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Single Nucleotide Polymorphisms in SULT1A1 and SULT1A2 in a Korean Population
Drug Metab. Pharmacokinet. 28 (4): 372–377 (2013).
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
SNP Communication Single Nucleotide Polymorphisms in SULT1A1 and SULT1A2 in a Korean Population Su-Jun L EE 1 , Woo-Young K IM 1 , Yazun B. JARRAR 1 , Yang-Weon K IM 1,2, Sang Seop L EE 1 and Jae-Gook S HIN 1,3, * 1
Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, Busan, South Korea 2 Department of Emergency Medicine, Inje University College of Medicine, Inje University, Busan, South Korea 3 Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Inje University College of Medicine, Inje University, Busan, South Korea
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: SULT1A1 and SULT1A2 are encoded on the same chromatid, and exhibit a 96% amino acid similarity. To screen for genetic variants in these two closely related genes, SULT1A1 and SULT1A2 were directly sequenced in 50 healthy Koreans. A total of 30 variations were identified in SULT1A1: eight in exons, thirteen in introns, and nine in the 5¤-untranslated region. With regard to SULT1A2, 21 variants were identified, comprising seven in exons, five in introns, and nine in the 5¤-untranslated region. Among these 51 variations, one in SULT1A1 and eight in SULT1A2 were previously unidentified, which include three coding variants (SULT1A2 R37Q, 110G>A; SULT1A2 G50S, 148G>A; SULT1A2 F286L, 3819C>A) and one null allele (SULT1A2 E217Stop, 3542G>T). Two LD blocks, major haplotype structures, and 7 haplotype-tagging SNPs were determined together for SULT1A1 and SULT1A2 as a single set. Frequencies of common functional variants were compared among ethnic groups. Since these two SULT enzymes are on the same chromatid in a parallel direction with overlapping substrate specificities, a combined analysis using LD and haplotype-tagging single-nucleotide polymorphisms (SNPs) will facilitate understanding of the variations in the sulfation reactions of a wide range of substrates, as compared with analysis of individual genes. Keywords: SULT1A1; SULT1A2; linkage disequilibrium; SNPs; tag SNPs
Based on their sequence similarities, a total of 13 human cytosolic SULT genes are classified into four families: SULT1, SULT2, SULT4, and SULT6.5) The SULT1 family is further divided into two subfamilies, SULT1A and SULT1E, which include SULT1A1, SULT1A2, SULT1A3, and SULT1E1. In the SULT1A subfamily, each gene has a preference in terms of its substrates and has a different inheritance pattern. For example, SULT1A1 and SULT1A2 exhibit a high amino-acid homology (96%) and preferentially catalyze the sulfation of small planar phenols, which include endogenous or exogenous estrogens and tamoxifen metabolites.1,6,7) However, SULT1A3 preferentially catalyzes sulfate conjugation with monoamines, such as dopamine and other neurotransmitters, and exhibits relatively lower amino-acid
Introduction Sulfotransferase (SULT) catalyzes transfer of a sulfate group from 3A-phosphoadenosine-5A-phosphosulfate (PAPS) to the nucleophilic moieties of a wide variety of human drugs.1) In addition to xenobiotic metabolism, SULT has an important role in the regulation of endogenous compounds, such as steroids, hormones, and catecholamines.2) Sulfation is also involved in the detoxification of environmental pollutants and inactivation of active drug metabolites, such as those of tamoxifen, 4-OH-tamoxifen and endoxifen.3) Although sulfated compounds usually exhibit increased water solubility and decreased cell-membrane penetratation, sulfation of some chemicals leads to generation of carcinogenic metabolites.4)
Received September 26, 2012; Accepted December 28, 2012 J-STAGE Advance Published Date: January 29, 2013, doi:10.2133/dmpk.DMPK-12-SC-110 *To whom correspondence should be addressed: Jae-Gook SHIN, Ph.D., M.D., Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, 633-165, Gaegum2-dong, Busanjin-gu, Busan 614-714, South Korea. Tel. ©82-51890-6709, Fax. ©82-51-893-1232, E-mail: [email protected] This work was supported by a grant from the Korea Science and Engineering Foundation (KOSEF), funded by the Ministry of Education, Science, and Engineering (MOEST) (No. R13-2007-023-00000-0) and by a grant from the National Project for Personalized Genomic Medicine (A111218-11-PG02), Korea Health 21 R&D Project, Ministry for Health & Welfare, Republic of Korea. 372
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SNPs of SULT1A1 and SULT1A2 in Koreans
sequence homologies with SULT1A1 (93%) and SULT1A2 (91%).8,9) Results from the Human Genome Project show that SULT1A3 is duplicated into two SULT1A3 genes (e.g., SULT1A3 and SULT1A4) on chromosome 16.10) Therefore, it appears that SULT1A1 and SULT1A2 share substrates, similar structures and functions, while SULT1A3 has a different substrate affinity and recombination events than SULT1A1 and SULT1A2. SULT1A1 and SULT1A2 are important genes, as they are involved in regulating estrogen levels and drug clearance in humans.2,11,12) Although SULT1A1 and SULT1A2 genetic polymorphisms have been reported in whites, blacks, and other ethnic populations,13–18) there is a lack of data regarding these genes in Koreans and other Asians. Therefore, we resequenced the SULT1A1 and SULT1A2 genes, which are located 8 kb apart, identified new variants, and analyzed them together in terms of allele frequencies, haplotype structures, linkage-disequilibrium (LD) blocks, and haplotype-tagging single-nucleotide polymorphisms (SNPs). Materials and Methods Subjects: Genomic DNA samples were obtained from 50 unrelated, healthy Koreans from the DNA repository bank at the INJE Pharmacogenomics Research Center (Inje University College of Medicine, Busan, Korea).19,20) All subjects provided written informed consent before participating in the present study. The research protocol for the use of human DNA from blood samples was approved by the Institutional Review Board (IRB) of Busan Paik Hospital, Inje University College of Medicine, Inje University, Busan, Korea. Variant identification: Genomic DNA was prepared from peripheral whole blood using a QIAamp blood kit (Qiagen, Valencia, CA). All exons, intron/exon junctions, 5A-untranslated regions (5A-UTRs), and 3AUTRs were amplified using SULT1A1and SULT1A2-specific primers, and the amplified fragments were directly sequenced to identify DNA variations. Polymerase chain reaction (PCR) primers were initiated approximately 50–100 bp from each intron-exon boundary, and spanned the intron/exon splice site. All primers used are described in Supplementary Table A. Since SULT1A1 and SULT1A2 exhibit a high sequence homology, these were amplified first, followed by individual fragments, for direct DNA sequencing. PCR was performed using primers (0.2 mmol l¹1 each), genomic DNA (100 ng), 2 U of r-Taq polymerase (Takara Bio, Shiga, Japan), and each dNTP at 0.2 mmol l¹1 per reaction. Amplification products were purified using a PCR purification kit (NucleoGen, Ansan, Korea) and directly sequenced according to the manufacturer’s instructions (ABI Prism 3700XL Genetic Analyzer, Applied Biosystems, Carlsbad, CA). All variants identified were confirmed by sequencing in both directions. In particular, the rare variants identified from a single individual were PCR amplified again and the mutation confirmed by resequencing in both directions to avoid artificial errors. A software package, PC Gene (Oxford Molecular, Campbell, CA), was used to identify variants with single-nucleotide substitutions in heterozygous or homozygous individuals. A software program available at http://www.fruitfly.org/seq_tools/splice.html was used to predict possible new splice sites introduced by mutations. Another software program (http://www.cbrc.jp/research/db/TFSEARCH.html) was used to detect changes in transcription factor-binding elements introduced by mutations. LD, haplotype analysis, and haplotype-tagging SNPs: Hardy-Weinberg equilibrium (HWE) and haplotype inference were
analyzed using SNPAlyze software (version 4.1; Dynacom, Yokohama, Japan). LD block and haplotype structure were constructed using Haploview software version 4.1 (http://www. broadinstitute.org/scientific-community/science/programs/medicaland-population-genetics/haploview/downloads). «DA« and rho square (r2) values were used to determine pairwise LD between SNPs, as described previously.21,22) Thirty-seven SNPs with frequencies of >5% were used to select haplotype-tagging SNPs (htSNPs). A tagger program that combined the simplicity of pairwise methods with the potential efficiency of multimarker approaches was used to select representative htSNPs on the basis of the exclusion of redundant SNPs displaying high levels of LD (http://www.broad.mit.edu/mpg/tagger/). Detailed methods for htSNP selection were described previously.22) Results and Discussion SULTs are important contributors to the conjugation of numerous xenobiotics and endogenous compounds. In general, conjugated compounds with a sulfate anion (SO3¹) from PAPS are more water soluble than the acceptor molecule itself, resulting in the increased elimination of substrates or increased toxicity of some chemicals.23) Genetic polymorphisms in SULTs can cause functional changes through various mutations in a number of genomic regions (e.g., expression level variation via mutations in regulatory regions and protein function alterations due to amino acid changes or insertions/deletions). Since SULTs are involved in the conjugation of steroidal hormones, drugs, and environmental compounds, their genetic polymorphism-induced altered functions may be associated with hormonal imbalance, adverse drug reactions, and/or an increased risk of chemical toxicity. Genetic polymorphisms in SULT1A1 and SULT1A2 in Caucasians have been evaluated.24,25) However, such information regarding Asian populations is limited compared to those regarding other ethnicities. Although a few studies of SULT variant frequencies have been published,13,15,26) no resequencing to comprehensively analyze SULT1A1 and SULT1A2 in Asians has been conducted. Therefore, the goals of our study were to identify further genetic variants and to analyze the LD and haplotype structures of the closely-related SULT1A1 and SULT1A2 genes. Direct DNA sequencing of SULT1A1 and SULT1A2 revealed a total of 51 variations in 50 Korean individuals. A summary of the identified frequencies is listed in Tables 1 and 2. All of the genotype frequencies were in line with HWE, with the exception of rs3743963 in SULT1A2 gene. Genotype error might be a potential source for this departure from HWE. However, further validation of sequencing data confirmed the genotype calling. Therefore, the deviation from HWE of this SNP was unlikely to be due to a sequencing error. For SULT1A1, eight SNPs were found within exons, including three that produced amino acid changes. Of these, SULT1A1 R213H, designated SULT1A1*2, was found at a frequency of 12%. Nine SNPs in the 5A-untranslated region (sequenced up to ¹1,200 bp) and 13 SNPs in introns were detected. None of these variations were implicated in the creation or disruption of splice sites or transcription factor-binding sites. Among SULT1A1 variants, SULT1A1*2 and SULT1A1*3 have been extensively investigated due to their enzymatic changes that occur at relatively high frequencies. SULT1A1*2 has a lower stability than the wild-type.15,27) SULT1A1*2 exhibited decreased activity in platelets28–30) and in a recombinant protein system15,29) when compared to the wild-type enzyme. The influence of SULT1A1*3 on
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Su-Jun LEE, et al. Table 1. Summary of SULT1A1 variations identified in a Korean population (n = 50) Positiona
¹1135G>A ¹904Tdelb ¹624G>C ¹396A>G ¹358A>C ¹341C>G ¹294T>C ¹66T>C ¹30G>A 57G>A 257T>C 266A>G 1940A>G 2106G>C 2109C>T 2112T>A 2130T>A 2140A>C 2159G>A 2166C>T 2582T>C 2603G>A 2605C>T 2625G>C 2663G>A 2670G>A 2816C>T 2869T>C 3049A>G 3120C>T
Amino acid substitution
Accession No.
Nucleotide change
rs2077412
ccccacacaG/Acacccacaa tgtttgttttttT/delccccgggg tgcctaggcctG/Ctgcttttgctgagt cagcaggaaA/Gtggtgagac ggagacagcA/Ccaggaaggtcc tcctagagC/Gttcctcagtgc acaccctgaT/Catctgggcct gcaagaatT/Cccactttcttgc taagggaacG/Aggcctggctct gggggtcccG/Actcatcaa caggcacT/Cacctgggt acctgggtA/Gagccaga gtggagctA/Gaggggtggt agaccacG/Ctgcgatgcttc accacgtgC/Tgatgcttc acgtgcgaT/Agcttccctc ctccatgtgacT/Acctggggg cctgggggcA/Cggcacctc agggacccG/Accaagg ccaaggC/Tcacccag tgaatcagT/Caatccgagcctc cactgagggG/Accctctgct tgaggggC/Tcctctgct agaacccG/Caaaagggag ttgtggggcG/Actccctgc gcgctccctG/Accagaggag gctggccagC/Tacgggggt cataggcactT/Cggggcctcc gggctcctggA/Ggtcactgcag ttgagggccC/Tgggacggta
rs3760091 rs750155 rs2925634 rs4149382 rs4149385 P19P T51T V54V
rs1126446 rs1126447 rs9282862
rs3020806 rs4149388
rs4149393
P200P R213H (*2) L215L
rs3176926 rs9282861
Subject number (n) wt/wt wt/mt mt/mt 19 20 11 49 1 0 27 20 3 14 19 17 26 21 3 47 3 0 7 27 16 47 3 0 32 14 4 47 3 0 39 11 0 38 12 0 39 10 1 36 13 1 36 13 1 36 13 1 36 13 1 36 13 1 36 13 1 36 13 1 31 19 0 30 20 0 48 2 0 31 19 0 48 12 0 43 7 0 48 12 0 43 17 0 39 11 0 33 17 0
Frequency
HW p-value
0.42 0.01 0.26 0.53 0.27 0.03 0.59 0.03 0.22 0.03 0.11 0.12 0.12 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.19 0.20 0.02 0.19 0.12 0.07 0.12 0.17 0.11 0.17
0.29 1.0 1.0 0.14 1.0 1.0 0.65 1.0 0.33 1.0 1.0 0.95 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.24 0.18 1.0 0.24 0.95 1.0 0.95 0.39 1.0 0.39
Frequency
HW p-value
0.11 0.01 0.12 0.37 0.02 0.01 0.12 0.06 0.01 0.11 0.11 0.01 0.01 0.11 0.33 0.01 0.01 0.14 0.01 0.14 0.14
1.0 1.0 0.95 0.63 1.0 1.0 0.95 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.01 1.0 1.0 0.82 1.0 0.82 0.82
The reference sequence used was GenBank accession no. NC_000016. Nucleotide changes are indicated in bold. a Position in relation to the ATG start codon of the SULT1A1 gene; the A in ATG is +1. b New variant allele indentified.
Table 2. Summary of SULT1A2 variations identified in a Korean population (n = 50) Positiona ¹1090A>G ¹981T>Gb ¹979C>T ¹366A>G ¹341G>C ¹283C>Tb ¹281C>T ¹119G>A ¹80C>Tb 20T>C 24T>C 110G>Ab 148G>Ab 182T>C 2566T>C 3424G>Ab 3542G>Tb 3597A>C 3819C>Ab 3910T>C 3917A>G
Amino acid substitution
Accession No.
Nucleotide change
rs762633
ggagggcacA/Gaggccaggt gcctctgctaT/Gccctgccctctc gcctctgctatcC/Tctgccctctc gctggcaggA/Gagacagca tcctagagG/Cttcctcag tgggcccC/Tgcgccacga tgggccccgC/Tgccacga tgtgagtgcG/Aggcaagtca tgacttccC/Ttgaaagca caggacaT/Cctctcgcc ggacatctcT/Ccgcccgcc caggcccG/Agcctgatg ccaagtccG/Agtaggtg tctcccaggT/Cggcagtccc gtgtcggcacT/Cccctgcc agagaagtG/Agaccccttt ctccctgccaG/Taggagact tgaagaagaA/Cccctatgac accaccttC/Aaccgtggc gaggggT/Ctcctggagtc gttcctggA/Ggtcactgcaga
rs743590 rs193652 rs1690403 rs4115668 rs4149403 I7T (*2) S8S R37Q G50S
rs1136703 rs16940475
rs4149406 rs3743963 E217Stop N235T (*2) F268L
rs1059491
Subject number (n) wt/wt wt/mt mt/mt 39 11 0 49 1 0 38 12 0 21 21 8 48 2 0 49 1 0 38 12 0 44 6 0 49 1 0 39 11 0 39 11 0 49 1 0 49 1 0 39 11 0 27 13 10 49 1 0 49 1 0 37 13 0 49 1 0 37 13 0 37 13 0
The reference sequence used was GenBank accession no. NC_000016. Nucleotide change is indicated in bold. Position is indicated in relation to the start codon ATG of the SULT1A2 gene; the A in ATG is +1. New variant alleles indentified.
a b
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SNPs of SULT1A1 and SULT1A2 in Koreans Table 3. Allele frequencies of SULT1A1 and SULT1A2 in various ethnic populations Population
Subject (n)
Frequency of SULT1A1 *2 *3
Frequency of SULT1A2 *2 *3
Reference
Asian Korean Japanese Chinese total White Germans Americans Turks total Black African-Americans Nigerians Black South Africans total
50 234 143 290 168 885
0.120 0.124 0.168 0.080 0.110 0.12 « 0.03
0.000 0.000 0.000 0.006 0.000 0.001 « 0.002
0.110 0.114 ND 0.076 0.110 0.103 « 0.018
0.000 ND ND ND ND 0.000
Present study Kim et al.26) Ozawa et al.15) Carlini et al.14) Glatt et al.33)
300 245 303 848
0.365 0.332 0.238 0.312 « 0.054
0.000 0.012 0 0.004 « 0.006
0.374 0.389 0.184 0.316 « 0.093
ND 0.104 0.125 0.115 « 0.011
Engelke et al.16) Carlini et al.14) Arslan17)
70 52 172 294
0.294 0.269 0.369 0.311 « 0.042
0.229 ND ND —
0.249 ND ND —
0.114 ND ND —
Carlini et al.14) Coughtrie et al.24) Dandara et al.18)
Frequency results from normal populations of differing ethnicities. ND, not determined.
enzyme activity appears to be less than that of SULT1A1*2, conferring activity similar to the wild-type.27,31) Frequencies of SUL1A1*2 and SULT1A1*3 differed among ethnic populations (Table 3). The frequency of SULT1A1*2 is higher in Caucasians (average 35%) and African-Americans (28%) than in Asians (12%).22,30–33) With regard to SULT1A1*3, African Americans exhibited higher frequencies compared to other ethnicities. For SULT1A2, a total of 21 SNPs were detected in the present study. Eight of the 21 were newly identified. Seven SNPs were found in exons, comprising three synonymous and five nonsynonymous mutations, including SULT1A2 I7T, R37Q, G50S, E217Stop, and F268L. All coding variants identified in the present study were heterozygous mutations in a single individual, with the exception of SULT1A2*2. Although the understanding of in vivo effects is limited due to the low frequency, the current study provides additional information on variants that may be helpful in understanding their influence in vivo by means of future global collaborations. For example, an individual with the E217Stop variant would have an impaired sulfation reaction, which may affect physiological changes or drug responses. Potential effects of amino acid substitutions on the SULT1A2 protein were assessed by an in silico method, polymorphism phenotyping (PolyPhen: http://genetics. bwh.harvard.edu/pph). PolyPhen algorithm prediction scores indicated that these variants were likely damaging for SULT1A2 G50S (Polyphen score 1.0) and F268L (Polyphen score 1.0), and benign for R37Q (Polyphen score 0.0). Functional studies using experimental approaches may provide greater clarity regarding functional changes than did in silico analysis. However, bioinformatic prediction analysis would facilitate prioritization of candidate variants according to the likely impact on protein function in vivo. In particular, the change from phenylalanine, which is an aromatic amino acid with a strong hydrophobic nature, to the aliphatic amino acid leucine, could cause structural change in its protein folding and this may cause the altered activity of SULT1A2. Decreased SULT1A2*2 and SULT1A2*3 activities have been reported.1,16,32) However, the relative contribution of these variants to the protein quantity in liver tissue remains to be determined. The frequencies of SUL1A2*2 and SULT1A2*3
differed according to ethnicity (Table 3). Briefly, the SULT1A2*2 frequency was higher in Caucasians than in Asians (38% vs. 10%).14,26,33) SULT1A2*3 was not detected in Asians, whereas African Americans and whites exhibited average frequencies of 10–11%.14) After identification of SNPs in both SULT1A1 and SULT1A2, pairwise LD was calculated using Haploview software, version 4.1 (Supplementary Fig. A and Fig. 1A) and seven haplotypetagging SNPs were determined using the program Tagger (Fig. 1B). These selected tagging SNPs comprehensively represented sequence variations in all exon regions and the 5A-UTR regions of SULT1A1 and SULT1A2, which are thought to be the main functional regions. Thirty-seven variants with a frequency >5% were included in the Haploview analysis for clear separation of an LD block. After the genotypes of all 37 SNPs were combined, seven haplotype alleles exhibited an allele frequency of >1%. Two LD blocks were found within the genomic DNA region containing SULT1A1 and SULT1A2. The locus containing SULT1A1 and SULT1A2 could be divided into two blocks between 3917A>G in SULT1A2 and 257T>C in SULT1A1. The distance between these two LD blocks is approximately 15 kb. Since limited inter-block recombination may occur in short-interval LD, recombination between these two blocks might be limited. Therefore, we analyzed the distribution of haplotype patterns within the entire SULT1A1 and SULT1A2 locus (Fig. 1B). The most frequent haplotype, designated H1, occurred at a rate of 33.6% in the present study. Among them, three haplotypes were inferred to occur at >70%. LD block patterns varied according to ethnicity when those with the same SNPs were analyzed in Caucasians and African Americans in the HapMap database (data not shown). These results suggest that the haplotype structures of the various ethnic groups differ, resulting in the requirement for different haplotype-tagging SNPs. A set of seven tagging SNPs determined for Korean populations could be useful for future association-mapping studies (e.g., hormone-related diseases, such as breast and prostate cancers). These haplotype-tagging SNPs would enable tracking of >85% of the haplotype structures in the SULT1A1 and SULT1A2 gene locus in the Korean population. The distribution of haplotypes containing SULT1A1*2 was ana-
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Su-Jun LEE, et al.
Fig. 1. Linkage disequilibrium (LD) map of SULT1A1 and SULT1A2 single nucleotide polymorphisms (SNPs) obtained from normal, healthy subjects (n = 50) A: SULT SNPs and their common occurrences in the LD blocks. The frequency of each haplotype is shown at the edge. The blue square indicates the most common allele, and pink the variant allele. B: Haplotype structures inferred from a combined analysis of SULT1A1 and SULT1A2. The frequency of each haplotype is shown at the edge. Seven haplotype-tag SNPs are marked by asterisks. The locations of SULT1A1*2 and SULT1A2*2 are underlined.
lyzed carefully. One haplotype was found to have a SULT1A1*2 allele, which comprised 4% of the total haplotype frequency in the combined analysis. The LD between SULT1A1*2 and SULT1A2*2 had DA and r2 values of 0.89 and 0.73, respectively, suggesting these two variants to be in positive LD in Koreans. In summary, we resequenced the SULT1A1 and SULT1A2 genes and identified 51 variations, including nine that were previously unknown. Allele frequencies, haplotype structures, LD blocks, and haplotype-tagging SNPs were determined. A linkage analysis of common alleles was performed. These results may contribute towards development of personalized therapies and enhance understanding of genetic inheritance of the SULT1A1 and SULT1A2 variants. Since SULT1A1 and SULT1A2 are closely related, being on the same chromatid, and their substrates overlap, these data will facilitate combined genetic analyses to gain an understanding of inter-individual differences in the sulfation reactions of their substrates. References 1)
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Shih, M. C., Chang, J. Y. and Chang, J. G.: The relationship among the polymorphisms of SULT1A1, 1A2 and different types of cancers in Taiwanese. Int. J. Mol. Med., 11: 85–89 (2003). Carlini, E. J., Raftogianis, R. B., Wood, T. C., Jin, F., Zheng, W., Rebbeck, T. R. and Weinshilboum, R. M.: Sulfation pharmacogenetics: SULT1A1 and SULT1A2 allele frequencies in Caucasian, Chinese and African-American subjects. Pharmacogenetics, 11: 57–68 (2001). Ozawa, S., Shimizu, M., Katoh, T., Miyajima, A., Ohno, Y., Matsumoto, Y., Fukuoka, M., Tang, Y. M., Lang, N. P. and Kadlubar, F. F.: Sulfatingactivity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians. J. Biochem., 126: 271–277 (1999). Engelke, C. E., Meinl, W., Boeing, H. and Glatt, H.: Association between functional genetic polymorphisms of human sulfotransferases 1A1 and 1A2. Pharmacogenetics, 10: 163–169 (2000). Arslan, S.: Genetic polymorphisms of sulfotransferases (SULT1A1 and SULT1A2) in a Turkish population. Biochem. Genet., 48: 987–994 (2010). Dandara, C., Li, D. P., Walther, G. and Parker, M. I.: Gene-environment interaction: the role of SULT1A1 and CYP3A5 polymorphisms as risk modifiers for squamous cell carcinoma of the oesophagus. Carcinogenesis, 27: 791–797 (2006). Lee, S. J., Jang, Y. J., Cha, E. Y., Kim, H. S., Lee, S. S. and Shin, J. G.: A haplotype of CYP2C9 associated with warfarin sensitivity in mechanical heart valve replacement patients. Br. J. Clin. Pharmacol., 70: 213–221 (2010). Lee, S. J., Kim, W. Y., Kim, H., Shon, J. H., Lee, S. S. and Shin, J. G.: Identification of new CYP2C19 variants exhibiting decreased enzyme activity in the metabolism of S-mephenytoin and omeprazole. Drug Metab. Dispos., 37: 2262–2269 (2009). Lee, S. J., Jung, I. S., Jung, E. J., Choi, J. Y., Yeo, C. W., Cho, D. Y., Kim, Y. W., Lee, S. S. and Shin, J. G.: Identification of P2Y12 singlenucleotide polymorphisms and their influences on the variation in ADPinduced platelet aggregation. Thromb. Res., 127: 220–227 (2011). Lee, S. J., Kim, W. Y., Choi, J. Y., Lee, S. S. and Shin, J. G.: Identification of CYP19A1 single-nucleotide polymorphisms and their haplotype distributions in a Korean population. J. Hum. Genet., 55: 189– 193 (2010). Pang, K. S., Schwab, A. J., Goresky, C. A. and Chiba, M.: Transport, binding, and metabolism of sulfate conjugates in the liver. Chem. Biol. Interact., 92: 179–207 (1994). Coughtrie, M. W., Gilissen, R. A., Shek, B., Strange, R. C., Fryer, A. A.,
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Jones, P. W. and Bamber, D. E.: Phenol sulphotransferase SULT1A1 polymorphism: molecular diagnosis and allele frequencies in Caucasian and African populations. Biochem. J., 337: 45–49 (1999). Nowell, S., Ratnasinghe, D. L., Ambrosone, C. B., Williams, S., Teague-Ross, T., Trimble, L., Runnels, G., Carrol, A., Green, B., Stone, A., Johnson, D., Greene, G., Kadlubar, F. F. and Lang, N. P.: Association of SULT1A1 phenotype and genotype with prostate cancer risk in African-Americans and Caucasians. Cancer Epidemiol. Biomarkers Prev., 13: 270–276 (2004). Kim, K. A., Lee, S. Y., Park, P. W., Ha, J. M. and Park, J. Y.: Genetic polymorphisms and linkage disequilibrium of sulfotransferase SULT1A1 and SULT1A2 in a Korean population: comparison of other ethnic groups. Eur. J. Clin. Pharmacol., 61: 743–747 (2005). Hildebrandt, M. A., Carrington, D. P., Thomae, B. A., Eckloff, B. W., Schaid, D. J., Yee, V. C., Weinshilboum, R. M. and Wieben, E. D.: Genetic diversity and function in the human cytosolic sulfotransferases. Pharmacogenomics J., 7: 133–143 (2007). Ohtake, E., Kakihara, F., Matsumoto, N., Ozawa, S., Ohno, Y., Hasegawa, S., Suzuki, H. and Kubota, T.: Frequency distribution of phenol sulfotransferase 1A1 activity in platelet cells from healthy Japanese subjects. Eur. J. Pharm. Sci., 28: 272–277 (2006). Nowell, S., Ambrosone, C. B., Ozawa, S., MacLeod, S. L., Mrackova, G., Williams, S., Plaxco, J., Kadlubar, F. F. and Lang, N. P.: Relationship of phenol sulfotransferase activity (SULT1A1) genotype to sulfotransferase phenotype in platelet cytosol. Pharmacogenetics, 10: 789–797 (2000). Raftogianis, R. B., Wood, T. C., Otterness, D. M., Van Loon, J. A. and Weinshilboum, R. M.: Phenol sulfotransferase pharmacogenetics in humans: association of common SULT1A1 alleles with TS PST phenotype. Biochem. Biophys. Res. Commun., 239: 298–304 (1997). Nagar, S., Walther, S. and Blanchard, R. L.: Sulfotransferase (SULT) 1A1 polymorphic variants *1, *2, and *3 are associated with altered enzymatic activity, cellular phenotype, and protein degradation. Mol. Pharmacol., 69: 2084–2092 (2006). Lu, J., Li, H., Zhang, J., Li, M., Liu, M. Y., An, X., Liu, M. C. and Chang, W.: Crystal structures of SULT1A2 and SULT1A1 *3: insights into the substrate inhibition and the role of Tyr149 in SULT1A2. Biochem. Biophys. Res. Commun., 396: 429–434 (2010). Glatt, H., Boeing, H., Engelke, C. E., Ma, L., Kuhlow, A., Pabel, U., Pomplun, D., Teubner, W. and Meinl, W.: Human cytosolic sulphotransferases: genetics, characteristics, toxicological aspects. Mutat. Res., 482: 27–40 (2001).
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
SNP Communication Single Nucleotide Polymorphisms in SULT1A1 and SULT1A2 in a Korean Population Su-Jun L EE 1 , Woo-Young K IM 1 , Yazun B. JARRAR 1 , Yang-Weon K IM 1,2, Sang Seop L EE 1 and Jae-Gook S HIN 1,3, * 1
Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, Busan, South Korea 2 Department of Emergency Medicine, Inje University College of Medicine, Inje University, Busan, South Korea 3 Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Inje University College of Medicine, Inje University, Busan, South Korea
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: SULT1A1 and SULT1A2 are encoded on the same chromatid, and exhibit a 96% amino acid similarity. To screen for genetic variants in these two closely related genes, SULT1A1 and SULT1A2 were directly sequenced in 50 healthy Koreans. A total of 30 variations were identified in SULT1A1: eight in exons, thirteen in introns, and nine in the 5¤-untranslated region. With regard to SULT1A2, 21 variants were identified, comprising seven in exons, five in introns, and nine in the 5¤-untranslated region. Among these 51 variations, one in SULT1A1 and eight in SULT1A2 were previously unidentified, which include three coding variants (SULT1A2 R37Q, 110G>A; SULT1A2 G50S, 148G>A; SULT1A2 F286L, 3819C>A) and one null allele (SULT1A2 E217Stop, 3542G>T). Two LD blocks, major haplotype structures, and 7 haplotype-tagging SNPs were determined together for SULT1A1 and SULT1A2 as a single set. Frequencies of common functional variants were compared among ethnic groups. Since these two SULT enzymes are on the same chromatid in a parallel direction with overlapping substrate specificities, a combined analysis using LD and haplotype-tagging single-nucleotide polymorphisms (SNPs) will facilitate understanding of the variations in the sulfation reactions of a wide range of substrates, as compared with analysis of individual genes. Keywords: SULT1A1; SULT1A2; linkage disequilibrium; SNPs; tag SNPs
Based on their sequence similarities, a total of 13 human cytosolic SULT genes are classified into four families: SULT1, SULT2, SULT4, and SULT6.5) The SULT1 family is further divided into two subfamilies, SULT1A and SULT1E, which include SULT1A1, SULT1A2, SULT1A3, and SULT1E1. In the SULT1A subfamily, each gene has a preference in terms of its substrates and has a different inheritance pattern. For example, SULT1A1 and SULT1A2 exhibit a high amino-acid homology (96%) and preferentially catalyze the sulfation of small planar phenols, which include endogenous or exogenous estrogens and tamoxifen metabolites.1,6,7) However, SULT1A3 preferentially catalyzes sulfate conjugation with monoamines, such as dopamine and other neurotransmitters, and exhibits relatively lower amino-acid
Introduction Sulfotransferase (SULT) catalyzes transfer of a sulfate group from 3A-phosphoadenosine-5A-phosphosulfate (PAPS) to the nucleophilic moieties of a wide variety of human drugs.1) In addition to xenobiotic metabolism, SULT has an important role in the regulation of endogenous compounds, such as steroids, hormones, and catecholamines.2) Sulfation is also involved in the detoxification of environmental pollutants and inactivation of active drug metabolites, such as those of tamoxifen, 4-OH-tamoxifen and endoxifen.3) Although sulfated compounds usually exhibit increased water solubility and decreased cell-membrane penetratation, sulfation of some chemicals leads to generation of carcinogenic metabolites.4)
Received September 26, 2012; Accepted December 28, 2012 J-STAGE Advance Published Date: January 29, 2013, doi:10.2133/dmpk.DMPK-12-SC-110 *To whom correspondence should be addressed: Jae-Gook SHIN, Ph.D., M.D., Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, 633-165, Gaegum2-dong, Busanjin-gu, Busan 614-714, South Korea. Tel. ©82-51890-6709, Fax. ©82-51-893-1232, E-mail: [email protected] This work was supported by a grant from the Korea Science and Engineering Foundation (KOSEF), funded by the Ministry of Education, Science, and Engineering (MOEST) (No. R13-2007-023-00000-0) and by a grant from the National Project for Personalized Genomic Medicine (A111218-11-PG02), Korea Health 21 R&D Project, Ministry for Health & Welfare, Republic of Korea. 372
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SNPs of SULT1A1 and SULT1A2 in Koreans
sequence homologies with SULT1A1 (93%) and SULT1A2 (91%).8,9) Results from the Human Genome Project show that SULT1A3 is duplicated into two SULT1A3 genes (e.g., SULT1A3 and SULT1A4) on chromosome 16.10) Therefore, it appears that SULT1A1 and SULT1A2 share substrates, similar structures and functions, while SULT1A3 has a different substrate affinity and recombination events than SULT1A1 and SULT1A2. SULT1A1 and SULT1A2 are important genes, as they are involved in regulating estrogen levels and drug clearance in humans.2,11,12) Although SULT1A1 and SULT1A2 genetic polymorphisms have been reported in whites, blacks, and other ethnic populations,13–18) there is a lack of data regarding these genes in Koreans and other Asians. Therefore, we resequenced the SULT1A1 and SULT1A2 genes, which are located 8 kb apart, identified new variants, and analyzed them together in terms of allele frequencies, haplotype structures, linkage-disequilibrium (LD) blocks, and haplotype-tagging single-nucleotide polymorphisms (SNPs). Materials and Methods Subjects: Genomic DNA samples were obtained from 50 unrelated, healthy Koreans from the DNA repository bank at the INJE Pharmacogenomics Research Center (Inje University College of Medicine, Busan, Korea).19,20) All subjects provided written informed consent before participating in the present study. The research protocol for the use of human DNA from blood samples was approved by the Institutional Review Board (IRB) of Busan Paik Hospital, Inje University College of Medicine, Inje University, Busan, Korea. Variant identification: Genomic DNA was prepared from peripheral whole blood using a QIAamp blood kit (Qiagen, Valencia, CA). All exons, intron/exon junctions, 5A-untranslated regions (5A-UTRs), and 3AUTRs were amplified using SULT1A1and SULT1A2-specific primers, and the amplified fragments were directly sequenced to identify DNA variations. Polymerase chain reaction (PCR) primers were initiated approximately 50–100 bp from each intron-exon boundary, and spanned the intron/exon splice site. All primers used are described in Supplementary Table A. Since SULT1A1 and SULT1A2 exhibit a high sequence homology, these were amplified first, followed by individual fragments, for direct DNA sequencing. PCR was performed using primers (0.2 mmol l¹1 each), genomic DNA (100 ng), 2 U of r-Taq polymerase (Takara Bio, Shiga, Japan), and each dNTP at 0.2 mmol l¹1 per reaction. Amplification products were purified using a PCR purification kit (NucleoGen, Ansan, Korea) and directly sequenced according to the manufacturer’s instructions (ABI Prism 3700XL Genetic Analyzer, Applied Biosystems, Carlsbad, CA). All variants identified were confirmed by sequencing in both directions. In particular, the rare variants identified from a single individual were PCR amplified again and the mutation confirmed by resequencing in both directions to avoid artificial errors. A software package, PC Gene (Oxford Molecular, Campbell, CA), was used to identify variants with single-nucleotide substitutions in heterozygous or homozygous individuals. A software program available at http://www.fruitfly.org/seq_tools/splice.html was used to predict possible new splice sites introduced by mutations. Another software program (http://www.cbrc.jp/research/db/TFSEARCH.html) was used to detect changes in transcription factor-binding elements introduced by mutations. LD, haplotype analysis, and haplotype-tagging SNPs: Hardy-Weinberg equilibrium (HWE) and haplotype inference were
analyzed using SNPAlyze software (version 4.1; Dynacom, Yokohama, Japan). LD block and haplotype structure were constructed using Haploview software version 4.1 (http://www. broadinstitute.org/scientific-community/science/programs/medicaland-population-genetics/haploview/downloads). «DA« and rho square (r2) values were used to determine pairwise LD between SNPs, as described previously.21,22) Thirty-seven SNPs with frequencies of >5% were used to select haplotype-tagging SNPs (htSNPs). A tagger program that combined the simplicity of pairwise methods with the potential efficiency of multimarker approaches was used to select representative htSNPs on the basis of the exclusion of redundant SNPs displaying high levels of LD (http://www.broad.mit.edu/mpg/tagger/). Detailed methods for htSNP selection were described previously.22) Results and Discussion SULTs are important contributors to the conjugation of numerous xenobiotics and endogenous compounds. In general, conjugated compounds with a sulfate anion (SO3¹) from PAPS are more water soluble than the acceptor molecule itself, resulting in the increased elimination of substrates or increased toxicity of some chemicals.23) Genetic polymorphisms in SULTs can cause functional changes through various mutations in a number of genomic regions (e.g., expression level variation via mutations in regulatory regions and protein function alterations due to amino acid changes or insertions/deletions). Since SULTs are involved in the conjugation of steroidal hormones, drugs, and environmental compounds, their genetic polymorphism-induced altered functions may be associated with hormonal imbalance, adverse drug reactions, and/or an increased risk of chemical toxicity. Genetic polymorphisms in SULT1A1 and SULT1A2 in Caucasians have been evaluated.24,25) However, such information regarding Asian populations is limited compared to those regarding other ethnicities. Although a few studies of SULT variant frequencies have been published,13,15,26) no resequencing to comprehensively analyze SULT1A1 and SULT1A2 in Asians has been conducted. Therefore, the goals of our study were to identify further genetic variants and to analyze the LD and haplotype structures of the closely-related SULT1A1 and SULT1A2 genes. Direct DNA sequencing of SULT1A1 and SULT1A2 revealed a total of 51 variations in 50 Korean individuals. A summary of the identified frequencies is listed in Tables 1 and 2. All of the genotype frequencies were in line with HWE, with the exception of rs3743963 in SULT1A2 gene. Genotype error might be a potential source for this departure from HWE. However, further validation of sequencing data confirmed the genotype calling. Therefore, the deviation from HWE of this SNP was unlikely to be due to a sequencing error. For SULT1A1, eight SNPs were found within exons, including three that produced amino acid changes. Of these, SULT1A1 R213H, designated SULT1A1*2, was found at a frequency of 12%. Nine SNPs in the 5A-untranslated region (sequenced up to ¹1,200 bp) and 13 SNPs in introns were detected. None of these variations were implicated in the creation or disruption of splice sites or transcription factor-binding sites. Among SULT1A1 variants, SULT1A1*2 and SULT1A1*3 have been extensively investigated due to their enzymatic changes that occur at relatively high frequencies. SULT1A1*2 has a lower stability than the wild-type.15,27) SULT1A1*2 exhibited decreased activity in platelets28–30) and in a recombinant protein system15,29) when compared to the wild-type enzyme. The influence of SULT1A1*3 on
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Su-Jun LEE, et al. Table 1. Summary of SULT1A1 variations identified in a Korean population (n = 50) Positiona
¹1135G>A ¹904Tdelb ¹624G>C ¹396A>G ¹358A>C ¹341C>G ¹294T>C ¹66T>C ¹30G>A 57G>A 257T>C 266A>G 1940A>G 2106G>C 2109C>T 2112T>A 2130T>A 2140A>C 2159G>A 2166C>T 2582T>C 2603G>A 2605C>T 2625G>C 2663G>A 2670G>A 2816C>T 2869T>C 3049A>G 3120C>T
Amino acid substitution
Accession No.
Nucleotide change
rs2077412
ccccacacaG/Acacccacaa tgtttgttttttT/delccccgggg tgcctaggcctG/Ctgcttttgctgagt cagcaggaaA/Gtggtgagac ggagacagcA/Ccaggaaggtcc tcctagagC/Gttcctcagtgc acaccctgaT/Catctgggcct gcaagaatT/Cccactttcttgc taagggaacG/Aggcctggctct gggggtcccG/Actcatcaa caggcacT/Cacctgggt acctgggtA/Gagccaga gtggagctA/Gaggggtggt agaccacG/Ctgcgatgcttc accacgtgC/Tgatgcttc acgtgcgaT/Agcttccctc ctccatgtgacT/Acctggggg cctgggggcA/Cggcacctc agggacccG/Accaagg ccaaggC/Tcacccag tgaatcagT/Caatccgagcctc cactgagggG/Accctctgct tgaggggC/Tcctctgct agaacccG/Caaaagggag ttgtggggcG/Actccctgc gcgctccctG/Accagaggag gctggccagC/Tacgggggt cataggcactT/Cggggcctcc gggctcctggA/Ggtcactgcag ttgagggccC/Tgggacggta
rs3760091 rs750155 rs2925634 rs4149382 rs4149385 P19P T51T V54V
rs1126446 rs1126447 rs9282862
rs3020806 rs4149388
rs4149393
P200P R213H (*2) L215L
rs3176926 rs9282861
Subject number (n) wt/wt wt/mt mt/mt 19 20 11 49 1 0 27 20 3 14 19 17 26 21 3 47 3 0 7 27 16 47 3 0 32 14 4 47 3 0 39 11 0 38 12 0 39 10 1 36 13 1 36 13 1 36 13 1 36 13 1 36 13 1 36 13 1 36 13 1 31 19 0 30 20 0 48 2 0 31 19 0 48 12 0 43 7 0 48 12 0 43 17 0 39 11 0 33 17 0
Frequency
HW p-value
0.42 0.01 0.26 0.53 0.27 0.03 0.59 0.03 0.22 0.03 0.11 0.12 0.12 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.19 0.20 0.02 0.19 0.12 0.07 0.12 0.17 0.11 0.17
0.29 1.0 1.0 0.14 1.0 1.0 0.65 1.0 0.33 1.0 1.0 0.95 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.24 0.18 1.0 0.24 0.95 1.0 0.95 0.39 1.0 0.39
Frequency
HW p-value
0.11 0.01 0.12 0.37 0.02 0.01 0.12 0.06 0.01 0.11 0.11 0.01 0.01 0.11 0.33 0.01 0.01 0.14 0.01 0.14 0.14
1.0 1.0 0.95 0.63 1.0 1.0 0.95 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.01 1.0 1.0 0.82 1.0 0.82 0.82
The reference sequence used was GenBank accession no. NC_000016. Nucleotide changes are indicated in bold. a Position in relation to the ATG start codon of the SULT1A1 gene; the A in ATG is +1. b New variant allele indentified.
Table 2. Summary of SULT1A2 variations identified in a Korean population (n = 50) Positiona ¹1090A>G ¹981T>Gb ¹979C>T ¹366A>G ¹341G>C ¹283C>Tb ¹281C>T ¹119G>A ¹80C>Tb 20T>C 24T>C 110G>Ab 148G>Ab 182T>C 2566T>C 3424G>Ab 3542G>Tb 3597A>C 3819C>Ab 3910T>C 3917A>G
Amino acid substitution
Accession No.
Nucleotide change
rs762633
ggagggcacA/Gaggccaggt gcctctgctaT/Gccctgccctctc gcctctgctatcC/Tctgccctctc gctggcaggA/Gagacagca tcctagagG/Cttcctcag tgggcccC/Tgcgccacga tgggccccgC/Tgccacga tgtgagtgcG/Aggcaagtca tgacttccC/Ttgaaagca caggacaT/Cctctcgcc ggacatctcT/Ccgcccgcc caggcccG/Agcctgatg ccaagtccG/Agtaggtg tctcccaggT/Cggcagtccc gtgtcggcacT/Cccctgcc agagaagtG/Agaccccttt ctccctgccaG/Taggagact tgaagaagaA/Cccctatgac accaccttC/Aaccgtggc gaggggT/Ctcctggagtc gttcctggA/Ggtcactgcaga
rs743590 rs193652 rs1690403 rs4115668 rs4149403 I7T (*2) S8S R37Q G50S
rs1136703 rs16940475
rs4149406 rs3743963 E217Stop N235T (*2) F268L
rs1059491
Subject number (n) wt/wt wt/mt mt/mt 39 11 0 49 1 0 38 12 0 21 21 8 48 2 0 49 1 0 38 12 0 44 6 0 49 1 0 39 11 0 39 11 0 49 1 0 49 1 0 39 11 0 27 13 10 49 1 0 49 1 0 37 13 0 49 1 0 37 13 0 37 13 0
The reference sequence used was GenBank accession no. NC_000016. Nucleotide change is indicated in bold. Position is indicated in relation to the start codon ATG of the SULT1A2 gene; the A in ATG is +1. New variant alleles indentified.
a b
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SNPs of SULT1A1 and SULT1A2 in Koreans Table 3. Allele frequencies of SULT1A1 and SULT1A2 in various ethnic populations Population
Subject (n)
Frequency of SULT1A1 *2 *3
Frequency of SULT1A2 *2 *3
Reference
Asian Korean Japanese Chinese total White Germans Americans Turks total Black African-Americans Nigerians Black South Africans total
50 234 143 290 168 885
0.120 0.124 0.168 0.080 0.110 0.12 « 0.03
0.000 0.000 0.000 0.006 0.000 0.001 « 0.002
0.110 0.114 ND 0.076 0.110 0.103 « 0.018
0.000 ND ND ND ND 0.000
Present study Kim et al.26) Ozawa et al.15) Carlini et al.14) Glatt et al.33)
300 245 303 848
0.365 0.332 0.238 0.312 « 0.054
0.000 0.012 0 0.004 « 0.006
0.374 0.389 0.184 0.316 « 0.093
ND 0.104 0.125 0.115 « 0.011
Engelke et al.16) Carlini et al.14) Arslan17)
70 52 172 294
0.294 0.269 0.369 0.311 « 0.042
0.229 ND ND —
0.249 ND ND —
0.114 ND ND —
Carlini et al.14) Coughtrie et al.24) Dandara et al.18)
Frequency results from normal populations of differing ethnicities. ND, not determined.
enzyme activity appears to be less than that of SULT1A1*2, conferring activity similar to the wild-type.27,31) Frequencies of SUL1A1*2 and SULT1A1*3 differed among ethnic populations (Table 3). The frequency of SULT1A1*2 is higher in Caucasians (average 35%) and African-Americans (28%) than in Asians (12%).22,30–33) With regard to SULT1A1*3, African Americans exhibited higher frequencies compared to other ethnicities. For SULT1A2, a total of 21 SNPs were detected in the present study. Eight of the 21 were newly identified. Seven SNPs were found in exons, comprising three synonymous and five nonsynonymous mutations, including SULT1A2 I7T, R37Q, G50S, E217Stop, and F268L. All coding variants identified in the present study were heterozygous mutations in a single individual, with the exception of SULT1A2*2. Although the understanding of in vivo effects is limited due to the low frequency, the current study provides additional information on variants that may be helpful in understanding their influence in vivo by means of future global collaborations. For example, an individual with the E217Stop variant would have an impaired sulfation reaction, which may affect physiological changes or drug responses. Potential effects of amino acid substitutions on the SULT1A2 protein were assessed by an in silico method, polymorphism phenotyping (PolyPhen: http://genetics. bwh.harvard.edu/pph). PolyPhen algorithm prediction scores indicated that these variants were likely damaging for SULT1A2 G50S (Polyphen score 1.0) and F268L (Polyphen score 1.0), and benign for R37Q (Polyphen score 0.0). Functional studies using experimental approaches may provide greater clarity regarding functional changes than did in silico analysis. However, bioinformatic prediction analysis would facilitate prioritization of candidate variants according to the likely impact on protein function in vivo. In particular, the change from phenylalanine, which is an aromatic amino acid with a strong hydrophobic nature, to the aliphatic amino acid leucine, could cause structural change in its protein folding and this may cause the altered activity of SULT1A2. Decreased SULT1A2*2 and SULT1A2*3 activities have been reported.1,16,32) However, the relative contribution of these variants to the protein quantity in liver tissue remains to be determined. The frequencies of SUL1A2*2 and SULT1A2*3
differed according to ethnicity (Table 3). Briefly, the SULT1A2*2 frequency was higher in Caucasians than in Asians (38% vs. 10%).14,26,33) SULT1A2*3 was not detected in Asians, whereas African Americans and whites exhibited average frequencies of 10–11%.14) After identification of SNPs in both SULT1A1 and SULT1A2, pairwise LD was calculated using Haploview software, version 4.1 (Supplementary Fig. A and Fig. 1A) and seven haplotypetagging SNPs were determined using the program Tagger (Fig. 1B). These selected tagging SNPs comprehensively represented sequence variations in all exon regions and the 5A-UTR regions of SULT1A1 and SULT1A2, which are thought to be the main functional regions. Thirty-seven variants with a frequency >5% were included in the Haploview analysis for clear separation of an LD block. After the genotypes of all 37 SNPs were combined, seven haplotype alleles exhibited an allele frequency of >1%. Two LD blocks were found within the genomic DNA region containing SULT1A1 and SULT1A2. The locus containing SULT1A1 and SULT1A2 could be divided into two blocks between 3917A>G in SULT1A2 and 257T>C in SULT1A1. The distance between these two LD blocks is approximately 15 kb. Since limited inter-block recombination may occur in short-interval LD, recombination between these two blocks might be limited. Therefore, we analyzed the distribution of haplotype patterns within the entire SULT1A1 and SULT1A2 locus (Fig. 1B). The most frequent haplotype, designated H1, occurred at a rate of 33.6% in the present study. Among them, three haplotypes were inferred to occur at >70%. LD block patterns varied according to ethnicity when those with the same SNPs were analyzed in Caucasians and African Americans in the HapMap database (data not shown). These results suggest that the haplotype structures of the various ethnic groups differ, resulting in the requirement for different haplotype-tagging SNPs. A set of seven tagging SNPs determined for Korean populations could be useful for future association-mapping studies (e.g., hormone-related diseases, such as breast and prostate cancers). These haplotype-tagging SNPs would enable tracking of >85% of the haplotype structures in the SULT1A1 and SULT1A2 gene locus in the Korean population. The distribution of haplotypes containing SULT1A1*2 was ana-
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Fig. 1. Linkage disequilibrium (LD) map of SULT1A1 and SULT1A2 single nucleotide polymorphisms (SNPs) obtained from normal, healthy subjects (n = 50) A: SULT SNPs and their common occurrences in the LD blocks. The frequency of each haplotype is shown at the edge. The blue square indicates the most common allele, and pink the variant allele. B: Haplotype structures inferred from a combined analysis of SULT1A1 and SULT1A2. The frequency of each haplotype is shown at the edge. Seven haplotype-tag SNPs are marked by asterisks. The locations of SULT1A1*2 and SULT1A2*2 are underlined.
lyzed carefully. One haplotype was found to have a SULT1A1*2 allele, which comprised 4% of the total haplotype frequency in the combined analysis. The LD between SULT1A1*2 and SULT1A2*2 had DA and r2 values of 0.89 and 0.73, respectively, suggesting these two variants to be in positive LD in Koreans. In summary, we resequenced the SULT1A1 and SULT1A2 genes and identified 51 variations, including nine that were previously unknown. Allele frequencies, haplotype structures, LD blocks, and haplotype-tagging SNPs were determined. A linkage analysis of common alleles was performed. These results may contribute towards development of personalized therapies and enhance understanding of genetic inheritance of the SULT1A1 and SULT1A2 variants. Since SULT1A1 and SULT1A2 are closely related, being on the same chromatid, and their substrates overlap, these data will facilitate combined genetic analyses to gain an understanding of inter-individual differences in the sulfation reactions of their substrates. References 1)
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