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[CANCER RESEARCH 61, 2307–2310, March 1, 2001] Distribution and Functional Consequences of Nucleotide Polymorphisms in the 3؅-Untranslated Region of the Human Sep15 Gene1

Ya Jun Hu, Konstantin V. Korotkov, Rajeshwari Mehta, Dolph L. Hatfield, Charles N. Rotimi, Amy Luke, T. Elaine Prewitt, Richard S. Cooper, Wendy Stock, Everett E. Vokes, M. Eileen Dolan, Vadim N. Gladyshev,2 and Alan M. Diamond2 Departments of Human Nutrition and Dietetics [Y. J. H., A. M. D.], Surgical Oncology [R. M.], and Hematology/Oncology [W. S.], University of Illinois at Chicago, Chicago, Illinois 60612; Department of Biochemistry, University of Nebraska, The Beadle Center, Lincoln, Nebraska 68588 [K. V. K., V. N. G.]; Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, NIH, Bethesda, Maryland 20892 [D. L. H.]; Department of Genetic Epidemiology, National Human Genome Center, Howard University, Washington DC 20059 [C. N. R.]; Department of Preventive Medicine and Epidemiology, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153 [A. L., T. E. P., R. S. C.]; and Department of Medicine [E. E. V., M. E. D.], University of Chicago, Chicago, Illinois 60637

ABSTRACT translation requires a recognition element within the 3Ј-UTR3 (3) of the corresponding mRNA referred to as a SECIS element (7). SECIS ele- Selenium has been shown to prevent cancer in a variety of animal ments are present in the mRNAs of all of the selenocysteine-containing model systems. Both epidemiological studies and supplementation trials proteins and share structural features, including an approximately 80- have supported its efficacy in humans. However, the mechanism by which nucleotide stem-loop structure containing both an internal and apical loop selenium suppresses tumor development remains unknown. Selenium is of unpaired nucleotides and a conserved 4-bp domain within the stem. present in known human selenoproteins as the amino acid selenocysteine Although the functions of several selenoproteins have been determined, (Sec). Sec is inserted cotranslationally in response to UGA codons within to date there has not been definitive evidence associating any of these -selenoprotein mRNAs in a process requiring a sequence within the 3؅ untranslated region (UTR), referred to as a Sec insertion sequence (SE- proteins with either cancer risk or development. A recent search for novel selenium-containing proteins resulted in the isolation and characterization CIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in of a new selenoprotein (8). Examination of data contained in the dbEST the 3؅-UTR. Examination of the available cDNA sequences for this protein databases resulted in the assembly of a 1268 nucleotide cDNA with an revealed two polymorphisms located at position 811 (C/T) and at position ORF of 162 amino acids. Within this ORF was an in-frame UGA codon G/A) located within the 3؅-UTR. Here, we demonstrate significant corresponding to amino acid 93. This and the observation that this protein) 1125 75 differences in Sep15 allele frequencies by ethnicity and that the identity of labels with Se and contains a SECIS element in the 3Ј-UTR strongly the nucleotides at the polymorphic sites influences SECIS function in a support the conclusion that the Mr 15,000 selenoprotein (Sep15) contains selenium-dependent manner. This, together with genetic data indicating selenocysteine at position 93. Further analysis of the Sep15 cDNA loss of heterozygosity at the Sep15 locus in certain human tumor types, sequence indicated that it was well conserved through evolution; homol- suggests that Sep15 may be involved in cancer development, risk, or both. ogous genes exist in mouse, rat, Brugia malayi, Caenorhabditis elegans, and other animals. On the basis of the frequency of recovery of Sep15 cDNA sequences from particular libraries included in the dbEST data- INTRODUCTION base, it was concluded that Sep15 is expressed in a wide variety of human tissues, with the highest levels being observed in the thyroid and prostate Selenium is an essential trace element currently being considered as a (8, 9). Examination of the available cDNA sequences for this protein candidate chemopreventive agent. Research spanning the last 25 years revealed two distinct forms of the Sep15 gene differing by polymor- has established that selenium is effective in the reduction of cancer phisms located at position 811 (C/T) and at position 1125 (G/A) within incidence when provided to animals as doses only 5- to 10-fold above the the 3Ј-UTR (8). Here, we demonstrate significant differences in Sep15 nutritional requirement (1). Chemoprevention studies with selenium in allele frequencies by ethnicity and that the identity of the nucleotides at animal model systems have indicated that this element is effective for the polymorphic sites influences SECIS function in a selenium-dependent most, if not all of the organ systems and is protective against the manner. This, together with genetic data indicating LOH at the Sep15 carcinogenic effects of a wide variety of insults (1). Both epidemiological locus in certain human tumor types, suggests that Sep15 may be involved studies and supplementation trials have also supported its efficacy in in cancer etiology. humans (2–5). However, the mechanism by which selenium suppresses tumor development remains unknown. Many of the biological activities MATERIALS AND METHODS of selenium are attributable to its role as a constituent of selenium- containing proteins. In all of the known mammalian selenoproteins, Sample Sources. The majority of the samples obtained from African Amer- selenium exists as the amino acid selenocysteine, which is inserted icans, 490 subjects, was recruited from the community of Maywood, IL, a suburb of Chicago. Unrelated individuals over the age of 18 years were identified through cotranslationally into the nascent protein in response to the UGA codon door-to-door canvassing and asked to participate in a study of diet and heart in selenoprotein mRNA (6). In mammals, the molecular choice to insert disease from an IRB-approved protocol, and signed consent was given from all of selenocysteine into the elongating selenoprotein rather than terminate the individuals. Exclusion criteria included any known cancer, non-insulin-dependent diabetes mellitus, and heart disease. The lymphocytes from normal volunteers Received 8/30/00; accepted 12/28/00. and colon tumor samples were collected anonymously from an IRB-approved The costs of publication of this article were defrayed in part by the payment of page protocol. All subjects were Ͼ18 years of age. Normal Caucasian volunteers were charges. This article must therefore be hereby marked advertisement in accordance with recruited from the University of Chicago. Colon tumor samples were obtained 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a grant from the American Institute for Cancer Research (to after surgical resection from patients with confirmed colon cancer. African- A. M. D.), a grant from the Cancer Research Foundation of America (to V. N. G.), and American patients with hematological malignancies were treated according to National Institute of Dental Research/National Cancer Institute Grant 11921 (to E. E. V.). institutional IRB-approved treatment studies for acute myeloid leukemia, acute 2 To whom requests for reprints should be addressed, Alan M. Diamond, Department of Human Nutrition and Dietetics, University of Illinois at Chicago, Chicago, IL 60612. Fax: (312) 413-0319; E-mail: [email protected]; or Vadim N. Gladyshev, Department of 3 The abbreviations used are: UTR, untranslated region; Sec, selenocysteine; SECIS, Biochemistry, University of Nebraska, The Beadle Center, Lincoln, NE 68588. Fax: selenocysteine insertion sequence; LOH, loss of heterozygosity; ORF, open reading (402) 472-4948; E-mail: [email protected]. frame; IRB, Institutional Review Board; EST, expressed sequence tag. 2307

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2001 American Association for Cancer Research. DISTRIBUTION AND FUNCTION OF Sep15 POLYMORPHISMS lymphoblastic leukemia, and chronic myeloid leukemia. All subjects were Ͼ18 for either SpeIorPstI(5Ј-AAAACTAGTGCTTTGTAACAGACTTGCG- years of age, were referred to the University of Illinois at Chicago for treatment, GTTAATTATGC-3Ј or 5Ј-AAAGTGCAGGGTCTTACAAATGATCACT- and consented to an IRB-approved study for the analysis of molecular abnormal- TTTAAATGGAC-3Ј, respectively). Amplification was as described above, ities present in leukemia cells. Bone marrow and blood samples were obtained at and the products were digested with SpeI and PstI and directionally cloned into specified time points before, during, and after completion of treatment. Breast the pBPLUGA plasmid. Transfection of mouse NIH 3T3 cells was performed carcinomas from African-American women were archived from the Department of using the lipofectin reagent at a ratio of 1:3 (v/v) as indicated by the vendor Surgical Oncology Tissue and Sera Bank at the University of Illinois at Chicago. (Life Technologies, Inc.). The cells were cotransfected with the reporter

Originally, tumors were obtained from women with confirmed histopathological construct and the pSV2Neo plasmid at a 10:1 molar ratio, and transfectants diagnosis of breast cancer who were undergoing lumpectomy or mastectomy. were selected with the antibiotic geneticin G418. G418-resistant colonies were After surgical excision, tumor tissue was transported to the Department of Pathol- pooled for further analysis. Selenium was added to the culture media in the ogy, an aliquot of the tissue required for the diagnostic purpose was reserved, and form of sodium selenite for 5 days, at which time the cells were collected and the remaining tumor tissue, otherwise to be discarded, was transported on ice to the lysates prepared. Cells were resuspended in Reporter Lysis Buffer (Promega) and Surgical Oncology Laboratory. Tumor was freed from nonmalignant tissue, allo- lysed by a single freeze-thaw cycle. Lysates were generated by suspension in cated, and frozen at Ϫ70°C. Only those women with accurate information of their Reporter Lysis Buffer and were either used immediately or stored at Ϫ70°C. race were included in the study. Women with more than one primary cancer were Luciferase and ␤-galactosidase activities were determined using the corresponding excluded. assay systems from Promega. Luciferase activity was quantified using a Fento- Determination of the Nucleotide Identity at Positions 811 or 1125 within master FB12 Luminometer from Zylux. Readthrough efficiencies were analyzed the Human Sep15 Gene. DNA from either lymphocytes or tissue was prepared by standard t test performed using SYSTAT version 8.0 software. using the protocols and procedures included in the Puragene DNA Isolation Kit (Gentra). The buffy coat plus red cells were isolated from a 10-ml draw into EDTA-containing vacutainers. Tubes were immediately placed in a refrigerator RESULTS and centrifuged between 30 min and 2 h postdraw. Plasma was drawn off and stored, and white cells plus 0.5–1.0 ml of red cells were stored in 2-ml cryovials Genotype of DNA from Human Samples. To determine the dis- and frozen at Ϫ80°C until DNA extraction. Remaining red cells were discarded. tribution of the Sep15 genetic variations within the human population, we From 0.1 to 1.0 ␮g of DNA was used as template for PCR amplification using the used PCR to amplify a region of ϳ400 nucleotides of the human Sep15 GeneAmp PCR Amplification Kit. Amplification of a 413-bp product was 3Ј-UTR that included positions 811 and 1125. In initial studies, we Ј Ј achieved with the Sep15 forward primer (5 -CAGACTTGCGGTTAATTATG-3 ) established the identity of the nucleotides at the polymorphic sites using Ј Ј and the Sep15 reverse primer (5 -GCCAAGTATGTATCTGATCC-3 ). PCR was primer extension. Subsequently, we took advantage of the observation performed at 95°C for 3 min, followed by 33 cycles of 95°C for 30 s, 55°C for that position 811 is potentially located within the recognition sequence 60 s, and 72°C for 90 s. Successful amplification was indicated by the appearance Ј of a DNA band of ϳ400 nucleotides on a 1% agarose gel. Radioactive primers for for the restriction endonuclease DraI (recognition sequence 5 -TT- Ј use in the determination of the nucleotide at position 811 (5Ј-GGCATAGTAAT- TAAA-3 ). C at position 811 results in the sequence TCTAAA, which is CATCTGTCTTGTT-3Ј) or 1125 (5Ј-GTATGTATCTGATCCACACAAATCC- not cleaved by DraI, whereas T at position 811 results in the recognition 3Ј) were obtained by 5Ј-end labeling with ␥-labeled ATP and polynucleotide sequence of the enzyme. To validate the use of DraI digestion, 100 kinase. Labeled primer and DNA were mixed in a solution of 40 mM Tris-HCl (pH samples were analyzed by both primer extension and DraI digestion of

7.5), 20 mM MgCl2, and 50 mM NaCl, heated to 95°C for 10 min, and then PCR-amplified DNA with perfect agreement. In addition, the results of transferred to a 37°C water bath for 1 h. Extension was accomplished by adding the primer extension and DraI digestions indicated that the only nucle- 5 ␮l of a mix containing 100 mM DTT, 1 mM each of 3 dideoxynucleotide otides at position 811 were eitheraTorC.Toextend the analysis of triphosphates, 5 mM dideoxynucleotide triphosphate, and 5 units of reverse tran- DNA samples to position 1125, we used differential digestion using BfaI scriptase. The mixture was incubated for 15 min at 42°C, ethanol precipitated, and (recognition sequence, 5Ј-CTAG-3Ј). G at position 1125 results in the resuspended in formamide-loading buffer, and the extension products separated on sequence 5Ј-TTTCTAGCCTAA-3Ј, which is cleaved by BfaI, whereas A a 10% polyacrylamide gel. Visualization of the extension products was accomplished by autoradiography. Nucleotide identity at positions 811 and 1125 was also results in a sequence not recognized by this enzyme. determined by restriction enzyme digestion of the PCR amplification product Using the techniques described above, we genotyped DNA samples described above. Amplified DNA was digested with DraI (Pharmacia) to evaluate from Ͼ700 individuals. All of the samples analyzed for nucleotide the nucleotide identity at position 811 or with BfaI (New England Biolabs) to identity at positions 811 and 1125 fell into one of two categories referred identify the nucleotide at position 1125 using buffers and conditions provided by to as either the TA or CG haplotype. This result was in agreement with those respective vendors. DNA digestion was evaluated by gel electrophoresis in previous observations made by the analysis of EST sequences (8), al- 2.5% agarose. Statistical analysis was performed using Statistical Analysis Sys- though the possibility of compound heterozygosity was never addressed tems (SAS version 6.0). As all of the data are categorical, and differences between in these studies. Unlike the database analysis, the methodology used in ␹2 cases and controls for the three genotypes were measured using statistics. this study permitted the assignment of DNA samples from individuals as Genetic analysis at the D1s207 and D1s223 loci was performed by Research either TA or CG homozygotes or heterozygotes, the later being indicated Genetics, Inc. (Huntsville, AL). In Vitro Mutagenesis. The plasmid containing the Sep15 cDNA by the presence of bands representing both alleles after restriction enzyme (AF051894; Ref. 8) was mutated with two pairs of primers, where the boldface digests of PCR amplification products (e.g., Fig. 1A). The genotypes for nucleotide indicates the site of the mutation, as follows: 1125F, 5Ј-CACA- all of the samples analyzed are shown in Table 1 and exhibit a classic GAAAACCTTTCTAAGGATTTGTGTGGATCAG-3Ј; 1125R, 5Ј-CTGATC- 1:2:1 distribution, following the Hardy-Weinberg equilibrium. However, CACACAAATCCTTAGAAAGGTTTTCTGTG-3Ј; and 811F, 5Ј-CCAGTT- when the data were subdivided by ethnicity, it was apparent that there TTACGAACAACAGATTTTTAAATTAGAGAGGTTAAC-3Ј; 811R, 5Ј- was a difference in allele distribution between DNAs obtained from GTTAACCTCTCTAATTTAAAAATCTGTTGTTCGTAAAACTGG-3Ј, us- Caucasians and African Americans (Table 1); e.g., the allele frequency ing the QuickChange site-directed mutagenesis kit (Stratagene). Mutagenesis for the TA/TA homozygous haplotype was 31% for African Americans, with primers 1125F/1125R yielded the CA (811:1125) variant of the Sep15 in contrast to 7% for Caucasians. No statistically significant differences in SECIS element, and mutagenesis with 811F/811R, the TG (811:1125) variant. genotype distribution were observed for sex or age (data not shown). Reporter Constructs and Assays. To generate pBPLUGA plasmids (10) containing a portion of the Sep15 3Ј-UTR, the 1535 CPTX human prostate cell Nucleotide Identity at Position 1125 Influences SECIS Func- line (11) and the STSAR90 human sarcoma cell line (12) were selected tion. Position 1125 resides within the apical loop of the Sep15 SECIS because their DNA contained either the CG or TA haplotype determined as element and thus could potentially influence the efficiency of Sec described above for human samples. DNA from these lines was prepared, and incorporation (8). Because of this and the allele distribution data the 3Ј-UTR was amplified with primers containing the recognition sequences described above, we used a reporter construct designed to quantify the 2308

plasmid; individual clones were isolated and both ␤-gal and luc activities were determined in cell extracts. Although both polymorphic sequences were indeed capable of functioning as a SECIS element, the TA sequence was approximately twice as efficient in stimulating the readthrough of the UGA codon, as measured by luc activity (Fig. 2). To determine whether both the 811 and 1125 nucleotide positions were contributing to the observed SECIS activity, two additional sequences (811:1125, CA or TG) were generated by in vitro mutagenesis and evaluated using the same reporter system. As seen in Fig. 2, the CG and TG pairs yielded similar activity as did the CA and TA pairs, indicating that the identity of the nucleotide at the 1125 position determines SECIS efficiency. It was also evaluated whether the naturally occurring haplotype sequences could influence SECIS efficiency as a function of increasing selenium avail- ability in the tissue culture media. Stably transfected NIH 3T3 cells were incubated in sodium selenite-supplemented media at concentrations between 30 nM and 120 nM, and reporter gene activity was determined. As seen in Table 2, the SECIS element containing T,A:811,1125 was less responsive to added selenium than was the CG-containing version of this sequence. LOH at the Sep15 Locus. The DNAs genotyped in Table 1 were derived either from lymphocytes obtained from cancer-free individuals or from malignant cells. We, therefore, determined whether allele frequencies at this locus were different for tumors versus lymphocytes from cancer-free individuals of a particular Fig. 1. A, identification of the nucleotide identities at positions 811 and 1125 within the ethnic background. These data (Table 1) indicate that there was a 3Ј-UTR of the Sep15 gene. Analysis of 16 DNA samples (Lanes 2–17) obtained from significant reduction in heterozygosity in tumors in African Amer- breast tumors. DNA was prepared from the provided tissue, the relevant region of the 3Ј-UTR amplified by PCR and the resulting DNA digested with either DraIorBfaI to icans, and this was true for both classes of tumors. Although there identify the nucleotides at positions 811 or 1125, respectively. In each case, the higher appears to be a trend toward LOH in head and neck tumors in molecular weight band represents the amplification product that is not cleaved by the restriction enzyme. Two bands in a particular lane indicate the presence of both Sep15 alleles. Lane 1, PCR amplification without DNA template; Lanes 2, 5, 7, 14, and 15, samples containing only the TA haplotype; Lanes 6, 13, and 17, only the GC haplotype; and Lanes 3, 4, 8–12, and 16, both alleles. B, detection of LOH at the Sep15 locus. LOH was detected at the Sep15 locus using the same approach as described above in DNA obtained from either a head and neck tumor (T) or lymphocytes (L) from the same patient, PCR-amplified, and digested with BfaI. Left, pair of samples indicating the same genotype in both tumor and lymphocyte DNAs (T,A:811,1125); right, analysis indicating LOH.

Table 1 Allelic distribution of the TA and GC haplotypes in human DNA samples DNAs were genotyped as described in the text at both the 811 and 1125 positions. DNA was prepared from lymphocytes obtained from cancer-free individuals or directly Fig. 2. The use of the pBPLUGA reporter plasmid to determine the consequences of from malignant tissue or cells. The difference in genotype at the Sep15 locus between the nucleotide at positions 811 and 1125 on SECIS function. Regions of the Sep15 3Ј-UTR African Americans and Caucasians was significant at P Ͻ 0.001, whether all samples, containing positions 811 and 1125 were cloned into the polycloning site of the pBLUGA only cancer-free individuals, or head and neck tumors were compared. The difference in reporter construct (10). The identities of the nucleotides at those positions are indicated on genotype between cancer-free African Americans and DNAs obtained from breast tumors the abscissa. Each plasmid was cotransfected into NIH 3T3 cells along with the pSV neo or head and neck tumors from African Americans was significant at P Ͻ 0.10. 2 plasmid, and colonies were selected in G418 and pooled. Cells were harvested, and CG/CG CG/TA TA/TA extracts were prepared and assayed for ␤-gal and luc activity as described in “Materials and Methods.” Readthrough efficiency represents the determined luc activity divided by Caucasians (all samples) 106 (57%) 67 (36%) 13 (7%) ␤ ϭ -gal activity to control for heterogeneity of expression attributable to integration into (n 186) different chromosomal sites. The data presented are the mean of three independently African Americans (all samples) 105 (18%) 297 (51%) 181 (31%) ϭ generated extracts; error bars, SD. The differences in readthrough efficiency for CG (n 583) versus CA, TA versus TG, and CA versus TG were significant at P Ͻ 0.05. Caucasians Cancer-free (n ϭ 33) 19 (58%) 13 (39%) 1 (3%) Head and neck cancer (n ϭ 94) 56 (57%) 30 (32%) 8 (8%) Chronic myelogenous leukemia 20 (54%) 15 (41%) 2 (5%) (n ϭ 37) Table 2 Relative SECIS element efficiency as a function of increasing Colon cancer (n ϭ 22) 11 (50%) 9 (41%) 2 (9%) selenium concentration African Americans NIH 3T3 cells containing the reporter constructs described above representing the Cancer-free (n ϭ 490) 81 (16%) 259 (53%) 150 (31%) naturally occurring haplotypes (TA, GC) were incubated in media containing the indicated Breast cancer (n ϭ 60) 17 (28%)a 27 (45%) 16 (27%) concentration of selenium in the form of sodium selenite. Cells were incubated for 5 days Head and neck cancer (n ϭ 33) 7 (21%) 11 (33%)a 15 (46%) and harvested, and extracts were prepared and analyzed as above. The data indicate the luc activity divided by ␤-gal levels and normalized to the readthrough efficiency determined a Statistical significance for that genetic class differed from the corresponding class in Ͻ in the absence of added selenium. The numbers in parentheses represent the average of cancer-free individuals at P 0.05. raw data obtained from six independent extracts. P is a measure of the statistical difference in the ability of the SECIS element to support readthrough of the TGA codon into the luc gene as a function of increasing selenium supplementation. ability of a SECIS element to direct the readthrough of an in-frame [Se] (nM) CG TA P UGA codon positioned between a ␤-galactosidase (␤-gal) gene into a 0 1.00 (7,542) 1.00 (14,142) — 30 1.31 Ϯ 0.14 (9,807) 1.31 Ϯ 0.12 (18,350) 0.49 downstream luciferase (luc) gene (10). Reporter constructs containing 60 1.59 Ϯ 0.11 (11,972) 1.44 Ϯ 0.12 (20,040) 0.23 the 3Ј-UTR sequences representing either the TA or CG haplotype were 90 1.92 Ϯ 0.09 (14,417) 1.48 Ϯ 0.16 (20,607) 0.02 120 2.24 Ϯ 0.13 (16,918) 1.46 Ϯ 0.10 (20,623) 0.006 cotransfected into NIH 3T3 mouse fibroblasts along with the pSV2neo 2309

Caucasians as well, the sample size was too small to permit the media is generally considered to be deficient in selenium compared statistical analysis to reach significance. with levels obtained in human tissues and plasma (18), the differences To evaluate whether LOH at the Sep15 locus could be detected during observed in SECIS function under conditions of selenium supplemen- tumor development, we obtained lymphocytes from three patients whose tation might suggest that a Sep15 gene with an A at 1125 may produce tumor DNAs genotyped to an apparently homozygous TA/TA haplotype. less of its protein product. Additionally, individuals with that geno- As seen in Fig. 1B, a single DNA pair demonstrated LOH at this locus. type might be less responsive to the protective benefits of dietary The Sep15 gene has been previously mapped to human chromosome 1 at selenium. The development of antibodies capable of quantifying the 121.9 interval (8). To verify LOH determined by restriction enzyme Sep15 in the relevant tissues will be required to quantify this protein analysis and to assess the extent of the sequences deleted in this tumor in human tissues to address this possibility. sample, we used highly polymorphic markers in the region of the Sep15 Although limited conclusions can be drawn from an uncontrolled gene. LOH was verified using the D1s207 and D1s223 markers located retrospective study, the observed differences in allele frequencies and at positions 117.6 and 137.3, respectively. Both markers were heterozy- the single example of LOH at the Sep15 locus presented suggest that gous in lymphocyte DNA, and only one allele was evident in the DNA this protein may play a role in tumor evolution. In addition, the data obtained from the tumor. presented indicating a functional consequence of the nucleotide identities at the polymorphic positions raise the possibility that the Sep15 DISCUSSION genotype may influence translation of that protein in response to dietary selenium and whether individuals can benefit from the che- The wealth of data indicating that selenium can reduce cancer inci- moprotective properties of this element. dence in animal models and humans has stimulated the search for selenoproteins that may contribute to the beneficial properties of this ACKNOWLEDGMENTS element. The observation that the Sep15 gene exists in humans as two allelic forms differing at nucleotide positions 811 and 1125 permitted the We thank Dr. Leopold Flohe´for kindly providing the pBPLUGA plasmid, evaluation of allele frequencies using the PCR amplification of the Dr. K. Stenson for help in collecting head and neck cancers, and Drs. C. polymorphic region, followed by diagnostic restriction enzyme digestion. Braunschweig and S. Kumanyika for helpful discussions. The examination of a large number of DNA samples clearly indicated that there was a significant difference in allele frequencies between REFERENCES African Americans and Caucasians. Therefore, subsequent analysis of the 1. El-Bayoumy, K. The role of selenium in cancer prevention. In: V. T. De Vita, S. distribution of polymorphic sequences within the Sep15 gene required the Hellman, and S. A. Rosenberg (eds.), Cancer Prevention, pp. 1–15. Philadelphia: J. B. sorting of comparative genetic data on the basis of ethnicity. When this Lippincott Co., 1991. 2. Yu, S. Y., Zhu, Y. J., and Li, W. G. Protective role of selenium against hepatitis B is done, differences in allele frequencies between DNAs obtained from virus and primary liver cancer in Qidong. Biol. Trace Elem. Res., 56: 117–124, 1997. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2001 American Association for Cancer Research. Distribution and Functional Consequences of Nucleotide Polymorphisms in the 3 ′-Untranslated Region of the Human Sep15 Gene

Ya Jun Hu, Konstantin V. Korotkov, Rajeshwari Mehta, et al.

Cancer Res 2001;61:2307-2310.

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