Environmental and Molecular Mutagenesis 56:594^608 (2015)

Research Article Mutagenicity Monitoring Following Battlefield Exposures: Molecular Analysis of HPRT Mutations in Gulf War I Veterans Exposed to Depleted Uranium Janice A. Nicklas,1* Richard J. Albertini,2,3 Pamela M.Vacek,4 Stephanie K. Ardell,5 Elizabeth W. Carter,6 Melissa A. McDiarmid,7 Susan M. Engelhardt,8 Patricia W. Gucer,7 and Katherine S. Squibb7 1

Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont 2 Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 3 Biostatistics Unit, University of Vermont College of Medicine, Burlington, Vermont 4 Center for Clinical and Translational Science – Biomedical Informatics Unit, University of Vermont, Burlington, Vermont 5 Division of Newborn Medicine, Department of Pediatrics, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 6 Center for Clinical and Translational Science – Biomedical Informatics Unit, University of Vermont, Burlington, Vermont 7 Occupational Health Program, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 8 Department of Veterans Affairs Medical Center, Baltimore, Maryland Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations,

deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures. Environ. Mol. C 2015 Wiley Mutagen. 56:594–608, 2015. V Periodicals, Inc.

Key words: depleted uranium; HPRT; HPRT mutations; DNA sequencing

INTRODUCTION We have had the opportunity over a period of eight years to longitudinally study Veterans of the 1991 Gulf War, who were exposed to depleted uranium (DU). The original exposures occurred in February of 1991 when a group of soldiers were mistakenly fired upon by US forces using DU penetrators. DU contains 60% of the radioactivity of naturally occurring uranium (U) primarily in the form of alpha particles (AEPI, 1995) and forms U oxide dust on impact because of its pyrophoric character. Acute exposures of the soldiers therefore, occurred by inhalation as well as wound contamination. In addition, many of the individuals have continuing systemic expoC 2015 Wiley Periodicals, Inc. V

Grant sponsors: U.S. Department of Veterans Affairs, Vermont Cancer Center, the Lake Champlain Cancer Research Organization, and the UVM College of Medicine. *Correspondence to: Janice A. Nicklas, Genetic Toxicology, University of Vermont, 665 Spear St., Burlington, VT 05405. E-mail: [email protected] Additional Supporting Information may be found in the online version of this article. Received 20 January 2015; provisionally accepted 8 April 2015; and in final form 00 Month 2015 DOI 10.1002/em.21956 Published online 12 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans

sure released from embedded DU containing shrapnel. This cohort of Veterans has been monitored since this incident for medical and genotoxic consequences of their exposures [Squibb and McDiarmid, 2006; McDiarmid et al., 2011, 2013]. The companion paper describes the frequency of somatic mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in peripheral blood lymphocytes (PBL) of 70 of these Veterans, including multiple cloning assays of the same individuals over an eight year time interval [Albertini et al., 2015]. Mutant colonies were isolated from these HPRT cloning assays for molecular analyses, the results of which were used for calculations of actual mutation frequencies (MutF), as described in that article. We now describe the complete results of these molecular analyses for 61 of these Veterans and present the frequencies of the various types of HPRT mutations observed, i.e. base substitution point mutations, deletions, insertions, complex mutations, etc. We compare the mutational spectra between DU exposure groups, comparing high to low DU exposures, to determine if the DU exposures left a mutational imprint on the spectrum. Finally, we compare the overall HPRT mutational spectrum identified in this large study with a HPRT mutation database of historic controls. MATERIALS AND METHODS Mutant Cells Mutants were obtained from the HPRT cloning assays as described in the companion paper [Albertini et al., 2015]. The protocol used in this work was approved by the Baltimore VAMC’s and University of Maryland School of Medicine’s IRB programs. Each participant completed an informed consent document. The University of Vermont (UVM) IRB reviewed the University of Maryland IRB approval; however, they decided that the UVM part of the studies did not require local approval because the UVM researchers had no access to patient identifiers.

Primer Design New primers (new multiplex and some exon specific) were designed using Primer3 from the Whitehead Institute (MIT) (http://frodo.wi.mit. edu/primer3/). The primers used are shown in Supporting Information 1 and the products they generated for the different types of analyses are shown in Supporting Information 2.

mRNA Extraction mRNA was isolated from frozen pellets of 50,000 cells using Qiagen RNeasy mini kits (for 2003, 2005, and 2007 samples) or Qiagen RNeasy 96 (for 2009 samples) (Valencia, CA) following the manufacturer’s directions. The mRNA was snap frozen in liquid N2 and kept at 270 C. This mRNA was also used as a source of genomic DNA for multiplex and exon specific amplifications.

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Fig. 1. Flow diagram for the molecular analysis of the HPRT mutations. Numbers indicate the number of mutants that had the type of mutation for each branch. workflow for molecular analysis. In addition, if a mutant isolate showed an exon 2–3 genomic deletion, it was tested to determine if the deletion had been created by a VDJ mediated mechanism [Fuscoe et al., 1991, 1992] by amplification with primers designed to amplify across the usual HPRT VDJ breakpoints. TCR gene rearrangement analysis [Albertini et al., 2008] was undertaken on mutants from the same Veteran with the same HPRT mutation to determine if they were siblings from an in vivo clone.

HPRT Reverse Transcription RT was performed in two ways. For the 2003, 2005, 2007, and some of the 2009 samples, 20 ll reaction RT was performed consisting of 1 ll RNase free H2O from Qiagen RNeasy kit, 2.0 ll Applied Biosystems PCR Buffer II (Foster City, CA), 4 ll of 25 mM MgCl2, 8 ll 2.5 mM (each) dNTPs, 1 ll primer Ex9A (oligo dT for the 2003 samples), 1 ll of RNase Inhibitor (Applied Biosystems), 1 ll Reverse Transcriptase (Applied Biosystems). Reverse transcription conditions were 25 C 15 min then 42 C 15 min then 99 C 5 min then a 10 C hold. Most of the 2009 samples used the High Capacity RT Kit (Invitrogen) following manufacturer’s directions.

1st Round HPRT PCR A 20 ll reaction PCR was performed consisting of 2.0 ll Qiagen 103 Buffer, 1.6 ll 2.5 mM (each) dNTPs, 12.7 ll HPLC Water, 0.8 ll 10 mM SSJ primer, 0.8 ll 10 mM 4B primer (RSJ primer for 2003 samples), 0.1 ll HotStar Taq (Qiagen) plus 2 ll of the cDNA preparation. PCR cycles were 95 C 15 min then 403 of 94 C-30 s, 58 C-30 s, 72 C-90 s then 72  C-10 min and a 4 C hold.

2nd Round HPRT PCR (Performed Only if the1st Round PCR Did Not Produce Sufficient Product) A 20 ll reaction PCR was performed consisting of 2.0 ll Qiagen 103 Buffer, 1.6 ll 2.5 mM (each) dNTPs, 14.5 ll HPLC Water, 0.4 ll 20 mM B primer, 0.4 ll 20 mM A primer (4B primer for 2003 samples), 0.1 ll HotStar Taq plus 1 ll of the 1st round PCR preparation. PCR cycles were 95 C 15 min then 283 of 94 C-30 s, 58 C-30 s, 72 C-90 s then 72 C-10 min and a 4 C hold.

Workflow for Molecular Analysis of HPRT Mutations

HPRT Specific Exon PCR (exon 2, exon 3, exon 4, exon 5, exon 6, exons 7^8, exon 9)

Molecular analysis of the HPRT mutant isolates was performed to determine the HPRT mutation in each isolate. Figure 1 diagrams the

A 20 ll reaction PCR was performed consisting of 2.0 ll Qiagen 103 Buffer, 1.6 ll 2.5 mM (each) dNTPs, 13.5 ll HPLC Water, 0.4 ll

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20 mM forward primer, 0.4 ll 20 mM reverse primer, 0.1 ll HotStar Taq plus 2 ll of mRNA preparation. PCR cycles were 95 C 15 min then 403 of 94 C-30 s, 58 C-30 s, 72 C-90 s then 72 C-10 min and a 4 C hold.

HPRT Specific Exon PCR (exon1) A 20 ll reaction PCR was performed consisting of 2.0 ll Qiagen 103 Buffer, 1.6 ll 2.5 mM (each) dNTPs, 9.3 ll HPLC Water, 4.0 ll Q buffer, 0.5 ll 20 mM 1S primer, 0.5 ll 20 mM 1A, 0.1 ll HotStar Taq plus 2 ll of the mRNA preparation. PCR cycles were 95 C 15 min then 403 of 94 C30 s, 55 C-30 s, 72 C-90 s then 72 C-10 min and a 4 C hold.

HPRT New Multiplex PCR (2005, 2005, and 2009 Samples) A 25 ll reaction PCR was performed consisting of 2.5 ll Qiagen 103 Buffer, 4.0 ll 2.5 mM (each) dNTPs, 14.77 ll HPLC Water, 1.0 ll of 25 mM MgCl2, 0.56 ll primer mix, 0.2 ll HotStar Taq plus 2 ll of mRNA preparation. Primer mix was 20 lM Ex2S, 20 lM Ex2A, 10.7 lM Ex3DFor, 10.7 lM Ex3CRev, 44.6 lM Ex4S, 44.6 lM Ex4A, 5.36 lM Ex5NewF, 5.36 lM Ex5NewR, 10.7 lM Ex6S, 10.7 lM Ex6A, 14.3 lM Ex7/8EFor, 14.3 lM Ex7/8CRev, 17.9 lM Ex9S2 and 17.9 lM Ex9A4. PCR cycles were 95 C 15 min then 363 of 94 C-30 s, 60 C-30 s, 72 C-90 s then 72 C-10 min and a 4 C hold.

HPRT Old Multiplex PCR (2003 Samples) For the 2003 samples, multiplex analysis was performed using Gitcher buffer (67 mM MgCl2, 166 mM (NH4)2SO4, 50 mM BME, 68 lM EDTA, 670 mM Tris HCL, pH 8.8) [Gibbs et al., 1990] in two reactions with two sets of primers. Set #1 had exons 1, 3, 7/8, and 9 while set #2 had exons, 2, 4, 5, and 6. A 25 ll reaction PCR was performed consisting of 2.5 ll Gitcher Buffer, 1.5 ll 25 mM dNTPs, 6.8 ll HPLC Water, 6.7 ll of 25 mM MgCl2, 2.5 ll DMSO and 2.5 ll of either primer mix plus 2.5 ll of DNA lysis. The primer mix #1 consisted of 3.5 lM Ex3s and 3.2 lM Ex3A, 8.0 lM Ex5S and Ex5A, 4.4 lM Ex7/8S and 5.0 lM Ex7/8A and 3.2 lM Ex9S and Ex9A. Primer mix #2 consisted of 2.0 lM Ex1S and Ex1A, 1.6 lM Ex2S and Ex2A, 5.0 lM Ex4S and Ex4A and 3.0 lM and Ex6A. After 5 min at 95 C, 0.4 ll of Taq polymerase (USB Corporation, Cleveland, OH) was added and then cycling proceeded for 33 cycles of 94 C-1 min, 59 C-1 min, 68 C-2 min then 68 C-5 min and 10 C hold.

HPRT Exon1-Exon9-Dystrophin PCR A 25 ll reaction PCR was performed consisting of 2.5 ll Gitcher Buffer, 1.5 ll 25 mM dNTPs, 4.5 ll HPLC Water, 6.7 ll of 25 mM MgCl2, 2.5 ll DMSO and 5.0 ll primer mix plus 2 ll of mRNA preparation. The primer mix consisted of 10 lM Ex1S, 10 lM Ex1A, 10 mM Ex9AS, 10 mM Ex9A, 10 lM dystrophin S and 10 lM dystrophin A. After 5 min at 95 C, 0.3 ll of Taq polymerase (USB) was added and then cycling proceeded for 40 cycles of 94C 1 min, 59 C 1 min, 68 C-2 min then 68 C for 7 min and a 10 C hold.

HPRT VDJ PCR A 25 ll reaction PCR was performed consisting of 2.5 ll Qiagen 103 Buffer, 5.0 ll Q buffer, 2.0 ll 25 mM dNTPs, 10.38 ll HPLC Water, 1.0 ll 10 mM primer A106, 1.0 ll 10 mM primer A107, 1.0 ll 10 mM primer 20304, 0.12 ll HotStar Taq plus 2 ll of mRNA preparation. PCR cycles were 95 C 15 min then 403 of 94 C-30 s, 58 C-30 s, 72 C-90 s then 72 C-10 min and a 4 C hold.

T-Cell Receptor (TCR) Gene Rearrangement Analysis The random primer RT product was PCR amplified using a mixture of V beta primers and a V beta constant region primer [Albertini et al., 2008]. A 20 ll reaction PCR was performed consisting of 2.0 ll Qiagen 103 Buffer, 1.6 ll 2.5 mM (each) dNTPs, 12 ll HPLC Water, 0.1 ll V beta primer mix (10 lM each primer), 0.2 ll 10 lM C beta primer, 0.1 ll HotStar Taq plus 4 ll of the RT product. PCR cycles were 95 C 15 min then 403 of 94 C-30 s, 60 C-30 s, 72 C-90 s then 72 C-7 min and a 4 C hold. Products were sequenced using TCRseq1 or TCRseq2. If no product was obtained, then an additional PCR using the same method except with a VB primer mix with six different V beta primers (representing V betas with low homology to the original primer set: VB5.4, VB5-8, VB7-9, VB12-5, and VB16-1) was performed.

Quick Lysis Method for DNA Extraction A frozen pellet of 50,000 cells was resuspended in 16 ll 10 mM Tris, 1.0 ll of a 1:10 dilution of NP40 (SIGMA, St. Louis, MO), 1.0 ll of a 1:10 dilution of Tween-20 (SIGMA, St. Louis, MO) and 2 ll a 1:10 dilution of Proteinase K (20 mg/ml solution from an Qiagen DNeasy kit). The sample was incubated at 56 C-60 min then 96 C-10 min followed by a 10 C hold. The sample was then kept frozen at 220 C.

Preparation of PCR Product for DNA Sequencing For most 2003 samples, PCR product was run on an agarose gel, the band excised and purified with QIAquick Gel Extraction Kit columns (Qiagen, Valencia, CA) according to manufacturer’s recommendations. The rest of the PCR products were ExoSAP-IT (USB Corporation, Cleveland, OH) treated according to manufacturer’s recommendations.

DNA Sequencing DNA sequencing was performed at the University of Vermont, Vermont Cancer Center DNA Analysis Facility utilizing an Applied Biosystems 3130XL.

Statistical Analysis Differences in the relative frequency of each type of mutation between groups of Veterans with different levels of exposure were examined using logistic regression with subject as a random effect to control for potential correlation between mutations from the same person. Logistic regression was also used to compare the relative frequencies of specific types of mutations in the DU exposed Veterans to published mutation data from other populations, but subject was not included as a random effect because subject-specific data were not available for comparison populations. To assess effects of low versus high U exposure, a lU value of 0.10 mg U/g creatinine was established as a cut-off level to separate participants into two groups; this cut-off level is slightly greater than 2-fold the 95th percentile lU concentration for adults (0.043 mg U/g creatinine) as determined during the 2001–2002 NHANES survey [NHANES, 2003]. Veterans in the high lU group had a mean lU value of 7.694 6 3.707 mg U/g creatinine, two orders of magnitude higher than the mean lU value of 0.012 6 0.003 mg U/g creatinine in the low group. Cutoffs using values of 10- and 100-fold greater were also investigated (1.0 mg U/g creatinine and 10.0 mg U/g creatinine).

RESULTS AND DISCUSSION A total of 1377 HPRT mutant isolates obtained from 61 Veterans were analyzed (282 mutants were analyzed from

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans

the 2003 samples, 357 from the 2005 samples, 341 from the 2007 samples and 397 from the 2009 samples) (Table I). Figure 1 gives a flow diagram of the molecular analysis and the number of mutants having different categories of

597

mutation and receiving each type of analysis. The complete list of mutations is given for these four sets of mutant isolates in the Appendix (Supporting Information 3) but results are summarized in the Tables and Figures below.

TABLE I. HPRT Mutants/Veteran

Mutants without determined mutation

2003

2005

2007

2009

Total

2003

2005

2007

2009

Total

Total no with discoverable mutation

11 11 11 – 11 – 11 – 10 – – 6 – – – 9 9 – 8 13 – 12 – – 10 – 10 11 9 – – – 10 9 – – – 4 10 11 6 – – 6 9 – 10 10 –

2 10 10 10 10 – 16 10 – – 5 10 – 4 – 11 14 9 – 10 9 10 – – 5 10 – – – 8 10 10 – 10 10 – 27 – 13 – 3 – – 15 – – 10 – –

10 9 11 – – 10 10 10 10 9 – 8 – – 10 6 10 10 – 10 10 10 – 8 8 10 5 – – – – 10 10 9 – – – 10 10 – – 9 – 10 – 7 10 – 10

10 – 10 10 10 10 10 – 10 10 10 – 10 – – 10 10 11 – 10 10 10 8 – 10 – – – – 10 – 12 10 10 10 10 – 10 10 10 – 9 10 10 10 – 10 – 8

33 30 42 20 31 20 47 20 30 19 15 24 10 4 10 36 43 30 8 43 29 42 8 8 33 20 15 11 9 18 10 32 30 38 20 10 27 24 43 21 9 18 10 41 19 7 40 10 18

2 4 2 – 3 – 1 – 0 – – 2 – – – 4 1

0 0 0 0 1 – 3 1 – – 1 0 – 1 – 1 1 1 – 0 0 0 – – 1 1 – – – 1 3 0 – 2 1 – 5 – 0 – 0 – – 4 – – 0 – –

0 0 1 – – 1 0 – 1 1 – 0 – – 1 0 7 0 – 2 0 1 – 1 0 1 0 – – – – 1 1 1 – – – 0 0 – – 2 – 1 – 0 2 – 1

1 – 4 2 0 0 1 0 2 0 2 – 2 – – 1 0 5 – 0 1 2 2 – 0 – – 0 – 3 – 2 2 4 4 3 – 0 0 0 – 3 2 1 0 – 4 – 0

3 4 7 2 4 1 5 1 3 1 3 2 2 1 1 6 9 6 0 4 1 5 2 1 2 2 0 3 0 4 3 3 7 10 5 3 5 4 1 1 4 5 2 8 0 0 9 1 1

30 26 35 18 27 19 42 19 27 18 12 22 8 3 9 30 34 24 8 39 28 37 6 7 31 18 15 8 9 14 7 29 23 28 15 7 22 20 42 20 5 13 8 33 19 7 31 9 17

No. of mutants analyzed Veteran 1153 1160 1232 1274 1336 1400 1415 1461 1515 1623 1836 1839 1865 2000 2027 2101 2117 2200 2270 2274 2333 2439 2466 2547 2647 2718 2733 2786 2897 2925 2949 3030 3041 3296 3579 3618 3621 3714 3795 3958 4102 4246 4432 4716 4893 4960 4998 5229 5392

0 2 – 2 – – 1 – 0 3 0 – – – 4 3 – – – 4 1 1 4 – – 2 0 – 3 1 –

Clones with the same mutation and same TCR rearrangement (size of clone)

Total with different HPRT mutations or different TCR rearrangments

12

19 26 32 16 26 19 40 19 25 17 12 21 8 3 8 27 7 22 8 22 27 33 5 5 24 13 10 8 9 14 7 25 19 27 14 7 22 20 41 20 5 13 8 33 12 7 21 9 16

2,2,2 3 2 3 2,2 2 2

2 2,3 14,15 3 3,9,2,4,4 2 2,3,2 2 3 5,2,3 3,4 4,2,2

3,3 3,3 2 2

2

8 11 2

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TABLE I. (continued). Mutants without determined mutation

Veteran

2003

2005

2007

2009

Total

2003

2005

2007

2009

Total

Total no with discoverable mutation

5494 5579 5590 5648 5689 5701 5758 5768 5779 5848 5859 5860 Total

10 3 12 – – – – – – 5 5 – 282

20 3 10 – 10 – – 13 1 15 – 4 357

10 – 10 8 4 3 10 – – 10 7 – 341

10 10 10 10 9 – – 10 – 10 – – 397

50 16 42 18 23 3 10 23 1 40 12 4 1377

3 0 1 –

2 0 0 – 0 – 0 1 0 0 – 1 32

1 – 0 0 0 0 1 – – 2 0 – 30

2 0 2 0 2 – – 0 – 4 – – 63

8 0 3 0 2 0 1 1 0 9 2 1 184

42 16 39 18 21 3 9 22 1 31 10 3 1193

No. of mutants analyzed

– – – – 3 2 – 59

Tables II and III show all of the mutations analyzed in this study according to type of DNA alteration and kind of mRNA alteration, respectively. Despite the extensive molecular analysis, no definitive mutation was identified in a minority of mutant isolates. There were three types of “nondefined” mutant isolates: 1. Those for which no product was obtained on either the 1st or 2nd round cDNA PCR and, in addition, there was no alteration on multiplex genomic DNA PCR (type 16A). 2. Those for which there was an alteration on the 1st or 2nd round cDNA sequence analysis, e.g., exon(s) loss, but no genomic alteration could be found to account for this loss (type 16B). 3. Those in which an RT-PCR product was found but that product exhibited no HPRT mutation on DNA sequencing (type 17). Our interpretation for the first type of result is that the responsible mutation was either a translocation involving portions of the HPRT gene, another major alteration of the gene affecting transcription or possibly epigenetic silencing. Since HPRT has large introns relative to the exons, a break within the gene is most likely in an intron and thus not detectable by multiplex PCR. Determination of the actual HPRT mutation in these mutants would require either Southern blotting, long PCR, cytogenetics or promoter methylation studies, which were not performed. Interpretation for the second type of result is that the mutation involved an intronic alteration, which affects splicing (exons 5 and 8 are particularly sensitive to alterations) or that the exon(s) in question is (are) present but not in the usual correct position. Mutant isolates of the third type may have had a nonHPRT mutation causing resistance to 6-thioguanine (e.g.,

Clones with the same mutation and same TCR rearrangement (size of clone)

Total with different HPRT mutations or different TCR rearrangments

3,2

39 16 35 15 18 3 9 21 1 29 10 3 1050

3,2,2 3,2 2,2,2

2 2,2

TABLE II. Mutant Types Used for Analysis Type

Description

With Mutation 1A G>A at non-CpG sites 1B C>T at non-CpG sites 2A A>G 2B T>C 3A G>A at CpG sites 3B C>T at CpG sites 4A G>T 4B C>A 5A G>C 5B C>G 6A A>T 6B T>A 7A A>C 7B T>G 8A single exon deletions 8B multiple exon deletions 8C total gene deletion (all exons deleted) 9A 21 frameshift (1bp deletion) 9B >1bp deletion (but less than 1 exon) 10A contraction of repeat 10B expansion of repeat 10C small duplication 11A 11 frameshift (1bp insertion) 11B small insertion (>1bp but less than 1 exon) 11C insertion of a whole exon or more (duplication) 12 has both an insertion and deletion (same place) 13 tandem (changes in two adjacent basepairs) 14 VDJ mediated 15 complex (two nonadjacent mutations) Total with mutation No Mutation Found 16A no RT product 16B change in mRNA but no mutation found 17 cDNA sequenced but there was no mutation TOTAL

#mutations %

195 33 60 19 11 30 55 15 50 31 39 32 12 49 48 76 21 38 116 34 12 6 4 5 0 29 12 11 7 1050

18.6 3.1 5.7 1.8 1.0 2.9 5.2 1.4 4.8 3.0 3.7 3.0 1.1 4.7 4.6 7.2 2.0 3.6 11.0 3.2 1.1 0.6 0.4 0.5 0.0 2.8 1.1 1.0 0.7 100.0

61 28 95 1234

4.9 2.3 7.7 100.0

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans TABLE III. Mutant Kinds Used in the Analysis Kind

mRNA alteration

F11 1bp insertion F-1 1bp deletion ID insertion/deletion IFD in frame small deletion IFI in frame insertion LD large deletion (one or more exons) LI large insertion/duplication M missense N nonsense NS no start codon NT no STOP OFD out of frame small deletion OFI out of frame insertion S splice/basepair change S/D splice/deletion S/F-1 splice/-1 frameshift S/ID splice/insertion S/T splice/tandem T tandem C complex total with mutation U unknown TOTAL

# mutations

%

12 57 19 29 3 156 3 340 81 25 0 56 8 205 34 1 8 5 6 2 1050 184 1234

1.1 5.4 1.8 2.8 0.3 14.9 0.3 32.4 7.7 2.4 0.0 5.3 0.8 19.5 3.2 0.1 0.8 0.5 0.6 0.2 100.0 14.9 100

mutations in transporter genes can lead to 6-TG resistance [Fotoohi et al., 2006]. Alternatively, wild-type (nonmutant) HPRT cDNA (thus mRNA) was due to either contamination of the mutant culture with wild-type cells or with residual wild-type mRNA derived from dying nonmutant T-cells. If the mutation in the mutant is such that it produces no mRNA (e.g., an exon 2–9 deletion), even a single wild-type mRNA left in dying non-TG resistant cells could give a product. Residual wild-type HPRT mRNA could not have resulted from the TK6 feeder cells in the original cloning assays as feeder cells have a total deletion of the HPRT gene. All mutants from 2005 and 2007 (and many from 2003) that were originally determined to be one of these three types were subsequently re-extracted and reanalyzed. In 50% of cases, a definitive mutation was determined the second time. For some of the 2005 samples where viable cells had been frozen, mutant isolates of Type 3 were thawed and regrown in 6-TG. Some did not grow, suggesting they were wild-type contaminants, not mutants. As noted, Type 17 had no defined mutations (and some might not be HPRT mutants). Therefore, they were not included in the statistical analysis of the mutational spectrum. Replicates of mutations (identical HPRT mutation

TABLE IV. HPRT TCR Clones Veteran

2003

2005

2007

2009

1153 1232 1232 1232 1274 1336 1415 1515 1515 1623 1839 2027 2101 2101 2117 2117

5 2 0 0 ND 1 1 1 0 ND 0 ND 1 0 3 3

0 0 0 0 2 1 0 ND ND ND 0 ND 1 0 8 4

1 0 1 2 ND ND 0 1 1 0 2 2 0 1 2 1

6 0 1 0 1 0 2 0 1 2 ND ND 0 2 1 7

2200 2274 2274 2274 2274 2274 2333 2439 2439 2439 2466

ND 0 3 1 0 1 ND 0 1 1 ND

0 2 1 0 2 2 0 2 1 0 ND

2 0 2 1 1 0 0 0 0 1 ND

1 1 3 0 1 1 2 0 1 0 2

Mutation 74C>G 551C>T del ex2-3 IVS2-1G>A 197G>A 100losA 551C>T 582C>A 612T>A 197G>A del ex2-3 del ex2-3 534dupT total 145C>T del ex4 IVS8-1 G>C 194T>C 544G>A 569G>A del ex2-3 (VDJ) IVS7 1 2_IVS7 1 62del61 151C>T 112–113CC>TT 3G>A IVS2-1G>A 400G>A

599

TRB V20-1 (25), 19, (20) J2.7 V18-1 (22), 18, (24) J2.7 V18 (23), 6, (24) J2.2 V7-3 (25), 16, (25) J2.1 V20-1 (23), 10, (27) J1.5 V14-1 (20), 15, (26) J2.7 V2-1 (24), 6, (20) J1.2 V20-1 (21), 10, (24) J1.5 V5-1 (22), 13, (24) J2.7 V6-2/3 (22), 9, (24) J2.1 V28-1 (28), 9, (25) J1.2 V5-1 (24), 16, (28) J2.1 V7-3 (24), 13, (24) J2.2 V28-1 (25), 13, (25) J2.7 V19-1 (25), 10, (23) J2.2 V6-1 (26), 15, (27) J1.4; V5-6/ 7 (21), 12, (25) J2.1 V18-1 (210), 12, (23) J1.4 V3-1 (21), 6, (0) J1.1 V2-1 (210), 10, (26) J2.7 V5-1 (20), 7, (21) J1.2 V12-3/4 (25), 9, (23) J2.3 V3-1 (21), 6, (20) J1.1 V4-1 1 V28-1 J1.1 1 J2.1 V30-1 (24), 19, (26) J2.7 V18-1(23), 25, (27) J1.5 V4-1 (23), 9, (0) 1 V7, J2.1 two overlapping hard to read

CDR3 (30 V-D -50 J) 2 STOP codons 1 OOF CASSPNTGTGLYEQ CASSPAITGE (IFFY) CASSPTRDRHNEQ CSASVGGQPQ CASSQ EFGQGG EQY CASSGVTNYG CSARGPGYNQP CASSFGLDSYEQ CASSYRGSYNE CASGNNYGY CASS AKGGFD EQF CASSSGQGFTGE CASSLGGRNEQY CASSFWGDTGE CASRSDGRGEKL; OOF CASSRDSNEK CASSQGNLNTE CATSYSEQY CASSLETVNYG CASSKLATDT CASSQGNLNTE CAWIKGRGIGEQY CASSPISTGGFGQPQ CASSQTGNSGN

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TABLE IV. (continued). Veteran

2003

2005

2007

2009

Mutation

2547 2647 2647

ND 0 2

0 1 0

3 3 0

ND 1 0

617G>A 212G>A 611A>G

2647 2718

1 ND

0 2

0 1

2 ND

del ex1 51T>G

2718

ND

2

2

ND

606G>C

2733 2733 2733 2897 3030

4 1 2 2 ND

ND ND ND ND 1

0 1 0 ND 2

ND ND ND ND 0

125T>A del ex1 51T>A 143G>A 113C>T

3030 3041 3041 3296 3579 3795 4893 4998 5392 5494 5494 5590 5590 5590 5648 5648 5689 5689 5689 5768 5848 5848

ND 2 0 2 ND 1 3 6 ND 2 1 1 0 0 ND ND ND ND ND ND 0 0

1 ND ND 0 2 0 ND 0 ND 1 0 0 2 0 ND ND 2 1 1 1 2 0

1 1 3 0 ND 0 ND 4 1 0 1 1 0 2 2 0 0 1 0 ND 0 1

1 0 0 0 0 1 5 1 1 0 0 1 0 0 1 2 0 0 1 1 0 1

del ex7-9 212G>A 388G>T IVS4-1G>A 527C>G del ex2-9 151C>T 527C>G 581 A>T 2_IVS1 1 4del30 22_IVS1 1 38del44 IVS8-16G>A IVS3 1 5delG IVS5 1 1G>A 39_76del38 total 617G>A IVS7-2 A>G IVS8 1 1G>A 400G>A 307A>T IVS4 1 1 G>C

and identical TCR gene rearrangement) among mutant isolates obtained from the same Veteran were deemed to be “sibling mutants” derived from the same in vivo HPRT in vivo mutant clone, as discussed in the previous companion paper [Albertini et al., 2015]. Sibling mutants were recovered from individual Veterans in a given year or even across years and were only counted as a single mutation in the statistical analysis of the mutational spectrum. Molecular analysis of a total of 1,377 mutant isolates were originally performed. Removing the type 16A, 16B, or 17 mutants left 1,193 mutants with unambiguously defined mutations (Table I). Results of the TCR gene analysis of mutants from the same Veteran with the same mutation left 1,050 unique mutations (Table I). Table IV

TRB very messy V27-1 (27), 15, (20) J1.1 V30-1 (25), 11, (25) J2.3 1 J2.7 V5-1 (25), 16, (25) J1.4 V19-1 (25) 1 V4-1 (24), 20, (24) J1.5 1 (25) J2.5 V7-3 (21), 17, (25) J2.4 (minor) 1 V5-6/7 (22), 5, (24) J2.3 (major) V3-1 (22), 8, (20) J2.7 V20-1 (29), 20, (29) J1.1 V30-1 (28), 11, (20) J1.6 ND V12-3/4 (26), 13, (210) J1.7; V19 (24), 16, (21) J1.1 Failed V19-1 (23), 15, (26) J2.1 V5-1 (24), 11, (21) J1.2 V20-1 (21), 13, (24) J1.4 V27-1 (24), 14, (24) J2.1 V3-1 (23), 3, (20) J2.7 V20-1 (25), 11, (24) J1.2 V27-1 (24), 14, (24) J2.1 V6-5 (24), 12, (25) J2.1 V7-3 AND V5-5/8 (-2) J2.2/J1.6 V20-1 (20), 21, (23) J2.7 V11-2 (20), 16, (21) J2.4 V20-1 (26), 15, (21) J1.4 V20-1 (23), 22, (25) J2.5 V11-2 (22), 9, (25) J1.1 V5-6/7 (20), 19, (23) J2.7 V7-6 (26), 13, (28) J2.2 V6-1 (28), 24, (25) J2.2 V20-1 (25), 16, (24) J2.3 V2-1 (27), 10, (20) J2.7 V9-1 (0), 8, (28) J1.2 V27-1 (26), 14, (26) J1.1

CDR3 (30 V-D -50 J)

CASKVQGAVNTE CAWRTSVDTQ CASRPQQGRNEK CASSLNGQGAPQGETQ; OOF

CASSQ MGI SYE CSAKDRRHKAFF CA GYRG SYN CASRQGVKSPL CASSTAWTRKNTE failed CASSIMRQGGNEQ CASSPERINYG CSARPPTPFNEK CASSSPRASYNE CASSQSSYE CSAADSRYGY CASSSPRASYNE CASSWVGTD NEQ CSARDSSGPSRGYEQ CASSL EENGRI AKN CSASVQGRTNE CSARSSAGQGWETQ CASSLITETEA CASSLALEVRTRYEQ CASSYSGVGEL CASGGTNRGITTGE CSAQPGGRSDTQ CASTGQISYE CASSVGVQGYT CASRTLQGSEAF

shows the TCR gene defined in vivo clones found in the four sets of HPRT data. For the HPRT mutants, three in vivo clones were represented at all four time-points, and an additional eight had a representative in 2003 and 2009, indicating persistence of the in vivo clone over at least six years. Note that other in vivo clones may have persisted but only 15 of the 61 Veterans were sampled all four times and an additional 24 Veterans had samples from at least 2003 and 2009 but not all four time-points. Figure 2 shows the exonic point mutations as distributed across the HPRT gene. There are a number of “hotspots” (27G>A, 143G>A, 197G>A, 508G>A, 551G>A, and 617G>A), which have been previously described [Cariello and Skopek, 1993; Burkhart-Schultz et al., 1996; Podlutsky et al., 1998]. Figure 3 shows the 1

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans

Fig. 2. Single base substitution mutations in the 657 bases of the HPRT cDNA. Mutations boxed in grey are changes in complex mutations and may not affect HPRT function. Bases that are in outline indicate that mutations at this site can cause a stop codon. A lowercase letter indicates that a point mutation at this site will not cause an amino acid change because of codon 3rd base redundancy.

601

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Fig. 3. Frameshifts and small insertions/duplications in the 657 bases of the HPRT cDNA. Bases that are in outline indicate a run of three or more of the same base (often sites of frameshift). Bases in grey are small duplications.

base-pair frame-shifts and insertions in the HPRT coding region; the only hotspots are frame-shifts in the run of six G’s in exon 3. Figure 4 shows the point mutations and frame-shifts in the splice junctions. The large deletions or deletion/insertions are shown in Supporting Information 4. There appears to be several clusters of breakpoints: at the end of exon 1 (9 breakpoints), between base 48 and 53 of exon 2 (16 breakpoints) and also a cluster between bases 460 and 470 of exon 6 (9 breakpoints). A cluster of breakpoints in exon 6 has been reported previously [Rainville et al., 1995] although many of the deletions reported here are slightly more 50 than those previously reported. Table V lists the large HPRT deletions (those encompassing an exon or more) and large duplications. Table VI lists the tandem mutations (two adjacent mutated base-pairs) and Table VII the complex mutations (have

two separate HPRT mutations). Four of the five tandem mutations are CC>TT, the hallmark of UV-induced mutation. Table VIII lists mutants that had two mutations but occurring at less than 100% as determined from sequencing patterns (but adding to 100%). These are the result of a mixed isolate, where two mutants grew in the same well. These HPRT molecular results were statistically analyzed in two ways. The first compared mutational spectrum versus the DU exposure concentration measured by urinary uranium excretion [Albertini et al., 2015]. For dose group comparisons, Veterans were classified as having urine uranium excretion values less than 0.10 mg U/g creatinine or greater than 0.10 mg U/g creatinine. Alternative groupings for statistical analysis were less than 1 mg U/g creatinine versus greater than 1 mg U/g creatinine

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans

Fig. 4. Point mutations and frameshifts at the splice junctions of the HPRT gene. Mutations boxed in gray are changes in complex mutations.

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TABLE V. HPRT Large Deletions

TABLE VII. Complex Mutations in the HPRT Gene

Type of mutation

No.

Single exon deletions Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Multiple exon deletions (external) Exons 1–2 Exons 1–3 Exons 1–9 Exons 2–9 Exons 3–9 Exons 4–9 Exons 5–9 Exons 6–9 Exons 7–9 Multiple exon deletions (internal) Exons 2–3 Exons 2–3 (VDJ) Exons 2–4 Exons 2–5 Exons 2–6 Exons 3–4 (1new) Exons 3–5 Exons 4–5 Exons 4–6 Exons 5–6 Exons 7–8 Undefined large deletion Duplications Duplication exons 2–3 Duplication exons 7–8

17 7 2 5 4 7 0 0 5 1 2 20 14 5 6 8 4 12 12 11 1 1 1 1 1 2 1 2 2 1 2 1

TABLE VI. Tandem Mutations in the HPRT Gene No.

Mutation

Veteran

Location(s)

5

CC>TT

4

GG>AA

1623 2274 2439 4716 5701 1153 1415 2027 3296 2101 5701 2439

550_551 463_464 112>113 550_551 463_464 27_IVS1 1 1 IVS1 1 52_IVS1 1 53 3_4 IVS5-1_403 384_IVS4 1 1 538_539 IVS8-1_610

1 1 1

GG>AC GG>CA AG>GC

or less than 0.1 mg U/g creatinine, 0.1–1.0 mg U/g creatinine or 1.0–10 mg U/g creatinine. These were the same groupings used in the companion paper. All but one of the Veterans with values > 0.1 had retained shrapnel (see Fig. 1 of the companion manuscript). The dose groups

Veteran 1336 1461 2925 3795 4246 5392 5648 5859

Mutations (both at 100%) 7_8insG 1 12_13insC 419G>A 1 424losA -17_6del23 1 10C>G 236T>C 1 239 A>G 27G>A 1 IVS1 1 5 G>T IVS6-1_498del14 1 506C>G 388G>T 1 394A>G 116A>C 1 33T>C

TABLE VIII. Isolates with Two Mutations at Less Than 100% (Mixed Isolates -Two Mutants Present in Same Well) Veteran 1153 1415 2439 3296 3795 4998 5859

Mutation(s) (less than 100%) 144_145insT (major) 1 48_49delTT (minor) 1/2 180_184delTCACA (major) 1/2142_144delCGT (minor) 1/2 131A>T (minor) 1/2 201_222del22 (50%) Mix 189G>A (val63>val [gta>gtg] (major) 1 210_213dupGGGC (minor) 1/2 67_71delTGCAT (50%)

were compared with respect to base-pair changes, frameshifts as well as larger alterations (deletions, insertions, etc. as listed in Tables II and III) and uncharacterized mutations (no mRNA but no mutation found and change in mRNA but no mutation found) were included in the denominator. There was no significant difference in the relative frequency of large events between the exposure groups. There were few significant differences in the less than 0.10 mgU/g creatinine versus the greater than 0.10 mgU/g creatinine analysis: the % of G>C mutations was lower with higher exposure (P 5 0.026), the % of multiple exon deletions was higher in the more highly exposed group (P 5 0.030) and the % of deletion/insertions was higher in the less exposed group (P 5 0.039) (Table IX). However, these differences were not significant when Veterans with urinary uranium excretion less than 1 mg U/g creatinine were compared to those with greater than 1 mg U/g creatinine. When dividing the Veterans into four exposure groups, there was a significant linear trend (P 5 0.033) for a decrease of G>C mutations with increasing dose (data not shown). There were no corrections for multiple comparisons and none of these differences are expected changes for ionizing radiation effects. The second analysis as compared the mutation spectrum in the DU exposed Veterans with historic mutation data from all published papers with the following criteria: mutants had to be from adult males (mutants from females are more difficult to analyze because of the

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans

605

TABLE IX. Mutation Distribution by DU Dose DU dose Mutation G>A C>T A>G T>C G>A (at CpG) C>T (at CpG G>T C>A G>C C>G A>T T>A A>C T>G single exon del mult exon del total gene del 1 bp del >1 bp del Repeat contraction Repeat expansion Small duplication Small insertion (>1 bp) >1 exon insertion Deletion/insertion Tandem mutation VDJ recomb mediated Complex

DU dose

No. with 0.1 lgM/ml (%)

112 (15.8) 22 (3.1) 44 (6.2) 4 (2.0) 7 (1.0) 17 (2.4) 32 (4.5) 11(1.5) 40 (5.6) 20 (2.8) 24 (3.4) 23 (3.2) 8 (1.1) 35 (4.9) 28 (3.9) 38 (5.3) 15 (2.1) 19 (2.7) 73 (10.3) 18 (2.5) 6 (0.8) 3 (0.4) 2 (0.3) 5 (0.7) 23 (3.2) 7 (1.0) 6 (0.8) 4 (0.6)

76 (18.9) 11 (2.7) 16 (4.0) 5 (1.2) 4 (1.0) 12 (3.0) 23 (5.7) 4 (1.0) 11 (2.7) 12 (3.0) 13 (3.2) 9 (2.2) 2 (0.5) 12 (3.0) 19 (4.7) 35 (8.7) 5 (1.2) 16 (4.0) 43 (10.7 16 (4.0) 6 (1.5) 2 (0.5) 2 (0.5) 0 6 (1.5) 5 (1.2) 4 (1.0) 1 (0.2)

presence of two X chromosomes and thus have two genomic copies of the HPRT gene) with no known exposure other than smoking (i.e., post-treatment chemotherapy patients were excluded but pretreatment samples were included) and a complete mutation analysis (mRNA and genomic) had been performed. The database includes 1,414 characterized mutants from 26 published papers [Recio et al., 1990; Andersson et al., 1992; Rossi et al., 1992; Vrieling et al., 1992; Burkhart-Schultz et al., 1993; Hou et al., 1993; Steingrimsdottir et al., 1993; Curry et al., 1995; Osterholm et al., 1995; Shimahara et al., 1995; Burkhart-Schultz et al., 1996; Osterholm et al., 1996; Burkhart-Schultz and Jones, 1997; Karnaoukhova et al., 1997; Osterholm and Hou, 1998; Podlutsky et al., 1998; Jones et al., 1999; Podlutsky et al., 1999; Curry et al., 2000; Diaz-Llera et al., 2000; Hackman et al., 2000; Horikawa et al., 2002; Albertini et al., 2003; Noori et al., 2005; Kendall et al., 2006; Nguyen et al., 2009]. A list of the mutants included can be found in Supporting Information 5. Uncharacterized mutation data was not available for all these studies and were therefore not included in the denominator when computing the relative frequency of different types of mutations. The only sig-

P (if A C>T A>G T>C G>A (at CpG) C>T (at CpG G>T C>A G>C C>G A>T T>A A>C T>G Single exon del Mult exon del Total gene del 1 bp del >1 bp del Repeat contraction Repeat expansion Small duplication Small insertion (>1bp) >1 exon insertion Deletion/insertion Tandem mutation VDJ recomb mediated Complex

No. with 0.1 lgM/ml (%)

195 (18.6) 33 (3.1) 60 (5.7) 19 (1.8) 11 (1.0) 30 (2.9) 55 (5.2) 15 (1.4) 51 (4.9) 30 (2.9) 39 (3.7) 32 (3.0) 12 (1.1) 49 (4.7) 48 (4.6) 76 (7.2) 21 (2.0) 38 (3.6) 117(11.1) 34 (3.2) 12 (1.1) 6 (0.6) 4 (0.4) 5 (0.5) 29 (2.8) 12 (1.1) 11 (1.0) 7 (0.7)

176 (17.3) 28 (2.7) 52 (5.1) 38 (3.7) 16 (1.6) 18 (1.8) 62 (6.1) 7 (0.7) 55 (5.4) 28 (2.7) 40 (3.9) 47 (4.6) 14 (1.4) 64 (6.3) 36 (3.5) 63 (6.2) 26 (2.6) 50 (4.9) 107 (10.5) 31 (3.0) 8 (0.8) 3 (0.3) 7 (0.7) 4 (0.4) 23 (2.3) 12 (1.2) 0 4 (0.4)

P (if < 0.05)

P 5 0.008

P 5 0.001

of several years. Significant differences not explicable by the numbers of statistical comparisons made were not found between the spectrum we describe here and that described in the several papers of HPRT mutations in normal males. Taken together, the background spectrum of mutations in this reporter gene can be considered to be well established, constituting an expected “normal distribution” for comparisons with spectra discovered in mutagenicity studies of humans exposed to various deleterious environmental agents. As noted in our companion paper, we have documented the persistence of in vivo HPRT mutant T-cell clones over many years. In the current paper, we show that this persistence includes a deletion mutation mediated by the V(D)J recombinase (subject #2274, Table IV), a mutation that is thought to arise within the thymus during T-cell maturation ([Albertini, 2001] and references therein). V(D)J recombinase mediated HPRT deletions are common in T-cells derived from placenta reflecting mutations in utero [McGinniss et al., 1989; Fuscoe et al., 1991]. They then decrease with age, becoming uncommon in adults [Fuscoe et al., 1992]. The mutation described here most likely arose in a progenitor cell that subsequently gave rise to an in vivo post-thymic T-cell clone.

The most important conclusion from molecular studies described here is that DU exposures in these Veterans, even though persistent, did not result in a molecular mutation spectrum suggestive of either ionizing radiation or oxidative DNA damage. Ionizing radiation results in a mutational spectrum that becomes increasingly dominated by deletion mutations [Nicklas et al., 1991a,1991b; Albertini et al., 1997]. Oxidative DNA damage, which mediates mutation induction by ionizing radiation, is also characterized by C>T transitions [Wallace, 2002]. Neither were significantly increased in these Veterans in association with DU exposure. Consistent with the quantitative results of HPRT mutations presented in the companion paper [Albertini et al., 2015], molecular analyses of HPRT mutations does not indicate mutation induction related to DU exposure. As discussed in the companion paper, the alpha particle emissions from DU will have a very short range and, thus, only cells in almost immediate contact with the retained particles will receive radiation exposure; however, T-cells circulate widely throughout the body, re-entering the circulation after having been in the tissues and any inflammation in the shrapnel containing tissue will be enriched for T-cells, which will then recirculate. Furthermore, it must be remembered that many of these T-cells are long lived, as shown by several-year persistence of some mutant clones, so there has been ample time for cells to circulate to the affected tissues. However, these are only our assumptions. We can only state that the circulating T-cells we studied manifested no molecular signatures of ionizing radiation mutagenesis. ACKNOWLEDGMENTS The authors would like to thank Terri Messier for protocols, primer samples and advice. The automated DNA sequencing was performed in the VT Cancer Center DNA Analysis Facility (special thanks to Mary Lou Shane and Jessica Hoffman). AUTHOR CONTRIBUTIONS RJA designed the studies and directed the work. SKA performed the molecular analysis for the 2003 samples. JAN performed the molecular analysis of the HPRT mutations for the 2005–2009 samples and the TCR analyses. PMV performed all of the statistical analysis. EWC performed the HPRT cloning assays and collated the MF data. MAM, as Director of the VA DU Follow-Up Program, designed the basic DU Health Surveillance Program that served as the basis for this study. SME organized and handled day-to-day logistics and data collection for the Health Surveillance visits. KSS worked with staff at the Joint Pathology Center (JPC) on the analysis of urine DU and other metal urine concentrations. PWG conducted data analysis for the Health Surveillance visits. All authors provided critical review and approved

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT Molecular Analysis in DU Exposed Veterans

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Accepted by— J. Fuscoe