese and Malays (0.40– 0.46; p ¯ 0.02), while Caucasians had the same allelic frequency as Indians. This variant was previously reported to be present at the allelic frequency of 0.40 in 548 healthy Japanese postmenopausal women. In the same study, this SNP was statistically associated with bone mineral density; however the functional basis is not known.26) The exonic polymorphism 540C>T (Pro180Pro; rs2307424)], which maps to the ligand binding domain,27) was previously screened in a cohort of local Asian breast cancer patients.28) The allelic frequency of the polymorphism in the patient population was similar to those of the local healthy populations
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Fig. 1. (Color online) Linkage disequilibrium plot of NR1I3 polymorphisms in healthy (A) Chinese, (B) Malay and (C) Indian populations LD values are denoted as «DA« = 100. Flags represent tag-SNPs that were selected based on MAF ² 0.05 and R2 ² 0.8.
(0.51 versus 0.45–0.55). While this SNP was shown to correlate with worse docetaxel-induced neutropenia in interaction with NR2A1 variant Met49Val, the effects of the SNPs located in
other regions were not investigated as the study limited the SNP screening to exons and splice-site junctions.28) The NR1I3 gene is known to have several alternatively spliced isoforms.4,5) Notice-
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Sin Chi CHEW, et al. Table 1. In-silico functional predictions of NR1I3 polymorphisms Possible functional effects SNP
rsID
FastSNPa
Region
¹3640_¹3639insG
NA
5A flanking
¹3492A>G
NA
5A flanking
¹3486G>A
rs2502805
5A flanking
¹2809C>T
rs79769623
5A flanking
¹2808G>A
NA
5A flanking
¹1184_¹1183insT
NA
5A flanking
¹1022T>C
rs75114882
5A flanking
¹350T>A
rs75090438
5A flanking
¹166_¹165ins
rs71701871
5A flanking
IVS2¹131C>A
rs6686001
Intron 2
Function Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Intronic enhancer
IVS3+457C>T IVS3+1099G>A IVS3+1211T>C
rs62001415 rs2501873 rs2501874
Intron 3 Intron 3 Intron 3
Intronic enhancer Intronic enhancer Intronic enhancer
IVS3¹1104_¹1086del
NA
Intron 3
Intronic enhancer
IVS3¹1089T>C
rs3003596
Intron 3
Intronic enhancer
540C>T (P180P; ligand binding domain) IVS5+41A>G
rs2307424
Exon 5
rs2307420
Intron 5
Sense/synonymous; Splicing regulation Intronic enhancer
IVS5+573G>C
rs112168438
Intron 5
Intronic enhancer
IVS8+223_+225del
rs71730831
Intron 8
Intronic with no known function
IVS8+327C>T
NA
Intron 8
Intronic enhancer
miRBaseb
PolymiRTSc
—
—
Disrupt CdxA and C/EBPb binding sites Disrupt C/EBP binding site
—
—
—
—
Create C/EBPb binding site
—
—
Disrupt GATA-1 binding site
—
—
Disrupt HFH-2 binding site
—
—
Disrupt USF binding site
—
—
Disrupt Pbx-1 and HNF-3b binding sites Disrupt SRY binding site
—
—
—
—
—
—
— — —
— — —
—
—
—
—
No function
—
—
—
—
—
—
Disrupt hsa-miR-30a-3p binding site and create hsa-miR-506-5p binding site in transcript variant 10 (NM_001077479) —
Description Disrupt TATA binding site
Create deltaE and C/EBP binding sites Create Lyf-1 binding site Create c-Ets binding site Create AML-1a, CRE-BP and CREB binding sites Disrupt CP2 and SRY binding sites Disrupt CP2 and SRY binding sites Affect SRp55 splicing factor binding Disrupt Nkx-2 and CdxA binding sites Disrupt deltaE binding site —
Create CdxA binding site
—
a
FastSNP prediction was restricted to SNPs located in 5A flanking region, 5A UTR, exons and introns. miRBase blast was restricted to SNPs located in exon, 5A UTR and 3A UTR. PolymiRTS database contains only information on 3A UTR SNPs in selected transcripts. — indicates that the SNP was not tested using the respective bioinformatics tool as described in footnotes a, b and c. No function indicates that no known function of the SNP was predicted by the bioinformatics tool. b c
ably, the intronic regions of CAR are rather polymorphic, in comparison to the well-conserved exonic regions, possibly suggesting the susceptibility of these genes to differential splicing effects. Among the four rare non-synonymous exonic variants [398T>G (Val133Gly), 737A>G (His246Arg), 923T>C (Leu308Pro) and 968A>G (Asn323Ser)] that mapped to ligand-binding domain of CAR, 737A>G (His246Arg) and 923T>C (Leu308Pro) led to significantly diminished transactivation activities of CAR.29) These variants were absent in our populations and present at extremely low frequencies in the Japanese (¯0.0030).29) A total of 19, 21 and 20 SNPs with MAF²0.05 were included in the LD analysis in Chinese, Malays and Indians, respectively, of which the following pairs of SNPs were in perfect linkage («DA« and R2 = 1): IVS3+1099G>A (rs2501873) with IVS3+1211T>C (rs2501874) in Chinese and Malays; and IVS3+1211T>C (rs2501874) with IVS3¹1089T>C (rs3003596) in Indians (Fig. 1). There were 8, 13 and 11 tag-SNPs selected in Chinese,
Malays and Indians, respectively and seven tag-SNPs [¹350T>A (rs75090438), ¹1022T>C (rs75114882), IVS3+587A>G (rs7414014), IVS3+1099G>A (rs2501873), IVS3¹499T>C (rs28738963), IVS5¹310C>T (rs9725457) and IVS8+116G>T (rs4073054)] were common across all three populations (Fig. 1). In-silico analysis (Table 1) revealed that the exonic SNP [540C>T (Pro180Pro; rs2307424)] may affect splicing regulation by altering the binding of splicing factor SRp55. Additionally, nine SNPs mapped to the 5A flanking region were predicted to alter promoter regulatory activity, and nine intronic SNPs may potentially affect intronic enhancer regions. The SNP IVS8+223_+225del (rs71730831) was predicted to alter the miRNA binding site in certain NR1I3 transcript variants. Population genetic analysis of NR2B1 in healthy Asian populations: Eighty-eight polymorphisms were identified in NR2B1. The majority of them were intronic SNPs (n = 45), nine were located in the 5A flanking region, and thirty-two were in
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Fig. 2. (Color online) Linkage disequilibrium plot of NR2B1 polymorphisms in healthy (A) Chinese, (B) Malay and (C) Indian populations LD values are denoted as «DA« = 100. Flags represent tag-SNPs that were selected based on MAF ² 0.05 and R2 ² 0.8.
3A UTR and flanking region (Supplementary Table S2). Similar to NR1I3, the exonic region of NR2B1 was well-conserved with only two exonic SNPs identified: 105G>A (Pro35Pro) which possibly
maps to the AF-1 region and 782C>T (Pro261Leu; rs2234960) located in the ligand binding domain.30) Only one heterozygous carrier was identified in the Indian population for each of
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Sin Chi CHEW, et al. Table 2. In-silico functional predictions of NR2B1 polymorphisms Possible functional effects SNP
rsID
FastSNPa
Region
miRBaseb
PolymiRTSc
—
—
Disrupt NF-E2 binding site
—
—
Disrupt Sox-5 binding site
—
—
— — Disrupt hsa-miR4763-3p binding site and create hsa-miR5088 binding site — — —
— — —
Function Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Promoter/regulatory region Intronic enhancer Intronic enhancer Exonic enhancer region
Description Disrupt Ik-2, Lyf-1 and Ik-1 binding sites Disrupt Oct-1 and USF binding sites Disrupt MZF1 binding site
Disrupt MZF-1 binding site Create v-Myb binding site Disrupt Ik-2 and c-Rel binding sites — Create GATA-2, YY1 and GATA-1 binding sites Create AML-1a binding site — Affect the binding of No miRNA SF2/ASF splicing factor binding site Create SRY binding site — Disrupt CP2 binding site — Disrupt p300 binding site — Create GR binding site — Disrupt Th1/E4 binding site — Create GATA-2 binding site — — Disrupt Tax/CR and MZF1 binding sites Create AML-1a binding site — Disrupt GATA-2 binding site — — No miRNA binding site
¹2650G>A
rs11185647
5A flanking
¹2637T>A
NA
5A flanking
¹2613C>T
NA
5A flanking
¹2147C>G
NA
5A flanking
¹1741G>A
rs62576288
5A flanking
IVS1+171C>T IVS1¹169C>T 105G>A (Pro35Pro; AF-1)
NA NA NA
Intron 1 Intron 1 Exon 2
IVS2+33G>A IVS2+57T>C IVS2¹46C>A
rs2234753 NA rs1805352
Intron 2 Intron 2 Intron 2
Intronic enhancer Intronic enhancer Intronic enhancer
IVS5+288C>T
rs7861779
Intron 5
Intronic enhancer
Create CDP binding site Create p300 binding site Create GATA-1 and GATA-2 binding sites
IVS5¹115G>T rs2234754 rs2234960 782C>T (P261; ligand binding domain) IVS6¹257T>G rs34502422 IVS7¹126G>A NA IVS8+106A>G rs35443779 IVS8+278T>C rs3132294 IVS8+308C>T NA IVS8¹126T>C rs3132293 IVS9+288C>G rs34703525
Intron 5 Exon 6
Intronic enhancer splicing site
Intron Intron Intron Intron Intron Intron Intron
Intronic Intronic Intronic Intronic Intronic Intronic Intronic
IVS9¹97G>A IVS9¹92C>G *294C>G
rs1805347 NA rs35438206
Intron 9 Intron 9 3A UTR
*846G>A
rs4240711
3A UTR
—
—
No miRNA binding site
*1848C>T
rs180869938 3A UTR
—
—
No miRNA binding site
*2100C>T
rs4842194
3A UTR
—
—
No miRNA binding site
*3649C>T
rs34109509
3A UTR
—
—
Disrupt hsa-miR4745-5p binding site
6 7 8 8 8 8 9
enhancer enhancer enhancer enhancer enhancer enhancer enhancer
Intronic enhancer Intronic enhancer —
— — — — — — — — — — — — — — — Disrupt hsa-miR-3425p, hsa-miR-4455, hsa-miR4651, hsa-miR-4664-5p and hsa-miR-608 binding sites and create hsa-miR4746-3p binding site Disrupt hsa-miR-3620 binding site and create hsamiR-3184-3p binding site Disrupt hsa-miR-4706 and hsa-miR-4749-5p binding sites Disrupt hsa-miR-298 and hsa-miR-3158-5p binding sites and create hsa-miR-548 binding site —
a
FastSNP prediction was restricted to SNPs located in 5A flanking region, 5A UTR, exons and introns. miRBase blast was restricted to SNPs located in exon, 5A UTR and 3A UTR. c PolymiRTS database contains only information on 3A UTR SNPs in selected transcripts. — indicates that the SNP was not tested using the respective bioinformatics tool as described in footnotes a, b and c. b
them. A total of 43 novel polymorphisms were identified (NCBI dbSNP database; information correct as of 22/2/2012) with allelic frequencies ranging from 1–4% (Supplementary Table S2). All SNPs conformed to HWE. Significant interethnic variations in the genotype distributions were detected in 25% of all identified polymorphisms. Similar to NR1I3, the pattern of genetic variability in Indians differed distinctly from those in Chinese and Malays
(Supplementary Table S2). Twenty-five SNPs with MAF ² 0.05 were included in the LD analysis in Chinese and Indians, while there were only twenty-three for Malays (Fig. 2). The following pairs of SNPs were in perfect linkage in Malays («DA« and R2 = 1): IVS9¹138T>C (rs6413514) with IVS9¹97G>A (rs1805347); IVS9¹27G>A (rs1805343) with *846G>A (rs4240711) and *+4768C>A (rs4842196) with
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
175
NR1I3 and NR2B1 Pharmacogenetics in Asians
*+4988A>G (rs4842198), but not in Chinese or Indians (Fig. 2). The number of tag-SNPs required to represent the genetic diversity of NR2B1 in Chinese, Malays and Indians was 12, 17, and 18, respectively, of which 5 tag-SNPs [¹2650G>A (rs11185647), ¹1741G>A (rs62576288), IVS2+33G>A (rs2234753), IVS2¹46C>A (rs1805352) and IVS8¹126T>C (rs3132293)] were common across all three populations. By virtue of its role as a heterodimerization partner of other regulatory receptors involved in cell proliferation, cholesterol metabolism or insulin sensitivity as mentioned above, the effects of NR2B1 polymorphisms have mostly been investigated in relation to disease susceptibilities such as Alzheimer’s disease, diabetes and various cancers to date.31–34) For instance, IVS2¹46C>A (rs1805352) was previously identified as one of the markers within the 6-SNP haplotype significantly associated with colon cancer.33) The remaining 5 SNPs were deep intronic SNPs which were not covered in the present study. Additionally, IVS8¹126T>C (rs3132293), when being in haplotypic linkage with IVS6¹99G>T (rs3118570) and IVS7+70A>G (rs1536475), was previously shown to correlate with Alzheimer’s disease.32) These three SNPs were also moderately linked in our three local populations, with pairwise «DA« values ranging from 0.62–0.96. In-silico analysis revealed the exonic polymorphism 782C>T (Pro261Leu; rs2234960) to be located at an exonic splicing site, while the novel polymorphism 105G>A (Pro35Pro) may affect transcription factor and/or miRNA binding sites. Five SNPs located in the 5A flanking region were predicted to exert altered regulatory function and sixteen intronic SNPs may be located within an intronic enhancer. Additionally, five 3A UTR SNPs may disrupt or create miRNA binding sites (Table 2). CAR/RXR¡ heterodimer is a master regulator of xenobiotic metabolism, in addition to governing various physiological processes. The collinearity between the mRNA levels of CAR and its target genes suggests that the variability in the expression level of the regulatory genes can possibly account for the altered induction and activity of the target genes.35) Nonetheless, the potential of the genetic variations in these regulatory genes to affect target gene expression and drug disposition has not been investigated extensively. It is also to be noted that the genetic polymorphisms in the NR1I3 and NR2B1 genes were confined to mostly the introns whilst the exons were highly conserved across the ethnic populations. Thus far, the studies on the effects of SNPs in these genes were mostly limited to disease susceptibility. The common cis-acting genetic variations in the Phase I to III drug disposition genes have often failed to account for the interindividual differences in drug pharmacokinetics and pharmacodynamics.36,37) Hence, exploration of the genes encoding orphan nuclear receptors that regulate the expression of downstream genes involved in drug disposition may provide useful insights into the mechanistic basis of inter-individual variability in drug pharmacokinetics and pharmacodynamics.38) Although several SNPs in our study were predicted to be functional, their biological relevance requires further experimental evaluation. In conclusion, our study provides a comprehensive overview of genetic variations present in NR1I3 and NR2B1 in the local Asian populations. The selection of tag-SNPs based on the LD pattern provides an economical means of representing the genetic variability of the genes without the loss of power for future studies. As these receptors are the focal components in xenobiotic detoxification, as well as in many developmental and metabolic pathways,
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