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Human SULT1A3 pharmacogenetics: gene duplication and functional genomic studies
BBRC Biochemical and Biophysical Research Communications 321 (2004) 870–878 www.elsevier.com/locate/ybbrc
Human SULT1A3 pharmacogenetics: gene duplication and functional genomic studies I Michelle A.T. Hildebrandt a, Oreste E. Salavaggione a, Yvette N. Martin a, Heather C. Flynn b, Syed Jalal b, Eric D. Wieben c, Richard M. Weinshilboum a,* a
Departments of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA b Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA c Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA Received 18 June 2004 Available online 24 July 2004
Abstract Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines. Inheritance is an important factor responsible for individual variation in SULT1A3 activity, and gene resequencing studies have shown the presence of one functionally significant SULT1A3 nonsynonymous cSNP. However, following completion of the Human Genome Project, it appeared that SULT1A3 might be duplicated. We used specific PCR-based assays and fluorescence in situ hybridization to verify that 2 SULT1A3 genes—SULT1A3 and SULT1A4—were present on chromosome 16 in all human DNA samples studied. Furthermore, reanalysis of previous gene resequencing data confirmed the presence of the SULT1A3 SNPs identified previously, but also revealed 11 novel polymorphisms, including 3 nonsynonymous cSNPs. Functional genomic studies showed that two of those cSNPs, C302T, and C302A, resulted in decreased enzyme activity without striking changes in substrate kinetics but with parallel changes in levels of immunoreactive protein. In addition, RT-PCR revealed that both SULT1A3 and SULT1A4 can be transcriptionally active. The duplication of SULT1A3 will have to be taken into account in future efforts to understand individual variation in SULT1A3 activity or properties. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Sulfotransferase; SULT1A3; SULT1A4; Pharmacogenetics; Gene duplication; Genetic polymorphism; SNPs; Functional genomics
Sulfotransferase (SULT) enzymes catalyze the sulfate conjugation of biogenic amine neurotransmitters, steroid hormones, drugs, and other xenobiotics [1–3]. SULT1A1, SULT1A2, and SULT1A3 are among the 11 known members of the human SULT gene family. I This work was supported in part by National Institutes of Health (NIH) Grants RO1 GM28157 (MATH, OES, YNM, and RMW), RO1 GM35720 (MATH, OES, YNM, and RMW), and UO1 GM61388, The Pharmacogenetics Research Network (MATH, OES, YNM, EDW, and RMW). * Corresponding author. Fax: 1-507-284-911. E-mail address: [email protected] (R.M. Weinshilboum).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.038
These 3 genes map to the short arm of chromosome 16 and the enzymes encoded by the 3 known SULT1A genes share over 93% amino acid sequence identity [4,5]. SULT1A3 catalyzes the sulfation of dopamine, other catecholamines, and structurally related drugs [6–9]. The high affinity sulfation of catecholamines catalyzed by this enzyme appears to be specific to higher primates, since no ortholog for SULT1A3 has been identified in nonprimate species [10,11]. In humans, over 95% of the circulating dopamine and approximately 70% of plasma norepinephrine are sulfate conjugated [12]. Because of the functional significance of catecholamines and drugs related to catecholamines, it would
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be important to determine the possible contribution of inheritance to variation in the activity and/or properties of SULT1A3. Early twin and family pharmacogenetic studies showed that variation in SULT1A3 enzyme activity in an easily accessible human tissue—the blood platelet—was due, in large part, to inheritance [13,14]. In the course of subsequent studies designed to explore molecular genetic mechanism(s) that might contribute to individual variation in SULT1A3 activity, the gene was resequenced and functional genomic studies were performed [15]. That gene resequencing effort resulted in the identification of only a single nonsynonymous cSNP that was present in DNA from African-American subjects which resulted in a Lys234Asn alteration in encoded amino acid. Functional genomic studies showed that this amino acid change resulted in decreased levels of SULT1A3 activity and immunoreactive protein as a result of accelerated proteasome-dependent degradation. SULT1A3 consists of 7 coding and a series of alternatively spliced 5 0 -noncoding exons [16]. In the course of the Human Genome Project, various chromosome 16p ‘‘builds’’ included as many as 5 copies of SULT1A3. Because of the highly repetitive nature of the DNA sequence within this region of chromosome 16, the presence of multiple copies of SULT1A3 was thought to result from difficulty in contig assembly. However, following ‘‘completion’’ of the Human Genome Project on April 11, 2003, a BLAST search of chromosome 16
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performed with a portion of the SULT1A3 sequence showed two copies of the gene. One had been annotated as SULT1A3, while the other location, approximately 700 kb telomeric, was annotated as MGC5178. Analysis of the sequences of these two regions showed that 3 SULT1A3 5 0 -noncoding exons—1D, 1C, and 1B—overlapped with MGC5178 coding exons 3, 4, and 5 with 100% identity (Fig. 1A). These observations suggested that both SULT1A3 and MGC5178 might be duplicated. In the present study, we set out to test that hypothesis—as well as the possible functional implications of such a duplication. As a first step, global alignments of sequence containing the two genes were performed, as were PCR-based and fluorescence in situ hybridization (FISH) assays designed to verify the presence of the duplication. Those experiments confirmed that an intrachromosomal duplication had occurred. On the basis of advice obtained from the SULT Nomenclature Committee [17], we would suggest that the two SULT1A3 genes in these duplicated segments be named SULT1A3 (16p11.2, the gene currently annotated as SULT1A3) and SULT1A4 (16p12.1, the region currently annotated as MGC5178). Both of these genes encode identical SULT1A3 proteins. We then reanalyzed our previous resequencing data for SULT1A3 as well as resequencing data for the overlapping gene, MGC5178—with the knowledge that the sequence chromatograms contained data for 4 rather
Fig. 1. SULT1A3/1A4 and MGC5178 gene structures and chromosome 16p duplication. (A) SULT1A3/1A4 and MGC5178 structures. Exons unique to SULT1A3/1A4 are shown in grey; MGC5178 exons are black; and 3 exons shared by the two genes are shown as hatched boxes. Exon and intron lengths are based on sequence from NT_024812.10 (B) Chromosome 16p duplication. The 146 kb duplications containing SULT1A3 and 1A4 are 98.5% identical in nucleotide sequence, and the regions containing SULT1A3 and 1A4 genes are 99.8% identical over 10 kb. Bold arrows ( ) show locations of primers used to perform the duplication-specific PCR assays. Numbers in italics are the nucleotide positions on NT_024812.10.
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Fig. 2. SULT1A3/1A4 and MGC5178 polymorphisms. The figure shows a schematic representation of human SULT1A3/1A4 and MGC5178 with the location of SNPs indicated by arrows. Rectangles represent exons, with exons unique to SULT1A3/1A4 shown in grey and MGC5178 exons in black. Three exons that are shared by both genes are shown as hatched boxes. Exons 2–8 of SULT1A3/1A4 encode protein. ‘‘AA’’ represents data obtained with DNA from African-American subjects, and ‘‘CA’’ represents data obtained with DNA from Caucasian-American subjects. Black arrows represent previously reported polymorphisms [15], and open arrows indicate polymorphisms identified in the course of present study. When applicable, amino acid changes are also indicated.
than 2 alleles. When that was done, we were able to confirm the presence of all SULT1A3 polymorphisms that had been identified previously [15], but we also detected 11 additional SULT1A3/SULT1A4/MGC5178 SNPs, including 3 novel nonsynonymous cSNPs, C302T, Pro101Leu; C302A, Pro101His; and C430T, Arg144Cys (Fig. 2). Functional genomic studies were performed for all 3 of these novel SULT1A3/SULT1A4 nonsynonymous cSNPs, and reverse transcription-PCR showed that both SULT1A3 and SULT1A4 can be transcriptionally active. Future studies of the possible effects of inheritance on SULT1A3 will have to take the duplication of this gene into account. Materials and methods DNA samples and cell lines. DNA samples from 60 CaucasianAmerican (CA), 60 African-American (AA), 60 Han Chinese-American (HCA), and 60 Mexican-American (MA) subjects were obtained from the Coriell Cell Repository (Camden, NJ) (Sample Sets HD100CAU, HD100AA, HD100CHI, and HD100MEX). Lymphoblastoid cell lines (GM17227, GM17109, GM17135, GM17159, GM17734, GM17758, GM17453, and GM17458) were also obtained for selected samples from these same subjects. These samples had been collected and anonymized by the National Institute of General Medical Sciences (NIGMS). All subjects had provided written informed consent for the use of their DNA for research purposes, and the present studies were reviewed and approved by the Mayo Clinic Institutional Review Board. PCR assays to verify SULT1A3 duplication. PCR assays were developed to verify the duplication of SULT1A3 by taking advantage of the unique 5 0 - and 3 0 -flanking sequences for the 2 duplicated regions. Specifically, primers were designed within the 5 0 - and 3 0 -ends of the duplications. These ‘‘common’’ primers were paired with primers specific for the sequence flanking each of the duplications, so that only the regions where the duplications began or ended would be amplified—with different length amplicons for the two duplications (primer
sequences and locations on NT_024812.10 are listed in Table 1 of the ‘‘Supplementary Material’’). Locations of the primers are indicated schematically in Fig. 1B. The PCR was then used to amplify each of these regions with 240 DNA samples from four ethnic groups (CA, AA, MA, and HCA) as template. Amplicons were analyzed on 1% agarose gels and selected amplicons were sequenced in the Mayo Molecular Biology Core Facility to verify that the amplifications were specific for the region of interest. SULT1A3 fluorescence in situ hybridization. To verify the SULT 1A3 duplication, a direct labeled FISH probe was designed using a bacterial artificial chromosome (BAC) which covered the region containing SULT1A3. It was anticipated that this probe would also detect the duplicated region containing SULT1A4 because of the very high sequence identity of these two regions. Specifically, a glycerol stock of BAC clone RP11-455F5 (Accession No. AC012645) was obtained from ResGen Invitrogen Corporation (Carlsbad, CA). After PCR amplification of the region of interest, nick translation was performed using the Vysis Nick Translation Kit (Vysis, Downers Groove, IL) to produce a fluorescence labeled DNA probe for FISH. Signal validation was verified by observing both interphase and metaphase cells. Metaphase preparations of 7 Coriell Institute lymphoblastoid cell lines from the 4 ethnic groups studied (GM17227, GM17109, GM17135, GM17734, GM17758, GM17453, and GM17458) were analyzed using the SULT1A3 probe together with a commercially available direct labeled centromere 16 probe (Vysis, Downers Groove, IL). The SULT1A3-centromere 16 probe mixture was applied to each cell line, and standard FISH pre-treatment and hybridization protocols for peripheral blood were followed [18]. A minimum of 2 metaphase spreads and 10 interphase cells were studied for each cell line analyzed. SULT1A3/1A4 RT-PCR analysis. mRNA was isolated from two lymphoblastoid cell lines (GM17109 and GM17159) using the RNeasy kit (Qiagen, Valencia, Ca). cDNAs were generated from these mRNA preparations with random hexamer reverse primers using the ThermalScript RT kit from Invitrogen (Carlsbad, CA). The PCR was then used to amplify SULT1A3/1A4 cDNA using SULT1A3/1A4-specific primers that would not cross-react with SULT1A1 or SULT1A2. The sequences of these primers are listed in Table 1 of the ‘‘Supplementary Material’’. Amplicons were then cloned into pCR2.1 (Invitrogen) and the inserts were sequenced. SULT1A/1A43 and MGC5178 resequencing. SULT1A3 exons and splice junctions had been resequenced in the course of a previous study
M.A.T. Hildebrandt et al. / Biochemical and Biophysical Research Communications 321 (2004) 870–878 [15]. During the present study, the PCR was used to amplify all MGC5178 and SULT1A3/1A4 exons that had not been included in the previous study. In addition, approximately 500 bp of sequence immediately upstream of MGC5178, as well as all splice junctions and portions of introns flanking each exon, were resequenced using dye primer chemistry as described previously [15]. Primer sequences and annealing temperatures are listed in ‘‘Table 1 of the Supplementary Material’’. Amplicons were sequenced in the Mayo Molecular Biology Core Facility. To verify that newly identified SNPs were not PCRinduced artifacts, DNA samples that included those polymorphisms were amplified and amplicons were subcloned into pCR2.1. The inserts for up to 20 clones were then sequenced to confirm the presence of variant nucleotides. SULT1A3/1A4 variant allozyme COS-1 cell expression. Expression constructs for the 3 novel variant SULT1A3/1A4 allozymes, SULT 1A3/1A4*3 (Leu101), SULT1A3/1A4*4 (His101), and SULT1A3/ 1A4*5 (Cys144), were created by site-directed mutagenesis of the wild type (WT) SULT1A3/1A4*1 cDNA sequence in the expression vector pCR3.1 (Invitrogen). DNA sequences of the inserts were verified by dye terminator sequencing. Sequences of primers used to perform sitedirected mutagenesis are listed in Table 1 of the ‘‘Supplementary Material.’’ These expression constructs were used to transfect COS-1 cells that had been cotransfected with a b-galactosidase construct (Promega, Madison, WI) to make it possible to control for transfection efficiency. Six separate plates were transfected with each allozyme construct, and COS-1 cell lysate from these transfections was used to assay enzyme activity, to perform substrate kinetic studies and to perform Western blot analyses. SULT1A3/1A4 allozyme enzyme activity and Western blot analysis. Recombinant SULT1A3/1A4 allozymes *1, *3, *4, and *5—as well as cells transfected with an empty vector control—were assayed for SULT1A3 activity using a modification of the radiochemical enzymatic assay of Foldes and Meek [19] as described previously [15] with dopamine and [35S]3 0 -phosphoadenosine 5 0 -phosphosulfate (PAPS) as cosubstrates. Substrate kinetic studies were also conducted with variable concentrations of these two substrates. Western blot analysis of the recombinant allozymes was also performed as described previously [15]. Data analysis. The EMBOSS tool ‘‘stretcher’’ was used to align nucleotide sequences. This tool performs global pair-wise alignments over the entire length of the sequences being studied to show alignments and to highlight similarities. BLAST [20] was also used to perform sequence comparisons. Dye primer DNA sequence chromatograms for the gene resequencing were analyzed using the PolyPhred 4.0 [21] and Consed 8.0 [22] programs. GenBank Accession numbers for reference sequences used in these studies were: chromosome 16 reference contig, NT_024812.10; SULT1A3 cDNA RefSeqs, NM_003166.2 and NM_177552.1; and MGC5178 cDNA RefSeqs, NM_024044.2 and NM_178044.1. Apparent Km values for the enzyme kinetic studies were calculated with the method of Wilkinson [23] using a computer program developed by Cleland [24]. Points that deviated from linearity on double inverse plots as a result of substrate inhibition were not included in these calculations. Statistical significance of differences between mean values was determined by the use of StudentÕs t test.
Results Introduction These experiments had a series of goals—all related to enhancing understanding of the possible contribution of variation in genomic sequence and/or structure to SULT1A3 function. The initial studies involved characterization and verification of the chromosome 16p dupli-
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cation that resulted in two nearly identical copies of SULT1A3 and MGC5178. Once the existence of the duplication—and its presence in 4 separate ethnic groups— had been verified, that fact had implications for the analysis and interpretation of the SULT1A3 gene resequencing data. As a result, we reanalyzed both our previous and new gene resequencing chromatograms and identified and confirmed a series of additional SULT1A3/1A4 cSNPs, including 3 novel nonsynonymous cSNPs, as well as polymorphisms for MGC5178. Studies were also conducted of the possible functional implication of those cSNPs—as were experiments designed to determine whether both SULT1A3 and SULT1A4 were transcriptionally active. Characterization of the SULT1A3 gene duplication Analysis of chromosome 16p sequence from the final build of the Human Genome Project showed that portions of SULT1A3 overlapped with MGC5178, a gene of unknown function. The ‘‘overlap’’ occurred in SULT1A3 5 0 -noncoding exons 1D, 1C, and 1B and MGC5178 coding exons 3, 4, and 5 (Fig. 1A). The SULT1A3 5 0 -noncoding exons were identified by performing 5 0 -RACE with cDNAs isolated from human brain, colon, kidney, and small intestine as well as by EST database searches. Although SULT1A3 exon 1E (see Fig. 1A) is included in NCBI SULT1A3 cDNA Ref Seq NM_0031662, the gene annotation on NT_024812.10 shows only exons 1A, 1B, and 1C. However, even though SULT1A3 and MGC5178 overlapped, the two genes were annotated at locations separated by approximately 700 kb—raising the possibility that SULT1A3 might be part of a intrachromosomal duplication (see Fig. 1B). As a first step in characterizing this possible duplication, the EMBOSS tool ‘‘stretcher’’ was used to identify and compare the 2 duplicated areas. The duplications were found to be over 146 kb in length and were 98.5% identical in sequence, with only 663 nucleotide differences (0.45%) and 1603 gaps (1.1%). SULT1A3 was located between nucleotides 1,605,080 and 1,615,497 on reference contig NT_024812.10 while SULT1A4 was located 5 0 -upstream, between nucleotides 867,119 and 877,526. The regions containing the 2 copies of SULT1A3 were 99.8% identical in sequence, with 7 nucleotide changes and 8 gaps over a total length of 10 kb (Fig. 1B). The open reading frames (ORFs) for SULT1A3 and SULT1A4 were 99.9% identical, with only a single nucleotide change that did not alter the encoded amino acid. This nucleotide location had been reported to be polymorphic during our previous resequencing study [15]. The 146 kb duplicated region on 16p was also reported to contain 5 other genes— LOC348197, LOC348196, LOC339088, LOC339091, and LAT1-3TM—that were duplicated, plus one gene, CORO1A, that was only partially duplicated.
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Fig. 3. SULT1A3/1A4 FISH analysis. The figure shows a metaphase spread of a lymphoblastoid cell line with a chromosome 16-specific centromere probe shown in green and with two SULT1A3/1A4 pink signals on each copy of chromosome 16. The insets show a close-up view of a single chromosome (upper right) and an interphase cell (lower right), both of which show two pink signals.
To verify the existence of the duplication, a requirement in light of the difficulty that had occurred in assembling this portion of the human genome, two PCR assays were developed based on the unique 5 0 - and 3 0 flanking regions which bordered the duplicated regions. When those assays were performed using 240 individual DNA samples from 4 ethnic groups—60 CA, 60 AA, 60 HCA, and 60 MA—all samples had both copies of the duplicated area. As a further verification of the existence of the duplication, 7 lymphoblastoid cell lines from the same 4 ethnic groups were used to perform FISH. FISH analysis showed two distinct signals on the short arm of chromosome 16 for at least 2 metaphase spreads and 10 interphase spreads from each cell line in the presence of the SULT1A3 probe (Fig. 3). These observations verified the existence of a near-perfect duplication of SULT1A3 in all ethnic groups studied—immediately raising the question of the implications of this observation for previous pharmacogenetic studies—including the recently published SULT1A3 gene resequencing study [15]. SULT1A3/1A4 resequencing analysis Dye primer sequence chromatograms [25] from the present study (MGC5178) and from the previous SULT1A3/1A4 resequencing study were analyzed or reanalyzed with the knowledge that the chromatograms included data for 4 not 2 alleles. This analysis was performed to verify the SNPs reported previously and to identify any additional SNPs which might have been ‘‘missed’’ based on peak height criteria appropriate for 2, but not for 4 alleles. As anticipated, all of the previously reported SULT1A3/1A4 SNPs were confirmed, but 11 new polymorphisms were identified, including 3 novel nonsynonymous cSNPs in SULT1A3/1A4 (Fig. 2 and Table 1): Pro101Leu (C302T), Pro101His
(C302A), and Arg144Cys (C430T). The analysis also showed that one DNA sample appeared to contain 3 nucleotides at one position (C302T/A) (‘‘Supplementary Material’’ Fig. 1). All of the novel SULT1A3/1A4 SNPs as well as the MGC5178 SNPs, including the nonsynonymous cSNPs, were verified as described in Materials and methods. Because both SULT1A3 and SULT1A4 would have been amplified and sequenced simultaneously during the resequencing studies, it was not possible to determine whether any individual polymorphism was present within SULT1A3 or SULT1A4. In addition, unequivocal determinations of allele frequencies could not be made and haplotype and linkage analysis could not be performed for these polymorphisms because it was not possible to determine which copy of the gene contained the polymorphism. Therefore, we have not included haplotype or linkage disequilibrium analyses, and the allele frequencies listed in Table 1 should be considered as estimates. However, our discovery of novel SULT1A3/ 1A4 nonsynonymous cSNPs provided an opportunity to determine whether these changes in amino acid sequence might influence function and also to determine whether SULT1A3 might be transcriptionally active in each of the duplicated areas. Novel SULT1A3/1A4 nonsynonymous cSNP functional genomics Expression constructs were created for SULT1A3/ 1A4*1 (WT), SULT1A3/1A4*3 (Leu101), SULT1A3/ 1A4*4 (His101), and SULT1A3/1A4*5 (Cys144) to make it possible to study the functional effects of the newly identified polymorphisms. Recombinant protein for the variant allozymes after transient expression in COS-1 cells was assayed for level of activity with dopamine as the substrate. Two of the variant allozymes— Leu101 (*3) and His101 (*4)—showed significant decreases in enzyme activity, while the level of activity for the Cys144 (*5) allozyme was slightly, but not significantly, elevated when compared with that for the WT (*1) allozyme. The Pro101Leu polymorphism resulted in a decrease in activity of approximately 87% and the Pro101His amino acid change resulted in a reduction of approximately 40% (Table 2 and Fig. 4A). One mechanism which might result in a decrease in enzyme activity as a result of a change in encoded amino acid would be an alteration in substrate kinetics. Therefore, we also determined apparent Km values for the WT and variant allozymes using both PAPS and dopamine as substrates. The apparent Km values with PAPS for the His101 (*4) and Cys144 (*5) polymorphisms were significantly different from that for the WT, although these differences were probably not large enough to be of physiological significance (Table 2). Another possible mechanism which could explain the decreased enzyme activity was
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Table 1 Human SULT1A3/1A4 and MGC5178 polymorphisms
The table lists the 11 newly identified SULT1A3/1A4 and MGC5178 polymorphisms in bold, with the previously reported polymorphisms in italics [15]. The estimated allele frequencies for each population are listed based on peak height ratios from dye primer sequencing for 2 alleles (previous study [15]) 4 alleles (present study). Polymorphisms located in exons are boxed. The numbering scheme for polymorphism locations is based on the cDNA sequence, with the ‘‘A’’ in the translation initiation codon designated (+1). Positions 5 0 and 3 0 to that location were assigned negative or positive numbers, respectively. Locations in introns were numbered from the intron–exon splice junctions, with ‘‘I’’ used to designate intron locations. Table 2 SULT1A3/1A4 functional genomics Allozyme
Polymorphism
SULT1A3 Activity, % of WT
Immunoreactive protein, % of WT
Dopamine Km (lM)
PAPS Km (lM)
SULT1A3/1A4*1 SULT1A3/1A4*3 SULT1A3/1A4*4 SULT1A3/1A4*5
WT Pro101Leu Pro101His Arg144Cys
100 ± 3.8 12.7 ± 2.0** 59.2 ± 6.2** 110 ± 4.8
100 ± 7.8 42.1 ± 3.6** 85.5 ± 2.8 120 ± 12
8.3 ± 0.6 7.2 ± 0.2 9.7 ± 1.4 8.8 ± 0.5
0.19 ± 0.003 0.14 ± 0.03 0.11 ± 0.01* 0.15 ± 0.004**
Average values for levels of activity (N = 6), immunoreactive protein (N = 3), and apparent Km values for dopamine and PAPS (N = 6) are listed. All values are means ± SEM. *P < 0.05 and **P < 0.001 when compared with the WT allozyme.
a decrease in protein quantity [15,30–35]. Quantitative Western blot analysis, corrected for transfection efficiency, showed a significant decrease in immunoreactive protein of approximately 60% for the Leu101 (*3) allozyme (Table 2 and Fig. 4B). There was also a slight decrease for the His101 (*4) and a slight increase for the Arg144 (*5) protein levels when compared with WT, but neither was statistically significant.
RT-PCR of SULT1A3/1A4 The final question that we asked was whether both SULT1A3 and SULT1A4 might be transcribed. We were fortunate that SULT1A3/1A4 is expressed in lymphoblastoid cell lines such as those created by the Coriell Institute for the DNA samples used in our experiments (Dr. V. Cheung, personal communication). Therefore,
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Fig. 4. SULT1A3/1A4 allozyme COS-1 cell expression. (A) Average levels of enzyme activity are shown for each of the recombinant SULT1A3/1A4 allozymes studied—*1, *3, *4, and *5—with dopamine as the sulfate acceptor substrate. Each bar represents the average value for 6 independent transfections (mean ± SEM), and all values have been corrected for transfection efficiency. **P < 0.001 when compared with the WT allozyme SULT1A3/1A4*1. (B) Average levels of SULT1A3/1A4 immunoreactive protein for each of the recombinant allozymes, expressed as a percentage of the level of SULT1A3/1A4*1 protein. Each bar represents the average of 3 independent transfections (mean ± SEM). The gels were loaded on the basis of b-galactosidase activity to correct for transfection efficiency. **P < 0.001 when compared with the value for SULT1A3/1A4*1.
reverse transcription-PCR was performed with mRNA from the lymphoblastoid cell line (GM17159) that corresponded to the DNA sample with three alleles at nucleotide 302 (codon 101) (‘‘Supplementary Material’’ Fig. 1). All three nucleotides were detected in cDNAs generated from this cell line, indicating that three distinct SULT1A3/1A4 mRNA species were present in the cell (‘‘Supplementary Material’’ Fig. 2). Furthermore, cDNAs from an additional cell line (GM17109), corresponding to a DNA sample that contained multiple SULT1A3/1A4 cSNPs, were also subjected to RTPCR. This cell line expressed mRNA species with 3 SULT1A3/1A4 haplotypes. These observations were all compatible with the conclusion that both SULT1A3 and SULT1A4 were transcribed in these lymphoblastoid cell lines.
Discussion SULT1A3 catalyzes the sulfation of dopamine as well as other catecholamines and is the only known SULT
isoform with a high affinity for these substrates [3]. This metabolic pathway for catecholamines is important, with over 95% of circulating dopamine in humans being sulfate conjugated [12]. Because of the importance of catecholamines and drugs related to catecholamines in human physiology, human pathophysiology, and the treatment of human disease, it is important that the possible contribution of inheritance to individual variation in this metabolic pathway be understood. The human SULT1A3 gene maps to 16p, a region that is highly repetitive [26,27]. During the assembly of chromosome 16p by the Human Genome Project, it was reported that as many as five copies of SULT1A3 were present. At that time, it was believed that these observations were most likely due to difficulty in contig assembly. The ‘‘completed’’ Human Genome Project has mapped SULT1A3 to 16p11.2, but a nearly identical copy of the gene is located at 16p12.1—a region currently annotated as MGC5178. Both copies of SULT1A3 overlap with 3 MGC5178 exons. In the present studies, we have confirmed the SULT1A3 gene duplication by performing duplication-specific PCR assays as well as FISH—in all cases using DNA from 4 ethnic groups. The duplicated regions were over 146 kb in length and 98.5% identical in sequence. Furthermore, the 10 kb portions of the duplications containing SULT1A3 and SULT1A4 were 99.9% identical in nucleotide sequence and encoded identical enzymes. We had previously resequenced SULT1A3/1A4 as part of a series of studies of human SULT pharmacogenetics [5,14,15,28–32]. However, analysis of those data was based on the assumption of a single copy of SULT1A3 with 2 alleles—and not a 2-gene, 4-allele system. To more accurately characterize genetic variation in SULT1A3 and SULT1A4, we have now subjected the original dye primer sequencing data, as well as additional resequencing that included MGC5178, to re-analysis based on an assumption of 4 rather than 2 alleles. That analysis resulted in the identification of 11 additional polymorphisms, including 3 novel SULT1A3/1A4 nonsynonymous cSNPs. The possible functional effects of these changes in amino acid sequence were determined by performing functional genomic studies designed to determine levels of activity, immunoreactive protein, and kinetic parameters for these new allozymes after expression in COS-1 cells. It is essential that functional genomic studies such as these be performed in mammalian cells since we have shown the need for mammalian protein degradation systems for accurate results [36], and we and others have consistently found that genotype– phenotype correlations observed in studies of this type correlate with in vivo observations [33,34,37]. Finally, based on RT-PCR data, both SULT1A3 and SULT1A4 appear to be transcriptionally active, although it remains to be determined whether both copies of the gene are regulated in a similar fashion.
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In summary, the presence in the genome of two copies of SULT1A3 may help to explain a portion of the individual variation in SULT1A3 activity in humans. It will be necessary that the SULT1A3/1A4 duplication be understood in order to interpret past or future studies of the pharmacogenetics of this important catecholamine and drug-metabolizing enzyme in humans.
Acknowledgments We thank Luanne Wussow for her assistance with the preparation of the manuscript; Bianca Thomae and Bruce Eckloff for generating the SULT1A3/1A4 and MGC5178 dye primer sequence chromatograms; and Dr. Vivien Cheung from the University of Pennsylvania for sharing her unpublished SULT1A3/1A4 lymphoblastoid cell line expression data.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2004.07.038.
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Human SULT1A3 pharmacogenetics: gene duplication and functional genomic studies I Michelle A.T. Hildebrandt a, Oreste E. Salavaggione a, Yvette N. Martin a, Heather C. Flynn b, Syed Jalal b, Eric D. Wieben c, Richard M. Weinshilboum a,* a
Departments of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA b Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA c Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA Received 18 June 2004 Available online 24 July 2004
Abstract Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines. Inheritance is an important factor responsible for individual variation in SULT1A3 activity, and gene resequencing studies have shown the presence of one functionally significant SULT1A3 nonsynonymous cSNP. However, following completion of the Human Genome Project, it appeared that SULT1A3 might be duplicated. We used specific PCR-based assays and fluorescence in situ hybridization to verify that 2 SULT1A3 genes—SULT1A3 and SULT1A4—were present on chromosome 16 in all human DNA samples studied. Furthermore, reanalysis of previous gene resequencing data confirmed the presence of the SULT1A3 SNPs identified previously, but also revealed 11 novel polymorphisms, including 3 nonsynonymous cSNPs. Functional genomic studies showed that two of those cSNPs, C302T, and C302A, resulted in decreased enzyme activity without striking changes in substrate kinetics but with parallel changes in levels of immunoreactive protein. In addition, RT-PCR revealed that both SULT1A3 and SULT1A4 can be transcriptionally active. The duplication of SULT1A3 will have to be taken into account in future efforts to understand individual variation in SULT1A3 activity or properties. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Sulfotransferase; SULT1A3; SULT1A4; Pharmacogenetics; Gene duplication; Genetic polymorphism; SNPs; Functional genomics
Sulfotransferase (SULT) enzymes catalyze the sulfate conjugation of biogenic amine neurotransmitters, steroid hormones, drugs, and other xenobiotics [1–3]. SULT1A1, SULT1A2, and SULT1A3 are among the 11 known members of the human SULT gene family. I This work was supported in part by National Institutes of Health (NIH) Grants RO1 GM28157 (MATH, OES, YNM, and RMW), RO1 GM35720 (MATH, OES, YNM, and RMW), and UO1 GM61388, The Pharmacogenetics Research Network (MATH, OES, YNM, EDW, and RMW). * Corresponding author. Fax: 1-507-284-911. E-mail address: [email protected] (R.M. Weinshilboum).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.038
These 3 genes map to the short arm of chromosome 16 and the enzymes encoded by the 3 known SULT1A genes share over 93% amino acid sequence identity [4,5]. SULT1A3 catalyzes the sulfation of dopamine, other catecholamines, and structurally related drugs [6–9]. The high affinity sulfation of catecholamines catalyzed by this enzyme appears to be specific to higher primates, since no ortholog for SULT1A3 has been identified in nonprimate species [10,11]. In humans, over 95% of the circulating dopamine and approximately 70% of plasma norepinephrine are sulfate conjugated [12]. Because of the functional significance of catecholamines and drugs related to catecholamines, it would
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be important to determine the possible contribution of inheritance to variation in the activity and/or properties of SULT1A3. Early twin and family pharmacogenetic studies showed that variation in SULT1A3 enzyme activity in an easily accessible human tissue—the blood platelet—was due, in large part, to inheritance [13,14]. In the course of subsequent studies designed to explore molecular genetic mechanism(s) that might contribute to individual variation in SULT1A3 activity, the gene was resequenced and functional genomic studies were performed [15]. That gene resequencing effort resulted in the identification of only a single nonsynonymous cSNP that was present in DNA from African-American subjects which resulted in a Lys234Asn alteration in encoded amino acid. Functional genomic studies showed that this amino acid change resulted in decreased levels of SULT1A3 activity and immunoreactive protein as a result of accelerated proteasome-dependent degradation. SULT1A3 consists of 7 coding and a series of alternatively spliced 5 0 -noncoding exons [16]. In the course of the Human Genome Project, various chromosome 16p ‘‘builds’’ included as many as 5 copies of SULT1A3. Because of the highly repetitive nature of the DNA sequence within this region of chromosome 16, the presence of multiple copies of SULT1A3 was thought to result from difficulty in contig assembly. However, following ‘‘completion’’ of the Human Genome Project on April 11, 2003, a BLAST search of chromosome 16
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performed with a portion of the SULT1A3 sequence showed two copies of the gene. One had been annotated as SULT1A3, while the other location, approximately 700 kb telomeric, was annotated as MGC5178. Analysis of the sequences of these two regions showed that 3 SULT1A3 5 0 -noncoding exons—1D, 1C, and 1B—overlapped with MGC5178 coding exons 3, 4, and 5 with 100% identity (Fig. 1A). These observations suggested that both SULT1A3 and MGC5178 might be duplicated. In the present study, we set out to test that hypothesis—as well as the possible functional implications of such a duplication. As a first step, global alignments of sequence containing the two genes were performed, as were PCR-based and fluorescence in situ hybridization (FISH) assays designed to verify the presence of the duplication. Those experiments confirmed that an intrachromosomal duplication had occurred. On the basis of advice obtained from the SULT Nomenclature Committee [17], we would suggest that the two SULT1A3 genes in these duplicated segments be named SULT1A3 (16p11.2, the gene currently annotated as SULT1A3) and SULT1A4 (16p12.1, the region currently annotated as MGC5178). Both of these genes encode identical SULT1A3 proteins. We then reanalyzed our previous resequencing data for SULT1A3 as well as resequencing data for the overlapping gene, MGC5178—with the knowledge that the sequence chromatograms contained data for 4 rather
Fig. 1. SULT1A3/1A4 and MGC5178 gene structures and chromosome 16p duplication. (A) SULT1A3/1A4 and MGC5178 structures. Exons unique to SULT1A3/1A4 are shown in grey; MGC5178 exons are black; and 3 exons shared by the two genes are shown as hatched boxes. Exon and intron lengths are based on sequence from NT_024812.10 (B) Chromosome 16p duplication. The 146 kb duplications containing SULT1A3 and 1A4 are 98.5% identical in nucleotide sequence, and the regions containing SULT1A3 and 1A4 genes are 99.8% identical over 10 kb. Bold arrows ( ) show locations of primers used to perform the duplication-specific PCR assays. Numbers in italics are the nucleotide positions on NT_024812.10.
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Fig. 2. SULT1A3/1A4 and MGC5178 polymorphisms. The figure shows a schematic representation of human SULT1A3/1A4 and MGC5178 with the location of SNPs indicated by arrows. Rectangles represent exons, with exons unique to SULT1A3/1A4 shown in grey and MGC5178 exons in black. Three exons that are shared by both genes are shown as hatched boxes. Exons 2–8 of SULT1A3/1A4 encode protein. ‘‘AA’’ represents data obtained with DNA from African-American subjects, and ‘‘CA’’ represents data obtained with DNA from Caucasian-American subjects. Black arrows represent previously reported polymorphisms [15], and open arrows indicate polymorphisms identified in the course of present study. When applicable, amino acid changes are also indicated.
than 2 alleles. When that was done, we were able to confirm the presence of all SULT1A3 polymorphisms that had been identified previously [15], but we also detected 11 additional SULT1A3/SULT1A4/MGC5178 SNPs, including 3 novel nonsynonymous cSNPs, C302T, Pro101Leu; C302A, Pro101His; and C430T, Arg144Cys (Fig. 2). Functional genomic studies were performed for all 3 of these novel SULT1A3/SULT1A4 nonsynonymous cSNPs, and reverse transcription-PCR showed that both SULT1A3 and SULT1A4 can be transcriptionally active. Future studies of the possible effects of inheritance on SULT1A3 will have to take the duplication of this gene into account. Materials and methods DNA samples and cell lines. DNA samples from 60 CaucasianAmerican (CA), 60 African-American (AA), 60 Han Chinese-American (HCA), and 60 Mexican-American (MA) subjects were obtained from the Coriell Cell Repository (Camden, NJ) (Sample Sets HD100CAU, HD100AA, HD100CHI, and HD100MEX). Lymphoblastoid cell lines (GM17227, GM17109, GM17135, GM17159, GM17734, GM17758, GM17453, and GM17458) were also obtained for selected samples from these same subjects. These samples had been collected and anonymized by the National Institute of General Medical Sciences (NIGMS). All subjects had provided written informed consent for the use of their DNA for research purposes, and the present studies were reviewed and approved by the Mayo Clinic Institutional Review Board. PCR assays to verify SULT1A3 duplication. PCR assays were developed to verify the duplication of SULT1A3 by taking advantage of the unique 5 0 - and 3 0 -flanking sequences for the 2 duplicated regions. Specifically, primers were designed within the 5 0 - and 3 0 -ends of the duplications. These ‘‘common’’ primers were paired with primers specific for the sequence flanking each of the duplications, so that only the regions where the duplications began or ended would be amplified—with different length amplicons for the two duplications (primer
sequences and locations on NT_024812.10 are listed in Table 1 of the ‘‘Supplementary Material’’). Locations of the primers are indicated schematically in Fig. 1B. The PCR was then used to amplify each of these regions with 240 DNA samples from four ethnic groups (CA, AA, MA, and HCA) as template. Amplicons were analyzed on 1% agarose gels and selected amplicons were sequenced in the Mayo Molecular Biology Core Facility to verify that the amplifications were specific for the region of interest. SULT1A3 fluorescence in situ hybridization. To verify the SULT 1A3 duplication, a direct labeled FISH probe was designed using a bacterial artificial chromosome (BAC) which covered the region containing SULT1A3. It was anticipated that this probe would also detect the duplicated region containing SULT1A4 because of the very high sequence identity of these two regions. Specifically, a glycerol stock of BAC clone RP11-455F5 (Accession No. AC012645) was obtained from ResGen Invitrogen Corporation (Carlsbad, CA). After PCR amplification of the region of interest, nick translation was performed using the Vysis Nick Translation Kit (Vysis, Downers Groove, IL) to produce a fluorescence labeled DNA probe for FISH. Signal validation was verified by observing both interphase and metaphase cells. Metaphase preparations of 7 Coriell Institute lymphoblastoid cell lines from the 4 ethnic groups studied (GM17227, GM17109, GM17135, GM17734, GM17758, GM17453, and GM17458) were analyzed using the SULT1A3 probe together with a commercially available direct labeled centromere 16 probe (Vysis, Downers Groove, IL). The SULT1A3-centromere 16 probe mixture was applied to each cell line, and standard FISH pre-treatment and hybridization protocols for peripheral blood were followed [18]. A minimum of 2 metaphase spreads and 10 interphase cells were studied for each cell line analyzed. SULT1A3/1A4 RT-PCR analysis. mRNA was isolated from two lymphoblastoid cell lines (GM17109 and GM17159) using the RNeasy kit (Qiagen, Valencia, Ca). cDNAs were generated from these mRNA preparations with random hexamer reverse primers using the ThermalScript RT kit from Invitrogen (Carlsbad, CA). The PCR was then used to amplify SULT1A3/1A4 cDNA using SULT1A3/1A4-specific primers that would not cross-react with SULT1A1 or SULT1A2. The sequences of these primers are listed in Table 1 of the ‘‘Supplementary Material’’. Amplicons were then cloned into pCR2.1 (Invitrogen) and the inserts were sequenced. SULT1A/1A43 and MGC5178 resequencing. SULT1A3 exons and splice junctions had been resequenced in the course of a previous study
M.A.T. Hildebrandt et al. / Biochemical and Biophysical Research Communications 321 (2004) 870–878 [15]. During the present study, the PCR was used to amplify all MGC5178 and SULT1A3/1A4 exons that had not been included in the previous study. In addition, approximately 500 bp of sequence immediately upstream of MGC5178, as well as all splice junctions and portions of introns flanking each exon, were resequenced using dye primer chemistry as described previously [15]. Primer sequences and annealing temperatures are listed in ‘‘Table 1 of the Supplementary Material’’. Amplicons were sequenced in the Mayo Molecular Biology Core Facility. To verify that newly identified SNPs were not PCRinduced artifacts, DNA samples that included those polymorphisms were amplified and amplicons were subcloned into pCR2.1. The inserts for up to 20 clones were then sequenced to confirm the presence of variant nucleotides. SULT1A3/1A4 variant allozyme COS-1 cell expression. Expression constructs for the 3 novel variant SULT1A3/1A4 allozymes, SULT 1A3/1A4*3 (Leu101), SULT1A3/1A4*4 (His101), and SULT1A3/ 1A4*5 (Cys144), were created by site-directed mutagenesis of the wild type (WT) SULT1A3/1A4*1 cDNA sequence in the expression vector pCR3.1 (Invitrogen). DNA sequences of the inserts were verified by dye terminator sequencing. Sequences of primers used to perform sitedirected mutagenesis are listed in Table 1 of the ‘‘Supplementary Material.’’ These expression constructs were used to transfect COS-1 cells that had been cotransfected with a b-galactosidase construct (Promega, Madison, WI) to make it possible to control for transfection efficiency. Six separate plates were transfected with each allozyme construct, and COS-1 cell lysate from these transfections was used to assay enzyme activity, to perform substrate kinetic studies and to perform Western blot analyses. SULT1A3/1A4 allozyme enzyme activity and Western blot analysis. Recombinant SULT1A3/1A4 allozymes *1, *3, *4, and *5—as well as cells transfected with an empty vector control—were assayed for SULT1A3 activity using a modification of the radiochemical enzymatic assay of Foldes and Meek [19] as described previously [15] with dopamine and [35S]3 0 -phosphoadenosine 5 0 -phosphosulfate (PAPS) as cosubstrates. Substrate kinetic studies were also conducted with variable concentrations of these two substrates. Western blot analysis of the recombinant allozymes was also performed as described previously [15]. Data analysis. The EMBOSS tool ‘‘stretcher’’ was used to align nucleotide sequences. This tool performs global pair-wise alignments over the entire length of the sequences being studied to show alignments and to highlight similarities. BLAST [20] was also used to perform sequence comparisons. Dye primer DNA sequence chromatograms for the gene resequencing were analyzed using the PolyPhred 4.0 [21] and Consed 8.0 [22] programs. GenBank Accession numbers for reference sequences used in these studies were: chromosome 16 reference contig, NT_024812.10; SULT1A3 cDNA RefSeqs, NM_003166.2 and NM_177552.1; and MGC5178 cDNA RefSeqs, NM_024044.2 and NM_178044.1. Apparent Km values for the enzyme kinetic studies were calculated with the method of Wilkinson [23] using a computer program developed by Cleland [24]. Points that deviated from linearity on double inverse plots as a result of substrate inhibition were not included in these calculations. Statistical significance of differences between mean values was determined by the use of StudentÕs t test.
Results Introduction These experiments had a series of goals—all related to enhancing understanding of the possible contribution of variation in genomic sequence and/or structure to SULT1A3 function. The initial studies involved characterization and verification of the chromosome 16p dupli-
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cation that resulted in two nearly identical copies of SULT1A3 and MGC5178. Once the existence of the duplication—and its presence in 4 separate ethnic groups— had been verified, that fact had implications for the analysis and interpretation of the SULT1A3 gene resequencing data. As a result, we reanalyzed both our previous and new gene resequencing chromatograms and identified and confirmed a series of additional SULT1A3/1A4 cSNPs, including 3 novel nonsynonymous cSNPs, as well as polymorphisms for MGC5178. Studies were also conducted of the possible functional implication of those cSNPs—as were experiments designed to determine whether both SULT1A3 and SULT1A4 were transcriptionally active. Characterization of the SULT1A3 gene duplication Analysis of chromosome 16p sequence from the final build of the Human Genome Project showed that portions of SULT1A3 overlapped with MGC5178, a gene of unknown function. The ‘‘overlap’’ occurred in SULT1A3 5 0 -noncoding exons 1D, 1C, and 1B and MGC5178 coding exons 3, 4, and 5 (Fig. 1A). The SULT1A3 5 0 -noncoding exons were identified by performing 5 0 -RACE with cDNAs isolated from human brain, colon, kidney, and small intestine as well as by EST database searches. Although SULT1A3 exon 1E (see Fig. 1A) is included in NCBI SULT1A3 cDNA Ref Seq NM_0031662, the gene annotation on NT_024812.10 shows only exons 1A, 1B, and 1C. However, even though SULT1A3 and MGC5178 overlapped, the two genes were annotated at locations separated by approximately 700 kb—raising the possibility that SULT1A3 might be part of a intrachromosomal duplication (see Fig. 1B). As a first step in characterizing this possible duplication, the EMBOSS tool ‘‘stretcher’’ was used to identify and compare the 2 duplicated areas. The duplications were found to be over 146 kb in length and were 98.5% identical in sequence, with only 663 nucleotide differences (0.45%) and 1603 gaps (1.1%). SULT1A3 was located between nucleotides 1,605,080 and 1,615,497 on reference contig NT_024812.10 while SULT1A4 was located 5 0 -upstream, between nucleotides 867,119 and 877,526. The regions containing the 2 copies of SULT1A3 were 99.8% identical in sequence, with 7 nucleotide changes and 8 gaps over a total length of 10 kb (Fig. 1B). The open reading frames (ORFs) for SULT1A3 and SULT1A4 were 99.9% identical, with only a single nucleotide change that did not alter the encoded amino acid. This nucleotide location had been reported to be polymorphic during our previous resequencing study [15]. The 146 kb duplicated region on 16p was also reported to contain 5 other genes— LOC348197, LOC348196, LOC339088, LOC339091, and LAT1-3TM—that were duplicated, plus one gene, CORO1A, that was only partially duplicated.
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Fig. 3. SULT1A3/1A4 FISH analysis. The figure shows a metaphase spread of a lymphoblastoid cell line with a chromosome 16-specific centromere probe shown in green and with two SULT1A3/1A4 pink signals on each copy of chromosome 16. The insets show a close-up view of a single chromosome (upper right) and an interphase cell (lower right), both of which show two pink signals.
To verify the existence of the duplication, a requirement in light of the difficulty that had occurred in assembling this portion of the human genome, two PCR assays were developed based on the unique 5 0 - and 3 0 flanking regions which bordered the duplicated regions. When those assays were performed using 240 individual DNA samples from 4 ethnic groups—60 CA, 60 AA, 60 HCA, and 60 MA—all samples had both copies of the duplicated area. As a further verification of the existence of the duplication, 7 lymphoblastoid cell lines from the same 4 ethnic groups were used to perform FISH. FISH analysis showed two distinct signals on the short arm of chromosome 16 for at least 2 metaphase spreads and 10 interphase spreads from each cell line in the presence of the SULT1A3 probe (Fig. 3). These observations verified the existence of a near-perfect duplication of SULT1A3 in all ethnic groups studied—immediately raising the question of the implications of this observation for previous pharmacogenetic studies—including the recently published SULT1A3 gene resequencing study [15]. SULT1A3/1A4 resequencing analysis Dye primer sequence chromatograms [25] from the present study (MGC5178) and from the previous SULT1A3/1A4 resequencing study were analyzed or reanalyzed with the knowledge that the chromatograms included data for 4 not 2 alleles. This analysis was performed to verify the SNPs reported previously and to identify any additional SNPs which might have been ‘‘missed’’ based on peak height criteria appropriate for 2, but not for 4 alleles. As anticipated, all of the previously reported SULT1A3/1A4 SNPs were confirmed, but 11 new polymorphisms were identified, including 3 novel nonsynonymous cSNPs in SULT1A3/1A4 (Fig. 2 and Table 1): Pro101Leu (C302T), Pro101His
(C302A), and Arg144Cys (C430T). The analysis also showed that one DNA sample appeared to contain 3 nucleotides at one position (C302T/A) (‘‘Supplementary Material’’ Fig. 1). All of the novel SULT1A3/1A4 SNPs as well as the MGC5178 SNPs, including the nonsynonymous cSNPs, were verified as described in Materials and methods. Because both SULT1A3 and SULT1A4 would have been amplified and sequenced simultaneously during the resequencing studies, it was not possible to determine whether any individual polymorphism was present within SULT1A3 or SULT1A4. In addition, unequivocal determinations of allele frequencies could not be made and haplotype and linkage analysis could not be performed for these polymorphisms because it was not possible to determine which copy of the gene contained the polymorphism. Therefore, we have not included haplotype or linkage disequilibrium analyses, and the allele frequencies listed in Table 1 should be considered as estimates. However, our discovery of novel SULT1A3/ 1A4 nonsynonymous cSNPs provided an opportunity to determine whether these changes in amino acid sequence might influence function and also to determine whether SULT1A3 might be transcriptionally active in each of the duplicated areas. Novel SULT1A3/1A4 nonsynonymous cSNP functional genomics Expression constructs were created for SULT1A3/ 1A4*1 (WT), SULT1A3/1A4*3 (Leu101), SULT1A3/ 1A4*4 (His101), and SULT1A3/1A4*5 (Cys144) to make it possible to study the functional effects of the newly identified polymorphisms. Recombinant protein for the variant allozymes after transient expression in COS-1 cells was assayed for level of activity with dopamine as the substrate. Two of the variant allozymes— Leu101 (*3) and His101 (*4)—showed significant decreases in enzyme activity, while the level of activity for the Cys144 (*5) allozyme was slightly, but not significantly, elevated when compared with that for the WT (*1) allozyme. The Pro101Leu polymorphism resulted in a decrease in activity of approximately 87% and the Pro101His amino acid change resulted in a reduction of approximately 40% (Table 2 and Fig. 4A). One mechanism which might result in a decrease in enzyme activity as a result of a change in encoded amino acid would be an alteration in substrate kinetics. Therefore, we also determined apparent Km values for the WT and variant allozymes using both PAPS and dopamine as substrates. The apparent Km values with PAPS for the His101 (*4) and Cys144 (*5) polymorphisms were significantly different from that for the WT, although these differences were probably not large enough to be of physiological significance (Table 2). Another possible mechanism which could explain the decreased enzyme activity was
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Table 1 Human SULT1A3/1A4 and MGC5178 polymorphisms
The table lists the 11 newly identified SULT1A3/1A4 and MGC5178 polymorphisms in bold, with the previously reported polymorphisms in italics [15]. The estimated allele frequencies for each population are listed based on peak height ratios from dye primer sequencing for 2 alleles (previous study [15]) 4 alleles (present study). Polymorphisms located in exons are boxed. The numbering scheme for polymorphism locations is based on the cDNA sequence, with the ‘‘A’’ in the translation initiation codon designated (+1). Positions 5 0 and 3 0 to that location were assigned negative or positive numbers, respectively. Locations in introns were numbered from the intron–exon splice junctions, with ‘‘I’’ used to designate intron locations. Table 2 SULT1A3/1A4 functional genomics Allozyme
Polymorphism
SULT1A3 Activity, % of WT
Immunoreactive protein, % of WT
Dopamine Km (lM)
PAPS Km (lM)
SULT1A3/1A4*1 SULT1A3/1A4*3 SULT1A3/1A4*4 SULT1A3/1A4*5
WT Pro101Leu Pro101His Arg144Cys
100 ± 3.8 12.7 ± 2.0** 59.2 ± 6.2** 110 ± 4.8
100 ± 7.8 42.1 ± 3.6** 85.5 ± 2.8 120 ± 12
8.3 ± 0.6 7.2 ± 0.2 9.7 ± 1.4 8.8 ± 0.5
0.19 ± 0.003 0.14 ± 0.03 0.11 ± 0.01* 0.15 ± 0.004**
Average values for levels of activity (N = 6), immunoreactive protein (N = 3), and apparent Km values for dopamine and PAPS (N = 6) are listed. All values are means ± SEM. *P < 0.05 and **P < 0.001 when compared with the WT allozyme.
a decrease in protein quantity [15,30–35]. Quantitative Western blot analysis, corrected for transfection efficiency, showed a significant decrease in immunoreactive protein of approximately 60% for the Leu101 (*3) allozyme (Table 2 and Fig. 4B). There was also a slight decrease for the His101 (*4) and a slight increase for the Arg144 (*5) protein levels when compared with WT, but neither was statistically significant.
RT-PCR of SULT1A3/1A4 The final question that we asked was whether both SULT1A3 and SULT1A4 might be transcribed. We were fortunate that SULT1A3/1A4 is expressed in lymphoblastoid cell lines such as those created by the Coriell Institute for the DNA samples used in our experiments (Dr. V. Cheung, personal communication). Therefore,
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Fig. 4. SULT1A3/1A4 allozyme COS-1 cell expression. (A) Average levels of enzyme activity are shown for each of the recombinant SULT1A3/1A4 allozymes studied—*1, *3, *4, and *5—with dopamine as the sulfate acceptor substrate. Each bar represents the average value for 6 independent transfections (mean ± SEM), and all values have been corrected for transfection efficiency. **P < 0.001 when compared with the WT allozyme SULT1A3/1A4*1. (B) Average levels of SULT1A3/1A4 immunoreactive protein for each of the recombinant allozymes, expressed as a percentage of the level of SULT1A3/1A4*1 protein. Each bar represents the average of 3 independent transfections (mean ± SEM). The gels were loaded on the basis of b-galactosidase activity to correct for transfection efficiency. **P < 0.001 when compared with the value for SULT1A3/1A4*1.
reverse transcription-PCR was performed with mRNA from the lymphoblastoid cell line (GM17159) that corresponded to the DNA sample with three alleles at nucleotide 302 (codon 101) (‘‘Supplementary Material’’ Fig. 1). All three nucleotides were detected in cDNAs generated from this cell line, indicating that three distinct SULT1A3/1A4 mRNA species were present in the cell (‘‘Supplementary Material’’ Fig. 2). Furthermore, cDNAs from an additional cell line (GM17109), corresponding to a DNA sample that contained multiple SULT1A3/1A4 cSNPs, were also subjected to RTPCR. This cell line expressed mRNA species with 3 SULT1A3/1A4 haplotypes. These observations were all compatible with the conclusion that both SULT1A3 and SULT1A4 were transcribed in these lymphoblastoid cell lines.
Discussion SULT1A3 catalyzes the sulfation of dopamine as well as other catecholamines and is the only known SULT
isoform with a high affinity for these substrates [3]. This metabolic pathway for catecholamines is important, with over 95% of circulating dopamine in humans being sulfate conjugated [12]. Because of the importance of catecholamines and drugs related to catecholamines in human physiology, human pathophysiology, and the treatment of human disease, it is important that the possible contribution of inheritance to individual variation in this metabolic pathway be understood. The human SULT1A3 gene maps to 16p, a region that is highly repetitive [26,27]. During the assembly of chromosome 16p by the Human Genome Project, it was reported that as many as five copies of SULT1A3 were present. At that time, it was believed that these observations were most likely due to difficulty in contig assembly. The ‘‘completed’’ Human Genome Project has mapped SULT1A3 to 16p11.2, but a nearly identical copy of the gene is located at 16p12.1—a region currently annotated as MGC5178. Both copies of SULT1A3 overlap with 3 MGC5178 exons. In the present studies, we have confirmed the SULT1A3 gene duplication by performing duplication-specific PCR assays as well as FISH—in all cases using DNA from 4 ethnic groups. The duplicated regions were over 146 kb in length and 98.5% identical in sequence. Furthermore, the 10 kb portions of the duplications containing SULT1A3 and SULT1A4 were 99.9% identical in nucleotide sequence and encoded identical enzymes. We had previously resequenced SULT1A3/1A4 as part of a series of studies of human SULT pharmacogenetics [5,14,15,28–32]. However, analysis of those data was based on the assumption of a single copy of SULT1A3 with 2 alleles—and not a 2-gene, 4-allele system. To more accurately characterize genetic variation in SULT1A3 and SULT1A4, we have now subjected the original dye primer sequencing data, as well as additional resequencing that included MGC5178, to re-analysis based on an assumption of 4 rather than 2 alleles. That analysis resulted in the identification of 11 additional polymorphisms, including 3 novel SULT1A3/1A4 nonsynonymous cSNPs. The possible functional effects of these changes in amino acid sequence were determined by performing functional genomic studies designed to determine levels of activity, immunoreactive protein, and kinetic parameters for these new allozymes after expression in COS-1 cells. It is essential that functional genomic studies such as these be performed in mammalian cells since we have shown the need for mammalian protein degradation systems for accurate results [36], and we and others have consistently found that genotype– phenotype correlations observed in studies of this type correlate with in vivo observations [33,34,37]. Finally, based on RT-PCR data, both SULT1A3 and SULT1A4 appear to be transcriptionally active, although it remains to be determined whether both copies of the gene are regulated in a similar fashion.
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In summary, the presence in the genome of two copies of SULT1A3 may help to explain a portion of the individual variation in SULT1A3 activity in humans. It will be necessary that the SULT1A3/1A4 duplication be understood in order to interpret past or future studies of the pharmacogenetics of this important catecholamine and drug-metabolizing enzyme in humans.
Acknowledgments We thank Luanne Wussow for her assistance with the preparation of the manuscript; Bianca Thomae and Bruce Eckloff for generating the SULT1A3/1A4 and MGC5178 dye primer sequence chromatograms; and Dr. Vivien Cheung from the University of Pennsylvania for sharing her unpublished SULT1A3/1A4 lymphoblastoid cell line expression data.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2004.07.038.
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