PALB2 variants in hereditary and unselected Finnish Prostate cancer cases
- Sanna Pakkanen†1,
- Tiina Wahlfors†1,
- Sanna Siltanen1,
- Mimmi Patrikainen1,
- Mika P Matikainen2,
- Teuvo L J Tammela2 and
- Johanna Schleutker1Email author
© Pakkanen et al; licensee BioMed Central Ltd. 2009
Received: 3 July 2009
Accepted: 15 December 2009
Published: 15 December 2009
PALB2 1592delT mutation is associated with increased breast cancer and suggestive prostate cancer (PRCA) risk in Finland. In this study we wanted to assess if any other PALB2 variants associate to increased PRCA risk and clinically describe patients with formerly found PALB2 1592delT mutation.
Finnish families with two or more PRCA cases (n = 178) and unselected cases (n = 285) with complete clinical data were initially screened for variants in the coding region and splice sites of PALB2. Potentially interesting variants were verified in additional set of unselected cases (n = 463).
From our clinically defined sample set we identified total of six variants in PALB2. No novel variants among Finnish PRCA cases were found. Clinical characteristics of the variant carriers, including the previously described family carrying PALB2 1592delT, revealed a trend towards aggressive disease, which also applied to a few non-familial cases. Hypersensitivity to mitomycin C (MMC) of lymphoblasts from individuals from the family with 1592delT revealed haploinsufficiency among carriers with altered genotype.
Though any of the detected PALB2 variants do not associate to PRCA in population level in Finland it cannot be ruled out that some of these variants contribute to cancer susceptibility at individual level.
Prostate cancer (PRCA) is the most frequently diagnosed cancer among men in the Western world . The incidence of PRCA has been increasing throughout the last decade, partly due to the earlier diagnosis provided by the prostate specific antigen (PSA) test, introduced in the 1990s. In Finland, the highest incidence of 115/100 000 was observed in 2005. In 2007 there were 4188 newly-diagnosed PRCA cases with the age-adjusted incidence rate of 85.9/100,000 . Similar high increase and now decline have been observed in most Western industrialized countries .
Extensive efforts have been made to reveal the genetics behind PRCA susceptibility . Early linkage studies and more recent genome-wide association analyses have revealed multiple susceptibility loci for PRCA [5–7]. However, both causal variants and genes of the associated loci in are still not known and the identified PRCA risk variants only account for a fraction of the overall genetic variance for PRCA risk [8–10].
DNA repair pathway genes have an undisputed role in cancer progression and inherited mutations in them have been strongly associated with different cancers. BRCA1 and BRCA2 are both tumor suppressor genes involved in DNA repair and mutations in these genes predominantly predispose carriers to breast and ovarian cancers . Potential links to PRCA have been studied for both of these genes and mutations in BRCA2 lead to an increased risk for the disease in multiple studies [12, 13]. In an Icelandic study, mutations in BRCA2 (999del5, Icelandic founder mutation) are associated with a poorly differentiated, advanced type of PRCA . These results are also consistent with results from a recent study in the UK, where a significantly higher Gleason score was observed among BRCA2 mutation carriers than with non-carriers. These findings suggest that BRCA2 is a high-risk PRCA susceptibility gene and mutational analysis could be used as a prognostic marker for aggressive PRCA . However, Finnish BRCA1/2 founder mutations do not associate with PRCA predisposition among Finnish PRCA patients . This does not exclude the possibility of these genes to be involved in PRCA susceptibility but no studies have been published where the whole genes would have been sequenced in large data sets. Nevertheless an increased risk for PRCA in Finnish breast cancer families carrying BRCA2 mutations has been observed . Moreover, mutations of CHEK2, a gene acting in the same DNA repair route as BRCA1/2, have been shown to have a significant role in PRCA susceptibility in Finland .
PALB2 is a BRCA2 binding protein and the BRCA2-PALB2 interaction is essential for BRCA2-mediated DNA repair . Recently it was shown that proper PALB2 function is necessary for the homologous recombination repair via interaction with BRCA1, revealing that PALB2 is actually a linker between BRCA1 and BRCA2 . In the Finnish study by Erkko and colleagues a novel PALB2 founder mutation (c.1592delT) was identified among Finnish breast cancer families (OR 11.3, CI 1.8-57.8) increasing the risk to breast cancer 4-fold . In the same study the c.1592delT mutation was observed also from a patient with familial PRCA (1/164) but none of 475 unselected cases were carriers of the mutation. No statistically significant association with PRCA and c.1592delT was detected and no other variants were screened for in PRCA patients. Here, to investigate the role of possible other PALB2 variants, we screened all PALB2 exons(exons 1-13) in a cohort of 938 Finnish PRCA patients including both familial and unselected cases.
Material and methods
Patients and controls
A population-based cohort was collected from 1999 to 2005 from patients diagnosed with PRCA in the Pirkanmaa Hospital District that serves a population of a half a million inhabitants. The mean age at diagnosis for the 760 unselected patients was 62 (range 43-77), The median primary PSA and Gleason score were 8.9 and 6.4, respectively. The controls (n = 760) consisted of DNA samples from anonymous male blood donors obtained from the Finnish Red Cross.
A detailed description of the collection of the PRCA families has been previously described elsewhere . In this study, we analysed 178 families (youngest affected male from each family) with two or more affected first-degree relatives. The average number of affected family members was three. Familial cancer data was attained from the Finnish Cancer Registry and detailed clinical information, including Gleason score, WHO grade, PSA at the time of diagnosis, TNM stage and primary treatment from hospital records.
To examine the association between the variants and the disease aggressiveness a subgroup of 380 with clinically aggressive disease was analyzed. Consequently clinically aggressive disease patients were selected according to Gleason score over seven and an age at diagnosis less than 61 years.
Mutation analysis of PALB2
All familial samples had been previously screened for the 11 Finnish BRCA1 and seven BRCA2 founder mutations . DNA samples from each family (n = 178), 285 unselected cases and 470 control samples were initially used for direct sequencing of the entire coding region and splice sites of PALB2. Unselected cases for screening the whole gene were early onset cases with Gleason score over seven. Additional analyses were carried out on four variants that showed a trend for association (PALB2 c.1592delT, 1674A>G, 2993G>A and 3300T>G). Unselected cases for additional analysis included 95 early onsets, aggressive cases and 368 non-aggressive cases with average age at diagnosis 67 years (range 63-77). Control samples in both primary and additional analysis were anonymous male blood donors from the Finnish Red Cross.
Mutation detection was performed through resequencing using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit with the ABI 3130xl sequencer (Applied Biosystems). Primers and the naming of sequence variants were produced to correspond to the GenBank reference sequences for PALB2 (NM_024675.3). Primer sequences are available on request. Variants were identified using Sequencher software 4.7 (Gene Codes Corporation, Ann Arbor, Mi). Loss of heterozygosity (LOH) analysis was carried out on two available tumor samples from the family with the segregating PALB2 1592delT. Selected sections of formalin fixed paraffin embedded tumors were obtained according to pathologist declaration. After tumor deparaffinization, DNA was extracted using a standard proteinase K protocol. Sequence from the tumor samples was compared to the patients' DNA sample from their peripheral blood.
DNA damage response in PALB2 deficient cell line
Peripheral blood leucocytes from two cancerous individuals carrying PALB2 1592delT mutation were immortalized with Epstein-Barr virus (EBV) and sensitized to DNA crosslinking agent mitomycin C (MMC). Cells were treated with 0 nM, 1 nM, 5 nM, 10 nM and 50 nM MMC for 96 h and their viability was assayed by CellTiter-Blue® Cell Viability Assay (Promega Corporation, Madison, WI, USA).
To validate significance of variants, the odds ratios and 95% confidence intervals were calculated and association analyses were made using the Fisher's exact test. Analyses were done using SPSS 12.0 statistical software. Pairwise linkage disequilibrium between three most frequently detected variants (1674A>G, 2993G>A and 3300T>G) in PALB2 were analysed with Haploview 4.1. To define the haplotypes of cases familial and unselected cases were combined.
Results and Discussion
Observed PALB2 variants among the Finnish familial and unselected prostate cancer cases.
Amino acid Change
Carrier Frequency (OR; 95% CI)
1/178 (0.6%) (4.3; 0.3-68.8)
2/748 (0.3%) (2.0; 0.2-22.2)
36/178 (20.2%) (1.2; 0.8-1.8)
153/748(20.5%) (1.2; 0.9-1.5)
4/178 (2.2%) (1.0; 0.3-3.4)
7/285(2.5%) (1.2; 0.4-3.1)
8/178 (4.5%) (1.6; 0.7-3.5)
35/748 (4.9%) (1.6; 0.9-2.8)
6/178 (3.4%) (0.8; 0.3-2.0)
30/748 (3.9%) (1.0: 0.6-1.7)
8/178 (4.5%) (1.8; 0.7-4.4)
14/285 (4.9%) (2.0; 0.9-4.3)
Clinical characteristics of family 310. The patient number refers to the number in the pedigree in Figure 1.
Age at diagnosis
Reason for diagnosis
Primary PSA value
2+3 = 5
2+3 = 5
We found that the 2993G>A variant exhibits a borderline significant odds ratios in a subgroup of 380 unselected cases with a Gleason score over seven and an age at diagnosis less than 61 years (20/380; OR 1.9; 95% CI 1.0-3.5). Edwards and colleagues  have presented similar age related observation with BRCA2 mutations in prostate cancer. They reported protein truncating mutations in BRCA2 to associate with PRCA diagnosed at or before age of 55. Our subgroup analysis also revealed that the variant 1674A>G has an OR of 1.4 (86/380; 95% CI 1.0-1.9) in a group of 368 patients with an average age at diagnosis of 67 years. However, the 1674A>G variant is commonly found in the Finnish population and well tolerated according to SIFT analysis, indicating no clear contribution to cancer formation. Similarly, the Val932 Met (2794G>A) change in exon 8 is well tolerated and has no effect on protein function .
In order to obtain information about loss of heterozygosity in the tumors from c.1592delT mutation positive patients, two available paraffin-embedded tumors were analysed. Repeated PCR-analysis did not reveal existence of LOH, which argues against the role as tumor suppressor gene. However the sample size is very small and therefore more tumor samples from PALB2-associated tumors needs to be characterized to be able to reliable assess PALB2 function in tumor formation. To date there is only one report indicating LOH in PALB2 related tumors. Unfortunately also this study lacks a statistical power to make an appropriate conclusion about the role of PALB2 in tumorigenesis .
DNA damage response in PALB2 deficient cell line
Geographical and ethnical differences in the PRCA susceptibility alleles have been previously reported in the CHEK2 gene that acts in the same pathway as PALB2, and therefore, variation in the PALB2 mutation spectrum in different populations would not be surprising. In the present study and in the study by Erkko et al. , the number of mutations found in PALB2 is relatively small among both Finnish breast and PRCA patients, with nine and six variants detected, respectively, compared to the British breast cancer family study by Rahman and colleagues  where a total of 50 variants and five frameshift mutations were reported. Recently, Tischkowitz and colleagues  reported a study of 95 US PRCA patients from Michigan, all diagnosed at < 55 years of age. Eleven variants in PALB2 were found, but none of them were truncating and no association with the disease was observed. When comparing our patients to those very early-onset patients studied by Tischkowitz and colleagues  there is a clear difference in the mean age of onset between the two datasets. On the other hand, no variants were found in the 14 Ashkenazi-Jewish and 21 French-Canadian PRCA cases with family history of cancer . In Finland, the smaller number of variation found likely reflects the known genetic homogeneity and founder effect of the population .
Our findings indicate that no other deleterious PALB2 variants, except 1592delT mutation, contribute even marginally to PRCA risk in Finland. However, it remains possible that other genes from the BRCA1-PALB2-BRCA2 pathway have PRCA-predisposing alleles
We thank all the patients and their families for participation. We also thank Riitta Vaalavuo, RN for assistance and Linda Enroth for skilful technical assistance. This study was financially supported by the Competitive research funding of the Pirkanmaa Hospital District, the Reino Lahtikari Foundation, the Finnish Cancer Organizations, the Sigrid Juselius Foundation and the Academy of Finland (grants 118413 and 1126714). The Lindell foundation, the Päivikki and Sakari Sohlberg foundation and the Pirkanmaa Regional Fund of the Finnish Cultural Foundation have financially supported author SP.
- Carter HB, Coffey DS: The prostate: an increasing medical problem. Prostate. 1990, 16: 39-48. 10.1002/pros.2990160105.View ArticlePubMed
- Finnish Cancer Registry. Cancer incidence and mortality in Finland. Cancer Statistics. 2007,http://www.cancerregistry.fi
- Kvale R, Auvinen A, Adami HO, Klint A, Hernes E, Moller B, Pukkala E, Storm HH, Tryggvadottir L, Tretli S, Wahlqvist R, Weiderpass E, Bray F: Interpreting trends in prostate cancer incidence and mortality in the five Nordic countries. J Natl Cancer Inst. 2007, 99: 1881-1887. 10.1093/jnci/djm249.View ArticlePubMed
- Schaid DJ: The complex genetic epidemiology of prostate cancer. Hum Mol Genet. 2004, 13 (Spec No 1): R103-21. 10.1093/hmg/ddh072.View ArticlePubMed
- Tavtigian SV, Simard J, Teng DH, Abtin V, Baumgard M, Beck A, Camp NJ, Carillo AR, Chen Y, Dayananth P, Desrochers M, Dumont M, Farnham JM, Frank D, Frye C, Ghaffari S, Gupte JS, Hu R, Iliev D, Janecki T, Kort EN, Laity KE, Leavitt A, Leblanc G, McArthur-Morrison J, Pederson A, Penn B, Peterson KT, Reid JE, Richards S, Schroeder M, Smith R, Snyder SC, Swedlund B, Swensen J, Thomas A, Tranchant M, Woodland AM, Labrie F, Skolnick MH, Neuhausen S, Rommens J, Cannon-Albright LA: A candidate prostate cancer susceptibility gene at chromosome 17p. Nat Genet. 2001, 27: 172-180. 10.1038/84808.View ArticlePubMed
- Carpten J, Nupponen N, Isaacs S, Sood R, Robbins C, Xu J, Faruque M, Moses T, Ewing C, Gillanders E, Hu P, Bujnovszky P, Makalowska I, Baffoe-Bonnie A, Faith D, Smith J, Stephan D, Wiley K, Brownstein M, Gildea D, Kelly B, Jenkins R, Hostetter G, Matikainen M, Schleutker J, Klinger K, Connors T, Xiang Y, Wang Z, De Marzo A, Papadopoulos N, Kallioniemi OP, Burk R, Meyers D, Gronberg H, Meltzer P, Silverman R, Bailey-Wilson J, Walsh P, Isaacs W, Trent J: Germline mutations in the ribonuclease L gene in families showing linkage with HPC1. Nat Genet. 2002, 30: 181-184. 10.1038/ng823.View ArticlePubMed
- Xu J, Zheng SL, Komiya A, Mychaleckyj JC, Isaacs SD, Hu JJ, Sterling D, Lange EM, Hawkins GA, Turner A, Ewing CM, Faith DA, Johnson JR, Suzuki H, Bujnovszky P, Wiley KE, DeMarzo AM, Bova GS, Chang B, Hall MC, McCullough DL, Partin AW, Kassabian VS, Carpten JD, Bailey-Wilson JE, Trent JM, Ohar J, Bleecker ER, Walsh PC, Isaacs WB, Meyers DA: Germline mutations and sequence variants of the macrophage scavenger receptor 1 gene are associated with prostate cancer risk. Nat Genet. 2002, 32: 321-325. 10.1038/ng994.View ArticlePubMed
- Rokman A, Ikonen T, Mononen N, Autio V, Matikainen MP, Koivisto PA, Tammela TL, Kallioniemi OP, Schleutker J: ELAC2/HPC2 involvement in hereditary and sporadic prostate cancer. Cancer Res. 2001, 61: 6038-6041.PubMed
- Rokman A, Ikonen T, Seppala EH, Nupponen N, Autio V, Mononen N, Bailey-Wilson J, Trent J, Carpten J, Matikainen MP, Koivisto PA, Tammela TL, Kallioniemi OP, Schleutker J: Germline alterations of the RNASEL gene, a candidate HPC1 gene at 1q25, in patients and families with prostate cancer. Am J Hum Genet. 2002, 70: 1299-1304. 10.1086/340450.PubMed CentralView ArticlePubMed
- Seppala EH, Ikonen T, Autio V, Rokman A, Mononen N, Matikainen MP, Tammela TL, Schleutker J: Germ-line alterations in MSR1 gene and prostate cancer risk. Clin Cancer Res. 2003, 9: 5252-5256.PubMed
- King MC, Marks JH, Mandell JB, New York Breast Cancer Study Group: Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science. 2003, 302: 643-646. 10.1126/science.1088759.View ArticlePubMed
- Sigurdsson S, Thorlacius S, Tomasson J, Tryggvadottir L, Benediktsdottir K, Eyfjord JE, Jonsson E: BRCA2 mutation in Icelandic prostate cancer patients. J Mol Med. 1997, 75: 758-761. 10.1007/s001090050162.View ArticlePubMed
- Friedenson B: BRCA1 and BRCA2 pathways and the risk of cancers other than breast or ovarian. Med Gen Med. 2005, 7: 60-
- Tryggvadottir L, Vidarsdottir L, Thorgeirsson T, Jonasson JG, Olafsdottir EJ, Olafsdottir GH, Rafnar T, Thorlacius S, Jonsson E, Eyfjord JE, Tulinius H: Prostate cancer progression and survival in BRCA2 mutation carriers. J Natl Cancer Inst. 2007, 99: 929-935. 10.1093/jnci/djm005.View ArticlePubMed
- Mitra AV, Bancroft EK, Eeles RA, IMPACT Steering Committee Collaborators: A review of targeted screening for prostate cancer: introducing the IMPACT study. BJU Int. 2007, 99: 1350-1355. 10.1111/j.1464-410X.2007.06759.x.View ArticlePubMed
- Ikonen T, Matikainen MP, Syrjakoski K, Mononen N, Koivisto PA, Rokman A, Seppala EH, Kallioniemi OP, Tammela TL, Schleutker J: BRCA1 and BRCA2 mutations have no major role in predisposition to prostate cancer in Finland. J Med Genet. 2003, 40: e98-10.1136/jmg.40.8.e98.PubMed CentralView ArticlePubMed
- Eerola H, Pukkala E, Pyrhonen S, Blomqvist C, Sankila R, Nevanlinna H: Risk of cancer in BRCA1 and BRCA2 mutation-positive and-negative breast cancer families (Finland). Cancer Causes Control. 2001, 12: 739-746. 10.1023/A:1011272919982.View ArticlePubMed
- Seppala EH, Ikonen T, Mononen N, Autio V, Rokman A, Matikainen MP, Tammela TL, Schleutker J: CHEK2 variants associate with hereditary prostate cancer. Br J Cancer. 2003, 89: 1966-1970. 10.1038/sj.bjc.6601425.PubMed CentralView ArticlePubMed
- Xia B, Sheng Q, Nakanishi K, Ohashi A, Wu J, Christ N, Liu X, Jasin M, Couch FJ, Livingston DM: Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2. Mol Cell. 2006, 22: 719-729. 10.1016/j.molcel.2006.05.022.View ArticlePubMed
- Zhang F, Ma J, Wu J, Ye L, Cai H, Xia B, Yu X: PALB2 links BRCA1 and BRCA2 in the DNA-damage response. Curr Biol. 2009, 19: 524-529. 10.1016/j.cub.2009.02.018.PubMed CentralView ArticlePubMed
- Erkko H, Xia B, Nikkila J, Schleutker J, Syrjakoski K, Mannermaa A, Kallioniemi A, Pylkas K, Karppinen SM, Rapakko K, Miron A, Sheng Q, Li G, Mattila H, Bell DW, Haber DA, Grip M, Reiman M, Jukkola-Vuorinen A, Mustonen A, Kere J, Aaltonen LA, Kosma VM, Kataja V, Soini Y, Drapkin RI, Livingston DM, Winqvist R: A recurrent mutation in PALB2 in Finnish cancer families. Nature. 2007, 446: 316-319. 10.1038/nature05609.View ArticlePubMed
- Matikainen MP, Schleutker J, Morsky P, Kallioniemi OP, Tammela TL: Detection of subclinical cancers by prostate-specific antigen screening in asymptomatic men from high-risk prostate cancer families. Clin Cancer Res. 1999, 5: 1275-1279.PubMed
- Rahman N, Seal S, Thompson D, Kelly P, Renwick A, Elliott A, Reid S, Spanova K, Barfoot R, Chagtai T, Jayatilake H, McGuffog L, Hanks S, Evans DG, Eccles D, Breast Cancer Susceptibility Collaboration (UK), Easton DF, Stratton MR: PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat Genet. 2007, 39: 165-167. 10.1038/ng1959.PubMed CentralView ArticlePubMed
- Edwards SM, Kote-Jarai Z, Meitz J, Hamoudi R, Hope Q, Osin P, Jackson R, Southgate C, Singh R, Falconer A, Dearnaley DP, Ardern-Jones A, Murkin A, Dowe A, Kelly J, Williams S, Oram R, Stevens M, Teare DM, Ponder BA, Gayther SA, Easton DF, Eeles RA, Cancer Research UK/Bristish Prostate Group UK Familial Prostate Cancer Study Collaborators British Association of Urological Surgeons Section of Oncology: Two percent of men with early-onset prostate cancer harbor germline mutations in the BRCA2 gene. Am J Hum Genet. 2003, 72: 1-12. 10.1086/345310.PubMed CentralView ArticlePubMed
- García MJ, Fernández V, Osorio A, Barroso A, Llort G, Lázaro C, Blanco I, Caldés T, de la Hoya M, Ramón Y, Cajal T, Alonso C, Tejada MI, San Román C, Robles-Díaz L, Urioste M, Benítez J: Analysis of FANCB and FANCN/PALB2 fanconi anemia genes in BRCA1/2-negative Spanish breast cancer families. Breast Cancer Res Treat. 2009, 113: 545-51. 10.1007/s10549-008-9945-0.View ArticlePubMed
- Tischkowitz M, Sabbaghian N, Ray AM, Lange EM, Foulkes WD, Cooney KA: Analysis of the gene coding for the BRCA2-Interacting protein PALB2 in hereditary prostate cancer. Prostate. 2008, 68: 675-678. 10.1002/pros.20729.PubMed CentralView ArticlePubMed
- Tischkowitz M, Xia B, Sabbaghian N, Reis-Filho JS, Hamel N, Li G, van Beers EH, Li L, Khalil T, Quenneville LA, Omeroglu A, Poll A, Lepage P, Wong N, Nederlof PM, Ashworth A, Tonin PN, Narod SA, Livingston DM, Foulkes WD: Analysis of PALB2/FANCN-associated breast cancer families. PNAS. 2007, 104: 6788-6793. 10.1073/pnas.0701724104.PubMed CentralView ArticlePubMed
- de la Chapelle A: Disease gene mapping in isolated human populations: the example of Finland. J Med Genet. 1993, 30: 857-865. 10.1136/jmg.30.10.857.PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.