Sequence Variant Discovery in DNA Repair Genes from Radiosensitive and Radiotolerant Prostate Brachytherapy Patients Tr E V O R J

Published OnlineFirst July 28, 2009; DOI: 10.1158/1078-0432.CCR-08-3357

Susceptibility and Prevention

Sequence Variant Discovery in DNA Repair Genes from Radiosensitive and Radiotolerant Prostate Brachytherapy Patients Tr e v o r J. Pu gh , 1Mira Keyes,2 Lorena Barclay,1Allen Delaney,1Martin Krzywinski,1DallasThomas,1 Karen Novik,1CindyYang,1AlexanderAgranovich,2 Michael McKenzie,2 W. JimMorris, 2 Peggy L. Olive,3 Marco A. Marra,1and Richard A. Moore1

Abstract Purpose: The presence of intrinsic radiosensitivity within prostate cancer patients may be an important factor contributing to development of radiation toxicity. We investigated whether variants in genes responsible for detecting and repairing DNA damage independently contribute to toxicity following prostate brachytherapy. Experimental Design: Genomic DNA was extracted from blood samples of 41prostate brachytherapy patients, 21with high and 20 with low late toxicity scores. For each patient, 242 PCR amplicons were generated containing 173 exons of eight candidate genes: ATM, BRCA1, ERCC2, H2AFX,LIG4,MDC1,MRE11A,andRAD50. These amplicons were sequenced and all sequence variants were subjected to statistical analysis to identify those associated with late radiation toxicity. Results: Across 41 patients, 239 sites differed fromthe human genomereference sequence; 170 of these corresponded to known polymorphisms. Sixty variants, 14 of them novel, affected protein coding regions and 43 of these were missense mutations. In our patient population, the high toxicity group was enriched for individuals with at least one LIG4 coding variant (P = 0.028). One synonymous variant in MDC1, rs28986317, was associated with increased radiosensitivity (P =0.048).AmissensevariantinATM , rs1800057, associated with increased prostate cancer risk, was found exclusively in two high toxicity patients but did not reach statistical significance for association with radiosensitivity (P =0.488). Conclusions: Our data revealed new germ-line sequence variants, indicating that existing sequence databases do not fully represent the full extent of sequence variation. Variants in three DNA repair genes were linked to increased radiosensitivity but require validation in larger populations.

Prostate brachytherapy is a standard treatment for early-stage use of prostate-specific antigen screening, the incidence of prostate cancer. Radioactive seeds are implanted into the prostate cancer has increased, and the age at diagnosis has prostate to deliver high doses of highly conformal radiation shifted toward younger men. Toxicity due to radiation exposure achieving excellent long-term results (1–3). Due to widespread of surrounding normal tissue includes acute and late urinary toxicity, acute and late rectal toxicity, and loss of sexual potency. Predictive clinical factors for severity of toxicity, such as baseline urinary and sexual function before procedure and Authors’ Affiliations: 1Genome Sciences Centre, 2Provincial Prostate Brachytherapy Program, and 3Medical Biophysics, British Columbia Cancer radiation dose, have been investigated by our group (3–9) and Agency,Vancouver, British Columbia, Canada others (10–12), but overall there is no consensus that any of Received 12/30/08; revised 3/24/09; accepted 4/9/09; published OnlineFirst these factors are effective predictors of late side effects of 7/28/09. prostate brachytherapy. Grant support: Abbott-CARO Uro-Oncologic Radiation Award. T.J. Pugh is a Senior GraduateTrainee of the Michael Smith Foundation for Health Research and The presence of intrinsic radiosensitivity may prove to be an the BC Cancer Foundation. M.A. Marra is a senior scholar of the Michael Smith important factor contributing to development of prostate Foundation for Health Research and aTerry FoxYoung Investigator. brachytherapy toxicity. Several observations support the hy- The costs of publication of this article were defrayed in part by the payment of page pothesis that an individual’s radiosensitivity is mediated by charges. This article must therefore be hereby marked advertisement in accordance genetic features. Radiosensitivity appears to be an inherited with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research trait, as monozygotic twins have greater intrapair correlation of Online (http://clincancerres.aacrjournals.org/). response than dizygotic twins (13) and two studies of breast Requests for reprints: Marco A. Marra, Genome Sciences Centre, British cancer patients have found that cells from first-degree relatives Columbia Cancer Agency, Suite 100, 570 West 7th Avenue, Vancouver, British of patients with high radiosensitivity are similarly sensitive to Columbia, Canada V5Z 4S6. Phone: 604-675-8000; Fax: 604-675-8178; E-mail: [email protected]. radiation (14, 15). Ionizing radiation such as that used in F 2009 American Association for Cancer Research. prostate brachytherapy has been well documented to cause doi:10.1158/1078-0432.CCR-08-3357 double-stranded breaks (DSB) in DNA, which are repaired

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DNA Repair Gene Variants in Brachytherapy Patients

prostate brachytherapy toxicity. To investigate this hypothesis, Translational Relevance we sequenced the coding and flanking intronic regions of eight DNA repair genes (ATM, BRCA1, ERCC2, H2AFX, LIG4, MDC1, Many genetic studies of radiosensitivity have focused on MRE11A, and RAD50) in 41 prostate cancer patients treated variants of ATM, a gene central to DNA damage detection with prostate brachytherapy at the British Columbia Cancer and repair, with mixed results. In an expanded search for Agency (BCCA). These genes were selected because each plays a variants predictive of radiosensitivity, we sequenced the role in the detection and repair of DNA damage from ionizing coding and flanking intronic regions of eight DNA repair radiation (16–18) and functional alterations of any of these genes, including ATM, in 41 prostate brachytherapy genes may result in reduced ability to repair double-stranded patients. Through this in-depth survey, genetic variants in DNA breaks caused by prostate brachytherapy. Although the three DNA repair genes were identified that may be able to effects of radiation can take several forms and involve several predict late side effects of radiation treatment.Validation of mechanisms (31), late side effects can develop months or years such variants inlarger patient populations may lead toprog- after irradiation due to the presence of unrepaired, damaged nostic tests to identify radiosensitive cancer patients before DNA. Therefore, we restricted our study to this set of DNA treatment. Given such knowledge, the clinical course of repair genes as the proteins they encode act directly at the site of these patients could be altered to consider their treatment DSBs and are the primary machinery for the detection and with nonradiation therapies. repair of these lesions. ATM kinase plays a central role as a sensor of DNA damage that activates signal transduction pathways to halt cell cycle through several DNA repair mechanisms (16–18). Defects in progression until the DNA damage is repaired. At the site of a DNA repair genes ATM, LIG4, and MRE11 lead to develop- DSB, ATM phosphorylates several proteins including H2AFX,a mental syndromes, all of which show increased radiosensitivity histone variant, to recruit a nuclease complex for DNA repair (18, 19). In vitro, cells with mutations in ATM have been shown (18). MRE11A nuclease and RAD50 ATPase are part of this to have increased radiosensitivity (20). complex and enzymatically process the ends of DSBs for repair In the treatment of cancer, positive correlations have been by homologous recombination (18, 32). MDC1 mediates the made between variants in ATM and radiosensitivity (21–27). recruitment of this complex by interacting with both H2AFX Such variants have been uncovered by two studies of prostate and MRE11A/RAD50 (32). BRCA1 acts as a scaffold for cancer patients. Hall et al. (25) used DNA sequencing to find replication and DNA repair proteins and forms a ‘‘BRCA1- ATM mutations in 3 of 17 prostate cancer patients with late associated genome surveillance complex’’ with ATM and the radiotherapy side effects. A study of 37 prostate brachytherapy MRE11A/RAD50 complex (18, 33). LIG4, also known as DNA patients by Cesaretti et al. (23) used denaturating high- ligase IV, plays a role in repairing DSBs by uniting broken ends performance liquid chromatography to identify 21 variants in through an alternative mechanism called nonhomologous end ATM in 16 patients and found a correlation between joining (18, 34). Damage to individual DNA bases is addressed possession of sequence variants in this gene, particularly by a third mechanism, nucleotide excision repair, in which missense variants, and late side effects of prostate brachyther- ERCC2 helicase, also known as XPD, is responsible for apy. Several studies of this kind have been limited by small unwinding the damaged DNA helical structure so repair can sample size, indirect or low-resolution variant detection take place (18). methods, and examination of only a single candidate gene (21). Current evidence suggests that radiosensitivity is a Methods and Materials complex genetic trait mediated by several genes, each of which may harbor lowfrequency variants that together modulate the Patient selection and toxicity metrics. The Prostate Brachy- radiosensitive phenotype (21, 27, 28). therapy Program at the BCCA was established in 1997. As of Two studies have examined the role of single nucleotide March 2008, >2,500 patients have undergone prostate brachy- polymorphisms (SNP) from multiple genes in predicting therapy as part of this program. Eligible patients included those radiosensitivity of prostate cancer patients treated with radia- with low-risk disease (clinical stage VT2a, initial prostate-specific tion. One study genotyped 49 SNPs from 24 genes in 83 antigen V10.0 ng/mL, and Gleason score V6) and "lowtier" LIG4, ERCC2 CYP2D6 patients and identified 3 genes, , and , intermediate-risk patients (stage VT2c and Gleason score V6 containing SNPs associated with radiation toxicity (29). The with initial prostate-specific antigen 10-15 ng/mL or Gleason second study genotyped 450 SNPs from 118 genes in 197 score 7 with initial prostate-specific antigen <10 ng/mL). Our patients and defined urinary toxicity ‘‘risk genotypes’’ associated implant technique is described in detail elsewhere (3, 4, 6). with SNPs in 5 genes, SART1, ID3, EPDR1, PAH, and XRCC6 Prostate and rectal dosimetry is obtained using day 30 post- (30). These studies genotyped an average of 1.8 and 6.1 known implant CT using VariSeed software. Post-implant contouring SNPs from each gene, respectively, and would not have was done by an implanting oncologist and tissue dosimetry was discovered novel variants in these genes that may directly recorded. Patients are seen at 6 weeks after the procedure, every mediate radiosensitivity. To date, no comprehensive sequenc- 6 months for 2 to 3 years, and then annually. Toxicity score ing-based survey of multiple candidate DNA repair genes has components are assessed by physician on each visit: Radiation been done in a set of high and lowtoxicity prostate Therapy Oncology Group (RTOG) urinary and rectal toxicity brachytherapy patients to discover and genotype such variants. scores (35), International Prostate Symptom Score (36), and We set out to perform such a survey to (a) discover new erectile function. To better reflect the specific brachytherapy variants and (b) investigate whether variants in genes respon- toxicity profile, the genitourinary and gastrointestinal RTOG sible for detecting and repairing DNA damage contribute to toxicity scale was modified at the inception of the program.

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Susceptibility and Prevention

Forty-one prostate brachytherapy patients living in the Vancouver lower mainland region with minimum 3 years of Table 1. Modified RTOG scoring system used to follow-up (mean, 6.6 years) were selected for study from the generate toxicity scores BCCA Prostate Brachytherapy database, agreed to participate, Category Description Score and provided informed consent. Ethical approval of the study was granted by the BCCA Research Ethics Board. Patients were IPSS* score Failed to normalize to within add 1 5 points of baseline after chosen based on development or lack of development of late 24 mo of follow-up normal tissue toxicity following brachytherapy and, to mini- Maximum RTOGc No symptoms add 0 mize radiation dose as a source of experimental variability, GI: rectum RTOG grade 1 their near-ideal rectal and prostate post-implant dosimetry: GU: urinary GI: Increased frequency or add 1 prostate D90 <175 Gy (dose covering 90% of the prostate change in quality of bowel habits not requiring <175 Gy), prostate V100 >85% (volume of the prostate covered medication/rectal discomfort 3 by >85% of the radiation dose), and rectal VR100 <1.0 cm not requiring analgesic (volume of the rectum receiving 145 Gy is <1.0 cm3). Median GU: Frequency, nocturia add 1 prostate D90, prostate V100, and rectal VR100 for lowand high twice pretreatment habits, toxicity patients were 154 Gy, 93%, and 0.26 cm3 and 148 Gy, dysuria, urgency not 3 requiring medication 92%, and 0.34 cm , respectively. Although some patients RTOG grade 2 received less than ideal dosimetry, neither the lowor high GI: pain or irritation requiring add 3 toxicity groups are enriched for these exceptions (P =1.00for medication, mucus or bloody V100, VR100, and D90) nor is there a linear, exponential, or up discharge, hemorrhoids requiring analgesics to sixth-order polynomial relationship between any of the alone, or change in bowel 2 dosimetry values and toxicity score (R < 0.4). habits requiring medication Usually, toxicity after radiation therapy is reported as a single GU: Frequency and nocturia add 3 organ or tissue toxicity score and the original RTOG toxicity less frequent than every hour, dysuria, and urgency, bladder scale is primarily used for external beam radiation therapy and spasm requiring medication not for brachytherapy. To adequately capture patients with RTOG grade 3 multiple organ or tissue toxicities, we have created a somewhat GI: hospitalization for severe add 5 arbitrary composite toxicity score listed in Table 1. Several peer- pain, bleeding from reviewed articles have been published regarding the toxicities thrombosed hemorrhoids, severe mucus or bloody determined using the modified RTOG toxicity scores (7–9). discharge, superficial From our analysis of 1,000 brachytherapy patients with a ulceration, minor minimum of 3 years of follow-up, patients with multiple severe surgical procedure toxicities are relatively rare, comprising 2% to 10% of the GU: frequency with urgency add 5 4 or nocturia hourly or more entire population. For this study, an attempt was made to frequently, dysuria, pelvic capture patients with the worse multiple toxicities, respecting pain or bladder spasm the limitation of geographical availability, patient willingness to requiring frequent narcotics, participate in the study, and self-imposed restrictions related to gross hematuria, and close to ideal post-implant dosimetry. As acute toxicity is likely obstruction requiring indwelling catheter or minor related to the prostate brachytherapy procedure itself (4–6, 9), surgical procedure this study is designed to account for late toxicity only, defined (transurethral resection or as a development of toxicity >1 year following the implant. incision of the prostate, The average follow-up time since implant was 78 F stricture dilatation) RTOG grade 4 14 months for patients with little or no evidence of late toxicity F GI: Ulceration, necrosis, add 7 and 81.2 12 months for patients with late toxicity. Twenty major surgical procedure patients with no late side effects of prostate brachytherapy were GU: Ulceration, necrosis, add 7 chosen based on a score of 0 or 1 for the criteria listed in Table 1. major surgical procedure Twenty-one patients with documented late side effects to Prolonged urinary Catheterization required add 3 retention for >3 wk brachytherapy were selected to score in at least two of the Table 1 Sexual potency Partially potent after add 2 categories and to have a total score of at least 2. Clinical and (for previously prostate brachytherapy DNA sequence variant data used in our analysis for all patients potent patients) Impotent after prostate are available in Supplementary Table S1. Supplementary Table S2 brachytherapy add 3 contains a summary of the DNA sequence variant data, a Maximum score: 21 breakdown of the toxicity scores, and additional clinical data including hormone usage, tumor stage, planning ultrasound Abbreviations: GI, gastrointestinal symptoms; GU, genitourinary target volume, Gleason score, and age at implant. symptoms. *IPSS, International Prostate Symptom Score (36), describes PCR amplification and sequencing of DNA repair genes. Each symptoms of prostate cancer. patient provided a 24 mL blood sample from which genomic cRTOG, RTOG late radiation morbidity scoring scale (35), DNA was extracted using the Gentra Puregene Blood kit describes radiation-induced side effects. This is an in-house modified RTOG toxicity scale to better reflect specific prostate brachytherapy rather than external beam radiation toxicity profile.

4 Unpublished data.

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DNA Repair Gene Variants in Brachytherapy Patients

Table 2. Number of variant sites detected in each DNA repair gene

Gene kbp sequenced Total per per coding In Novel In- Amino acid change Synonymous kbp kbp dbSNP Dels (novel) Total Coding Conservative Nonconservative (novel) (novel) ATM 40.9 13.0 62 1.5 0.7 41 21 16 4 (2) 4 (0) 1 (0) BRCA1 18.2 7.1 47 0.4 2.1 43 4 6 8 (0) 4 (0) 3 (0) ERCC2 8.8 2.3 37 4.2 1.7 28 9 5 2 (0) 0 (0) 2 (0) H2AFX 1.8 1.4 5 2.8 0.7 2 3 0 0 (0) 0 (0) 1 (1) LIG4 5.3 4.0 19 3.6 1.5 10 9 6 1 (1) 2 (0) 3 (2) MDC1 9.7 6.5 36 3.7 3.5 22 14 0 5 (2) 12 (4) 6 (2) MRE11A 14.3 5.1 15 1.0 0 13 2 1 0 (0) 0 (0) 0 (0) RAD50 15.8 5.8 18 1.1 0.3 11 7 2 1 (0) 0 (0) 1 (0) Overall 114.8 45.2 239 2.3 1.1 170 69 36 21 (5) 22 (4) 17 (5)

(Qiagen) and quantified using a NanoDrop spectrophotometer with radiosensitivity, we conventionally set our P value (NanoDrop Technologies). PCR and sequencing of the target threshold for significance at 0.05 with the understanding that amplicons were carried out by the BCCA Genome Sciences false-positives are a real possibility given the number of tests Centre sequencing group using previously published reaction done. More stringent corrections for multiple testing are chemistries (37). PCR volumes were scaled down to 10 AL, warranted for future validation studies in larger patient 10 ng genomic DNA was used for each reaction, and reactions populations. were done using a 60jC annealing temperature. PCR primer sequences and the genome coordinates of each amplicon are Results available in Supplementary Table S3. To facilitate sequencing with universal sequencing primers, each forward primer was DNA sequencing summary. We selected eight DNA repair ordered with the prefix sequence TGTAAAACGAGGCCAGT and genes in 41 individuals for sequencing. These eight genes each reverse primer was ordered with the prefix sequence contained 173 exons, which were covered by 242 PCR CAGGAAACAGCTATGAC. Variants were detected using Muta- amplicons. The amplicons targeted 115 kbp of genomic tion Surveyor version 3.2 (SoftGenetics) and mutation reports sequence, of which 45.2 kbp were exonic (Table 2). Across 41 were summarized using custom scripts. Genome coordinates patients, 239 sites were shown to differ from the human and dbSNP accession numbers reported correspond to human genome reference sequence and 170 of these corresponded to 5 genome build 36.1/hg18 (March 2006) and dbSNP build 128. known variants listed in dbSNP.7 Sixty of the variants, 14 of Statistical analysis. Statistical analyses were done using them novel, fell in a protein coding region (Table 3) and 43 of custom Perl and shell scripts. A Perl module implementation these result in an amino acid substitution (Table 3). Twenty- 6 of the two-tailed Fisher’s exact test was used to assess two of the variants that affect an amino acid change are differences in allele distribution between low and high toxicity expected to be nonconservative, which we defined as a score <0 patients at each variant site. To investigate the contribution of on the BLOSUM62 alignment score matrix (39, 40). Thirty-six individual alleles to intrinsic radiosensitivity, four statistical variants were small (1-5 bp) insertions or deletions, none of tests were done using different allele distributions between high which affected protein coding regions. Twenty-three of these and lowtoxicity groups at each variant site (A = reference allele, were known polymorphisms recorded in dbSNP, 14 of 25 B = nonreference allele): p1 = AA versus AB + BB, p2 = BB versus deletions and 9 of 11 insertions. Variants within exon splicing AA + AB, p3 = A versus B, and p4 = AA versus BB (heterozygotes sites were rare and only a single variant fell within fell within discarded). Patients without sequence coverage of a particular 2 bp of an annotated exon end, a novel intronic MRE11A variant were not included in the statistical tests of that site. We variant located at chr11:93,866,690 in a single heterozygous also used this approach to investigate correlations between lowtoxicity individual. these variants and residual gH2AX measured in blood cells The largest gene, ATM, contained the greatest number of from these patients following radiation (38). We used the variants, 53 in noncoding and 9 in coding regions. However, combined ranks of the monocyte and lymphocyte scores the 1.5 variants/kbp sequenced was near the average of published by Olive et al. (38) to divide our patients into two 2.3 variants/kbp for the eight genes sequenced, suggesting that groups; 19 ‘‘low gH2AX’’ individuals (sum of ranks < 41) and this gene does not have increased variant density compared z 21 ‘‘high gH2AX’’ individuals (sum of ranks 41). A higher with the other genes in our study despite harboring the greatest expression of residual gH2AX measured 24 h after radiation can number of variants. The BRCA1 gene contained the second suggest decreased DNA repair capacity and, we hypothesize, an largest number of variants, 47, and the greatest proportion of increased likelihood of late side effects. As this is a hypothesis- known variants recorded in dbSNP, 91%. This likely reflects the generating study seeking to identify subtle genetic relationships intense research interest in this gene due to its association with breast cancer risk. MRE11A and RAD50 contained the lowest number of variants per kbp in coding regions, 0 and 0.3, 5 http://www.ncbi.nlm.nih.gov/ 6 http://search.cpan.org/dist/Text-NSP/lib/Text/NSP/Measures/2D/Fisher/ twotailed.pm 7 http://www.ncbi.nlm.nih.gov/projects/SNP/

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Susceptibility and Prevention

Table 3. Coding variant genotypes observed in high and low toxicity prostate brachytherapy patients

Gene Genome coordinate Amino Conservation dbSNP High toxicity Low toxicity (build 36, hg18) acid change score* accession AA AB BB AA AB BB ATM chr11:107,603,786 S49C -1 rs1800054 21 0 0 19 1 0 ATM chr11:107,626,842 L480F 0 novel 20 1 0 20 0 0 ATM chr11:107,629,971 S707P -1 rs4986761 21 0 0 17 1 0 ATM chr11:107,648,666 P1054R -2 rs1800057 19 2 0 20 0 0 ATM chr11:107,668,697 P1526P 7 rs1800889 3 1 0 1 1 0 ATM chr11:107,669,347 V1570A 0 novel 20 1 0 19 0 0 ATM chr11:107,680,672 D1853N 1 rs1801516 11 2 0 9 3 0 ATM chr11:107,680,673 D1853V -3 rs1801673 11 0 0 9 1 0 ATM chr11:107,688,377 N1983S 1 rs659243 0 0 21 0 0 20 BRCA1 chr17:38,476,501 M1652I 1 rs1799967 9 9 2 9 6 3 BRCA1 chr17:38,476,574 M1628T -1 rs4986854 6 6 4 6 7 1 BRCA1 chr17:38,476,620 S1613G 0 rs1799966 11 7 3 10 5 1 BRCA1 chr17:38,487,996 S1436S 4 rs1060915 14 3 3 9 10 1 BRCA1 chr17:38,497,526 K1183R 2 rs16942 7 8 5 4 11 5 BRCA1 chr17:38,497,955 S1040N 1 rs4986852 20 1 0 19 0 0 BRCA1 chr17:38,497,961 E1038G -2 rs16941 19 0 0 13 1 0 BRCA1 chr17:38,498,462 L871P -3 rs799917 11 9 0 16 3 0 BRCA1 chr17:38,498,553 R841W -3 rs1800709 19 1 0 16 0 0 BRCA1 chr17:38,498,763 L771L 4 rs16940 4 0 0 7 1 0 BRCA1 chr17:38,498,992 S694S 4 rs1799949 4 7 1 7 3 0 BRCA1 chr17:38,498,997 D693N 1 rs4986850 4 0 0 7 2 0 BRCA1 chr17:38,499,587 R496H 0 rs28897677 19 1 0 18 1 0 BRCA1 chr17:38,499,693 F461L 0 rs62625300 19 1 0 18 1 0 BRCA1 chr17:38,500,007 Q356R 1 rs1799950 15 1 0 18 1 0 ERCC2 chr19:50,546,759 K751Q 1 rs13181 15 5 0 18 0 0 ERCC2 chr19:50,547,364 D711D 6 rs1052555 18 1 0 17 0 0 ERCC2 chr19:50,559,099 D312N 1 rs1799793 18 0 0 17 1 0 ERCC2 chr19:50,560,149 R156R 5 rs238406 18 0 0 17 1 0 H2AFX chr11:118,471,119 L66L 4 novel 13 1 0 13 0 0 LIG4 chr13:107,659,299 Q773Q 5 novel 13 0 0 13 1 0 LIG4 chr13:107,659,914 D568D 6 rs1805386 16 0 0 12 1 0 LIG4 chr13:107,660,726 Y298H 2 novel 16 0 0 12 1 0 LIG4 chr13:107,661,333 E95E 5 novel 16 0 0 12 1 0 LIG4 chr13:107,661,592 T9I -2 rs1805388 9 0 0 8 1 0 LIG4 chr13:107,661,610 A3V -2 rs1805389 9 0 0 8 1 0 MDC1 chr6:30,779,291 D1855E 2 rs28994874 9 1 0 8 0 0 MDC1 chr6:30,779,567 V1791E -2 rs28994873 16 1 0 16 1 0 MDC1 chr6:30,779,705 P1745R -2 rs28994871 14 1 0 14 0 0 MDC1 chr6:30,779,968 A1657A 4 rs28986317 14 1 0 14 0 0 MDC1 chr6:30,780,543 T1466A -1 novel 14 1 0 14 0 0 MDC1 chr6:30,780,702 K1413E 1 novel 14 6 0 14 5 1 MDC1 chr6:30,780,736 G1401G 6 rs28994870 14 1 0 13 1 0 MDC1 chr6:30,780,992 M1316T -1 rs61733213 14 1 0 13 0 0 MDC1 chr6:30,781,326 T1205A -1 novel 14 1 0 13 0 0 MDC1 chr6:30,781,604 S1112F -2 rs28987085 17 1 0 17 0 0 MDC1 chr6:30,781,641 P1100A -1 rs28994869 19 1 0 20 0 0 MDC1 chr6:30,781,660 P1093P 7 rs28994868 21 0 0 19 1 0 MDC1 chr6:30,783,584 R917S -1 rs28986467 20 1 0 20 0 0 MDC1 chr6:30,783,903 D811G -1 novel 21 0 0 17 1 0 MDC1 chr6:30,783,969 Q789P -1 novel 19 2 0 20 0 0 MDC1 chr6:30,784,025 T770T 4 rs28986466 3 1 0 1 1 0 MDC1 chr6:30,788,541 P386L -3 rs28986465 20 1 0 19 0 0 MDC1 chr6:30,788,587 E371K 1 rs2075015 11 2 0 9 3 0 MDC1 chr6:30,788,777 S307S 4 novel 11 0 0 9 1 0 MDC1 chr6:30,788,895 R268K 2 rs9262152 0 0 21 0 0 20 MDC1 chr6:30,789,456 R179C -3 rs28986464 9 9 2 9 6 3 MDC1 chr6:30,789,662 P138P 7 novel 6 6 4 6 7 1 MDC1 chr6:30,789,910 V56I 3 novel 11 7 3 10 5 1 RAD50 chr5:131,951,572 V315L 1 rs28903090 14 3 3 9 10 1 RAD50 chr5:132,005,895 I1293I 4 rs28903094 7 8 5 4 11 5

Abbreviations: A, reference allele; B, nonreference allele. *Conservation score of an amino acid substitution as determined from the BLOSUM62 alignment score matrix (39, 40). We defined scores <0 as nonconservative substitutions and all others as conservative substitutions.

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DNA Repair Gene Variants in Brachytherapy Patients respectively. This high level of sequence conservation likely population, the high toxicity group was enriched for individ- reflects the fact that the products of these genes interact with a uals harboring at least one LIG4 coding variant. large number of proteins. In contrast, ERCC2, LIG4, and MDC1 Using specific DNA repair gene variants to predict radio- had the greatest number of variants per kbp sequenced, 4.2, sensitivity. We hypothesized that specific DNA repair gene 3.6, and 3.7, respectively. Although MDC1 had 23 coding variants, not the number of such variants, would be associated variants, the greatest of any of the genes studied, 5 of these were with radiosensitivity. To assess genetic associations with only present in a single individual with no late side effects radiation toxicity, the genotype and allele distribution between (patient 33) and 4 were shared exclusively between this high and lowtoxicity groups wereanalyzed at every variant site individual and a patient with high toxicity (patient 43). using four two-tailed Fisher’s exact tests (Materials and H2AFX, the smallest gene, contained the fewest number of Methods). One coding synonymous variant in MDC1, variants, 5, and the single coding variant, although novel, did 4178C>CG, A1657AA, located at chr6:30,779,968, returned a not result in an amino acid change. P < 0.05 (p1 = 0.048, p2 = 1.00, p3 = 0.056, and p4 = 1.00). All ATM variants detected by previous studies of radiosensitivity. five patients with the minor allele (patients 4, 18, 21, 26, and We detected five known coding variants observed in previous 34) were heterozygous and had high radiation toxicity scores studies relating ATM to radiosensitivity: (a) 2119T>C, P707S (6, 3, 4, 5, and 13). This variant has been previously recorded in (chr11:107,629,971, rs4986761; refs. 22, 24); (b) 5558A>T, dbSNP as rs28986317 and minor allele frequencies have been D1853V (chr11:107,680,673, rs1801673; ref. 22); (c) observed from 1% to 6% in four populations. All other variant 3161C>G, P1054R (chr11:107,648,666, rs1800057; refs. 22, sites returned P values > 0.05 and none appeared to be 23, 26); (d) 4578C>T, P1526P (chr11:107,668,697, rs1800889; associated with increased radiation toxicity at a statistically refs. 22, 23, 26); and (e) 5557G>A, D1853N (chr11:107, significant level in our patient population. 680,672, rs1801516; refs. 22, 23). Coding sequence coordi- Relationship of DNA repair gene variants with residual gH2AX nates listed are relative to the ATM transcript record following irradiation. Assessments of DNA repair ability, ENST00000278616 accessed through the Ensembl Web site.8 represented by the relative expression of gH2AX remaining The first and second variants, resulting in amino acid changes 24 h after exposure to 2 Gy, were taken for 40 of 41 of the S707P and D1853V, respectively, were observed in a single low patients (38). Although residual gH2AX following irradiation toxicity patient heterozygous at both sites. The third variant, did not correlate with late side effects of prostate brachytherapy resulting in the amino acid change P1045R, has been found (38), we did observe 15 intronic variants to be correlated with previously to double the risk of developing prostate cancer (41) decreased residual gH2AX (increased DNA repair activity; P V and was observed in two heterozygous high toxicity patients in 0.049) and 1 coding, missense variant to be correlated with our population (patients 9 and 17). The fourth variant, a increased residual gH2AX (decreased DNA repair ability; P = synonymous change retaining P1526, was observed in two 0.042; Table 4). Fourteen of the 15 low gH2AX variants were in heterozygous patients, one with high toxicity and one with low BRCA1, and 13 of these were documented in dbSNP, suggesting toxicity. The fifth variant, resulting in the amino acid change a primary role for this protein in addressing DSBs marked by D1853N and suggested previously to mediate radiosensitivity gH2AX. The remaining intronic variant is a known variant in in breast cancer patients (22), was observed in five heterozy- MDC1 (rs9405048), but none of these variants were correlated gous patients, three lowtoxicity and twohigh toxicity. None of with fewer late side effects of prostate brachytherapy (P z these variants were statistically associated with high prostate 0.127). The single variant associated with increased gH2AX brachytherapy toxicity in our population (P > 0.46). results in a conservative amino acid change in ERCC1 (D312N, Using quantity of DNA repair gene variants to predict BLOSUM62 score = 1) and is documented in dbSNP radiosensitivity. Previous studies have postulated that the (rs1799793). Twelve of the 17 patients harboring the minor number of variants in a DNA repair gene can be used to allele had high expression of residual H2AX (sum of ranks z 41) distinguish radiotherapy patients with high toxicity from low and 5 of these had toxicity scores >2. Patient 10 was an toxicity (22–24, 30). In our study, every patient had at least exception as he harbored the minor allele and received a high one variant in each of five DNA repair genes (ATM, ERCC2, toxicity score of 7 and yet had the lowest residual gH2AX. This H2AFX, MDC1, and RAD50; Fig. 1). Three genes had more patient did not harbor any of the 15 variants correlated with variants on average in the high toxicity patients than in the low decreased gH2AX, suggesting that clearance of DSBs is also toxicity patients (BRCA1, H2AFX, and MDC1). However, there mediated by genes outside of our candidate set. was no statistically significant enrichment to either side of the mean in high or lowtoxicity groups for any of the eight genes P P studied ( > 0.10). This was also true for missense variants ( > Discussion 0.09) and nonconservative variants (P > 0.16). However, when all coding variants were taken as a group regardless of amino To the best of our knowledge, this study represents the first acid conservation score, we did observe an enrichment of such direct sequencing study of multiple DNA repair genes in variants in the LIG4 gene in high toxicity patients (P = 0.03 for radiosensitive and radiotolerant prostate brachytherapy LIG4 and P > 0.34 for all other genes). We tested all possible patients. This survey uncovered 239 variants distributed across quantity thresholds (from 1 to 32 variants) of all four variant eight DNA repair genes, of which 69 were novel and had not classes to distinguish lowtoxicity and high toxicity groups and been recorded in dbSNP. Of the 46 coding variants, 32 of found only one that met statistical significance. In our which resulted in an amino acid change, 14 were not in dbSNP. These results suggest that the genetic diversity of these genes is not fully captured in existing databases and that sequencing of 8 http://www.ensembl.org/ genes in larger populations is necessary to uncover lower

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Susceptibility and Prevention

Fig. 1. Toxicity scores, radiation dosimetry, count of DNA variants, and gH2AX rank expression from41prostate brachytherapy patients. Xaxis,patient numbers ordered by toxicity score (top).This panel indexes the subsequent panels and shows the toxicity score for each patient determined using the scoring system shown inTable 1. Dashed vertical line (middle) separates data of low toxicity patients (left ; toxicity score V1) fromhigh toxicity patients (right ; toxicity score >1).The next three panels present similar post-implant radiation dosimetry for each patient.Dashed red lines, thresholds for ‘‘ideal’’dosimetry (D90 <175 Gy,V100 >85%, and VR100 <1cm3). Panels 5 to 12, number of all variants (black, left axis) and coding variants (red, right axis) in each gene fromeach patient. Genes are ordered first by the magnitude of the left Yaxis (the maximum count of total variants) and then by the magnitude of the right Yaxis (the maximum count of coding variants). Note the enrichment of individuals with at least one LIG4 coding mutation in high versus low toxicity individuals (P = 0.028). Counts that include specific variants associated with radiosensitivity, ATM rs1800057 (P1054R) and MDC1 rs28986317, are indicated by wireframe data markers.The last panel displays sum of rank data from Olive et al. (38) and represents the combined rank of residual gH2AX scores fromlymphocytes and monocytes from40 of the 41patients. The numbersused to generate these plots are available in SupplementaryTable S3 . Lines between each point are for ease of comparison and do not represent continuous variables or an explicit relationship between data points. frequency variants that may define complex radiosensitive observed no statistically significant relationship with late side phenotypes. effects of prostate brachytherapy. Contrary to previous reports This survey identified five ATM variants analyzed in previous (23, 24), the number of variants in ATM could not predict investigations of ATM and radiosensitivity (22–24, 26) but radiation toxicity. However, a specific variant that doubles the

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DNA Repair Gene Variants in Brachytherapy Patients risk of prostate cancer, P1054R (41), was seen exclusively in results associating the MDC1 variant with increased radiosen- two high toxicity individuals, which suggests that aspects of sitivity and the observation of the ATM variant P1054R DNA repair ability may underlie a predisposition for prostate exclusively in high toxicity patients need to be validated in cancer. Although the frequency of this variant in the high larger populations particularly as no correction for multiple toxicity group did not reach statistical significance (P = 0.488), testing was done and there is a possibility of false-positives. The the fact that this variant was found exclusively in high toxicity finding that no single LIG4 variant was statistically associated patients warrants further investigation in larger populations as a with radiosensitivity despite the observation that high toxicity potential predictor of radiosensitivity. Of 239 variants detected patients were more likely to contain LIG4 coding variants across eight DNA repair genes, only one variant, rs28986317 suggests that this gene may contain a class of lowfrequency in MDC1, was statistically associated with late side effects of variants with similar effect on protein behavior. As LIG4 prostate brachytherapy (P = 0.048). The biological effect of this functions as a ligase, variants resulting in codon or amino acid variant on protein structure is not immediately obvious, as it substitutions in this protein may decrease its ability to join does not result in an amino acid change. However, synony- broken ends of DNA or reduce the amount of enzyme available mous changes have been shown to affect protein translation to perform DNA repair. due to changes in codon usage, altered mRNA stability, Variants in DNA repair genes were both positively and disrupted miRNA binding, and exon skipping (42). negatively associated with residual gH2AX that signifies the Despite representing <10% of the sequence targeted, coding presence of unrepaired or misrepaired DSBs following irradi- variants in MDC1 represented >33% of the synonymous ation (38). Noncoding variants, 14 in BRCA1 and 1 in MDC1 variants, >25% of the conservative variants, and >50% of the correlated with lower levels of gH2AX (P V 0.049), whereas a nonconservative variants, including all of the novel nonconser- coding, nonsynonymous variant in ERCC2 (rs1799793) was vative variants detected in our study. The large amount of per- correlated with higher levels of gH2AX (P = 0.042; Table 4). kbp variation present in this gene and others may explain the The ERCC2 minor allele was found in 42% of patients (5 of 12) wide range of toxicities observed following radiation treatment. with high levels of residual gH2AX and high toxicity. Whereas In the case of MDC1, a recruiter of DNA damage repair gH2AX levels did not correlate with development of late side complexes, different variants may preferentially recruit com- effects of prostate brachytherapy (38), variants in specific DNA plexes specific to each individual. Similarly, the high number of repair genes, particularly BRCA1 and ERCC1, appear to mediate variants per kbp in ERCC2 and LIG4, proteins that directly carry the clearance of DSBs, which may play a factor in the eventual out repair, may reflect a spectrum of activation efficiencies or development of toxicity. enzymatic activities that manifest as a range of toxicity levels. This initial survey has identified several promising candidate Our study’s small patient population limits the statistical variants that may showan ability to predict increased power of our analysis to resolve cumulative genetic effects on radiosensitivity in a larger population and serves to illustrate radiosensitivity at a population level. Regardless, even from this the genetic diversity present in several DNA repair genes. small population, a large amount of genetic diversity was Although the hypothesis that DNA repair gene variants mediate observed in the eight candidate genes sequenced. The positive radiosensitivity has not been disproven, it is likely that variants

Table 4. Variants associated with residual gH2AX levels following irradiation

Gene Genome coordinate Amino acid change, dbSNP High Low Possible effect P values < 0.05 (build 36, hg18) conservation score* accession ;H2AX ;H2AX on DNArepair activity AA ABBBAA ABBB MDC1 chr6:30778271 Intronic rs9405048 16 3 0 8 9 2 Increase p1 = 0.009, p3 = 0.006 BRCA1 chr17:38449934 Intronic rs12516 8 2 0 5 6 3 Increase p1 = 0.047, p3 = 0.023 BRCA1 chr17:38453439 Intronic rs817630 14 5 2 5 10 3 Increase p1 = 0.025 BRCA1 chr17:38469351 Intronic rs3092994 10 4 0 3 8 3 Increase p1 = 0.021, p3 = 0.009, p4 = 0.036 BRCA1 chr17:38469731 Intronic rs8176257 10 2 0 3 8 1 Increase p1 = 0.012, p3 = 0.017 BRCA1 chr17:38469732 Intronic rs8176256 10 1 0 3 4 0 Increase p1 = 0.047 BRCA1 chr17:38472867 Intronic rs11654396 14 3 1 6 8 1 Increase p1 = 0.038 BRCA1 chr17:38480127 Intronic rs8176212 12 5 2 5 10 3 Increase p1 = 0.049 BRCA1 chr17:38480201 Intronic rs2236762 12 5 2 5 11 3 Increase p1 = 0.049 BRCA1 chr17:38480270- Intronic novel 14 4 2 5 7 3 Increase p1 = 0.044 38480274 BRCA1 chr17:38496716 Intronic rs799916 14 5 2 5 10 3 Increase p1 = 0.025 BRCA1 chr17:38501690 Intronic rs8176147 11 3 0 3 9 2 Increase p1 = 0.007, p3 = 0.007 BRCA1 chr17:38502890 Intronic rs8176144 13 3 0 6 8 3 Increase p1 = 0.013, p3 = 0.004 BRCA1 chr17:38510660 Intronic rs799912 11 4 1 5 10 3 Increase p1 = 0.037, p3 = 0.037 BRCA1 chr17:38530713 Intronic rs799905 13 5 2 5 11 3 Increase p1 = 0.025 ERCC2 chr19:50559099 D312N, 1 rs1799793 8 9 3 14 4 1 Decrease p3 = 0.042

*Conservation score of an amino acid substitution as determined from the BLOSUM62 alignment score matrix (39, 40). We defined scores <0 as nonconservative substitutions and all others as conservative substitutions.

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Susceptibility and Prevention outside of this set of candidate genes may also play a role in Acknowledgments mediating radiosensitivity. We thank the sequencing group of the BCCA Genome Sciences Centre for Disclosure of Potential Conflicts of Interest expert technical assistance, Prab Dhaliwhal (Prostate Brachytherapy Program, BCCA) for administrative assistance, and Noushin Farnoud for consultation on No potential conflicts of interest were disclosed. statistical testing.

References 1. Grimm PD, Blasko JC, Sylvester JE, Meier RM, JB, Evans DG, Scott D. Heritability of cellular radio- polymorphisms in candidate genes. Radiother Oncol Cavanagh W. 10-year biochemical (prostate-specific sensitivity: a marker of low-penetrance predisposition 2003;69:127^ 35. antigen) control of prostate cancer with (125)I bra- genes in breast cancer? AmJ HumGenet 1999;65: 29. Damaraju S, Murray D, Dufour J, et al. Association chytherapy. Int J Radiat Oncol Biol Phys 2001;51: 784 ^ 94. of DNA repair and steroid metabolism gene polymor- 31 ^ 40. 15. Burrill W, Barber JB, Roberts SA, Bulman B, Scott phisms with clinical late toxicity in patients treated 2. Potters L, Morgenstern C, Calugaru E, et al. 12-year D. Heritability of chromosomal radiosensitivity in with conformal radiotherapy for prostate cancer. Clin outcomes following permanent prostate brachyther- breast cancer patients: a pilot study with the lym- Cancer Res 2006;12:2545 ^ 54. apy in patients with clinically localized prostate can- phocyte micronucleus assay. Int J Radiat Biol 2000; 30. SugaT, Iwakawa M,Tsuji H, et al. Influence of multi- cer. J Urol 2005;173:1562 ^ 6. 76:1617^ 9. ple genetic polymorphisms on genitourinary morbidity 3. MorrisWJ, Keyes M, Palma D, et al. Population-based 16. WoodRD,MitchellM,LindahlT.HumanDNArepair after carbon ion radiotherapy for prostate cancer. Int J study of biochemical and survival outcomes after per- genes, 2005. Mutat Res 2005;577:275 ^ 83. Radiat Oncol Biol Phys 2008;72:808 ^ 13. manent (125)I brachytherapy for low- and intermedi- 17. WoodRD,MitchellM,SgourosJ,LindahlT.Human 31. Bentzen SM. Preventing or reducing late side effects ate-risk prostate cancer. Urology 2009;4:860 ^5. DNA repair genes. Science 2001;291:1284 ^9. of radiation therapy: radiobiology meets molecular pa- 4. Keyes M, Miller S, MoravanV, et al. Predictive factors 18. De la Torre C, Pincheira J, Lopez-Saez JF. Human thology. Nat Rev Cancer 2006;6:702^ 13. for acute and late urinary toxicity after permanent syndromes with genomic instability and multiprotein 32. Spycher C, Miller ES, Townsend K, et al. Constitu- prostate brachytherapy: long-termoutcomein 712 machines that repair DNA double-strand breaks. His- tive phosphorylation of MDC1 physically links the consecutive patients. Int J Radiat Oncol Biol Phys tol Histopathol 2003;18:225^ 43. MRE11-50-NBS1 complex to damaged chromatin. 2008;73:1023^ 32. 19. O’Driscoll M, Cerosaletti KM, Girard PM, et al. DNA JCellBiol2008;181:227^40. 5. Bucci J, Morris WJ, Keyes M, Spadinger I, Sidhu S, ligase IV mutations identified in patients exhibiting de- 33.WangY, Cortez D,Yazdi P, Neff N, Elledge SJ, Qin J. Moravan V. Predictive factors of urinary retention fol- velopmental delay and immunodeficiency. Mol Cell BASC, a super complex of BRCA1-associated proteins lowing prostate brachytherapy. Int J Radiat Oncol Biol 2001;8:1175 ^ 85. involved in the recognition and repair of aberrant DNA Phys 2002;53:91 ^8. 20. Gutierrez-Enriquez S, Fernet M, DorkT, et al. Func- structures. Genes Dev 2000;14:927 ^ 39. 6. Keyes M, Schellenberg D, MoravanV, et al. Decline in tional consequences of ATM sequence variants for 34. Robins P, Lindahl T. DNA ligase IV fromHeLa cell urinary retention incidence in 805 patients after pros- chromosomal radiosensitivity. Genes Chromosomes nuclei. J Biol Chem1996;271:24257 ^ 61. tate brachytherapy: the effect of learning curve? Int J Cancer 2004;40:109 ^ 19. 35. Cox JD, Stetz J, Pajak TF. Toxicity criteria of the Radiat Oncol Biol Phys 2006;64:825^ 34. 21. Andreassen CN, Alsner J, Overgaard J. Does vari- Radiation Therapy Oncology Group (RTOG) and the 7. KeyesM,MillerS,MoravanV,etal.Acuteandlate ability in normal tissue reactions after radiotherapy European Organization for Research and Treatment of urinary toxicity in 606 prostate brachytherapy have a genetic basiswhere and how to look for it? Cancer (EORTC). Int J Radiat Oncol Biol Phys 1995; patients. Radiother Oncol 2006;80:S41 ^2. Radiother Oncol 2002;64:131 ^ 40. 31:1341 ^ 6. 8. Keyes M, Moravan V, Liu M, Jankovic B, Morris WJ. 22. Angele S, Romestaing P, Moullan N, et al. ATM 36. Barry MJ, Fowler FJ, Jr., O’Leary MP, et al. The Rectal toxicity after I125 permanent. Radiother Oncol haplotypes and cellular response to DNA damage: as- American Urological Association symptom index for 2005;76:S5^6. sociation with breast cancer risk and clinical radiosen- benign prostatic hyperplasia. The Measurement Com- 9. Macdonald AG, Keyes M, Kruk A, Duncan G, sitivity. Cancer Res 2003;63:8717^ 25. mittee of the American Urological Association. J Urol Moravan V, Morris WJ. Predictive factors for erectile 23. Cesaretti JA, Stock RG, Lehrer S, et al. ATM se- 1992;148:1549^57; discussion 64. dysfunction in men with prostate cancer after brachy- quence variants are predictive of adverse radiotherapy 37. Pugh TJ, Bebb G, Barclay L, et al. Correlations of therapy: is dose to the penile bulb important? Int J response among patients treated for prostate cancer. EGFR mutations and increases in EGFR and HER2 Radiat Oncol Biol Phys 2005;63:155^ 63. Int J Radiat Oncol Biol Phys 2005;61:196 ^ 202. copy number to gefitinib response in a retrospective 10. Bottomley D, Ash D, Al-Qaisieh B, et al. Side effects 24. Iannuzzi CM, Atencio DP, Green S, Stock RG, analysis of lung cancer patients. BMC Cancer 2007; of permanent I125 prostate seed implants in 667 Rosenstein BS. ATM mutations in female breast can- 7:128. patients treated in Leeds. Radiother Oncol 2007;82: cer patients predict for an increase in radiation- 38. Olive PL, Banath JP, Keyes M. Residual gH2AX 46^9. induced late effects. Int J Radiat Oncol Biol Phys after irradiation of human lymphocytes and mono- 11. Lehrer S, Cesaretti J, Stone NN, Stock RG. Urinary 2002;52:606 ^ 13. cytes in vitro and its relation to late effects after symptom flare after brachytherapy for prostate cancer 25. Hall EJ, Schiff PB, Hanks GE, et al. A preliminary re- prostate brachytherapy. Radiother Oncol 2008;86: is associated with erectile dysfunction and more uri- port: frequency of A-T heterozygotes among prostate 336^46. nary symptoms before implantation. BJU Int 2006; cancer patients with severe late responses to radiation 39. Henikoff S, Henikoff JG. Amino acid substitution 98:979^81. therapy. Cancer J Sci Am1998;4:385 ^ 9. matrices from protein blocks. Proc Natl Acad Sci 12. Wust P,von Borczyskowski DW, HenkelT, et al. Clin- 26. Andreassen CN, Overgaard J, Alsner J, et al. ATM U S A 1992;89:10915^ 9. ical and physical determinants for toxicity of 125-I sequence variants and risk of radiation-induced sub- 40. Eddy SR. Where did the BLOSUM62 alignment seed prostate brachytherapy. Radiother Oncol 2004; cutaneous fibrosis after postmastectomy radiothera- score matrix come from? Nat Biotechnol 2004;22: 73:39 ^ 48. py. Int J Radiat Oncol Biol Phys 2006;64:776 ^ 83. 1035 ^ 6. 13. Finnon P, Robertson N, Dziwura S, et al. Evidence 27.WestCM,ElliottRM,BurnetNG.Thegenomicsrev- 41. MeyerA,WilhelmB, DorkT, et al. ATM missense var- for significant heritability of apoptotic and cell cycle olution and radiotherapy. Clin Oncol (R Coll Radiol) iant P1054R predisposes to prostate cancer. Radio- responses to ionising radiation. HumGenet 2008; 2007;19:470 ^ 80. ther Oncol 2007;83:283 ^ 8. 12 3 :4 8 5 ^ 9 3. 28. Andreassen CN, Alsner J, Overgaard M, Overgaard 42. Parmley JL, Hurst LD. How do synonymous muta- 14. Roberts SA, Spreadborough AR, Bulman B, Barber J. Prediction of normal tissue radiosensitivity from tions affect fitness? Bioessays 2007;29:515^ 9.

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Sequence Variant Discovery in DNA Repair Genes from Radiosensitive and Radiotolerant Prostate Brachytherapy Patients

Trevor J. Pugh, Mira Keyes, Lorena Barclay, et al.

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