PALB2 mutations in European familial pancreatic cancer families

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1 Clin Genet 2010: 78: Printed in Singapore. All rights reserved Short Report 2010 John Wiley & Sons A/S CLINICAL GENETICS doi: /j x PALB2 mutations in European familial pancreatic cancer families Slater EP, Langer P, Niemczyk E, Strauch K, Butler J, Habbe N, Neoptolemos JP, Greenhalf W, Bartsch DK. PALB2 mutations in European pancreatic cancer families. Clin Genet 2010: 78: John Wiley & Sons A/S, 2010 Recently, PALB2 was reported to be a new pancreatic cancer susceptibility gene as determined by exomic sequencing, as truncating PALB2 mutations were identified in 3 of 96 American patients with familial pancreatic cancer (FPC). Representing the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC) and the German National Case Collection for Familial Pancreatic Cancer (FaPaCa), we evaluated whether truncating mutations could also be detected in European FPC families. We have directly sequenced the 13 exons of the PALB2 gene in affected index patients of 81 FPC families. An index patient was defined as the first medically identified patient, stimulating investigation of other members of the family to discover a possible genetic factor. None of these patients carried a BRCA2 mutation. We identified three (3.7%) truncating PALB2 mutations, each producing different stop codons: R414X, 508-9delAG and 3116delA. Interestingly, each of these three families also had a history of breast cancer. Therefore, PALB2 mutations might be causative for FPC in a small subset of European families, especially in those with an additional occurrence of breast cancer. EP Slater a, P Langer a, E Niemczyk b, K Strauch c, JButler b, N Habbe a, JP Neoptolemos b, W Greenhalf b and DK Bartsch a a German National Case Collection of Familial Pancreatic Cancer (FaPaCa), Department of Surgery, Philipps-University, Marburg, Germany, b EUROPAC-Registry, Department of Surgery, University of Liverpool, United Kingdom, and c Institute of Medical Biometry and Epidemiology, Philipps-University, Marburg, Germany Key words: BRCA2 familial pancreatic cancer mutation PALB2 Corresponding author: Emily P. Slater, PhD, Department of Visceral, Thoracic and Vascular Surgery, Philipps-University, Baldingerstrasse, Marburg, Germany. Tel.: ; fax: ; slater@med.uni-marburg.de Received 09 October 2009, revised and accepted for publication 10 March 2010 Hereditary pancreatic cancer is a rare, but established, inherited tumor predisposition syndrome. Families with FPC have two or more firstdegree relatives with pancreatic carcinoma (PC) and do not fulfill the criteria for another inherited syndrome. International and national tumor registries have been established to collect families in order to determine the characteristics and underlying gene defect(s) of FPC. The comprehensive clinical and genetic analyses of FPC families within these registries have revealed important data about FPC. The vertical pattern of cancer observed in the majority of the FPC families is consistent with an autosomal dominant trait. The most commonly mutated gene for FPC is BRCA2 (1 2). However, recent studies indicate that the prevalence of BRCA2 mutations is lower than initially reported (3). Another gene in this pathway is PALB2, which acts as a bridge between BRCA2 and BRCA1 in a complex that is critical for homologous recombination and double-strand break repair (4). Germline PALB2 mutations have previously been associated with Fanconi anemia and breast cancer predisposition (5 7). Jones et al. recently described the exomic sequencing of PALB2 in patients with FPC (8). They identified truncating mutations in 3 (3.1%) of 96 American FPC patients. Each of these mutations produces a different stop codon. Representing the EUROPAC and the FaPaCa, we evaluated whether truncating mutations could be detected in our FPC families. 490

2 PALB2 mutations in European FPC families Materials and methods As Europe can be considered to comprise a single large genetic pool, we included families from Germany (n = 41), the United Kingdom (n = 30), Latvia (n = 3), Italy (n = 3), Greece (n = 2), Hungary (n = 1) and Spain (n = 1). To determine whether PALB2 mutations occur in our patients with FPC, we directly sequenced the 13 exons of this gene in affected index pancreatic cancer patients of 81 FPC families, of whom 57 had at least one affected first-degree relative and 24 had at least two affected relatives with PC. None of these patients carried a BRCA2 mutation as evaluated by denaturing high-peformance liquid chromatography (DHPLC) analysis and/or direct sequencing of the whole gene. All participants provided written informed consent and the study was approved by the local Ethics Committees of either the Philipps-University, Marburg, or the North West Multi-centre Research Ethics Committee in the United Kingdom. Results and Discussion We identified three (3.7%, standard error 2.1%, 95% CI %) PALB2 germline mutations, one nonsense and two frameshift mutations, in three families. These mutations are considered to be pathogenic, because each creates a stop codon that is predicted to cause a truncation of the PALB2 protein. Truncating mutations in PALB2 are rare in individuals without cancer; none was reported in 1084 normal participants in a previous study (7). Family was found to carry the mutation c.1240c>t, p.r414x (Fig. 1). This is a nonsense mutation located in exon 4, resulting in truncation at position 414. This family has four cases of PC: three sisters and one brother. Another sister had breast cancer, as did her daughter (Fig. 2). This large family was screened and seven as of yet unaffected members were found to be carriers of this mutation. They are presently enrolled in our prospective PC screening Fig. 1. PALB2 mutation c.1240c>t, p.r414x identified in FPC family Arrow, mutation; II3, family member identification. 491

3 Slater et al. Family I:1 I:2 II:1 II:2 wt II:3 II:4 II:5 wt * * * * 75y dg 53y dg 70y dg 65y 67y dg 54y 64y dg 39y 60y 60y 58y II:6 II:7 II:8 II:9 II:10 II:11 III:1 III:2 III:3 III:4 III:5 III:6 III:7 III:8 III:9 III:10 III:11 III:12 III:13 III:14 III:15 III:16 III:17 III:18 III:19 III:20 III:21 III:22 III:23 III:24 III:25 III:26 III:27 III:28 III:29 wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt * * * * * 58y 51y 39y 49y 42y 46y 40y 38y 41y 40y 39y 35y 29y 26y 38y 33y 29y 39y 38y 25y 33y 32y 40y 37y 32y unaffected pancreatic cancer breast cancer Fig. 2. Pedigree of FPC family ; *, mutation carrier; wt, wild type; y, years; dg, diagnosis. The arrow indicates the index case (II:3). program (9). The penetrance of the mutation in family was estimated by means of a model-maximized logarithm of odds (MOD) score analysis (10) as implemented in the program GENEHUNTER-MODSCORE (11, 12). In the context of this linkage-based method, the parametric logarithm of odds (LOD) score is maximized with respect to the trait-model parameters, i.e. the three penetrances and the disease allele frequency. As the LOD score represents the conditional probability of the observed genotypes given the trait phenotypes (rather than the probability of the observed genotypes and the trait phenotypes), the maximization yields unbiased estimates of these parameters (13). Hence, a MOD-score analysis is an ascertainment-assumption-free method, that is the parameter estimates are independent of the ascertainment scheme and the degree to which affected persons are enriched in the studied sample compared with the general population. Because of the younger age of the persons in the third generation who have a mutation-carrying parent (46 years or younger), we decided to include only individuals of the parental and grandparental generation, to avoid a bias due to an age-of-onset effect. The genotyped persons III:23 and III:24 were nevertheless included, with their phenotype set to unknown, to allow for reconstructing the genotype of their unaffected but untyped father II:9 as far as possible in the calculation. As PALB2 mutations are expected to be extremely rare in the general population, we assumed that one grandparent carries the mutation but not the other, thus excluding a recessive mode of inheritance. With this setting, the MOD-score analysis yielded a dominant disease model with an arbitrary low disease allele frequency, no phenocopies, and a penetrance of This estimate represents the cumulative risk of developing PC at an age of 58 years. Family was found to carry the mutation c.508-9delag, p.r170i, 183X. This deletion located in exon 4 causes a frameshift mutation, resulting in a truncation at amino acid position 183. In this family, the male index patient and his mother were diagnosed with PC at ages 58 and 68 years, respectively. His aunt had breast cancer at age 65 years and another aunt had a brain tumor at age 64 years. Segregation of the PALB2 492

4 PALB2 mutations in European FPC families mutation could not be analyzed, because DNA of the other deceased affected family members was not available. The two unaffected siblings of the affected PALB2 mutation carrier showed wild-type PALB2 sequences. Family P666 was found to carry the mutation c.3116dela, p.n1039fs. This deletion located in exon 11 causes a frameshift mutation. It has been described in Fanconi anemia and familial breast cancer as well as in one of the FPC families described by Jones et al. (1). This family has two cases of breast cancer (members aged 60 and 56 years) on the index patient s maternal side, two additional cases of PC (members aged 56 and 64 years) and one gastric cancer case (member aged 80 years) on the index patient s paternal side. Because of the lack of DNA from the parents of the index patient, we could not determine from which side the mutation was inherited. Interestingly, each of these three FPC families also had a history of breast cancer. Of the 41 German families screened in this study, 14 (34%) had a history of breast cancer and of these 14, 2 had truncating mutations (14.3%). Of the 40 EUROPAC families, only family P666 with the PALB2 mutation had cases of breast cancer. This underscores the observations by both Jones et al., where three of four FPC families in their report also had cases of breast cancer (8), and Tischkowitz et al., who described a 6.7-kb PALB2 germline deletion in a patient affected by both breast and pancreatic cancer (14). However, PALB2 mutations in the setting of FPC do not seem to predispose to early age onset of PC or breast cancer as previously suggested for breast cancer in a Finnish study (15). Conclusions Therefore, we conclude that PALB2 mutations might contribute to hereditary pancreatic cancer in a small subset of European FPC families, most probably in those with concomitant breast cancer. The prevalence of PALB2 mutations appears to be comparable to that of BRCA2 mutations in FPC families. As we did not analyze exonic deletions, the observed prevalence of PALB2 mutations might even be a modest underestimate. Although the absolute and relative risk for the development of PC in PALB2 mutation carriers still remains unknown, PALB2 mutation carriers of FPC families have to be considered as high-risk individuals with at least 10- to 32-fold increased risk depending on the number of affected family members (16). According to an expert consensus statement, such high-risk family members should be offered participation in board-approved, prospective screening programs for the early detection and potentially curative operative treatment of PC or even better its precursor lesions (16), as it has been shown that PC screening of high-risk individuals might be effective (9, 17). Acknowledgements We are grateful to all the families who participated in the study. We thank Brunhilde Chaloupka and Elvira Matthäi for their excellent technical assistance. This work was supported by a grant from the Deutsche Krebshilfe to FaPaCa and the European Union, Cancer Research UK and the NIHR via the Liverpool Pancreatic Biomedical Research Unit to EUROPAC. Conflict of interest The authors have no conflict of interest. References 1. Murphy KM, Brune KA, Griffin C et al. Evaluation of candidate genes MAP2K4, MADH4, ACVR1B and BRCA2 in familial pancreatic cancer: deleterious BRCA2 mutations in 17%. Cancer Res 2002: 62: Hahn SA, Greenhalf W, Ellis I et al. BRCA2 germline mutations in familial pancreatic carcinoma. J Natl Cancer Inst 2003: 95: Couch FJ, Johnson MR, Rabe KG et al. The prevalence of BRCA2 mutations in familial pancreatic cancer. Cancer Epidemiol Biomarkers Prev 2007: 16: Sy SM, Huen MS, Chen J. PALB2 is an integral component of the BRCA complex required for homologous recombination repair. 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