The Pharmacogenomics Journal (2007) 7, 133–143 & 2007 Nature Publishing Group All rights reserved 1470-269X/07 $30.00 www.nature.com/tpj ORIGINAL ARTICLE Genetic diversity and function in the human cytosolic sulfotransferases MAT Hildebrandt1, Amino-acid substitutions, which result from common nonsynonymous (NS) 2 1 polymorphisms, may dramatically alter the function of the encoded protein. DP Carrington , BA Thomae , Gaining insight into how these substitutions alter function is a step toward 3 2 BW Eckloff , DJ Schaid , acquiring predictability. In this study, we incorporated gene resequencing, VC Yee4, RM Weinshilboum1 functional genomics, amino-acid characterization and crystal structure and ED Wieben3 analysis for the cytosolic sulfotransferases (SULTs) to attempt to gain predictability regarding the function of variant allozymes. Previously, four 1Division of Clinical Pharmacology, Department SULT genes were resequenced in 118 DNA samples. With additional of Molecular Pharmacology and Experimental resequencing of the remaining eight SULT family members in the same Therapeutics, Mayo Clinic College of Medicine, DNA samples, a total of 217 polymorphisms were revealed. Of 64 Mayo Foundation, Rochester, MN, USA; 2Department of Health Sciences Research, Mayo polymorphisms identified within 8785 bp of coding regions from SULT Clinic College of Medicine, Mayo Foundation, genes examined, 25 were synonymous and 39 were NS. Overall, the Rochester, MN, USA; 3Department of proportion of synonymous changes was greater than expected from a Biochemistry and Molecular Biology, Mayo Clinic random distribution of mutations, suggesting the presence of a selective College of Medicine, Mayo Foundation, Rochester, MN, USA; 4Department of pressure against amino-acid substitutions. Functional data for common Biochemistry, Case Western Reserve University variants of five SULT genes have been previously published. These data, School of Medicine, Cleveland, OH, USA together with the SULT1A1 variant allozyme data presented in this paper, showed that the major mechanism by which amino acid changes altered Correspondence: function in a transient expression system was through decreases in Dr ED Wieben, Department of Biochemistry and Molecular Biology, Mayo Clinic College of immunoreactive protein rather than changes in enzyme kinetics. Additional Medicine, Mayo Foundation, Rochester, MN insight with regard to mechanisms by which NS single nucleotide 55985, USA. E-mail: [email protected] polymorphisms alter function was sought by analysis of evolutionary conservation, physicochemical properties of the amino-acid substitutions and crystal structure analysis. Neither individual amino-acid characteristics nor structural models were able to accurately and reliably predict the function of variant allozymes. These results suggest that common amino-acid substitutions may not dramatically alter the protein structure, but affect interactions with the cellular environment that are currently not well understood. The Pharmacogenomics Journal (2007) 7, 133–143. doi:10.1038/sj.tpj.6500404; published online 27 June 2006 Keywords: amino-acid substitutions; functional genomics; single nucleotide polymorphisms Introduction The human genome contains an estimated 11 million common single nucleotide changes with allele frequencies greater than 1%.1 A vast majority of these sequence variations are believed to be functionally neutral, yet a subset alter the Received 5 January 2006; revised 28 April 2006; accepted 24 May 2006; published structure of a gene product. Such structural changes are often detrimental to online 27 June 2006 function, but some variant proteins have normal, or even increased, function. Predicting the function of amino-acid substitutions MAT Hildebrandt et al 134 With the goals of better understanding of how molecular 11 evolution influences the function of variant allozymes and 6 how specific substitutions affect protein function, we have et al. incorporated resequencing data, functional genomics, ami- et al. no-acid characterization and crystal structure analysis to Reference analyze structure–function relationships within the human cytosolic sulfotransferase (SULT) gene family. The members of the SULT gene family play an important D 1.331.06 Thomae Current study 0.043 Current study 0.303 Current1.16 study 0.214 Current study Current1.18 study Adjei À À À À À À À role in the biotransformation of a variety of substrates, Tajima’s including steroid hormones, neurotransmitters, drugs and other xenobiotics.2–4 To date, 12 human SULT genes have p been identified.5 The family members share a high degree of sequence identity and are found in clusters on chromo- somes.5 Because of the importance of sulfation in the metabolism of a wide variety of substrates, much work has y been carried out in characterizing the genetic variation and functional consequences of amino-acid substitutions in the SULT family6–13 and crystal structures have been solved for nine of the 12 members.14–22 Previously, four SULT genes – SULT1A3, 1A4, 1E1 and 2A1 Non-coding – were resequenced from 59 Caucasian-American (CA) and polymorphisms 59 African-American (AA) DNA samples.6,8,11,12 To deter- mine the nature and degree of sequence variation within the entire gene family for the analyses reported here, the S remaining members of SULT gene family – SULT1A1, 1A2, 1B1, 1C2, 1C4, 2B1, 4A1 and 6B1 – were resequenced in the polymorphisms same DNA samples (Table 1). In total, over 8.5 million base pairs of sequence were analyzed from regions on six chromosomes. Functional genomic studies have been completed for 14 NS nonsynonymous (NS) single nucleotide polymorphism (SNPs) as a step toward understanding how variation in polymorphisms nucleotide sequence translates into variation in enzyme function in the SULT gene family.6-9,11,12 These previous studies showed that alterations in amino-acid sequence can cause decreased enzyme activity owing to decreased levels of immunoreactive protein. In this study, additional functional Coding genomic studies were performed for three variant allozymes polymorphisms of SULT1A1 to determine the functional consequences of the amino-acid substitutions in a recombinant expression system. Previous genotype–phenotype correlation studies in both human liver and platelet samples showed that one common variant allele, SULT1A1*2 (Arg213His), was Frequency associated with low enzyme activity.9,10 The present work extends this analysis to additional SULT1A1 variants and (polymorphism/bp) presents new data on levels of immunoreactive proteins for all SULT1A1 variants. The SULT gene family is an ideal candidate for studying the relationship between sequence variation, structure and function by combining functional genomic data with amino-acid characterization and structural modeling. Un- polymorphisms derstanding of the consequences of amino-acid changes may aid in characterizing the mechanisms responsible for 2417313335585298 15 22 5 1/161.1 36 1/142.4 1/711.6 1/147.2 6 12 0 11 3 5 10 0 3 7 1 0 9 10 25 5 10.28 11.10 11.25 4.82 6.68 11.08 2.33 2.40 0.060 Current study decreases in enzyme function and ultimately gaining 2430401036084423 213734 363648 17 1/115.7 29 1/111.4 14 1/212.2 22 1/152.5 1/266.7 1/165.8 10 10 2 7 1 5 3 7 2 5 1 3 7 3 0 2 0 11 2 26 15 14.31 22 14.87 18.60 13 18.07 17 7.80 0.794 10.86 Current 0.613 study 6.88 6.21 Current 6.33 study 9.99 5.67 5.59 predictability regarding these effects. Our results suggest Summary of SULT gene sequence variation that commonly used methods for predicting the effect of amino-acid substitutions on protein function have only Table 1 SULT2A1 SULT2B1 SULT4A1 SULT6B1 NS, nonsynonymous; S, synonymous; SULT, sulfotransferase. limited utility when applied to SULT allozymes and better Gene Total bpSULT1A1 SULT1A2 SULT1B1 Total SULT1C2 SULT1C4 SULT1E1 The Pharmacogenomics Journal Predicting the function of amino-acid substitutions MAT Hildebrandt et al 135 ‘tools’ are needed in order to predict how variation at the polymorphisms – an average of one polymorphism every nucleotide level translates into altered function of variant 712 bp. All of SULT4A1 polymorphisms are located in allozymes within the cellular environment. intronic regions (Table 1). The extent of nucleotide variation found in members of the SULT gene family was similar to 23–27 Results that observed for other groups of human genes. Genetic variation in the SULT gene family Genetic diversity across gene regions All human SULT genes were resequenced in DNA samples Neutral theory predicts that nucleotide changes occur from AA and CA populations (236 alleles total) to catalog randomly throughout the genome with no selective pres- the genetic diversity within this gene family. A total of sure as to location.28,29 In general, this appeared to hold 36 259 bp were amplified from the human SULT genes in true for the regions of the SULT genes analyzed, with the each DNA sample, including 8785 bp (24%) of coding and proportion of base pairs sequenced roughly equaling the 27 474 bp (76%) of non-coding sequences (Table 2). Se- proportion of polymorphisms identified for the total quence analysis identified 212 SNPs and five insertion and population (Figure 1) (w2 ¼ 9.13, df ¼ 5, P ¼ 0.104). The deletions with an overall minor allele frequency (MAF) of notable deviation from this pattern was for nucleotide 0.102. Of the 217 polymorphisms identified, 64 (29.5%) changes within the coding region of the genes. Although were located in coding regions and 153 (70.5%) were non- 80% of the coding region nucleotides were NS sites where coding sequence variants (Table 2). On average, one nucleotide changes have the potential to change the protein polymorphism was identified every 167 bp of total se- sequence, only 61% of the observed polymorphisms in the quence. The most polymorphic SULT gene was SULT1A2, coding region actually did lead to a change in the encoded with an average of one nucleotide change per every 111 bp amino acid. This excess of synonymous (S) changes screened.