Mutational Analysis of Thirty-Two Double-Strand DNA Break Repair Genes in Breast and Pancreatic Cancers

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Mutational Analysis of Thirty-two Double-Strand DNA Break Repair Genes in Breast and Pancreatic Cancers

Xianshu Wang,1 Csilla Szabo,1 Chiping Qian,3 Peter G. Amadio,1 Stephen N. Thibodeau,1 James R. Cerhan,2 Gloria M. Petersen,2 Wanguo Liu,3 and Fergus J. Couch1

Departments of 1Laboratory Medicine and Pathology and 2Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota and 3Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Abstract that mutations in other DNA damage repair genes may predispose and/or contribute to breast cancer. Similarly, the recent discovery Inactivating mutations in several genes that encode compo- BRCA2, FANCC FANCG nents of the DNA repair machinery have been associated with that mutations in , and (2, 11, 12) are an increased risk of breast cancer. To assess whether associated with pancreatic cancer suggests that mutations in other alterations in other DNA repair genes contribute to breast repair genes may contribute to pancreatic cancer risk. To identify cancer and to further determine the relevance of these genes other DNA repair genes associated with breast and pancreatic to pancreatic cancer, we performed mutational analysis of 32 cancer, we performed a mutation screen of the coding regions of 32 genes involved in DSB signaling and repair in 38 breast tumors, DNA double-strand break repair genes in genomic DNA from BRCA1/ BRCA1/ 48 pancreatic tumors, and germline DNA from 10 non- 38 breast tumors, 48 pancreatic tumors, and 10 non- BRCA2 BRCA2 hereditary breast cancer patients. A total of 494 coding hereditary breast cancer patients. We report the identifi- exons were screened by denatured high-performance liquid cation of several truncating and missense mutations. chromatography and direct DNA sequencing. Two inactivating mutations were identified in breast tumor samples, a germline Materials and Methods single-nucleotide deletion in POLQ (c.3605delT) and a somatic Samples for mutation screen. Genomic DNA was obtained from an nonsense change in PRKDC (c.2408C>A, p.Ser803X). Two unselected series of 48 pancreatic tumors and 38 breast tumors and from germline-inactivating mutations in RAD50 (c.1875C>G, blood samples from probands of 10 high-risk breast cancer families without p.Tyr625X and IVS14+1G>A) were also detected in separate BRCA1 and BRCA2 mutations collected at Mayo Clinic. In addition, 48 pancreatic tumor samples. In addition, 35 novel nonsynon- control DNAs were obtained from blood samples of noncancer patients ymous amino acid substitutions, resulting from two in-frame attending Internal Medicine clinics. DNA was extracted from cryocut sections of frozen tumors or buffy coats with Easy-DNA kits (Invitrogen). deletions and 33 single nucleotide alterations, were identified. Mutation screening by denatured high-performance liquid chro- Seven of these were predicted to influence protein function. A matography. PCR primers were designed for all coding exons of 32 genes CLSPN separate analysis of the c.3839C>T (rs35490896) variant (Supplementary Table S1). PCR products were generated using AmpliTaq that was observed more frequently in breast tumors than in Gold (Roche) or HotStar Taq (Invitrogen), 10 ng genomic DNA, and 35 to 40 pancreatic tumors or normal controls failed to detect a cycles of amplification. Denatured high-performance liquid chromatography significant association with breast cancer risk in a Mayo Clinic (DHPLC) analysis was performed on automated DHPLC instruments (Trans- breast cancer case-control study. In conclusion, this screen of genomic or Varian). DNA samples displaying alterations on DHPLC were DNA repair genes implicates PRKDC and POLQ as candidate reamplified and sequenced on an ABI Prism 377 Sequencer (Perkin-Elmer). tumor suppressor genes involved in breast cancer and Coding single-nucleotide polymorphisms selection and PMut suggests that inactivating mutations in RAD50 predispose to analysis. All known single-nucleotide polymorphisms (SNP) in the coding regions of the 32 genes were obtained from Hapmap release 21 (13). Only pancreatic cancer as well as breast cancer. [Cancer Res SNPs with validation and defined minor allele frequency in Caucasian 2008;68(4):971–5] populations were used as positive controls to validate the sensitivity of the DHPLC analysis. Amino acid substitutions were predicted to be neutral or Introduction pathologic using PMut4 prediction scores and an associated reliability Double-strand DNA break (DSB) repair pathways are essential index, which compares favorably to PolyPhen and SIFT (14). Loss of heterozygosity and mRNA transcription analysis. Loss of for the prevention of genomic instability. Previous studies have BRCA1 BRCA2 heterozygosity (LOH) analysis was performed on laser capture micro- shown that genetic defects in and , key components dissected pancreatic tumor and matched normal stromal tissues. DNA and of homologous recombination repair, are associated with breast, RNA were isolated from 4,000 tumor cells captured on a Veritas Laser ovarian, and pancreatic cancer risk (1–3). Rare inactivating Capture Microdissection instrument (Molecular Devices Corporation) using mutations in several DNA damage repair and signaling genes, QIAamp DNA Microkit (Qiagen) and PicoPure RNA Isolation kit (Molecular ATM (4), BRIP1 (5), CHEK2 (6), PALB2 (7), NBS1 (8), RAD50 (9), and Devices Corporation). PCR and reverse transcription–PCR (RT-PCR) were TP53 (10), have also been implicated in breast cancer. This suggests conducted using primers shown in Supplementary Table S2. Case and control population. Genotyping of the CLSPN rs35490896 SNP was conducted using 798 invasive breast cancer cases and 843 matched controls from an Institutional Review Board–approved Mayo Clinic breast Note: Supplementary data for this article are available at Cancer Research Online cancer case-control series (15). Collection and characteristics of cases and (http://cancerres.aacrjournals.org/). controls are described in the Supplementary Materials and Methods. Requests for reprints: Fergus J. Couch, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. Phone: 507-284-3623; Fax: 507-538-1937; E-mail: [email protected]. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-6272 4 http://mmb2.pcb.ub.es:8080/PMut/ www.aacrjournals.org 971 Cancer Res 2008; 68: (4). February 15, 2008

TaqMan genotyping and data analysis. Genomic DNAs from breast sufficiently sensitive to detect the majority of putative disease- cancer cases and controls were genotyped by the 5¶ nuclease assay associated mutations. (TaqMan), using the 7900HT Real-Time PCR System (Applied Biosystems), Novel deleterious mutations. The mutation screen of 96 in 384-well format. Duplicate samples (5%) were included and all displayed samples resulted in the identification of 73 unique alterations in 100% concordance for the CLSPN c.3839C>T genotype. Unconditional 17 genes. These variants included three truncating mutations, an logistic regression was used to estimate odds ratios and 95% confidence intervals (CI) under general (df, 2) and ordinal models. Analyses were in-frame splicing variant, two in-frame three-nucleotide deletions, adjusted for common breast cancer risk factors and performed using and 33 novel nonsynonymous changes. In addition, 154 intronic SAS software, version 8.2 (SAS Institute, Inc.). changes were detected. A summary of the results of this extensive mutation screen is shown in Tables 1 and 2. One truncating mutation, resulting in a premature stop codon Results p.Ser803X, was detected in PRKDC in a breast tumor sample Sensitivity of DHPLC analysis. We screened a total of 1.7 Â 107 (Table 2). PCR and DNA sequencing of blood-based DNA from the bases of DNA in 494 coding exons from 32 genes to identify same patient confirmed that the mutation was of somatic origin. mutations associated with breast and pancreatic cancer. To assess Another truncating mutation, c.3605delT, was identified in POLQ in the sensitivity of the DHPLC screen, we compared the variants blood DNA from a proband of a high-risk breast cancer family detected in the mutation screen with all known SNPs from these (Table 2). The single-nucleotide deletion caused a frameshift and exons. Among 60 SNPs listed in dbSNP as validated in at least two truncation of the protein and exclusion of the POLQ C-terminal studies of Caucasians, 54 were detected in this mutation screen. DNA polymerase and 3¶-5¶ exonuclease domains. These mutations Four of the six SNPs not identified exhibited minor allele likely inactivate the DNA-PK and POLQ DNA repair proteins. frequencies of <2% and were excluded. The detection rate of Two deleterious mutations were also identified in the RAD50 96.4% for known SNPs indicated that the DHPLC technique was gene in DNA from pancreatic tumors (Table 2). A c.1875C>G

Table 1. Mutation screening of DNA damage repair genes in breast and pancreatic cancers

DNA repair genes Coding exons screened Known SNPs in databases Known SNPs detected Novel alterations detected

ABL1 11/11 4 4 6 ATF2 12/12 2 0 2 ATR 47/47 7 7 7 ATRX 35/35 1 0 4 CLSPN 25/25 1 1 4 EEF1E1 4/4 0 0 0 FAAP24 4/4 4 4 0 H2AFX 1/1 0 0 0 HUS1 8/8 1 1 0 LIG4 1/1 3 3 0 MRE11 19/19 0 0 0 NBN 16/16 4 3 1 POLQ 31/31 10 10 13 RAD1 5/5 1 1 1 RAD50 25/25 0 0 2 RAD51 9/9 0 0 0 RAD51C 9/9 0 0 0 RAD51L3 10/10 2 2 0 RPA1 17/17 2 2 1 SFN 1/1 0 0 0 SMC1A 25/25 0 0 0 TERF2 10/10 0 0 1 TP53BP1 27/27 5 5 7 TREX1 1/1 1 1 2 XPA 6/6 0 0 0 XPC 16/16 4 4 6 XRCC2 3/3 1 1 0 XRCC3 7/7 1 1 0 XRCC4 7/7 3 3 0 XRCC5 21/21 2 1 4 XRCC6 12/12 1 0 2 XRCC7* 69/86 NA NA 10 Total 494 60 54 73

*For XRCC7 (PRKDC), only 69 of 86 coding exons were completed. NA: rs7830743, rs8178216, rs8178017 and rs8177999 of XRCC7 are validated in Caucasian populations but are not included in the calculation of sensitivity because the gene is not completely finished.

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Table 2. Novel truncating and missense variants detected in DNA repair genes in breast and pancreatic cancers

Genes Mutation detected Mutation incidence PMut

Nucleotide Amino acid Breast (n = 48) Pancreas (n = 48) Prediction score Reliability index

ABL1 c.589G>A p.Glu197Lys 1 0.70 3 ATR c.2290A>G p.Lys764Glu 1 0.63 2 c.4405A>G p.Thr1469Ala 1 0.43 1 c.4835A>G p.Asn1612Asp 1 0.18 6 CLSPN c.17G>A p.Gly6Asp 5 3 0.90 7 c.3595_3597delGAA p.Glu1199del 12 3 NA NA c.3671G>A p.Arg1224His 1 0.68 3 c.3839C>T p.Ser1280Leu 12 3 0.68 3 NBS1 c.1405G>T p.Asp469Tyr 1 0.83 6 POLQ c.1312T>G p.Ser438Ala 1 0.04 9 c.1328T>G p.Val443Gly 1 0.09 8 c.1397C>T p.Thr466Met 1 0.20 6 c.3605delT p.Leu1202Tyrfs 1 NA NA c.4300A>G p.Thr1434Ala 1 0.10 7 c.7795G>A p.Ala2599Thr 4 1 0.14 7 RAD50 c.1875C>G p.Tyr625X 1 NA NA IVS14+1G>A p.Val787_Gln799del 1 NA NA RPA1 c.125A>T p.Tyr42Phe 1 0.07 8 TERF2 c.1366G>A p.Glu456Lys 1 0.57 1 TP53BP1 c.1829C>T p.Thr610Met 1 0.39 3 c.2941G>A p.Val981Met 1 0.06 8 c.3041A>G p.Glu1014Gly 1 0.71 4 c.3940C>T p.Arg1314Cys 1 0.88 7 c.5242C>T p.Arg1748Cys 1 0.94 8 TREX1 c.962A>G p.Glu321Gly 1 0.87 7 XPC c.191G>C p.Gly64Ala 1 0.35 2 c.859T>C p.Phe287Leu 1 0.44 1 c.872C>G p.Ser291Cys 1 0.31 3 c.1177C>T p.Arg393Trp 1 0.92 8 c.1442G>T p.Lys481Asn 1 0.19 6 XRCC5 c.984G>C p.Glu328Asp 1 0.05 8 c.1345G>A p.Ala449Thr 1 0.30 3 XRCC6 c.1540A>G p.Met514Val 1 0.36 2 XRCC7 c.1526G>A p.Arg509His 1 0.65 2 c.2408C>A p.Ser803X 1 NA NA c.6338_6340delGAG p.Gly2113del 1 NA NA c.9446G>A p.Gly3149Asp 1 0.78 5 c.7825A>C p.Thr2609Pro 1 0.50 0

NOTE: The mutations are described based on HGVS nomenclature. The cDNA sequences referenced here are NM_005157.3 (ABL1), NM_001184.3 (ATR), NM_022111.2 (CLSPN), NM_002485.4 (NBS1), NM_006596.3 (POLQ), NM_005732.2 (RAD50), NM_002945.2 (RPA1), NM_005652.2 (TERF2), NM_005657.1 (TP53BP1), NM_016381.3 (TREX1), NM_004628.3 (XPC), NM_021141.2 (XRCC5), NM_001469.3 (XRCC6), and NM_006904.6 (XRCC7/PRKDC). The range of the PMut prediction score is from 0 to 1. A prediction score over 0.5 suggests an effect on protein function. A prediction score <0.5 indicates that the substitution is likely neutral. The range of the reliability index is from 0 to 9. The higher the number, the more reliable is the prediction.

variant resulting in a premature stop codon p.Tyr625X was occurred in tumor cells harboring the two mutations, we identified in a tumor from a 64-year-old male. The germline origin performed LOH analysis using genomic DNA from laser micro- of the mutation was confirmed by analysis of blood DNA. An dissected tumor cells. As shown in Fig. 1A, equal amounts of the IVS14+1G>A mutation in a tumor from a 67-year-old male was also 1875C and 1875G alleles were present in the tumor cells, suggesting detected in blood DNA. This alteration was predicted to disrupt the that the 1875C wild-type allele was not lost. Similarly, no LOH of splice donor site resulting in aberrant splicing of intron 14. These the wild-type IVS14+1G allele was detected. mutations represent the first descriptions of inherited mutations in Analysis of RNA from microdissected tumor cells was also the RAD50 gene in pancreatic cancer patients and strongly suggest performed to determine whether the IVS14+1G>A mutation induced that RAD50 functions as a pancreatic cancer tumor suppressor. aberrant splicing. Interestingly, RT-PCR analysis detected two pro- LOH and RNA analysis of RAD50 mutations in pancreatic ducts differing by 39 bp in size in both microdissected tumor cells tumors. To check whether loss of the RAD50 wild-type allele and microdissected adjacent stromal cells (Fig. 1B). Sequencing www.aacrjournals.org 973 Cancer Res 2008; 68: (4). February 15, 2008

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. Cancer Research showed that the larger product represented the wild-type allele, protein function by PMut analysis (Table 2). Seven of these variants whereas the smaller product resulted from use of a cryptic ‘‘GT’’ in displayed a reliability index >5 and seem most likely to affect the exon 14 located 39 bp upstream from the original donor splice site. normal protein function. Thus, the splicing mutation results in an in-frame deletion of 13 Association of CLSPN p.Ser1280Leu with breast cancer risk. amino acids, p.Val787_Gln799del, in the functionally important The p.Ser1280Leu (rs35490896) and p.Glu1199del variants in EzrA domain of RAD50. This rearrangement was not detected in CLSPN were found to be in strong linkage disequilibrium because RNA from lymphoblastoid cell lines derived from 20 unaffected they cooccurred in 15 tumor samples but were never identified controls. In parallel, we evaluated the expression of the c.1875C>G independently. We also noted a higher prevalence of these (p.Tyr625X) mutant by RT-PCR. The mutant c.1875G mRNA tran- mutations in breast samples (25%) than in pancreatic samples script was not detected in two separate tumor cell specimens or (6%) and an unselected series of 48 normal controls (2%). Based on the associated stromal cells, suggesting that the mutant transcript this observation, we further examined the association of was eliminated by nonsense mediated mRNA decay (Fig. 1C). p.Ser1280Leu and p.Glu1199del with breast cancer risk by Unclassified missense variants. A total of 29 of 33 novel genotyping rs35490896 in a Mayo breast cancer case-control study. missense variants were found once. These variants were equally As shown in Table 3, no significant association (P < 0.05) was distributed between breast and pancreatic cancer samples (Table 2). observed between rs35490896 and breast cancer risk (odds ratio, In addition, multiple variants were found to coexist in the same 0.99; 95% CI, 0.67–1.20) under a log-additive model. breast or pancreatic tumor samples (Supplementary Table S3). Fifteen of 33 variants located in nine genes were predicted to disrupt Discussion Mutation studies of cancer susceptibility genes suggest that defective DNA damage repair is an important etiologic factor underlying a subset of breast and pancreatic cancer. In an effort to identify defects in DNA damage response genes that contribute to these cancers, we screened 1.7 Â 107 bases of DNA from 32 genes that cover a wide spectrum of DNA damage and signaling pathway components in 96 samples. As described above, a germline POLQ c.3605delT frameshift mutation that is expected to completely disrupt POLQ DNA polymerase activity was identified in the blood of an individual with a family history of breast cancer. Cosegregation of the mutation with breast cancer in the family could not be confirmed because of the small size of the family and the absence of a sample from the affected mother (Supplementary Fig. S1). It was also not possible to conduct LOH analysis of the mutation due to the absence of any remaining tumor tissue. Whereas five missense variants in POLQ were identified in breast tumors (Table 2), PMut analysis predicted that none of these variants influence protein function. Although the possibility that POLQ is a tumor suppressor is fully consistent with its role in DNA damage repair and maintenance of genomic stability, expanded genetic studies of the POLQ gene in breast cancer–prone families and unselected breast cancer populations are needed to determine whether inherited mutations in POLQ predispose women to breast cancer. The PRKDC nonsense mutation detected in a breast tumor was predicted to eliminate the phosphoinositide 3-kinase domain that is essential for the nonhomologous end-joining repair activity of PRKDC and the FAT domain that is associated with ATM mediated protein-protein interactions (16, 17). In addition, two other novel missense variants, p.Arg509His and p.Gly3149Asp, identified in pancreatic tumor samples, were predicted by PMut to influence the DNA-dependent protein kinase activity of this protein. Because not all PRKDC exons were analyzed in this study (69 of 86; Table 1), it Figure 1. RAD50 truncating mutations c.1875C>G and IVS14+1G>A in DNA and RNA purified from laser capture microdissected pancreatic tumors. A, remains possible that other novel mutations remain to be LOH analysis by PCR and direct DNA sequencing of genomic DNA isolated from identified. However, whereas PRKDC may have tumor suppressor B, pure population of pancreatic tumor cells. variant splicing of mRNA in tumor activity, the absence of the identified truncating mutation from the cells and adjacent stroma harboring the IVS14+1G>A germline mutation. LCM-T1 and LCM-T2 represent tumor cell populations laser capture microdissected from germline DNA of the relevant breast cancer patient means that we the IVS14+1G>A mutant tumor. The alternative splicing of exon 14 (exon 14S) cannot propose PRKDC as a breast cancer predisposition gene. beginning at a cryptic ‘‘GT’’ donor site 39 bp upstream from the mutated splice RAD50 donor site is shown along with the sequence chromatograms for the normal and We also identified c.1875C>G (p.Tyr625X) and IVS14+1G>A aberrantly sized RT-PCR products. C, nonsense mediated mRNA decay in tumor germline mutations in pancreatic tumors. These are likely to disrupt cells and adjacent stroma containing the c.1875C>G germline mutation. LCM-T1 the well-established DSB repair function of RAD50. The 13 amino acid and LCM-T2 represent tumor cell populations laser capture microdissected from the c.1875C>G mutant tumor. The sequence chromatogram based on cDNA in-frame deletion caused by IVS14+1G>A removes a section of the derived from c.1875C>G tumor cells shows only wild-type sequence. EzrA domain close to the CXXC motif essential for RAD50

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Table 3. Association between rs35490896 (p.Ser1280Leu) of CLSPN and risk of breast cancer among Caucasians in the Mayo Clinic breast cancer case-control study

SNP Genotype No. copies of Breast cancer case Control Odds ratio Lower 95% CI Upper 95% CI representation variant allele n = 797 n = 840

CLSPN MAF 0.06 rs35490896 Ordinal 0.9 0.67 1.2 General 0 705 739 1.0 REF REF 1 87 90 0.99 0.72 1.35 2 2 7 0.3 0.06 1.48 HWE P 0.036

Abbreviations: HWE P, P value for departures of Hardy-Weinberg equilibrium in control population; CI, confidence interval; REF, reference group with 0 copies of the minor allele; MAF, minor allele frequency.

dimerization and DNA repair function (18). Similarly, the c.1875C>G of CLSPN rs35490896 showed that p.Ser1280Leu was not associated truncating mutation is expected to disrupt MRN complex formation with increased breast cancer risk, in contrast to the pathologic and DNA repair activity. The detection of two mutations in 48 prediction by PMut. This finding illustrates the importance of pancreatic tumors strongly suggests that RAD50 is a pancreatic genetic association studies, together with functional analysis, for cancer predisposition gene, especially when considering that further validation of results obtained from high-throughput truncating mutations in RAD50 have previously been associated mutation screening studies. with a moderate risk of breast and ovarian cancer in the Finnish Overall, the POLQ, the RAD50, and, perhaps, the PRKDC genes population (8, 9). The absence of RAD50 LOH from the mutant warrant further analysis on the basis of our comprehensive screen pancreatic tumors is consistent with a similar observation in breast of DNA damage repair genes. Our findings are consistent with the and ovarian tumors with RAD50 mutations and does not exclude notion that a large number of genes containing rare genetic RAD50 as a tumor suppressor. In fact, a substantial increase in alterations that confer small to moderate risk of complex diseases, chromosomal instability in lymphoblastoid cells carrying RAD50 such as breast and pancreatic cancer may exist (19, 20). mutations has been observed, suggesting that haploinsufficiency for Collectively, these rare variants may constitute a significant portion RAD50 predisposes to cancer (8). Thus, a single RAD50 mutation may of the genetic components of these malignancies. Additional be associated with a small increased risk of pancreatic cancer, much studies are needed to define their contribution to the development like mutations in the PALB2, BRIP1, and ATM genes in breast cancer. and progression of these diseases. In addition to truncating mutations, 15 missense variations in nine different DNA repair genes may be associated with increased risk of breast and/or pancreatic cancer based on PMut analysis Acknowledgments (Table 2). Although the functional effects of some mutations can be Received 11/21/2007; revised 1/2/2008; accepted 1/8/2008. The costs of publication of this article were defrayed in part by the payment of page predicted with very high reliability, the actual effects on protein charges. This article must therefore be hereby marked advertisement in accordance function need to be determined. Interestingly, the genotyping result with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. Mutational Analysis of Thirty-two Double-Strand DNA Break Repair Genes in Breast and Pancreatic Cancers

Xianshu Wang, Csilla Szabo, Chiping Qian, et al.

Cancer Res 2008;68:971-975.

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