Analysis of the Gene Coding for the BRCA2-Interacting Protein PALB2 in Familial and Sporadic Pancreatic Cancer

Abstract

Current evidence indicates that about 5%–10% of pancreatic cancer has a familial component, although the vast majority of pancreatic cancer families remain unexplained.1Shi C. Hruban R.H. Klein A.P. Familial pancreatic cancer.Arch Pathol Lab Med. 2009; 133: 365-374PubMed Google ScholarPALB2 is a recently identified breast cancer susceptibility gene whose protein is closely associated with BRCA2, and is essential for BRCA2 anchorage to nuclear structures. This functional relationship made PALB2 a candidate gene for susceptibility to BRCA2-related cancers such as pancreas cancer. Recently, Jones et al2Jones S. Hruban R.H. Kamiyama M. et al.Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.Science. 2009; 324: 217Crossref PubMed Scopus (626) Google Scholar screened 96 familial pancreatic cancer patients, 16 of whom had 1 first-degree relative with pancreatic cancer and 80 had ≥2 additional relatives with pancreatic cancer, ≥1 of which was first degree.2Jones S. Hruban R.H. Kamiyama M. et al.Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.Science. 2009; 324: 217Crossref PubMed Scopus (626) Google Scholar Truncating PALB2 mutations were identified in 3 patients (3.1%) and there was no difference in average age of cancer onset between mutation-positive and -negative families. We sought to screen a larger cohort of pancreatic cancer cases, including both familial and sporadic types, to determine the wider contribution of PALB2 gene mutations in pancreatic cancer. We selected a total of 254 individuals with pancreas cancer (148 male, 106 female) at a median age of diagnosis of 61 years. Patients were identified between 1997 and 2007 via clinic-based recruitment in Toronto and Montreal and through either the Familial Gastrointestinal Cancer Registry at Mount Sinai Hospital in Toronto or the population-based Ontario Pancreas Cancer Study. In total, 203 patients were recruited in Toronto and 51 in Montreal. All probands were confirmed to have pancreatic adenocarcinoma by pathology report; 101 probands had a family history of pancreatic cancer, of which 32 had 2 affected first-degree relatives. In these 101 cases, 74 had 1 affected relative, 18 had 2 affected relatives, 7 had 3 affected relatives, and 2 cases had >3 affected relatives (Table 1). Sixty probands had a family history of breast/ovarian cancer, including 21 cases with a family history of pancreatic cancer (included above). Because the cases were ascertained through pancreas cancer studies, the family history of breast cancer or ovarian cancer was not strong, with most families having a single relative affected, and no family having a BRCAPRO score >0.12 (ie, these were not primarily familial breast/ovarian cancer families with 1 or 2 additional cases of pancreas cancer). The median age at diagnosis of pancreatic cancer cases with no family history was 49 years (range, 31–85); 55% of these were <50 years old and 66% were <60 years old at diagnosis. Genomic DNA was obtained from blood, saliva, or buccal cells using standard extraction methods. Before analysis, 20 ng of total genomic DNA was used for whole genome amplification according to the manufacturer's instructions using the Repli-g Mini Kit (Qiagen, Mississauga, Ontario, Canada). We screened the 13 coding exons of PALB2 by sequencing (n = 83) or high-resolution melt analysis (n = 171), which has similar sensitivity to sequencing.3Wittwer C.T. High-resolution DNA melting analysis: advancements and limitations.Hum Mutat. 2009; 30: 857-859Crossref PubMed Scopus (361) Google Scholar Samples with variants were reamplified by polymerase chain reaction (PCR) using the original, non–whole-genome-amplified DNA as template and the PCR product was sequenced in forward and reverse directions for confirmation. We performed multiplex ligation-dependent probe amplification assay (MLPA) for PALB2 on 228 samples where we had sufficient DNA of adequate quality, as previously described by Foulkes et al.4Foulkes W.D. Ghadirian P. Akbari M.R. et al.Identification of a novel truncating PALB2 mutation and analysis of its contribution to early-onset breast cancer in French-Canadian women.Breast Cancer Res. 2007; 9: R83Crossref PubMed Scopus (120) Google ScholarTable 1Pancreas Cancer CasesType of caseNo. of cases (total = 254)No. of relatives with pancreas cancerNo. of relatives with breast/ovarian cancerSporadic pancreas114NANAFamily history of pancreas cancer801 relative: 742 relatives: 183 relatives: 7>3 relatives: 2NAFamilial pancreas with breast/ovary cases211 relative: 342 relatives: 173 relatives: 7>3 relatives: 2Sporadic pancreas with breast/ovary cases39NANA, not applicable. Open table in a new tab We identified a heterozygous, 6.7-kb germline deletion including exons 12 and 13 of PALB2 in a patient who was affected by breast and then pancreas cancer (ages 47 and 61, respectively) and whose mother died of pancreas cancer at age 83 (Figure 1). This result was confirmed by long range PCR (Takara Bio Inc., Madison, WI) using 2 different primer pairs, which determined the deleted region to span a region from the middle of intron 11 (2.7 kb from the beginning of exon 12), up to 1.8kb after exon 13. This deletion would disrupt the PALB2 WD40 motif, which is required for interaction with the BRCA2 protein.5Xia B. Sheng Q. Nakanishi K. et al.Control of BRCA2 Cellular and Clinical Functions by a Nuclear Partner, PALB2.Mol Cell. 2006; 22: 719-729Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar Aside from the exonic deletion, 2 previously unreported missense variants (S285L and T911I) were identified, but neither were predicted to be pathogenic. Both these variants were seen in young-onset pancreas cancer cases (41 years and 48 years) with no family history. A number of previously reported variants were also identified (Table 2).Table 2Other Variants Detected in PALB2Sequence variantProtein changeNo. of times observedAdditional informationc.1 −47 G>A5′ UTR13rs8053188aPreviously reported.9c.212 −58 A>CIntron 316non-codingaPreviously reported.9c.1684 +42 ins.TGAIntron 41non-codingcPreviously unreported.c.854 C>TS285L1possibly damaging,cPreviously unreported.,dPolyphen classification. toleratedeSorting intolerant from tolerant (sift) classification.c.1010 T>CL337S4rs45494092aPreviously reported.9c.1572 A>GS524S1rs45472400aPreviously reported.9c.1194 G>AV398V1SilentaPreviously reported.9c.1676 A>GQ559R40rs152451aPreviously reported.9c.1743 A>GL581L1SilentcPreviously unreported.c.2014 G>CE672Q18rs45532440aPreviously reported.9c.2586 +58 C>TIntron 657rs249954bPreviously reported.10c.2590 C>TP864S1rs45568339aPreviously reported.9c.2732 C>TT911I1possibly damaging,cPreviously unreported.,dPolyphen classification. toleratedeSorting intolerant from tolerant (sift) classification.c.2794 G>AV932M4rs45624036aPreviously reported.9c.2816 T>GL939W2rs45478192aPreviously reported.9c.2993 G>AG998E11rs45551636aPreviously reported.9c.3300 T>GT1100T16rs45516100aPreviously reported.9a Previously reported.9Garcia M.J. Fernandez V. Osorio A. et al.Analysis of FANCB and FANCN/PALB2 fanconi anemia genes in BRCA1/2-negative Spanish breast cancer families.Breast Cancer Res Treat. 2009; 113: 545-551Crossref PubMed Scopus (79) Google Scholarb Previously reported.10Chen P. Liang J. Wang Z. et al.Association of common PALB2 polymorphisms with breast cancer risk: a case-control study.Clin Cancer Res. 2008; 14: 5931-5937Crossref PubMed Scopus (38) Google Scholarc Previously unreported.d Polyphen classification.e Sorting intolerant from tolerant (sift) classification. Open table in a new tab The main purpose of the study by Jones et al2Jones S. Hruban R.H. Kamiyama M. et al.Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.Science. 2009; 324: 217Crossref PubMed Scopus (626) Google Scholar was to illustrate that exomic sequencing could be used to identify genes conferring a substantial risk of cancer, in the absence of linkage or other localizing information. After identification of a PALB2 germ-line mutation in a pancreas cancer case with a family history of pancreatic cancer, truncating mutations were found in 3 other individuals, all of whom had a personal and family history of pancreas cancer. Here, we studied a more varied set of pancreas cancer cases, with fewer probands with very strong family histories of pancreas cancer, but more cases with family histories of other BRCA2-related cancers, and 13 cases diagnosed under the age of 40. We found only 1 deleterious mutation, a 2-exon deletion, which is only the second described case of a PALB2 exonic deletion, the other being reported in a case of Fanconi anemia.6Xia B. Dorsman J.C. Ameziane N. et al.Fanconi anemia is associated with a defect in the BRCA2 partner PALB2.Nat Genet. 2007; 39: 159-161Crossref PubMed Scopus (370) Google Scholar To date, PALB2 exonic deletions have not been reported in breast cancer families.7Ameziane N. van den Ouweland A.M. Adank M.A. et al.Lack of large genomic deletions in BRIP1, PALB2, and FANCD2 genes in BRCA1/2 negative familial breast cancer.Breast Cancer Res Treat. 2009 Jun 6; ([Epub ahead of print])PubMed Google ScholarTaking the 2 sets of data together, we conclude that germ-line mutations in PALB2 are a rare cause of pancreas cancer, whether hereditary, familial, or sporadic. Of note, 4 of the 5 PALB2-related pancreas families identified to date include ≥1 case of breast cancer and in 2 families mutations were seen in individuals with both breast and pancreatic cancer (a total of 9 cases in our study had both breast and pancreas cancer). It may be the case that investigating PALB2 will be worthwhile in individuals with very strong family histories of pancreas cancer, or where there is a concomitant history of breast cancer, but based on the findings presented herein, routine screening of PALB2 in individuals with, or at risk of pancreas cancer is difficult to justify. If PALB2 mutation screening is undertaken, it should include MLPA for exonic deletions. As is seen in breast cancer,8Erkko H. Dowty J.G. Nikkila J. et al.Penetrance analysis of the PALB2 c.1592delT founder mutation.Clin Cancer Res. 2008; 14: 4667-4671Crossref PubMed Scopus (86) Google Scholar the penetrance for these very rare disease-causing variants seems to be high. Current evidence indicates that about 5%–10% of pancreatic cancer has a familial component, although the vast majority of pancreatic cancer families remain unexplained.1Shi C. Hruban R.H. Klein A.P. Familial pancreatic cancer.Arch Pathol Lab Med. 2009; 133: 365-374PubMed Google ScholarPALB2 is a recently identified breast cancer susceptibility gene whose protein is closely associated with BRCA2, and is essential for BRCA2 anchorage to nuclear structures. This functional relationship made PALB2 a candidate gene for susceptibility to BRCA2-related cancers such as pancreas cancer. Recently, Jones et al2Jones S. Hruban R.H. Kamiyama M. et al.Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.Science. 2009; 324: 217Crossref PubMed Scopus (626) Google Scholar screened 96 familial pancreatic cancer patients, 16 of whom had 1 first-degree relative with pancreatic cancer and 80 had ≥2 additional relatives with pancreatic cancer, ≥1 of which was first degree.2Jones S. Hruban R.H. Kamiyama M. et al.Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.Science. 2009; 324: 217Crossref PubMed Scopus (626) Google Scholar Truncating PALB2 mutations were identified in 3 patients (3.1%) and there was no difference in average age of cancer onset between mutation-positive and -negative families. We sought to screen a larger cohort of pancreatic cancer cases, including both familial and sporadic types, to determine the wider contribution of PALB2 gene mutations in pancreatic cancer. We selected a total of 254 individuals with pancreas cancer (148 male, 106 female) at a median age of diagnosis of 61 years. Patients were identified between 1997 and 2007 via clinic-based recruitment in Toronto and Montreal and through either the Familial Gastrointestinal Cancer Registry at Mount Sinai Hospital in Toronto or the population-based Ontario Pancreas Cancer Study. In total, 203 patients were recruited in Toronto and 51 in Montreal. All probands were confirmed to have pancreatic adenocarcinoma by pathology report; 101 probands had a family history of pancreatic cancer, of which 32 had 2 affected first-degree relatives. In these 101 cases, 74 had 1 affected relative, 18 had 2 affected relatives, 7 had 3 affected relatives, and 2 cases had >3 affected relatives (Table 1). Sixty probands had a family history of breast/ovarian cancer, including 21 cases with a family history of pancreatic cancer (included above). Because the cases were ascertained through pancreas cancer studies, the family history of breast cancer or ovarian cancer was not strong, with most families having a single relative affected, and no family having a BRCAPRO score >0.12 (ie, these were not primarily familial breast/ovarian cancer families with 1 or 2 additional cases of pancreas cancer). The median age at diagnosis of pancreatic cancer cases with no family history was 49 years (range, 31–85); 55% of these were <50 years old and 66% were <60 years old at diagnosis. Genomic DNA was obtained from blood, saliva, or buccal cells using standard extraction methods. Before analysis, 20 ng of total genomic DNA was used for whole genome amplification according to the manufacturer's instructions using the Repli-g Mini Kit (Qiagen, Mississauga, Ontario, Canada). We screened the 13 coding exons of PALB2 by sequencing (n = 83) or high-resolution melt analysis (n = 171), which has similar sensitivity to sequencing.3Wittwer C.T. High-resolution DNA melting analysis: advancements and limitations.Hum Mutat. 2009; 30: 857-859Crossref PubMed Scopus (361) Google Scholar Samples with variants were reamplified by polymerase chain reaction (PCR) using the original, non–whole-genome-amplified DNA as template and the PCR product was sequenced in forward and reverse directions for confirmation. We performed multiplex ligation-dependent probe amplification assay (MLPA) for PALB2 on 228 samples where we had sufficient DNA of adequate quality, as previously described by Foulkes et al.4Foulkes W.D. Ghadirian P. Akbari M.R. et al.Identification of a novel truncating PALB2 mutation and analysis of its contribution to early-onset breast cancer in French-Canadian women.Breast Cancer Res. 2007; 9: R83Crossref PubMed Scopus (120) Google Scholar NA, not applicable. We identified a heterozygous, 6.7-kb germline deletion including exons 12 and 13 of PALB2 in a patient who was affected by breast and then pancreas cancer (ages 47 and 61, respectively) and whose mother died of pancreas cancer at age 83 (Figure 1). This result was confirmed by long range PCR (Takara Bio Inc., Madison, WI) using 2 different primer pairs, which determined the deleted region to span a region from the middle of intron 11 (2.7 kb from the beginning of exon 12), up to 1.8kb after exon 13. This deletion would disrupt the PALB2 WD40 motif, which is required for interaction with the BRCA2 protein.5Xia B. Sheng Q. Nakanishi K. et al.Control of BRCA2 Cellular and Clinical Functions by a Nuclear Partner, PALB2.Mol Cell. 2006; 22: 719-729Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar Aside from the exonic deletion, 2 previously unreported missense variants (S285L and T911I) were identified, but neither were predicted to be pathogenic. Both these variants were seen in young-onset pancreas cancer cases (41 years and 48 years) with no family history. A number of previously reported variants were also identified (Table 2). The main purpose of the study by Jones et al2Jones S. Hruban R.H. Kamiyama M. et al.Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene.Science. 2009; 324: 217Crossref PubMed Scopus (626) Google Scholar was to illustrate that exomic sequencing could be used to identify genes conferring a substantial risk of cancer, in the absence of linkage or other localizing information. After identification of a PALB2 germ-line mutation in a pancreas cancer case with a family history of pancreatic cancer, truncating mutations were found in 3 other individuals, all of whom had a personal and family history of pancreas cancer. Here, we studied a more varied set of pancreas cancer cases, with fewer probands with very strong family histories of pancreas cancer, but more cases with family histories of other BRCA2-related cancers, and 13 cases diagnosed under the age of 40. We found only 1 deleterious mutation, a 2-exon deletion, which is only the second described case of a PALB2 exonic deletion, the other being reported in a case of Fanconi anemia.6Xia B. Dorsman J.C. Ameziane N. et al.Fanconi anemia is associated with a defect in the BRCA2 partner PALB2.Nat Genet. 2007; 39: 159-161Crossref PubMed Scopus (370) Google Scholar To date, PALB2 exonic deletions have not been reported in breast cancer families.7Ameziane N. van den Ouweland A.M. Adank M.A. et al.Lack of large genomic deletions in BRIP1, PALB2, and FANCD2 genes in BRCA1/2 negative familial breast cancer.Breast Cancer Res Treat. 2009 Jun 6; ([Epub ahead of print])PubMed Google Scholar Taking the 2 sets of data together, we conclude that germ-line mutations in PALB2 are a rare cause of pancreas cancer, whether hereditary, familial, or sporadic. Of note, 4 of the 5 PALB2-related pancreas families identified to date include ≥1 case of breast cancer and in 2 families mutations were seen in individuals with both breast and pancreatic cancer (a total of 9 cases in our study had both breast and pancreas cancer). It may be the case that investigating PALB2 will be worthwhile in individuals with very strong family histories of pancreas cancer, or where there is a concomitant history of breast cancer, but based on the findings presented herein, routine screening of PALB2 in individuals with, or at risk of pancreas cancer is difficult to justify. If PALB2 mutation screening is undertaken, it should include MLPA for exonic deletions. As is seen in breast cancer,8Erkko H. Dowty J.G. Nikkila J. et al.Penetrance analysis of the PALB2 c.1592delT founder mutation.Clin Cancer Res. 2008; 14: 4667-4671Crossref PubMed Scopus (86) Google Scholar the penetrance for these very rare disease-causing variants seems to be high. William D. Foulkes is the corresponding author. Department of Oncology, McGill University, 546 Pine Avenue West, Montreal, Quebec, Canada H2W 1S6; phone: (514) 934-1934 ext. 44121; fax: (514) 934-8273; e-mail: [email protected] .