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Cancer Research 68, 971, February 15, 2008. doi: 10.1158/0008-5472.CAN-07-6272
© 2008 American Association for Cancer Research
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Priority Reports

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

Xianshu Wang1, Csilla Szabo1, Chiping Qian3, Peter G. Amadio1, Stephen N. Thibodeau1, James R. Cerhan2, Gloria M. Petersen2, Wanguo Liu3 and Fergus J. Couch1

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

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: couch.fergus{at}mayo.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inactivating mutations in several genes that encode components of the DNA repair machinery have been associated with an increased risk of breast cancer. To assess whether alterations in other DNA repair genes contribute to breast cancer and to further determine the relevance of these genes to pancreatic cancer, we performed mutational analysis of 32 DNA double-strand break repair genes in genomic DNA from 38 breast tumors, 48 pancreatic tumors, and 10 non-BRCA1/BRCA2 hereditary breast cancer patients. A total of 494 coding exons were screened by denatured high-performance liquid chromatography and direct DNA sequencing. Two inactivating mutations were identified in breast tumor samples, a germline single-nucleotide deletion in POLQ (c.3605delT) and a somatic nonsense change in PRKDC (c.2408C>A, p.Ser803X). Two germline-inactivating mutations in RAD50 (c.1875C>G, p.Tyr625X and IVS14+1G>A) were also detected in separate pancreatic tumor samples. In addition, 35 novel nonsynonymous amino acid substitutions, resulting from two in-frame deletions and 33 single nucleotide alterations, were identified. Seven of these were predicted to influence protein function. A separate analysis of the CLSPN c.3839C>T (rs35490896) variant that was observed more frequently in breast tumors than in pancreatic tumors or normal controls failed to detect a significant association with breast cancer risk in a Mayo Clinic breast cancer case-control study. In conclusion, this screen of DNA repair genes implicates PRKDC and POLQ as candidate tumor suppressor genes involved in breast cancer and suggests that inactivating mutations in RAD50 predispose to pancreatic cancer as well as breast cancer. [Cancer Res 2008;68(4):971–5]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Double-strand DNA break (DSB) repair pathways are essential for the prevention of genomic instability. Previous studies have shown that genetic defects in BRCA1 and BRCA2, key components of homologous recombination repair, are associated with breast, ovarian, and pancreatic cancer risk (1–3). Rare inactivating mutations in several DNA damage repair and signaling genes, ATM (4), BRIP1 (5), CHEK2 (6), PALB2 (7), NBS1 (8), RAD50 (9), and TP53 (10), have also been implicated in breast cancer. This suggests that mutations in other DNA damage repair genes may predispose and/or contribute to breast cancer. Similarly, the recent discovery that mutations in BRCA2, FANCC, and FANCG (2, 11, 12) are associated with pancreatic cancer suggests that mutations in other repair genes may contribute to pancreatic cancer risk. To identify other DNA repair genes associated with breast and pancreatic cancer, we performed a mutation screen of the coding regions of 32 genes involved in DSB signaling and repair in 38 breast tumors, 48 pancreatic tumors, and germline DNA from 10 non-BRCA1/BRCA2 hereditary breast cancer patients. We report the identification of several truncating and missense mutations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples for mutation screen. Genomic DNA was obtained from an unselected series of 48 pancreatic tumors and 38 breast tumors and from blood samples from probands of 10 high-risk breast cancer families without BRCA1 and BRCA2 mutations collected at Mayo Clinic. In addition, 48 control DNAs were obtained from blood samples of noncancer patients attending Internal Medicine clinics. DNA was extracted from cryocut sections of frozen tumors or buffy coats with Easy-DNA kits (Invitrogen).

Mutation screening by denatured high-performance liquid chromatography. PCR primers were designed for all coding exons of 32 genes (Supplementary Table S1). PCR products were generated using AmpliTaq Gold (Roche) or HotStar Taq (Invitrogen), 10 ng genomic DNA, and 35 to 40 cycles of amplification. Denatured high-performance liquid chromatography (DHPLC) analysis was performed on automated DHPLC instruments (Transgenomic or Varian). DNA samples displaying alterations on DHPLC were reamplified and sequenced on an ABI Prism 377 Sequencer (Perkin-Elmer).

Coding single-nucleotide polymorphisms selection and PMut analysis. All known single-nucleotide polymorphisms (SNP) in the coding regions of the 32 genes were obtained from Hapmap release 21 (13). Only SNPs with validation and defined minor allele frequency in Caucasian populations were used as positive controls to validate the sensitivity of the DHPLC analysis. Amino acid substitutions were predicted to be neutral or pathologic using PMut4 prediction scores and an associated reliability index, which compares favorably to PolyPhen and SIFT (14).

Loss of heterozygosity and mRNA transcription analysis. Loss of heterozygosity (LOH) analysis was performed on laser capture microdissected pancreatic tumor and matched normal stromal tissues. DNA and RNA were isolated from 4,000 tumor cells captured on a Veritas Laser Capture Microdissection instrument (Molecular Devices Corporation) using QIAamp DNA Microkit (Qiagen) and PicoPure RNA Isolation kit (Molecular Devices Corporation). PCR and reverse transcription–PCR (RT-PCR) were 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 cancer case-control series (15). Collection and characteristics of cases and controls are described in the Supplementary Materials and Methods.

TaqMan genotyping and data analysis. Genomic DNAs from breast cancer cases and controls were genotyped by the 5' nuclease assay (TaqMan), using the 7900HT Real-Time PCR System (Applied Biosystems), in 384-well format. Duplicate samples (5%) were included and all displayed 100% concordance for the CLSPN c.3839C>T genotype. Unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals (CI) under general (df, 2) and ordinal models. Analyses were adjusted for common breast cancer risk factors and performed using SAS software, version 8.2 (SAS Institute, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sensitivity of DHPLC analysis. We screened a total of 1.7 x 107 bases of DNA in 494 coding exons from 32 genes to identify mutations associated with breast and pancreatic cancer. To assess the sensitivity of the DHPLC screen, we compared the variants detected in the mutation screen with all known SNPs from these exons. Among 60 SNPs listed in dbSNP as validated in at least two studies of Caucasians, 54 were detected in this mutation screen. Four of the six SNPs not identified exhibited minor allele frequencies of <2% and were excluded. The detection rate of 96.4% for known SNPs indicated that the DHPLC technique was sufficiently sensitive to detect the majority of putative disease-associated mutations.

Novel deleterious mutations. The mutation screen of 96 samples resulted in the identification of 73 unique alterations in 17 genes. These variants included three truncating mutations, an in-frame splicing variant, two in-frame three-nucleotide deletions, and 33 novel nonsynonymous changes. In addition, 154 intronic changes were detected. A summary of the results of this extensive mutation screen is shown in Tables 1 and 2 .


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

 

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

 
One truncating mutation, resulting in a premature stop codon p.Ser803X, was detected in PRKDC in a breast tumor sample (Table 2). PCR and DNA sequencing of blood-based DNA from the same patient confirmed that the mutation was of somatic origin. Another truncating mutation, c.3605delT, was identified in POLQ in blood DNA from a proband of a high-risk breast cancer family (Table 2). The single-nucleotide deletion caused a frameshift and truncation of the protein and exclusion of the POLQ C-terminal DNA polymerase and 3'-5' exonuclease domains. These mutations likely inactivate the DNA-PK and POLQ DNA repair proteins.

Two deleterious mutations were also identified in the RAD50 gene in DNA from pancreatic tumors (Table 2). A c.1875C>G variant resulting in a premature stop codon p.Tyr625X was identified in a tumor from a 64-year-old male. The germline origin of the mutation was confirmed by analysis of blood DNA. An IVS14+1G>A mutation in a tumor from a 67-year-old male was also detected in blood DNA. This alteration was predicted to disrupt the splice donor site resulting in aberrant splicing of intron 14. These mutations represent the first descriptions of inherited mutations in the RAD50 gene in pancreatic cancer patients and strongly suggest that RAD50 functions as a pancreatic cancer tumor suppressor.

LOH and RNA analysis of RAD50 mutations in pancreatic tumors. To check whether loss of the RAD50 wild-type allele occurred in tumor cells harboring the two mutations, we performed LOH analysis using genomic DNA from laser microdissected tumor cells. As shown in Fig. 1A , equal amounts of the 1875C and 1875G alleles were present in the tumor cells, suggesting that the 1875C wild-type allele was not lost. Similarly, no LOH of the wild-type IVS14+1G allele was detected.


Figure 1
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  		Figure 1. RAD50 truncating mutations c.1875C>G and IVS14+1G>A in DNA and RNA purified from laser capture microdissected pancreatic tumors. A, LOH analysis by PCR and direct DNA sequencing of genomic DNA isolated from pure population of pancreatic tumor cells. B, variant splicing of mRNA in tumor cells and adjacent stroma harboring the IVS14+1G>A germline mutation. LCM-T1 and LCM-T2 represent tumor cell populations laser capture microdissected from the IVS14+1G>A mutant tumor. The alternative splicing of exon 14 (exon 14S) beginning at a cryptic "GT" donor site 39 bp upstream from the mutated splice donor site is shown along with the sequence chromatograms for the normal and aberrantly sized RT-PCR products. C, nonsense mediated mRNA decay in tumor cells and adjacent stroma containing the c.1875C>G germline mutation. LCM-T1 and LCM-T2 represent tumor cell populations laser capture microdissected from the c.1875C>G mutant tumor. The sequence chromatogram based on cDNA derived from c.1875C>G tumor cells shows only wild-type sequence.

 
Analysis of RNA from microdissected tumor cells was also performed to determine whether the IVS14+1G>A mutation induced aberrant splicing. Interestingly, RT-PCR analysis detected two products differing by 39 bp in size in both microdissected tumor cells and microdissected adjacent stromal cells (Fig. 1B). Sequencing showed that the larger product represented the wild-type allele, whereas the smaller product resulted from use of a cryptic "GT" in exon 14 located 39 bp upstream from the original donor splice site. Thus, the splicing mutation results in an in-frame deletion of 13 amino acids, p.Val787_Gln799del, in the functionally important EzrA domain of RAD50. This rearrangement was not detected in RNA from lymphoblastoid cell lines derived from 20 unaffected controls. In parallel, we evaluated the expression of the c.1875C>G (p.Tyr625X) mutant by RT-PCR. The mutant c.1875G mRNA transcript was not detected in two separate tumor cell specimens or the associated stromal cells, suggesting that the mutant transcript was eliminated by nonsense mediated mRNA decay (Fig. 1C).

Unclassified missense variants. A total of 29 of 33 novel missense variants were found once. These variants were equally distributed between breast and pancreatic cancer samples (Table 2). In addition, multiple variants were found to coexist in the same breast or pancreatic tumor samples (Supplementary Table S3). Fifteen of 33 variants located in nine genes were predicted to disrupt protein function by PMut analysis (Table 2). Seven of these variants displayed a reliability index >5 and seem most likely to affect the normal protein function.

Association of CLSPN p.Ser1280Leu with breast cancer risk. The p.Ser1280Leu (rs35490896) and p.Glu1199del variants in CLSPN were found to be in strong linkage disequilibrium because they cooccurred in 15 tumor samples but were never identified independently. We also noted a higher prevalence of these mutations in breast samples (25%) than in pancreatic samples (6%) and an unselected series of 48 normal controls (2%). Based on this observation, we further examined the association of p.Ser1280Leu and p.Glu1199del with breast cancer risk by genotyping rs35490896 in a Mayo breast cancer case-control study. As shown in Table 3 , no significant association (P < 0.05) was observed between rs35490896 and breast cancer risk (odds ratio, 0.99; 95% CI, 0.67–1.20) under a log-additive model.


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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

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 x 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 remains possible that other novel mutations remain to be identified. However, whereas PRKDC may have tumor suppressor activity, the absence of the identified truncating mutation from the germline DNA of the relevant breast cancer patient means that we cannot propose PRKDC as a breast cancer predisposition gene.

We also identified RAD50 c.1875C>G (p.Tyr625X) and IVS14+1G>A germline mutations in pancreatic tumors. These are likely to disrupt the well-established DSB repair function of RAD50. The 13 amino acid in-frame deletion caused by IVS14+1G>A removes a section of the EzrA domain close to the CXXC motif essential for RAD50 dimerization and DNA repair function (18). Similarly, the c.1875C>G truncating mutation is expected to disrupt MRN complex formation and DNA repair activity. The detection of two mutations in 48 pancreatic tumors strongly suggests that RAD50 is a pancreatic cancer predisposition gene, especially when considering that truncating mutations in RAD50 have previously been associated with a moderate risk of breast and ovarian cancer in the Finnish population (8, 9). The absence of RAD50 LOH from the mutant pancreatic tumors is consistent with a similar observation in breast and ovarian tumors with RAD50 mutations and does not exclude RAD50 as a tumor suppressor. In fact, a substantial increase in chromosomal instability in lymphoblastoid cells carrying RAD50 mutations has been observed, suggesting that haploinsufficiency for RAD50 predisposes to cancer (8). Thus, a single RAD50 mutation may be associated with a small increased risk of pancreatic cancer, much like mutations in the PALB2, BRIP1, and ATM genes in breast cancer.

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 (Table 2). Although the functional effects of some mutations can be predicted with very high reliability, the actual effects on protein function need to be determined. Interestingly, the genotyping result of CLSPN rs35490896 showed that p.Ser1280Leu was not associated with increased breast cancer risk, in contrast to the pathologic prediction by PMut. This finding illustrates the importance of genetic association studies, together with functional analysis, for further validation of results obtained from high-throughput mutation screening studies.

Overall, the POLQ, the RAD50, and, perhaps, the PRKDC genes warrant further analysis on the basis of our comprehensive screen of DNA damage repair genes. Our findings are consistent with the notion that a large number of genes containing rare genetic alterations that confer small to moderate risk of complex diseases, such as breast and pancreatic cancer may exist (19, 20). Collectively, these rare variants may constitute a significant portion of the genetic components of these malignancies. Additional studies are needed to define their contribution to the development and progression of these diseases.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

4 http://mmb2.pcb.ub.es:8080/PMut/ Back

Received 11/21/07. Revised 1/ 2/08. Accepted 1/ 8/08.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online