Proteasome B Subunit Pharmacogenomics: Gene Resequencing ... · Proteasome B Subunit...

Proteasome B Subunit Pharmacogenomics: Gene Resequencingand Functional GenomicsLiewei Wang,1Shaji Kumar,2 Brooke L. Fridley,3 Krishna R. Kalari,3 IreneMoon,1Linda L. Pelleymounter,1

Michelle A.T. Hildebrandt,1Anthony Batzler,3 BruceW. Eckloff,4 Eric D.Wieben,4 and Philip R. Greipp2

Abstract Purpose: The proteasome is a multisubunit cellular organelle that functions as a nonlysosomalthreonine protease. Proteasomes play a critical role in the degradation of proteins, regulating avariety of cellular processes, and they are also the target for antineoplastic proteasome inhibitors.Genetic variation in proteasome subunits could influence bothproteasome function and responseto drug therapy.Experimental Design:We resequencedgenes encoding the three active proteasomeh subunitsusing 240 DNA samples from four ethnic groups and the h5 subunit gene in 79 DNA samplesfrom multiple myeloma patients who had been treated with the proteasome inhibitor bortezomib.Resequencing was followed by functional studies of polymorphisms identified in the codingregion and 3¶-flanking region (3¶-FR) of PSMB5, the gene encoding the target for clinically usefulproteasome inhibitors.Results:Resequencing of 240DNA samples identified a series of novel ethnic-specificpolymor-phisms that are not represented in public databases.The PSMB5 3¶-FR1042 Gallele significantlyincreased transcription during reporter gene studies, observations confirmed by genotype-phenotype correlations between single nucleotide polymorphisms (SNP) in PSMB5 and mRNAexpression in the 240 lymphoblastoid cell lines fromwhich the resequenced DNAwas obtained.Studies with patient DNA samples identified additional novel PSMB5 polymorphisms, includinga SNP and an insertion in the 3¶-FR. Reporter-gene studies indicated that these two novel poly-morphisms might decrease transcription.Conclusions: These results show that nonsynonymous coding SNPs in the PSMB5 gene didnot show significant effects on proteasome activity, but SNPs did influence transcription. Futurestudies might focus on regulatory region polymorphisms.

Protein degradation regulates a variety of critical cellularprocesses, including cell division, signal transduction, andapoptosis (1–4). The proteasome is a multisubunit cellularorganelle that functions as a nonlysosomal threonine protease.

It plays a critical role in protein degradation and is the target forantineoplastic proteasome inhibitors (1–5). The 26S protea-some consists of a 20S barrel-shaped core particle and two 19Sregulatory complexes that cap the 20S core particle. The 20Score particle is a multisubunit enzyme complex that consists offour heptameric rings arranged in a7h7h7a7 fashion, surround-ing a central cavity where the catalytic sites are found (6–8).Three of the seven h subunits, h1, h2, and h5, areproteolytically active with different substrate specificities. Theh1 subunit catalyzes a postglutamyl peptidyl hydrolytic-likeactivity; the h2 subunit catalyzes a trypic-like activity; and theh5 subunit catalyzes a chymotryptic-like activity (9).

Proteasome inhibitors have been tested for the treatment ofa variety of types of cancers. One of those drugs, bortezomib,with activity directed mainly against the h5 subunit, has beenapproved for the treatment of refractory multiple myeloma(10, 11). However, clinical response to bortezomib therapyvaries widely (12–14). Because the proteasome has criticalimportance for cellular processes and because it is also a drugtarget, it would be important to identify common sequencevariation in genes encoding the three active h subunits and todetermine the possible functional implications of that sequencevariation. However, no systematic pharmacogenomic studieshave been done of genes encoding human proteasome hsubunits. Therefore, in the present study, we did comprehensive

CancerTherapy: Preclinical

Authors’Affiliations: 1Divisionof Clinical Pharmacology,Department ofMolecularPharmacology and Experimental Therapeutics and Departments of 2InternalMedicine, 3Health Sciences Research, and 4Biochemistry and Molecular Biology,Mayo Clinic, MayoMedical School, Rochester, MinnesotaReceived12/12/07; revised1/15/08; accepted1/28/08.Grant support:NIH grantsGM61388 (ThePharmacogenetics ResearchNetwork)and CA102701 (The Pancreatic Cancer Specialized Programs of ResearchExcellence), an American Society for Pharmacology and ExperimentalTherapeutics-Astellas Award, and a PhRMA Foundation Center of Excellence inClinical PharmacologyAward.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).Requests for reprints: Liewei Wang, Division of Clinical Pharmacology,Department of Molecular Pharmacology and Experimental Therapeutics, MayoClinic, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-284-5264;Fax: 507-507-284-4455; E-mail: [email protected].

F2008 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-07-5150

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resequencing studies of PSMB1, PSMB2 , and PSMB5 genesencoding the three active h subunits, followed by functionalcharacterization of nonsynonymous coding single nucleotidepolymorphisms (cSNP) in PSMB5 (the gene encoding the h5subunit), the major proteasome inhibitor therapeutic target, aswell as a correlation of level of PSMB5 expression with genesequence variation. We also resequenced the PSMB5 gene usingDNA from patients with multiple myeloma who had beentreated with bortezomib, using both tumor and germ line DNA,resulting in the identification of additional novel PSMB5polymorphisms. This series of studies represents a step towardunderstanding sequence variation in genes encoding the threeactive proteasome h subunits, as well as the potentialimplications of that DNA sequence variation for individualdifferences in response to treatment with proteasome inhibitorsand/or contribution to disease pathophysiology.

Materials andMethods

DNA samples. DNA samples from 60 Caucasian American, 60African American, 60 Han Chinese American, and 60 Mexican Americansubjects (sample sets HD100CAU, HD100AA, HD100CHI, andHD100MEX) were obtained from the Coriell Cell Repository. TheseDNA samples have been widely used for human gene resequencingstudies (15–20). Immortalized lymphoblastoid cell lines from thesesame subjects are available from the Coriell Institute, and those celllines were also used in the experiments described subsequently. All ofthese DNA samples and cell lines had been obtained and anonymizedby the National Institute of General Medical Sciences before deposit,and all subjects had provided written consent for the use of their DNAand cells for experimental purposes. In addition, 79 DNA samples frompatients with multiple myeloma were isolated from either bone marrowor peripheral blood. Specifically, these 79 clinical DNA samples wereobtained from 61 multiple myeloma patients who had been treatedwith the proteasome inhibitor bortezomib. These patients had alsoprovided written consent for the use of their samples for researchpurposes. The clinical DNA samples included 11 paired bone marrowand peripheral blood samples from the same patient. A total of 39 DNAsamples were isolated from bone marrow plasma cells, and 40 sampleswere isolated from peripheral blood obtained from these patients. All ofthese experiments were reviewed and approved by Mayo ClinicInstitutional Review Board.

Human PSMB1, PSMB2, and PSMB5 gene resequencing. DNAsamples were used to perform PCR amplifications to resequence genesencoding the three active proteasome h subunits. PCR primer sequencesand amplification conditions are listed in Supplementary Table S1.Resequencing was done using dye termination sequencing chemistry asdescribed previously (18–20). Amplicons were sequenced on bothstrands in the Mayo Molecular Biology Core Facility with an ABI 377DNA sequencer. To exclude PCR-induced artifacts, independentamplifications were done for samples in which a SNP was observedonly once or for any sample with an ambiguous chromatogram. Thechromatograms were analyzed with Mutation Surveyor (SoftGenetics).

Transient transfection and expression. The wild-type (WT) cDNAsequence for PSMB5 was cloned into pcDNA3.1/V5-HisTOPO TA to useas a template for site-directed mutagenesis done using circular PCR tocreate variant allozyme expression constructs that encoded PSMB5Cys24, Cys212, and Met238. The sequences of primers used to performsite-directed mutagenesis are also listed in Supplementary Table S1.Sequences of all constructs were confirmed by sequencing the insert inboth directions. These constructs were transiently transfected into HeLacells using the TransFast reagent (Promega) at a charge ratio of 1:1. Cellswere harvested after 48 h, and proteasomes were isolated and used toperform proteasome activity assays.

Proteasome isolation. 26S proteasomes were isolated as described

elsewhere (21) from cells transfected with constructs for the PSMB5 WTand three variant allozymes, as well as from lymphoblastoid cells that

naturally expressed these nonsynonymous cSNPs. Specifically, cells

were collected and resuspended in 4 volumes of homogenization buffer[50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L MgCl2, 2 mmol/L ATP,

1 mmol/L DTT, 0.5 mmol/L EDTA] containing freshly prepared0.025% digitonin. The resuspended cells were incubated on ice for

5 min, and were then centrifuged at 20,000 � g at 4jC for 15 min. The

supernatants were separated, and protein concentrations were deter-mined. The cytosolic 26S proteasomes obtained in this fashion were

used to perform enzyme activity and inhibition studies.Proteasome activity and inhibition studies. Proteasome activity and

inhibition assays were done as described previously (21). Specifically,cytosolic 26S proteasomes isolated as described above were incubatedat 37jC with assay buffer [50 mmol/L Tris-HCl (pH 7.5), 40 mmol/LKCl, 5 mmol/L MgCl2, 0.5 mmol/L ATP, 1 mmol/L DTT, and0.05 mg/mL bovine serum albumin], and increasing concentrationsof the fluorogenic substrate Suc-LLVY-amc (0, 12.5, 25, 50, and100 Amol/L) and fluorescence was measured with a Safire2 plate reader(Tecan). For inhibition studies, concentrations of the proteasomeinhibitor MG262 ranging from 10 to 60 nmol/L were premixed withassay buffer 10 min before the addition of substrate. All assays weredone at least three times, and results were expressed as mean F SE forthree determinations.

Proteasome inhibitor cytotoxicity studies. Inhibitors of the protea-some h5 subunit are used clinically to treat cancer because of theircytotoxic properties. Therefore, cytotoxicity studies were done using theh5 subunit inhibitor MG262 with lymphoblastoid cells having knowngenotypes for the PSMB5 gene. Specifically, increasing concentrationsof MG262 (10, 20, 25, and 30 nmol/L) were incubated with the celllines in 96-well plates for 3 d, followed by MTS assays as a measure ofcytotoxicity. Each experiment was repeated three times, and results wereexpressed as GI50 values, the concentration of MG262 that inhibitedgrowth by 50%.

Expression array genotype-phenotype correlation. Expression array

analyses were done using Affymetrix U133 Plus 2.0 GeneChips.

Specifically, RNA was isolated from the same lymphoblastoid cells fromwhich the DNA used to resequence PSMB1, PSMB2 , and PSMB5 had

been obtained. Expression array data were normalized using the GCRMAmethod (22), and levels of PSMB5 expression in all 240 cell lines were

used to perform genotype-phenotype correlation studies. Before per-

forming the genetic association studies, Hardy-Weinberg equilibriumwas analyzed for each SNP using a stratified Hardy-Weinberg equili-

brium test. Because there were significant differences among ethnic

groups in minor allele frequencies for a given SNP, the genotype-phenotype association analyses were done by ethnic group for SNPs

with a minor allele frequency of >5% within that ethnic group. Theassociation of each SNP with expression level was evaluated using a

linear model in which SNP genotypes were evaluated with the use of

two indicators as covariates (e.g., a 2-degree-of-freedom test for eachSNP). In addition to the 2-degree-of-freedom test, a 1-degree-of-freedom

test for trend was also used, with SNP genotypes coded as 0, 1, or 2according to the number of rare variants present. PSMB5 expression

values were analyzed on a log 2 scale, and the data were adjusted for

gender, race, and the time of cell storage at the Coriell Institute.Luciferase reporter gene assays. To create reporter gene constructs,

f500 bp of PSMB5 3¶-flanking region (3¶-FR) sequence was amplifiedfrom either the Coriell or the patient genomic DNA samples. Forwardand reverse primers for these amplifications contained ACC65I andXhoI restriction sites, respectively, to make it possible to subclone theamplicons into pGL-3 Basic (Promega) upstream of the firefly luciferasegene open-reading frame. Specifically, reporter gene constructs werecreated by the amplification of a portion of the PSMB5 3¶-FR thatincluded either SNPs or a 17-bp insertion using DNA samples thatcontained these polymorphisms, followed by the cloning of ampliconsinto pGL-3 Basic, with or without subsequent site-directed mutagenesis.


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All of these reporter gene constructs were used to transfect HEK293Tand COS-1 cells, and luciferase activity was determined as describedpreviously (23). All of the primers used to create the reporter geneconstructs are also listed in Supplementary Table S1.

Data analysis. DNA sequence obtained during the gene resequenc-ing studies was compared with PSMB1, PSMB2 , and PSMB5 genomicand cDNA genomic consensus sequences (PSMB1 NM_144662 andNT_007583.11; PSMB2, NM_002794 and NT_004511.17; and PSMB5,D29011 and NT_026437.11). IC50 and GI50 values for WT and variantallozymes, as well as the luciferase activity data for WT and variantconstructs, were compared by ANOVA done with the Prism program.Linkage disequilibrium among PSMB1, PSMB2 , and PSMB5 poly-morphisms was determined by calculating D ¶ values for all possiblepairwise combinations of polymorphisms. This method for determin-ing linkage disequilibrium is independent of allele frequency (2, 24).Haplotypes for alleles that contained only one heterozygous polymor-

phism were determined unequivocally, and other haplotypes wereinferred computationally as described by Schaid et al. (25).

Results

PSMB1, PSMB2, and PSMB5 gene resequencing. PSMB1,PSMB2 , and PSMB5 were resequenced using 240 DNA samplesobtained from 60 African American, 60 Caucasian American,60 Han Chinese American, and 60 Mexican American subjects.The areas resequenced included all exons, exon-intron splicejunctions, and f1 to 2 kb of 5¶-FR for each gene. These rese-quencing data have been deposited in the NIH databasePharmGKB. Fig. 1 shows the locations of the polymorphismsobserved, and individual polymorphisms are listed inTable 1. Twenty-six polymorphisms were identified in PSMB1 ,

Fig.1. Human PSMB1, PSMB2, and PSMB5 genetic polymorphisms; a schematic representation of the (A) PSMB1, (B) PSMB2 , and (C) PSMB5 gene structures. Arrows,locations of polymorphisms. Rectangles, exons; black rectangles, open reading frame; open rectangles, untranslated regions. AA, African American subjects; CA, CaucasianAmerican subjects; HCA, Han ChineseAmerican subjects; MA, Mexican American subjects. SNPs identified in PSMB5 for DNA samples from multiple myeloma (MM)patients are also listed. *, SNPs altering encoded amino acids.

Proteosome Pharmacogenomics




including two nonsynonymous cSNPs; 40 polymorphisms, butno nonsynonymous SNPs, were identified in PSMB2 ; and 21polymorphisms were identified in PSMB5, including threenonsynonymous cSNPs. None of the nonsynonymous cSNPschanged amino acids located within the catalytic sites for any of

the subunits (9). The vast majority of these polymorphismswere not available in public databases. For example, 21 of 26polymorphisms that we identified in PSMB1 , 36 of 40 poly-morphisms in PSMB2 , and 14 of 21 in PSMB5—including twonovel nonsynonymous cSNPs—were not publicly available.

Table 1. Human PSMB1, PSMB2, and PSMB5 genetic polymorphisms

Location Nucleotide Sequence Amino acid Frequency of variant allele

change changeAfrican

AmericanCaucasianAmerican

Han ChineseAmerican

MexicanAmerican

PSMB15¶-FR -1199 A!G 0.017 0.000 0.000 0.0005¶-FR -1043 A!G 0.133 0.308 0.767 0.3425¶-FR -933 C!G 0.008 0.000 0.000 0.0005¶-FR -779 A!C 0.000 0.017 0.000 0.0005¶-FR -767 to -769 GAT deletion 0.000 0.008 0.000 0.0085¶-FR -696 G!T 0.000 0.000 0.008 0.0005¶-FR -695 C!T 0.000 0.000 0.000 0.0085¶-FR -621 A!C 0.008 0.000 0.000 0.0005¶-FR -573 G!A 0.017 0.000 0.000 0.0005¶-FR -213 A!C 0.008 0.000 0.000 0.0005¶-FR -192 G!C 0.000 0.000 0.000 0.0085¶-FR -126 C!A 0.008 0.050 0.000 0.0425¶-FR -62 C!T 0.008 0.000 0.000 0.0005¶-UTR -6 C!T 0.100 0.025 0.008 0.042Exon 1 31 C!G Pro11Ala 0.300 0.383 0.800 0.417Exon 1 73 C!T Pro25Ser 0.000 0.000 0.008 0.000Intron 1 -27 G!A 0.000 0.033 0.000 0.025Intron 2 49 A!G 0.233 0.550 0.200 0.475Intron 3 15 A!G 0.008 0.042 0.000 0.042Intron 3 23 A!G 0.000 0.000 0.000 0.008Intron 4 35 Deletion of G 0.208 0.000 0.000 0.017Intron 4 128 T!C 0.042 0.167 0.008 0.308Intron 4 136 A!G 0.000 0.000 0.000 0.008Intron 4 154 A!G 0.133 0.000 0.000 0.008Intron 4 -30 A!G 0.000 0.000 0.017 0.0003¶-FR 862 C!A 0.008 0.000 0.000 0.000

PSMB25¶-FR -1041 A!G 0.033 0.000 0.000 0.0005¶-FR -976 to -975 Deletion of TA 0.033 0.000 0.000 0.0005¶-FR -859 T!G 0.000 0.008 0.000 0.0005¶-FR -826 (TC)n

n = 2 0.782 1.000 0.917 0.967n = 3 0.182 0.000 0.083 0.017n = 4 0.036 0.000 0.000 0.000

5¶-FR -716 A!G 0.125 0.000 0.008 0.0175¶-FR -707 to -706 Deletion of CT 0.092 0.000 0.017 0.0085¶-FR -555 G!A 0.000 0.008 0.000 0.0005¶-FR -477 G!T 0.192 0.917 0.208 0.4925¶-FR -425 G!A 0.117 0.000 0.000 0.0175¶-FR -405 T!C 0.000 0.008 0.000 0.0005¶-FR -381 G!C 0.000 0.000 0.008 0.0005¶-FR -224 G!A 0.008 0.000 0.000 0.0005¶-FR -197 C!A 0.033 0.000 0.000 0.0005¶-FR -146 Deletion of A 0.025 0.000 0.000 0.0005¶-UTR -109 Deletion of T 0.017 0.000 0.000 0.000Intron 1 20 G!T 0.008 0.000 0.000 0.000Intron 1 43 G!C 0.008 0.000 0.000 0.000Intron 1 -27 T!C 0.008 0.000 0.000 0.000Exon 2 133 C!T 0.000 0.000 0.008 0.000Intron 2 57 A!C 0.008 0.000 0.000 0.000Intron 2 123 C!G 0.000 0.000 0.000 0.008Intron 2 145 G!A 0.000 0.008 0.000 0.008Intron 3 6 T!G 0.000 0.000 0.000 0.017

(Continued on the following page)





Allele frequencies for the nonsynonymous cSNPs differedgreatly among ethnic groups (Table 1). For example, thePSMB5 Arg24Cys polymorphism was present in all ethnicgroups except Han Chinese American; PSMB5 Try212Cys wasobserved only in one Mexican American subject; and the

PSMB5 Val238Met polymorphism was observed only in a HanChinese American subject (Table 1). It should also be notedthat we arbitrarily selected the least common allele in theAfrican American data as the variant allele but, in several cases,that was the most common allele in other populations. All of

Table 1. Human PSMB1, PSMB2, and PSMB5 genetic polymorphisms (Cont’d)


change changeAfrican

AmericanCaucasianAmerican

Han ChineseAmerican

MexicanAmerican

Intron 3 7446 A!G 0.000 0.000 0.000 0.008Intron 3 7763 to 7766 Deletion of CAGA 0.008 0.000 0.000 0.000Intron 3 7772 C!G 0.092 0.000 0.017 0.008Intron 3 7776 C!T 0.092 0.000 0.017 0.008Intron 3 7937 T!C 0.092 0.000 0.017 0.008Intron 3 -68 C!T 0.017 0.000 0.000 0.000Intron 3 -30 T!C 0.008 0.000 0.000 0.000Intron 4 30 Deletion of G 0.017 0.000 0.000 0.000Intron 4 65 T!C 0.058 0.000 0.000 0.000Intron 4 72 T!C 0.125 0.000 0.017 0.008Intron 4 93 G!A 0.117 0.000 0.008 0.017Intron 4 -26 T!C 0.000 0.025 0.000 0.0083¶-UTR 611 A!G 0.092 0.000 0.017 0.0083¶-UTR 641 T!C 0.033 0.000 0.000 0.0003¶-UTR 683 G!A 0.000 0.000 0.008 0.0003¶-UTR 684 C!T 0.092 0.000 0.017 0.0083¶-FR 758 T!C 0.008 0.000 0.000 0.000


change changeAfricanAmerican

CaucasianAmerican

Han ChineseAmerican

MexicanAmerican

Multiplemyelomasamples

PSMB55¶-FR -693 C!T 0.117 0.658 0.825 0.717 0.5385¶-FR -534 G!A 0.067 0.000 0.000 0.000 0.0135¶-FR -438 G!A 0.008 0.000 0.000 0.000 0.0005¶-FR -384 T!C 0.000 0.000 0.000 0.000 0.0005-FR -310 T!C 0.158 0.000 0.000 0.000 0.0135¶-FR -187 C!G 0.008 0.000 0.000 0.000 0.0005¶-FR -178 to -177 Insertion of C 0.000 0.000 0.042 0.000 0.0005¶-FR -57 G!T 0.000 0.000 0.000 0.008 0.000Exon 1 70 C!T Arg24Cys 0.025 0.083 0.000 0.017 0.114Intron 1 230 A!T 0.000 0.008 0.000 0.000 0.000Intron 1 269 G!T 0.000 0.000 0.008 0.000 0.000Intron 1 331 C!G 0.000 0.008 0.000 0.000 0.000Exon 3 588 G!C 0.000 0.000 0.008 0.000 0.000Exon 3 635 A!G Tyr212Cys 0.000 0.000 0.000 0.008 0.000Exon 3 712 G!A Val238Met 0.000 0.000 0.008 0.000 0.0003¶-UTR 821 C!T 0.008 0.000 0.000 0.000 0.0003¶-UTR 847 G!A 0.075 0.000 0.000 0.000 0.0133¶-UTR 938 to 939 AT deletion 0.000 0.000 0.008 0.000 0.0003¶-FR 1042 G!A 0.200 0.683 0.932 0.792 0.6083¶-FR 1094 G!A 0.000 0.000 0.000 0.000 0.0063¶-FR 1103 G!A 0.175 0.533 0.825 0.700 0.4873¶-FR 1209 Insertion of

GAGAAGGAGAGAGAGGC0.000 0.000 0.000 0.000 0.013

3¶-FR 1131 to 1133 AGT deletion 0.008 0.025 0.000 0.000 0.000

NOTE: Polymorphism locations, alterations in nucleotide and amino acid sequences, and minor allele frequencies for the polymorphismsobserved during the gene resequencing studies are listed for each of the four ethnic groups studied. The least common allele in African Americansamples has been designated as the minor allele. Polymorphisms identified in the PSMB5 gene in multiple myeloma patient samples were alsolisted. The table also indicates whether the polymorphism is represented in dbSNP. Highlighted and bold-faced SNPs are found in dbSNP.Polymorphisms in exons and UTRs and FRs have been numbered with respect to the ‘‘A’’ in the ‘‘ATG’’ translation initiation codon, with positivenumbers located 3- and negative numbers 5- to that position. Nucleotides located within introns are numbered based on their distance fromsplice junctions, using negative or positive numbers, respectively, for distance to 5¶- and 3¶-splice sites.





the polymorphisms identified during the resequencing effortwere in Hardy-Weinberg equilibrium (P > 0.05).We also used our gene resequencing data to calculate

nucleotide diversity, a quantitative measure of genetic variation,

adjusted for the number of alleles studied. Two standardmeasures of nucleotide diversity are p, average heterozygosityper site, and u , a population mutation measure that istheoretically equal to the neutral mutation parameter (26).

Table 2. PSMB1, PSMB2, and PSMB5 haplotypes

PSMB1

Observedor inferred

Haplotypedesignation

AfricanAmerican

CaucasianAmerican

Han ChineseAmerican

MexicanAmerican

5¶-FR(-1199)

5¶-FR(-1043)

5¶-FR(-779)

5¶-FR(-573)

5¶-FR(-126)

5¶UTR(-6)

o *1A 0.184 A A A G C Co *1B 0.183 0.386 0.192 0.167 A A A G C Co *1C 0.117 A A A G C Co *1D 0.067 A A A G C Ci *1E 0.083 0.025 0.033 A A A G C Ti *1F 0.033 0.156 0.300 A A A G C Ci *1G 0.033 0.025 A A A G A Co *2A 0.150 0.050 0.025 0.050 A A A G C Co *2B 0.116 0.300 0.733 0.333 A G A G C Ci *2C 0.017 G A A A C Ci *2D 0.017 A A C G C Ci *2E 0.014 0.025 A A A G C Ci *2F 0.017 A G A G C Ci *2 and *3 0.008 A A A G C C

PSMB2

Observedor inferred


AfricanAmerican

CaucasianAmerican

Han ChineseAmerican

MexicanAmerican

5¶-FR(-1041)

5¶-FR(-826)(TC)n

5¶-FR(-716)

5¶-FR(-707)

5¶-FR(-477)

5-FR(-425)

o *1A 0.316 0.075 0.725 0.450 A 2 A I G Go *1B 0.123 0.867 0.158 0.467 A 2 A I T Gi *1C 0.091 0.025 A 3 A I G Go *1D 0.083 A 2 G I G Go *1E 0.075 0.017 A 2 A I G Ao *1F 0.050 A 2 A I G Gi *1G 0.026 0.017 A 3 A D G Gi *1H 0.025 A 3 A D T Go *1I 0.018 A 4 A I G Gi *1J 0.017 G 2 A I G Gi *1K 0.017 A 3 A D G Ai *1L 0.014 A 2 G I G Gi *1M 0.014 A 2 G I G Go *1N 0.025 A 2 A I T Go *1O 0.042 A 3 A I T Gi *1P 0.017 A 2 G I G G

PSMB5

Observedor inferred


AfricanAmerican

CaucasianAmerican

Han ChineseAmerican

MexicanAmerican

5¶-FR(-693)

5¶-FR(-534)

5¶-FR(-310)

5¶-FR(-178)

Exon 1(70) *2

Intron 1(269)

o *1A 0.537 0.081 0.067 0.166 C G T D C Go *1B 0.139 C G C D C Go *1C 0.108 0.489 0.761 0.683 T G T D C Gi *1D 0.065 C A T D C Go *1E 0.057 0.020 C G T D C Go *1F 0.025 0.142 0.105 0.083 C G T D C Go *1G 0.025 T G T D C Go *1H 0.144 0.025 T G T D C Go *1I 0.039 T G T I C Gi *2A 0.022 0.075 0.008 C G T D T Gi *2B 0.003 C G C D T Gi *2C 0.008 0.008 C G T D T Gi *3A 0.008 T G T D C Gi *4A 0.008 T G T D C T

NOTE: Nucleotide positions are numbered as described in Table 1. Variant nucleotides compared to the reference sequence, that is, the mostcommon sequence in African American subjects, are highlighted as white on black. Initial haplotype designations (*1, *2, *3, etc.) were madeon the basis of amino acids that vary, with the WT sequence designated *1. Subsequent assignments (letter designations) were made withinethnic groups, based on decreasing frequencies. o, unequivocal haplotypes; i, inferred haplotypes; I, insertion; D, deletion.





These values for PSMB1, PSMB2, and PSMB5 are listed inSupplementary Table S2. For all three genes, DNA from AfricanAmerican subjects showed greater apparent diversity insequence than DNA obtained from other ethnic groups,probably reflecting the greater antiquity of these sequences(27). In addition, values for Tajima’s D, a test of the neutralmutation hypothesis (28), were also estimated for each

population (Supplementary Table S2). Under conditions ofneutrality, Tajima’s D should equal zero; none of these valuesdiffered significantly from zero in any of the populationsstudied.

PSMB1, PSMB2, and PSMB5 haplotype and linkage disequi-librium analyses. Haplotype and pairwise linkage disequilib-rium analyses were done for all three genes because it is

Table 2. PSMB1, PSMB2, and PSMB5 haplotypes (Cont’d)

PSMB1

Exon 1(31) *2

Exon 1(73) *3

Intron 1(-27)

Intron 2(49)

Intron 3(15)

Intron 4(35)

Intron 4(128)

Intron 4(154)

Intron 4(-30)

C C G A A I T A AC C G G A I T A AC C G A A D T G AC C G A A D T A AC C G A A I T A AC C G G A I C A AC C G A G I T A AG C G A A I T A AG C G A A I T A AG C G A A I T A AG C A A A I T A AG C A A A I T A AG C G A A I T A GG T G A A I T A A

PSMB2

5¶-FR(-197)

5¶-FR(-146)

5¶-UTR(-109)

Intron3 (6)

Intron 3(7772)

Intron 3(7776)

Intron 3(7937)

Intron4 (30)

Intron4 (65)

Intron4 (72)

Intron4 (93)

Intron4 (-26)

3¶-UTR(611)

3¶-UTR(641)

3¶-UTR(684)

C I I T C C T I T T G T A T CC I I T C C T I T T G T A T CC I I T C C T I T T G T A T CC I I T C C T I T T A T A T CC I I T C C T I T T G T A T CC I I T C C T I C T G T A T CC I I T G T C I T C G T G T TC I I T G T C I T C G T G T TC I I T C C T I T T G T A T CA D I T C C T I T C G T A C CC I I T G T C I T C G T G T TC I D T C C T I T T A T A T CC I I T C C T D T T A T A T CC I I T C C T I T T G C A T CC I I T C C T I T T G T A T CC I I G C C T I T T A T A T C

PSMB5

Exon 3 (635) *4 Exon 3 (712) *5 3¶UTR (847) 3¶-FR (1042) 3¶-FR (1103) 3¶-FR (1131)

A G G G G IA G G G G IA G G A A IA G A G G IA G G A A IA G G A G IA G G A A DA G G G G IA G G A A IA G G G G IA G G G G IA G G A G IG G G A A IA A G A A I





becoming increasingly clear that the determination of haplo-type may be more helpful than the assay of individual SNPs foruse in association studies (29). A total of 14 haplotypes wereidentified for PSMB1, including 6 observed and 8 inferredhaplotypes; 16 haplotypes were identified in PSMB2, including8 observed and 8 inferred; and there were 14 PSMB5haplotypes, 8 of which were observed (Table 2). Haplotypefrequencies also differed among ethnic groups. African Amer-ican subjects had more unique haplotypes than other ethnicgroups. For example, four unique PSMB1 haplotypes wereobserved for African American subjects, including one that wasthe most common haplotype in this ethnic group, with afrequency of f18%; however, that haplotype was not observedin other ethnic groups (Table 2). Similar observations weremade for PSMB5 , with African American subjects having threehaplotypes that were unique when compared with the otherethnic groups (Table 2). Haplotype designations were assignedbased on the amino acid sequences of the encoded allozyme,with the WT amino acid sequence designated as *1 . Subsequentnumber designations referred to variant haplotypes thatcontained nonsynonymous cSNPs, beginning at the NH2

terminus and proceeding to the COOH terminus of theencoded protein. Letters were then added for haplotypes thatincluded variant nucleotides that did not alter amino acidsequence, ranked from most common to least common.Linkage disequilibrium analysis was also done for all pairwisecombinations of SNPs in these three genes by calculating D ¶values (2, 24). D ¶ values can range from +1.0, when twopolymorphisms are maximally associated, to zero, when theyare randomly associated. However, no clearly defined haplo-type blocks were observed in any of these relatively short geneswhen those data were displayed graphically (data not shown).

Proteasome activity and inhibitor studies. Because the h5subunit is the major therapeutic target for proteasomeinhibitors used in the clinic (10, 11), and because threenonsynonymous cSNPs were observed in that gene with minorallele frequencies that differed among ethnic groups, functionalcharacterization of those three nonsynonymous cSNPs wasdone after transient transfections of HeLa cells. HeLa cells havebeen used extensively to study proteasome activity (21, 30). Inaddition, lymphoblastoid cell lines that naturally expressedthese nonsynonymous cSNPs, either homozygous or heterozy-gous, were also used to perform functional studies. Specifically,proteasomes isolated from HeLa cells transfected with fourdifferent constructs and from lymphoblastoid cell lines wereused to perform assays with the fluorogenic substrate Suc-LLVY-amc to measure the chymotrypsin-like activity of the h5subunit. There were no significant differences among the fourallozymes (WT plus three variants) studied with regard to levelsof enzyme activity with either type of cell extract (data notshown).Because the h5 subunit is the target for clinically used

proteasome inhibitors, we also did inhibition studies using aspecific h5 proteasome inhibitor, MG262. Those experimentswere done with proteasomes isolated from HeLa cellstransiently transfected with constructs for PSMB5 variantallozymes, as well as proteasomes isolated from lymphoblas-toid cells that naturally expressed these allozymes. Because thePSMB5 WT gene sequence for the African American sampleswas different from that for the other three ethnic groups, weused two WT samples, one for African American and the otherfor the remaining ethnic groups. Concentrations of MG262 thatranged from 10 to 60 nmol/L were tested, but IC50 values didnot differ significantly among proteasomes isolated from either

Fig. 2. MG262 inhibition studies and cytotoxicity studies. MG262 was used to perform (A) inhibition studies with cell extracts from HeLa cells transfected with expressionconstructs for PSMB5WTand variant allozymes, (B) inhibition studies with lymphoblastoid cell extracts from cells expressingWTand variant PSMB5 allozymes, and (C)cytotoxicity studies with lymphoblastoid cells expressingWTand variant allozymes. Columns, mean for three independent determinations; bars, SE.





transfected cells or from cells that naturally expressed theseallozymes (Fig. 2A and B).Proteasome inhibitors are used clinically because of their

cytotoxic properties; therefore, we also did MG262 cytotoxicitystudies with the lymphoblastoid cell lines from which theproteasomes with known PSMB5 genotypes had been isolated.GI50 values calculated from the cytotoxicity data also failed toshow significant differences among cells expressing theseallozymes (Fig. 2C). All of these functional genomic resultsindicated that the three nonsynonymous PSMB5 cSNPs did notseem to have significant effects on proteasome enzyme activity,inhibition by MG262, or MG262-induced cytotoxicity. How-ever, a h5 proteasome inhibitor, bortezomib, has beenapproved by the Food and Drug Administration to treatrefractory multiple myeloma. To determine whether theremight be additional sequence variation in the PSMB5 gene inpatients with multiple myeloma that might influence responseto proteasome inhibitor therapy, we also resequenced PSMB5using 79 DNA samples obtained from 61 patients with multiplemyeloma who had been treated with bortezomib. Thosesamples included both DNA isolated from bone marrowmyeloma cells and from peripheral blood.PSMB5 resequencing using DNA from multiple myeloma

patients. The same primers that had been used to amplifyPSMB5 from Coriell Institute DNA samples were used toamplify the gene using DNA from multiple myeloma patients.A total of 9 PSMB5 polymorphisms were identified in these79 DNA samples (Table 1; Fig. 1). When we compared theresequencing results for 11 individuals for whom we had twoDNA samples, one from bone marrow myeloma cells and theother isolated from peripheral blood, no differences in PSMB5sequence were observed between the two sources of DNA.However, we observed novel PSMB5 polymorphisms in theseDNA samples, including a 3¶-FR 1094 G/A polymorphism andan insertion of GAGAAGGAGAGAGAGGC at nucleotide 1209,also located in the 3¶-FR of the gene, neither of which had beenidentified in the 240 Coriell DNA samples that we hadresequenced (Table 1). The 1094 G/A polymorphism waspresent in one DNA sample extracted from bone marrowmyeloma cells, and the nucleotide 1209 insertion was presentin peripheral blood DNA obtained from the two patients. Bothof these novel polymorphisms were located in an area thatshared 73% sequence identity with the mouse gene, indicatingthat it might be of functional significance. Therefore, we createdluciferase reporter gene constructs that contained these twopolymorphisms to test their possible effect on the regulation oftranscription. We also attempted to determine whether agenotype-phenotype correlation might exist among SNPs inthe PSMB5 gene and clinical response to bortezomib therapy ofmultiple myeloma as the phenotype. However, perhapsbecause of the relatively small number of samples, nosignificant associations were observed.PSMB5 genotype-phenotype correlation for expression. To

determine whether any of the SNPs that we had identified inPSMB5 might be associated with the level of gene expression,RNA isolated from the same 240 lymphoblastoid cells fromwhich DNA had been isolated to perform the gene resequenc-ing was used to perform expression array studies usingAffymetrix U133 Plus 2.0 GeneChips. PSMB5 expression variedf2-fold among these 240 cell lines (Fig. 3A). We thencorrelated PSMB5 genotype in these cells with level of

expression using seven SNPs with minor allele frequencies>5% in at least one ethnic group. One SNP at position 1042 inthe PSMB5 3¶-FR showed a significant correlation with level ofexpression for all cell lines, with G at that position beingassociated with elevated expression (Fig. 3B). However, thisassociation was statistically significant only in CA subjects,perhaps because of differences in allele frequencies amongethnic groups, with P = 0.00038 for normalized data adjustedfor covariates. The P value for CA subjects remained significanteven after correction for multiple comparisons (P = 0.012). TheG nucleotide at position 1042 was the WT sequence in theAfrican American population, but G was the variant sequencein the other three populations studied (see Table 1). Thispolymorphism was located very close to the area of PSMB5 thatshares >70% sequence identity with the mouse gene sequence.Therefore, we created luciferase constructs that contained the1042 SNP as well as the unique 3¶-FR polymorphisms identifiedin the patient samples to test their possible effect ontranscription.

Luciferase reporter gene assays. Luciferase reporter geneconstructs were created; these constructs contained the twonovel 3¶-FR polymorphisms that we had identified in samplesfrom patients with multiple myeloma as well as the SNP atnucleotide 1042 that was associated with level of mRNAexpression in the lymphoblastoid cell lines. WT and these threevariant constructs were transiently transfected into HEK293Tand COS-1 cells with Renilla luciferase (Promega) as a controlfor transfection efficiency. Patterns of luciferase activity forvariant constructs were very similar in the two cell lines studied(Fig. 4A and B). Luciferase activity was significantly elevated inboth cell lines after transfection with the construct containing aG at position 1042 (P < 0.001), consistent with the results ofthe genotype-phenotype correlation study in the lymphoblas-toid cell lines. These results were compatible with theconclusion that the SNP at nucleotide 1042 can influencePSMB5 expression. The two polymorphisms identified insamples from patients with multiple myeloma, 1094A andthe insertion at position 1209, showed slightly decreasedactivity in both cell lines when compared with the WT sequence(Fig. 4A and B); however, those differences were not statisticallysignificant.

Discussion

The proteasome plays a major role in the degradation ofproteins that regulate critical cellular processes and it is also thetarget for antineoplastic proteasome inhibitors such as borte-zomib (1, 3, 4, 10, 31). Because of the importance of theproteasome and because it is a drug target, we set out todetermine whether sequence variation in genes encoding thethree active proteasome h subunits might influence proteasomeactivity or expression. This sequence variation might alsocontribute to individual variation in disease pathophysiologyor response to drug therapy. The proteasome inhibitorbortezomib is used to treat refractory multiple myeloma andnon–small cell lung cancer. There are large variations inresponse to this drug (12–14). For example, only f35% to50% of multiple myeloma patients respond to bortezomibtherapy (32). Obviously, multiple factors can influence drugresponse, including genetic factors, age, gender, and environ-mental factors. In the treatment of cancer, both tumor DNA





and germ line DNA are important for explaining variation inresponse to drug therapy. Therefore, we resequenced the genesencoding the three active proteasome h subunits using DNAsamples from four ethnic groups and characterized thefunctional effects of sequence variation in PSMB5, the geneencoding the h5 subunit, the major clinical target forproteasome inhibitors (10, 31).We identified a series of SNPs in genes encoding the three

active proteasome h subunits (Table 1), and the majority ofthese SNPs were not available in any public database. The h5subunit of the proteasome has chymotryptic-like proteaseactivity, and this subunit is the target for clinical proteasomeinhibitors such as bortezomib (9, 10, 13, 31, 33). Therefore, wefocused our functional genomic studies, including activityassays, inhibition studies, and cytotoxicity studies done withthe specific proteasome inhibitor, MG262, on three non-synonymous cSNPs in the PSMB5 gene (Fig. 2A-C). All of theseresults were compatible with the conclusion that these non-synonymous cSNPs in PSMB5 did not significantly affectproteasome function. Obviously, we cannot rule out thepossibility that these polymorphisms might alter the proteolysisof specific substrates, including endogenous substrates, or theresponse to inhibitors other than MG262. However, geneticpolymorphisms can also influence transcription, RNA splicing,and mRNA stability. Therefore, we next turned our attention totranscription. Specifically, we did genotype-phenotype correla-tion analyses using expression array data (Fig. 3A) and ourPSMB5 gene resequencing data for all 240 lymphoblastoid cell

lines. That study identified one SNP, a polymorphic nucleotideat position 1042 in the 3¶-FR of PSMB5 (near a regionconserved in the mouse) that was significantly associated withlevel of expression in the lymphoblastoid cell lines (Fig. 3B).These genotype-phenotype association results were supportedby data from reporter gene assays (Fig. 4).Finally, because patients with refractory multiple myeloma

are treated with bortezomib (13, 33), we also resequencedPSMB5 using 79 DNA samples from both tumor cells andperipheral blood obtained from patients with multiplemyeloma who had been treated with this drug. We were notable to detect significant associations between SNPs in PSMB5and response to bortezomib therapy in this group of patients,although these results must be viewed as preliminary because ofthe relatively small number of samples studied and theheterogeneous nature of this disease. However, resequencingof these 79 patient DNA samples resulted in the identificationof two additional novel PSMB5 polymorphisms, both locatedin the 3¶-FR of the gene in the same conserved region where thefunctionally significant 1042 SNP was located. Therefore, wecreated luciferase reporter gene constructs to test the possibleimplications for transcription of these two novel polymor-phisms together with the nucleotide 1042 SNP. As anticipatedon the basis of the genotype-phenotype correlation analysisdone with the lymphoblastoid cell lines, a G at nucleotide 1042significantly increased luciferase activity (Fig. 4). Although the1094 SNP and the insertion at nucleotide 1209 that we hadidentified by resequencing DNA samples from the patientsshowed slightly decreased luciferase activity when compared

Fig. 3. PSMB5 expression and association with theA1042GSNP in 240lymphoblastoid cell lines. A, PSMB5 expression in 240 lymphoblastoid cells.PSMB5 expression was assayed with Affymetrix U133 Plus2 GeneChips.Expression levels were normalized with GCRMA. Columns, individual cell line;colors, ethnic groups. B, PSMB5 A1042GSNP genotypes were correlated withcorrespondingnormalized expression array data for each of the 240 cell lines shownin A. Columns, mean; bars SE.

Fig. 4. PSMB5 3¶-FR luciferase reporter gene studies. Reporter gene studieswere done with (A) COS-1and (B) HEK293Tcells transfected with reporter geneconstructs containingWTsequence as well as variant sequence at nucleotides1042and1094 and the insertion at position1209. Luciferase activity was corrected forRenilla luciferase activity and is expressed as a percent of theWTactivity. Columns,mean for three independent experiments; bars, SE.





with the WT sequence, those differences were not statisticallysignificant (Fig. 4).In summary, we have done a comprehensive series of studies

of the pharmacogenomics of genes encoding the three activeproteasome subunits. Those experiments resulted in theidentification of a large number of novel SNPs and haplotypesin both control subjects and patients with multiple myelomathat were not represented in the HapMap or other publicdatabases. Functional characterization of nonsynonymouscSNPs and SNPs in the 3¶-FR of the PSMB5 gene showed thatthe common nucleotide 1042 SNP in the 3¶-FR of the genesignificantly increased transcription, confirming the results ofgenotype-phenotype correlation studies between SNPs andPSMB5 expression in 240 lymphoblastoid cell lines. Theseresults significantly increase our knowledge of common genetic

variation in these important genes and represent a step towardfuture translational pharmacogenomic studies of patientstreated with proteasome inhibitors.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Luanne Wussow for her assistance with the preparation of themanuscript.

The PSMB1, PSMB2, and PSMB5 gene sequence data reported in this articlehave been deposited in the NIH-supported database PharmGKB with accessionnumbers PS205879, PS205881, and PS205880.

References1. Adams J. The proteasome: structure, function, androle in the cell. CancerTreat Rev 2003;29 Suppl1:3^9.

2. Hendrick PW. Genetics of populations. 2nd ed.Sudbury (MA): Jones and Bartlett Publ.; 2000. p.396^405.

3.Mani A, Gelmann EP.The ubiquitin-proteasomepath-way and its role in cancer. J Clin Oncol 2005;23:4776^89.

4. Matthews W, Driscoll J, Tanaka K, Ichihara A,Goldberg AL. Involvement of the proteasome in vari-ous degradative processes in mammalian cells. ProcNatl Acad Sci US A1989;86:2597^601.

5. Glickman MH, Ciechanover A. The ubiquitin-protea-some proteolytic pathway: destruction for the sake ofconstruction. Physiol Rev 2002;82:373^428.

6. Coux O,Tanaka K, Goldberg AL. Structure and func-tions of the 20S and 26S proteasomes. Annu RevBiochem1996;65:801^47.

7. Groll M, Ditzel L, LoweJ, et al. Structure of 20S pro-teasome from yeast at 2.4 A resolution. Nature 1997;386:463^71.

8. Groll M, Bajorek M, Kohler A, et al. A gated channelinto the proteasome core particle. Nat Struct Biol2000;7:1062^7.

9. Heinemeyer W, Fischer M, Krimmer T, Stachon U,Wolf DH. The active sites of the eukaryotic 20 S pro-teasome and their involvement in subunit precursorprocessing. JBiol Chem1997;272:25200^9.

10. AdamsJ.The development of proteasome inhibitorsas anticancer drugs. Cancer Cell 2004;5:417^21.

11. AhmadK. Proteasome inhibitor for treatment ofmul-tiple myeloma. Lancet Oncol 2005;6:546.

12. Richardson PG, Barlogie B, Berenson J, et al. Aphase 2 study of bortezomib in relapsed, refractorymyeloma. NEngl JMed 2003;348:2609^17.

13. Jagannath S, Barlogie B, BerensonJ, et al. A phase2 study of two doses of bortezomib in relapsed or re-fractory myeloma. BrJHaematol 2004;127:165^72.

14. Richardson PG, Sonneveld P, Schuster MW, et al.Bortezomib or high-dose dexamethasone for relapsedmultiplemyeloma.NEnglJMed2005;352:2487^98.

15. JiY, Salavaggione OE,Wang L, et al. Humanphenyl-ethanolamine N-methyltransferase pharmacogenom-ics: gene resequencing and functional genomics. JNeurochem 2005;95:1766^76.

16. Ma CX, Adjei AA, Salavaggione OE, et al. Humanaromatase : gene resequencing and functionalgenomics. Cancer Res 2005;65:11071^82.

17. Martin YN, Salavaggione OE, Eckloff BW,WiebenED, Schaid DJ,Weinshilboum RM. Humanmethylene-tetrahydrofolate reductase pharmacogenomics: generesequencing and functional genomics. Pharmacoge-net Genomics 2006;16:265^77.

18. Mukherjee B, Salavaggione OE, Pelleymounter LL,et al. Glutathione S-transferase N1and N2 pharmaco-genomics. Drug Met Dispos 2006;34:1237^46.

19.Wang L, Salavaggione E, Pelleymounter L, Eckloff B,Wieben E,Weinshilboum R. Human 3h-hydroxyste-roid dehydrogenase types1and 2: gene sequencevar-iation and functional genomics. J Steroid BiochemMol Biol 2007;107:88^99.

20.Wood TC, Salavagionne OE, Mukherjee B, et al.Human arsenic methyltransferase (AS3MT) pharma-cogenetics : gene resequencing and functionalgenomics studies. JBiol Chem 2006;281:7364^73.

21. Deshaies RJ. Methods in enzymology ubiquitin andprotein degradation, part A; San Diego, CA: Elsevier,2005. p. 364^78.

22.Wu Z, Irizarry RA, Gentleman R, Martinez-Murillo F,Spencer F. A model-based background adjustmentfor oligonucleotide expression arrays. J Am StatAssoc 2004;99:909.

23. Wang L, Thomae B, Eckloff B, Wieben E,WeinshilboumR.HumanhistamineN-methyltransferasepharmacogenetics: gene resequencing, promotercharacterization, and functional studies of a common

5¶-flanking region single nucleotide polymorphism(SNP). Biochem Pharmacol 2002;64:699^710.

24. Hartl DL, Clark AG. Organization of genetic varia-tion. Principles of population genetics. Chapter 3. 3rded. Sunderland (MA): SinauerAssociates, Inc.; 2000.p. 95^107.

25. Schaid DJ, Rowland CM,Tines DE, Jacobson RM,Poland GA. Score tests for association between traitsand haplotypes when linkage phase is ambiguous.AmJHum Genet 2002;70:425^34.

26. Fullerton SM, Clark AG,Weiss KM, et al. Apolipo-protein E variation at the sequence haplotype level:implications for the origin and maintenance of a majorhuman polymorphism. Am J Hum Genet 2000;67:881^900.

27. Kaessmann H, Heissig F, von Haeseler A, Paabo S.DNA sequence variation in a non-coding regionof lowrecombination on the human X chromosome. NatGenet 1999;22:78^81.

28. Tajima F. Statistical method for testing the neutralmutation hypothesis by DNA polymorphism. Genetics1989;123:585^95.

29. Brodde OE, Leineweber K. h2-adrenoceptor genepolymorphisms. Pharmacogenet Genomics 2005;15:267^75.

30. Kisselev AF, Goldberg AL. Proteasome inhibitors:from research tools to drug candidates. Chem Biol2001;8:739^58.

31. AdamsJ. Proteasome inhibition in cancer: develop-ment of PS-341. Semin Oncol 2001;28:613^9.

32.Richardson PG,Mitsiades C, HideshimaT,AndersonKC. Bortezomib: proteasome inhibition as an effec-tive anticancer therapy. Annu Rev Med 2006;57:33^47.

33. HideshimaT, Chauhan D, Podar K, Schlossman RL,Richardson P, Anderson KC. Novel therapies targetingthe myeloma cell and its bone marrow microenviron-ment. Semin Oncol 2001;28:607^12.





2008;14:3503-3513. Clin Cancer Res Liewei Wang, Shaji Kumar, Brooke L. Fridley, et al. Resequencing and Functional Genomics

Subunit Pharmacogenomics: GeneβProteasome

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