Genomics 84 (2004) 707–714 www.elsevier.com/locate/ygeno

Characterization of a common deletion polymorphism of the UGT2B17 linked to UGT2B15 $

Willie Wilson IIIa, Fernando Pardo-Manuel de Villenab, Beverly D. Lyn-Cookc, Pradeep K. Chatterjeed, Timothy A. Bellb, David A. Detwilerb, Rodney C. Gilmorea, Isis C. Valladerasa, Camille C. Wrighta, David W. Threadgillb, Delores J. Granta,*

aCancer Research Program, JLC-Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA bDepartment of Genetics, Lineberger Comprehensive Cancer Center and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA cDivision of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, AK, USA dGenomics Research Program, JLC-Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA Received 20 February 2004; accepted 23 June 2004 Available online 3 August 2004

Abstract

Members of the human UDP- 2B family are located in a cluster on 4q13 and code for whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two , it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians. D 2004 Elsevier Inc. All rights reserved.

Keywords: UDP Glucuronosyltransferase; Gene deletion; Polymorphisms (genetics); Duplicate genes; Genetic variation; Genetic diversity

Introduction identified in humans: UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28 [2–5].As Based on sequence similarity and evolutionary divergence, part of the phase II liver detoxification system, these genes are the human UDP- (EC 2.4.1.17) have responsible for maintaining steady-state levels of steroid been subdivided into two families (UGT1 and UGT2) and four hormones and other endogenous substances by catalyzing subfamilies (UGT1A, UGT1B, UGT2A, and UGT2B) [1]. the transfer of glucuronic acid moieties to these molecules and Seven cDNAs from the UGT2B subfamily have been rendering them hydrophilic [6]. This reaction, glucuronidation, facilitates the excretion of steroid compounds into the circulatory system and their elimination from the body through $ SNPs data from this article have been deposited with NCBI dbSNP bile and urine [7]. UGT2B17 and UGT2B15 both bind under Accession No. 23140548 and 23140172. * Corresponding author. JLC-Biomedical/Biotechnology Research testosterone and 5-a reductase metabolites in liver and Institute, 1801 Fayetteville Street, Durham, NC 27707. Fax: +1 919 530 7998. extrahepatic tissue such as the prostate, testis, breast, lung, E-mail address: [email protected] (D.J. Grant). placenta, skin, uterus, kidney, and adrenal gland [8–10]. Their

0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.06.011 708 W. Wilson III et al. / Genomics 84 (2004) 707–714 expression in steroid target tissues such as the breast, uterus, the SNP Discovery Repository was screened with and prostate is suggestive of a role in hormonally influenced UGT2B17-and UGT2B15-specific primers from exon 1 diseases. designated markers C and G, respectively. Fig. 1 and UGT2B17 cDNA shares over 95% sequence identity Table 1 show the locations and sequence of the primers with UGT2B15 cDNA and similar substrate specificity [7]. on the genomic BAC clones that are annotated in the While both are reactive with androgen metabolites NCBI database and positioned so that gene orientation on at the 17h position, UGT2B17 conjugates at both the 17h both BACs is the same. The PCR amplification of and the 3a positions due to a single amino acid change that UGT2B15-specific marker G generated the expected causes a broader range of substrate specificity [8,11]. The 329-bp product from all 40 individual DNA samples high level of sequence identity between UGT2B15 and (Fig. 2A). Unexpectedly, amplification of the UGT2B17- UGT2B17 suggests that the genes may have arisen from a specific marker C generated the predicted 316-bp product duplication event [2,7]. The high level of identity and tight from only 31 samples and failed to amplify from the physical clustering of the known UGT2B subfamily genes remaining 9 samples (Fig. 2B). PCR products from within 1.0 Mb on chromosome 4q13.2 indicates the markers C and G were sequenced and aligned to confirm presence of multiple gene duplication events. specificity for UGT2B17 and UGT2B15, respectively We used gene-specific markers to characterize the (Fig. 2C). C and G marker specificity was further genomic relationship between UGT2B15 and UGT2B17and validated by sequence alignments of PCR products with to investigate genetic variation at this locus in different ethnic that of marker I, revealing the same sequence differences groups. Our results show for the first time that UGT2B17 (Fig. 2C). occurs as a deletion polymorphism that is linked to UGT2B15 The lack of amplification and the sequence differences and differs in its frequency between two ethnic groups. suggest the existence of a deletion spanning UGT2B17 in some human samples. To test this hypothesis the same DNA panel was screened with two additional markers, Results marker D, WIAF-2471-STS, and marker E (exon 6) (Fig. 1). The ability to amplify both markers was concordant Identification of a UGT2B17 polymorphism with the amplification of marker C. Markers that fail to amplify span 64 kb from WIAF-2471-STS (marker D), To determine the genomic organization of UGT2B17 located over 30 kb upstream of exon 1, to marker E, and UGT2B15, a human DNA panel of 40 samples from located in exon 6 of UGT2B17; markers downstream of

Fig. 1. Gene-specific marker locations. Portions of NCBI annotated BACs RP11-597F18 and RP11-185H6 are shown so that the genes are oriented in the same direction. Gene-specific marker locations are indicated and abbreviated ends are indicated by // marks. A sequence gap is indicated as designated in the University of California at Santa Cruz Genome Database. UGT2B17-specific markers A–E were derived from BAC clone RP11-597F18, while UGT2B15- specific markers F–H were designed from RP11-185H6. Markers C and G map to the exon 1 regions of UGT2B17 and UGT2B15, respectively. Markers D and H were located upstream of the transcriptional start sites of UGT2B17 and UGT2B15, respectively. The locations of markers A and B are within the intron 3 regions of UGT2B17, while marker F is located within the intron 3 region of UGT2B15. Marker E is located within exon 6 of UGT2B17. *Amplification of markers B and F was performed with a single primer pair and distinguished on the basis of fragment size (marker B 640 bp, marker F 706 bp). **Marker I maps to the exon 1 regions of both UGT2B15 and UGT2B17. Marker J amplification was used to assay for the presence or absence of the genomic interval containing UGT2B17. W. Wilson III et al. / Genomics 84 (2004) 707–714 709

Fig. 2. Gene-specific amplification of UGT2B15 and UGT2B17. (A) UGT2B15-specific marker G and (B) UGT2B17-specific marker C were amplified in a panel of 40 unrelated, genetically diverse human DNA samples. Amplification of marker G was observed in all 40 samples, while only 31 of 40 samples amplified marker C. Arrows indicate the 9 samples that failed to amplify marker C. (C) Sequence alignments between markers G and C were performed to illustrate primer specificity for each marker. Alignment between both markers reveals 17 misaligned bases with an overall 93% sequence similarity. Marker I represents amplification of both UGT2B15 and UGT2B17. The sequences from markers G and C were equally represented in the sequence of marker I (arrows indicate four representative misaligned bases). exon 6 amplify from all individuals. These results suggest The results for Pedigree 13291 are summarized in Fig. that there is a deletion polymorphism spanning 3B. The father and his seven offspring (F, 1, 2, 3, 4, 5, 6, UGT2B17. and 7) showed amplification with UGT2B17-specific markers A through E, while the mother and maternal Segregation analysis of UGT2B15/UGT2B17 markers in grandparents did not amplify any of these markers. All CEPH families samples amplified UGT2B15-specific markers F through H. The maternal grandmother was heterozygous for both To determine the segregation patterns of UGT2B17 and markers F and H, while the mother and father were UGT2B15, PCR was performed on genomic DNA samples heterozygous for marker H. The presence of amplification from two CEPH families (Pedigrees 1344 and 13291) using for all UGT2B17-specific markers (A–E) was concordant in seven primer pairs described in Table 1 and Fig. 1.In individuals from both pedigrees. addition to the deletion polymorphism detected by An analysis of marker segregation was performed in UGT2B17-specific primers, two UGT2B15-specific markers both pedigrees (Fig. 4).Only the mother in Pedigree 1344 (F and H, Figs. 1 and 3) were polymorphic. (Fig. 4A) was informative for the segregation of the The results for Pedigree 1344 are summarized in Fig. UGT2B17 deletion polymorphism; she is heterozygous 3A. The mother and three offspring (M, 1, 4, and 8) for a UGT2B15 polymorphism in marker F and for the first showed amplification with UGT2B17-specific markers A SNP identified within marker H (see below). Three through E, while the father, his parents, and remaining six offspring inherited UGT2B17and allele 2 at marker F and offspring (F, GM, GF, 2, 3, 5, 6, 7, and 9) failed to a G at all of the alleles for the SNPs from their mother, amplify these five markers. As with the previous samples, while six offspring inherited allele 1 at marker F and the A all individuals amplified with UGT2B15-specific markers allele at SNP3 but not UGT2B17. The predicted phase of F through H. The paternal grandmother was heterozygous the alleles is shown in Fig. 4. Segregation analysis indicates for marker H, while the mother was heterozygous for that UGT2B17 and UGT2B15 are closely linked (LOD = marker F. 2.41, u = 0). 710 W. Wilson III et al. / Genomics 84 (2004) 707–714

In Pedigree 13291 (Fig. 4B), the segregation of UGT2B17 is not informative in either parent. However, both parents are 271 (bp) 200 327 580 136 884 329 640 316 706 heterozygous for two SNPs in marker H and transmit the alleles according to Mendelian expectations. Importantly, UGT2B17 is observed in all offspring independent of the

V paternal UGT2B15 allele inherited, indicating that the father was homozygous for the presence of UGT2B17.We

V conclude from these analyses that the UGT2B17 deletion V

V polymorphism is closely linked to UGT2B15andthat

V individuals with two, one, or zero copies of UGT2B17 exist. V V V V V

Allele frequency of the UGT2B17 deletion polymorphism

Successful amplification with marker C identifies the presence of the UGT2B17 gene. To test reliably the deletion of UGT2B17 in different ethnic groups, we developed a second PCR assay that spans the putative breakpoint of the deletion (marker J). Successful amplification of a 1.2-kb product with this assay identifies with the -GATTGTAGGTATATTCTATAGATTAG-3 -ACAGGCAACATTTTGTGATC-3 -GACTTCAAGTGTATTCTGCATACTAC-3 -CTGGTCCCACTTCTTCAGAT-3 -GATCATCCTATATCCTGACAGAATTCTTTTG-3 -CCCAACAAAGACTATGCTCT-3 -CTGCAGAATATGTCAATAATTGGC-3 -CCCAACAAAGACTATGCTCT-3 -CTGCATCTTCACAGAGCTTT-3 -CTGCCAGAATGACATCAAAC-3 deletion. Chromosomes lacking the deletion fail to amplify V V V V V V V V V V 5 5 5 5 5 5 5 5 5 5 marker J due to the presence of an 150-kb interval spanning UGT2B17. The assay for marker J was validated in the two pedigrees. All individuals amplify marker J except the father

V in Pedigree 13291, which is the only individual homozy- gous for the presence of the UGT2B17. The combination of V V both assays allows the unambiguous genotyping of the deletion polymorphism. V V V V We screened 85 African Americans and 103 Caucasians V V V for markers C and J, which specifically recognize the presence or absence of the UGT2B17 gene, respectively. The results in Table 2 show that the distribution of genotypes is significantly different between these two ethnic groups (Ho, test for independence between genotype and ethnic background;m2 = 7.41, 2 df, p b 0.05). The absence of the UGT2B17 gene was five times more frequent in Caucasians than in African Americans (11 and 2%, respectively). The deletion polymorphism is at Hardy– -CCCACCTAGCCAAGTAGATTTAGAG-3 -CCTGGAAGAGCTTGTTCAGA-3 -TGCACAGAGTTAAGAAATGGAGAGATGTG-3 -GAATTCATCATGATCAACCG-3 -CCACTTGTCTACTAGTCATA-3 -CCACTTGTCTACTAGTCATA-3 -CCCACCTAGCCAAGTAGATTTAGAG-3 -CCTGGAAGAGCTTGTTCAGA-3 -TCACAAGTCAATCTCCCATCC-3 -CCTGGAAGAGCTTGTTCAGA-3 V V V V V V V V V V Weinberg equilibrium in both populations (Table 2). 5 5 5

Discussion

Here we report that DNA spanning the UGT2B17 gene is of V V V absent in 9 of 40 human DNA samples, while all samples Intron 3 5 Exon 13 5 UGT2B15 Exon 6 5 Intron 3 5 Intron 3 5 5 Upstream Exon 15 Upstream 5 Exon 1 5 contain the UGT2B15 gene. Segregation analysis demon- strates that UGT2B15 and UGT2B17 are linked and that the absence of UGT2B17 in some individuals is the result of a deletion polymorphism. Therefore, these genes are distinct and inherited in Mendelian fashion. The deletion poly- SpecificityUGT2B17 Location Forward primer Reverse primer Amplicon UGT2B15/ UGT2B17 UGT2B17 deletion UGT2B17 UGT2B17 UGT2B15 UGT2B15 UGT2B17 UGT2B17 UGT2B15 morphism is common in humans (deletion allele frequency = 0.27) and the allele frequency varies between two ethnic groups, African Americans and Caucasians (deletion allele frequencies are 0.21 and 0.33, respectively; Table 2). a a The UGT2B gene cluster on chromosome 4q13 appears to be the site of numerous duplication events that may have Primers for markers B and F are identical. Marker B Table 1 Gene-specific markers Marker A a Marker F Marker I Marker J Marker E Marker H Marker C Marker D Marker G led to intergene redundancy and neofunctionalization. Work W. Wilson III et al. / Genomics 84 (2004) 707–714 711

Fig. 3. CEPH family marker amplification. Amplifications of markers A–E were used to genotype CEPH families A and B for UGT2B15 and UGT2B17. (A) Family A (CEPH/UTAH Pedigree 1344): paternal grandfather (GM), NA12056; paternal grandmother (GF), NA12057; father (F), NA10851; mother (M), NA10850; and offspring (1–9; d, daughter; s, son), NA12047 (d), NA12048 (d), NA12049 (d), NA12050 (s), NA12051 (s), NA12052 (s), NA12053 (d), NA12054 (d), and NA12055 (s). (B) Family B (CEPH/UTAH Pedigree 13291): maternal grandmother, NA07045; maternal grandfather, NA06986; father, NA06995; mother, NA06997; and offspring (1–7), NA07018 (s), NA07036 (s), NA06981 (d), NA06980 (d), NA07047 (s), NA07433 (s), and NA07058 (s). Agarose gel electrophoresis was performed for markers C, D, E, and G. SSCP polyacrylamide gel electrophoresis was performed for markers A, B, F, and H. Amplification results for markers A–E are indicated by + (amplification) and À (no amplification). Amplifications of marker F are designated 1 (top band) and 2 (lower band). Amplifications of marker H were analyzed by DNA sequence analysis and the haplotypes of two SNPs (SNP3, G to A, and SNP4, G to A) are indicated. by Riedy and co-workers [2] showed that there are amino acid change has been reported [16]. The poly- numerous pseudogenes, orphan exons, and related sequen- morphism leads to proteins with different levels of ces surrounding UGT2B4 and UGT2B7. UGT2B15 and enzymatic activity and is a possible risk factor for prostate UGT2B17 probably arose from a tandem duplication event cancer [16,17]. Population studies have shown that this by unequal recombination. Comparative sequence analysis polymorphism has different distributions among various suggests that this duplication is fairly ancient. Within exons ethnic groups [2,18]. Notably Asians have a higher the sequence identity between the genes is only 95%, while prevalence for the D85 homozygous genotype compared average sequence identity genome-wide between humans to Caucasians and about half the proportion of Y85 and chimpanzees is 98.8% [12–14]. Therefore, the duplica- homozygotes compared to Caucasians [18]. Small studies tion should predate the divergence of the human and have been done to determine whether the polymorphism is chimpanzee lineages and is likely to be present in other associated with prostate cancer. Genotyping of DNA primates. The different levels of sequence conservation samples from a case–control group (64 cases and 64 between introns (approximately 91%) and exons indicate controls) revealed that prostate cancer patients were that both genes have been under positive selection. The significantly more likely to be homozygous for the lower deletion polymorphism described here represents an addi- activity D85 UGT2B15 allele than controls [19]. Similar tional and more recent genomic rearrangement in the results were obtained in another study with a larger case– UGT2B cluster. The deletion encompasses 150 kb and control group (155 cases and 155 controls) in which a probably also originated by unequal recombination. The significant association was found between D85 homozygo- allele frequency of the deletion and its presence in two sity and increased risk of prostate cancer [20]. However, in a ethnic groups suggests that it arose early in the human- third study, the D85 polymorphism was not associated with specific lineage. prostate cancer risk (190 cases and 190 controls) [21]. We have identified two SNPs in the promoter region of Unlike the first two studies, in the third study the controls UGT2B15 and discovered that most of the UGT2B15 SNPs were patients with benign prostatic hyperplasia, which may currently available in public databases are in fact paralogous have confounded the association. Additionally, Murata et al. sequence variants [15]. These SNPs were validated by [22] have shown that the absence of UGT2B17 alters segregation analysis and resequencing. In addition to these immunogenicity in the ’s role as a human minor polymorphisms, a SNP in UGT2B15 resulting in a D-Y histocompatibility antigen. 712 W. Wilson III et al. / Genomics 84 (2004) 707–714

Fig. 4. UGT2B15 and UGT2B17 inheritance. Marker amplifications from Fig. 3 were used to construct the haplotypes for UGT2B15 and UGT2B17 in CEPH families A and B. (A) Family A (CEPH/UTAH Pedigree 1344): paternal grandmother, NA12056; paternal grandfather, NA12057; father, NA10851; mother, NA10850; and offspring, NA12047 (d), NA12048 (d), NA12049 (d), NA12050 (s), NA12051 (s), NA12052 (s), NA12053 (d), NA12054 (d), and NA12055 (s). (B) Family B (CEPH/UTAH Pedigree 13291): maternal grandmother, NA07045; maternal grandfather, NA06986; father, NA06995; mother, NA06997; offspring, NA07018 (s), NA07036 (s), NA06981 (d), NA06980 (d), NA07047 (s), NA07433 (s), and NA07058 (s). Shaded and boxed regions indicate individual haplotypes of UGT2B15 and UGT2B17 markers. *Haplotypes for family B members NA07036 and NA07058 could not be determined. **Haplotypes for grandparents of both families were predicted from their respective offspring.

In addition to the UGT2B15 functional polymorphism, allele frequencies between ethnic groups complicates asso- UGT2B17 has been shown to conjugate a broader range of ciation analyses between polymorphisms within this gene steroid molecules than UGT2B15 as a result of a single complex and disease phenotypes. Amino acid substitutions, amino acid difference in exon 1 [8,11]. The added substrate gene copy number, and expression differences either alone or specificity range of the UGT2B17 gene product may play a in combination may influence disease susceptibility. Studies significant biological role in maintaining steroid levels in combining genotyping with SNP quantitation are being used the body. to facilitate the discovery of cancer-causing genes and cancer The presence of two common and functionally relevant diagnosis [23]. Recently, deletion polymorphisms in gluta- polymorphisms in two closely related genes with different thione S- T1 (GSTT1) were examined in con- junction with SNPs to determine association with risk for Table 2 lung and brain cancer [24,25]. In both studies the deletion UGT2B17 genotype distribution polymorphism was associated with increased risk for cancer Insertion/ Insertion/ Deletion/ m2 in Caucasians. The same deletion, however, was associated insertion deletion deletion with increased risk for breast cancer in Tunisians [26] but not Caucasians 47 45 11 3.35 in Chinese [27]. Moreover, an insertion/deletion polymor- African- 52 31 2 4.05 phism of the angiotensin-converting enzyme (ACE) that Americans results in higher levels of circulating and tissue ACE activity p b 0.05. was found to be significantly associated with advanced W. Wilson III et al. / Genomics 84 (2004) 707–714 713 prostate cancer [28]. The fact that UGT2B17 copy number City, CA, USA) incubated with forward and reverse primers differs significantly between African Americans and Cau- at 100 pmol and 30 ng of template DNA. The incubation casians along with the prevalence of prostate cancer warrants was carried out at 948C for 4 min followed by 33 cycles of further research into the role of the UGT2B gene family in 948C for 30 s, 608C for 1 min, and 728C for 3 min in a ethnic-based disease disparities. GeneAmp PCR System 9700 thermocycler (Applied Bio- systems). All PCR products were analyzed on 1% agarose/ TBE gels and photographed with an Alpha Innotech Imager Materials and methods (Alpha Innotech Corp., San Leandro, CA, USA). PCR products from UGT2B15-specific markers F and H DNA samples and UGT2B17-specific markers A and B were analyzed by SSCP. PCR amplifications were performed on genomic A panel of 40 unrelated, genetically diverse human DNA DNA samples using a 10-Al reaction volume that contained samples (subset M44PDR) was obtained from the Human Taq polymerase buffer and polymerase (Promega, Madison, SNP Discovery Resource of the Coriell Cell Repositories WI, USA), dNTP’s (Promega), 2 pmol of primers, 2ACi (Camden, NJ, USA). The panel of DNA represents the a-32P-radiolabeled dCTP, and 50 ng genomic DNA. The following ethnic backgrounds: European, African, Mexican, PCR was performed as described above except 35 cycles at Native, and Asian-American. Human DNA samples from 958C for 30 s, 558C for 30 s, and 728C for 30 s were used. the members of two Centre d’Etude du Polymorphisme Amplification of markers B and F was followed by a 1-h Humain (CEPH) pedigrees were obtained from the NIGMS digestion with 5–20 units of Fnu4HI, EcoRI, or HindIII Human Genetic Cell Repository (CEPH/UTAH Pedigree restriction enzyme (New England BioLabs). SSCP analysis Subcollection). CEPH/UTAH Pedigree 1344 consists of of PCR products was performed in accordance with the paternal grandfather, NA12056; paternal grandmother, MDE Polyacrylamide Gel Solution protocol (BioWhittaker NA12057; father, NA10851; mother, NA10850; and off- Molecular Applications, Rockland, ME, USA). Electro- spring, NA12047 (d), NA12048 (d), NA12049 (d), phoresis of PCR products was for 16–18 h at 2.5 W. The NA12050 (s), NA12051(s), NA12052 (s), NA12053 (d), gels were dried prior to an overnight autoradiography NA12054 (s), and NA12055. CEPH/UTAH Pedigree 13291 exposure on Kodak BioMax X-ray film (Rochester, NY, (Family B) consists of maternal grandmother, NA07045; USA). maternal grandfather, NA06986; father, NA06995; mother, NA06997; and offspring, NA07018 (s), NA07036 (s), Analysis of UGT2B17 deletion polymorphism NA06981 (d), NA06980 (d), NA07047 (s), NA07433 (s), and NA07058 (s). PCR amplification of marker J was performed on DNA DNA samples from African Americans and Caucasians samples in a 10-Al reaction volume that contained Taq were obtained from frozen pancreatic and liver tissues that polymerase buffer and polymerase (Promega), dNTP’s were purchased from the Southern Division of the Human (Promega), 2 pmol of primers, 2ACi a-32P-radiolabeled Tissue Network (Birmingham, AL, USA) and the Interna- dCTP, and 50 ng genomic DNA. Touchdown PCR tional Institute for the Advancement of Medicine (Exton, amplification was performed with an initial 2 cycles at PA, USA). All experiments were conducted under an 958C for 30 s, followed by 59–568C for 30 s, 728C for 30 s; approved IRB protocol at the National Center for Toxico- then 20 cycles at 958C for 30 s, 558C for 30 s, and 728C for logical Research. Genomic DNA was prepared from tissue 30 s. SSCP analysis of PCR products was performed as samples using Qiagen DNA isolation kits (Qiagen, Valencia, described above. CA, USA). Primers and product sizes for gene-specific markers A–I Sequencing are indicated in Table 1. Marker D is a UGT2B17-specific sequence-tagged site (WIAF-2471-STS) identified on the PCR products were purified with QIAquick PCR University of California at Santa Cruz Genomic Database. purification kit (Qiagen) and sequenced with a 3100 genetic Primer sequences for markers A, E, and H were previously analyzer (Applied Biosystems). The sequencing reaction described [22,29]. A single pair of primers designed from was performed using the ABI Prism BigDye Terminator the NCBI dSNP database (Cluster ID: rs5029389) was used Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA in the amplification of markers B and F. polymerase, FS (Applied Biosystems) and primers from markers C, G, H, and I. Analysis of UGT2B15 and UGT2B17 markers

PCR products from markers C, D, E, and G were Acknowledgments analyzed by agarose gel electrophoresis. PCR amplification was performed on all human genomic DNA samples using The authors thank Beverly Koller (Department of AmpliTaq Gold MasterMix (Applied Biosystems, Foster Genetics, University of North Carolina at Chapel Hill), 714 W. Wilson III et al. / Genomics 84 (2004) 707–714

Ingrid Hall (Division of Cancer Prevention and Control, [13] F.C. Chen, W.H. Li, Genomic divergences between human and other National Center for Chronic Disease Prevention and Health hominoids and the effective population size of the common ancestor of human and chimpanzees, Am. J. Hum. Genet. 68 (2001) 444–456. Promotion, Centers for Disease Control and Prevention), [14] I. Ebersberger, D. Metzler, C. Schawrz, S. Paabo, Genomewide and Leroy Worth (DERT, SRB, NIEHS/NIH) for critically comparison of DNA sequences between human and chimpanzees, reading the manuscript. We appreciate additional technical Am. J. Hum, Genet. 70 (2002) 1490–1497. assistance from Elena Arthur, Keoshia Johnson, and Beverly [15] E. Xavier, J. Cheung, M.A. Pujana, K. Nakabayashi, S.W. Scherer, Word. This work was supported by grants from the NCI/ L.-C. Tsui, Chromosomal regions containing high-density and ambiguously mapped putative single nucleotide polymorphisms NIH (CA92075-02 UNC, CA992077-02 NCCU), NIGMS/ (SNPs) correlate with segmental duplications in the human genome, NIH (S06-GM08049), and NSF (MCB-0133526 to Hum. Mol. Genet. 11 (2002) 1987–1995. F.P.M.V.). [16] E. Levesque, M. Beaulieu, M.D. Green, T.R. Tephly, A. Belanger, D.W. Hum, Isolation and characterization of UGT2B15 (Y85): a UDP-glucuronosyltransferase encoded by a polymorphic gene, Pharmacogenetics 7 (1997) 317–325. References [17] J.O. Miners, R.A. McKinnon, P.I. Mackenzie, Genetic polymorphisms of UDP-glucuronosyltransferases and their functional significance, [1] P.I. Mackenzie, et al., The UDP gene superfamily: Toxicology 181-182 (2002) 453–456. recommended nomenclature update on evolutionary divergence, [18] J.W. Lampe, J. Bigler, A.C. Bush, J.D. Potter, Prevalence of Pharmacogenetics 7 (1997) 255–296. Polymorphisms in the Human UDP-Glucuronosyltransferase 2B [2] M. Reidy, J.Y. Wang, A.P. Miller, A. Buckler, J. Hall, M. Guida, Family: UGT2B4 (D458E), UGT2B7 (H268Y), and UGT2B15(D85Y), Genomic organization of the UGT2B gene cluster on human Cancer Epid. Biomark. Prev. 9 (2000) 329–333. chromosome 4q13, Pharmacogenetics 10 (2000) 251–260. [19] S.L. MacLeod, S. Nowell, J. Paxco, N.P. Lang, An allele-specific [3] E. Levesque, et al., Isolation and characterization of the UGT2B28 polymerase chain reaction method for the determination of the D85Y cDNA encoding a novel human steroid conjugating UDP-glucurono- polymorphism in the human UDP-glucuronosyltransferase 2B15 gene syltransferase, Biochemistry 4 (2001) 3869–3881. in a case–control study of prostate cancer, Ann. Surg. Oncol. 7 (2000) [4] D. Turgeon, J.S. Carrier, S. Chouinard, A. Belanger, Glucuronidation 777–782. activity of the UGT2B17 enzyme toward xenobiotics, Drug Metab. [20] J. Park, et al., ASP85TYR polymorphism in the UDP-Glucuronosyl- Dispos. 31 (2003) 670–676. transferase (UGT) 2B15 Gene and the risk of prostate cancer, J. Urol. [5] D. Turgeon, et al., Glucuronidation of arachidonic and linoleic acid 171 (2004) 2484–2488. metabolites by human UDP-glucuronosyltransferases, J. Lipid Res. 44 [21] A. Gsur, et al., A polymorphism in the UDP-glucuronosyltransferase (2003) 1182–1191. 2B15 Gene (D85Y) is not associated with prostate cancer risk, Cancer [6] B. Burchell, C.H. Brierley, D. Rance, Specificity of human UDP- Epidemiol. Biomarkers Prev. 11 (2002) 497–498. glucuronosyltransferases and xenobiotic glucuroniation, Life Sci. 57 [22] M. Murata, E.H. Warren, S.R. Riddell, A human minor histocompat- (1995) 1819–1831. ibility antigen resulting from differential expression due to a gene [7] D. Turgeon, et al., Isolation and characterization of the human deletion, J. Exp. Med. 197 (2003) 1279–1289. UGT2B15 gene, localized within a cluster of UGT2B genes [23] X. Zhao, et al., An integrated view of copy number and allelic and pseudogenes on , J. Mol. Biol. 295 (2000) alterations in the cancer genome using single nucleotide polymor- 489–504. phism arrays, Cancer Res. 64 (2004) 3060–3071. [8] M. Beaulieu, E. Levesque, A. Tchernof, B.G. Beatty, A. Belanger, [24] M. Sorensen, H. Antrup, A. Tjonneland, K. Overvad, O. Raaschou- D.W. Hum, Chromosomal localization, structure, and regulation of the Nielsen, Glutathione S-transferase T1 null-genotype is associated with UGT2B17 gene, encoding a C19 steroid metabolizing enzyme, DNA an increased risk of lung cancer, Intl. J. Cancer 110 (2004) 219–224. Cell Biol. 16 (1997) 1143–1154. [25] M. Wrensch, et al., Glutathione-S-transferase variants and adult [9] F. Chen, J.K. Ritter, M.G. Wang, O.W. McBride, R.A. Lubet, I.S. glioma, Cancer Epidemiol. Biomarkers Prev. 13 (2004) 461–467. Owen, Characterization of a cloned human dihydrotestosterone/ [26] A. Khedhaier, et al., Glutathione S- (GSTTI and GSTMI) androstanediol UDP-glucuronosyltransferase and its comparison to gene deletions in Tunisians: susceptibility and prognostic implications other steroid isoforms, Biochem. 32 (1993) 10648–10657. in breast carcinoma, Br. J. Cancer 89 (2003) 1502–1507. [10] D. Turgeon, J. Carrier, E. Levesques, D.W. Hum, A. Belanger, [27] K.M. Egan, et al., Genetic polymorphisms in GSTM1, GSTP1, and Relative enzymatic activity, stability, and tissue distribution of GSTT1 and the risk for breast cancer: results from the Shanghai Breast human steroid-metabolizing UGT2B subfamily members, Endocri- Cancer Study and meta-analysis, Cancer Epidemiol. Biomarkers Prev. nology 142 (2001) 778–787. 13 (2004) 197–204. [11] S.G. Dubois, M. Beaulieu, E. Levesque, D.W. Hum, A. Belanger, [28] R. Medeiros, et al., Linkage of angiotensin I-converting enzyme gene Alteration of human UDP-glucuronosyltranssferase UGT2B17 regio- insertion/deletion polymorphism to the progression of human prostate specificity by a single amino acid substitution, J. Mol. Biol. 289 cancer, J. Pathol. 202 (2004) 330–335. (1999) 29–39. [29] A. Iida, et al., Catalog of 86 single-nucleotide polymorphisms (SNPs) [12] R. Sakate, et al., Analysis of 5V-end sequences of chimpanzee cDNAs, in three uridine diphosphate glycosltransferase genes: UGT2A1, Genome Res. 13 (2003) 1022–1026. UGT2B15, and UGT8, J. Hum. Genet. 47 (2002) 505–510. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。 图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具