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The study was designed by N.R. and M.R.S. The molecular analyses were performed by S.S., P.K., A.R., S.R., K.S., R.B., T.C., H.J. and S.H. under the direction of N.R. The statistical analyses were performed by D.T., A.E. and L.M. under the direction of D.F.E. The familial collections were initiated by D.G.E. and D.E. and were collected by the Breast Cancer Susceptibility Collaboration (UK). The manuscript was written by N.R. and M.R.S.
PALB2 interacts with BRCA2, and biallelic mutations in PALB2 (also known as FANCN), similar to biallelic BRCA2 mutations, cause Fanconi anemia. We identified monoallelic truncating PALB2 mutations in 10/923 individuals with familial breast cancer compared with 0/1,084 controls (P = 0.0004) and show that such mutations confer a 2.3-fold higher risk of breast cancer (95% confidence interval (c.i.) = 1.4–3.9, P = 0.0025). The results show that PALB2 is a breast cancer susceptibility gene and further demonstrate the close relationship of the Fanconi anemia–DNA repair pathway and breast cancer predisposition.
PALB2 (for ‘partner and localizer of BRCA2’) encodes a recently discovered protein that interacts with BRCA2, is implicated in its nuclear localization and stability and is required for some functions of BRCA2 in homologous recombination and double-strand break repair1. In a paper in this issue, we show that biallelic PALB2 mutations are responsible for a subset of Fanconi anemia cases characterized by a phenotype similar to that caused by biallelic BRCA2 mutations2. Prompted by these observations, we investigated whether monoallelic PALB2 mutations confer susceptibility to breast cancer by sequencing the gene in individuals with breast cancer from familial breast cancer pedigrees that were negative for mutations in BRCA1 and BRCA2 and controls (Supplementary Methods online).
We identified truncating PALB2 mutations in 10/923 (1.1%) independently ascertained individuals with familial breast cancer from separate families compared with 0/1,084 (0%) controls (P = 0.0004) (Table 1 and Fig. 1a). Nine of the PALB2 mutations were in the 908 families with female breast cancer only (1.0%). One occurred in the 15 families (6.7%) with cases of both female and male breast cancer (P = 0.15). Although this observation requires further investigation, it suggests that PALB2 mutations may confer a higher relative risk of male breast cancer than female breast cancer, and BRCA2 mutations are known to confer a high relative risk of male breast cancer3. One proband with a PALB2 mutation developed melanoma at 47 years of age in addition to breast cancer at 56 years. Apart from this individual, there were no other malignancies other than breast cancer in individuals with PALB2 mutations. Two of four first-degree affected relatives of probands with PALB2 mutations also carried a PALB2 mutation. This pattern of incomplete segregation in affected relatives is typical of susceptibility alleles that confer modestly increased risks and is similar to that reported in breast cancer families carrying CHEK2, ATM or BRIP1 mutations4-6.
Segregation analysis incorporating the information from controls and the full pedigrees of the affected individuals estimated the relative risk of PALB2 mutations to be 2.3 (c.i. = 1.4–3.9, P = 0.0025). The relative risk for women under 50 years was 3.0 (95% c.i. = 1.4–5.5), and for women over 50 years it was 1.9 (95% c.i. = 0.8–3.7, P = 0.35 for difference in relative risk between the age groups). The median age at diagnosis of individuals with PALB2 mutations was 46 years (interquartile range (IQR) = 40–51) compared with a median age at diagnosis of 49 years (IQR = 42–55) in individuals with breast cancer without PALB2 mutations (P = 0.24 for difference). These data suggest that the risks of breast cancer associated with PALB2 mutations may be age dependent, but additional studies will be required to address this question. There was no difference in the extent of familial clustering of breast cancer (P = 0.69) or in the probability of being a bilateral case (P = 0.23) in families with PALB2 mutations compared with families without mutations. Assuming a conservative sensitivity of 90% for mutation detection, we estimate the breast cancer population attributable fraction of PALB2 mutations to be 0.23% (95% c.i.: 0.072%–0.52%) and the percentage of the familial relative risk due to PALB2 to be 0.24% (0.02%–1.16%).
We identified 50 nontruncating variants within the PALB2 coding sequence, including 35 nonsynonymous and 15 synonymous variants (Supplementary Table 1 online). There was no overall evidence that PALB2 missense variants confer susceptibility to breast cancer, with 215 (23%) affected individuals and 265 (24%) controls carrying at least one nonsynonymous missense variant. Only four missense variants had an allele frequency greater than 1%, and there was no evidence that any of these were breast cancer susceptibility alleles. This result is consistent with the data from individuals with Fanconi anemia in which all reported PALB2 mutations result in premature protein truncation2,7.
Fanconi anemia is a genetically heterogeneous recessive condition that currently includes 13 subtypes, 12 of which have been attributed to distinct genes2,8. The known Fanconi anemia genes encode proteins that interact in an incompletely understood fashion to facilitate recognition and repair of DNA double-strand breaks. A key process in the pathway involves eight of the known Fanconi anemia proteins forming a nuclear core complex that mediates monoubiquitination and activation of FANCD2. Activated FANCD2 is translocated to DNA repair foci, where it colocalizes with BRCA2 and other proteins that effect DNA repair by homologous recombination (Fig. 1b)8.
Biallelic mutations of BRCA2 and PALB2 cause Fanconi anemia subtypes FA-D1 and FA-N, respectively2,7,9. The phenotypes associated with biallelic BRCA2 and PALB2 mutations are markedly similar to each other and differ from the other ten known Fanconi anemia genes. In particular, FA-D1 and FA-N are associated with high risks of solid childhood malignancies such as Wilms tumor and medulloblastoma, which occur very rarely in other subtypes2,8,10. Heterozygous mutations in BRIP1, which encodes a BRCA1-interacting protein, also confer an elevated risk of breast cancer6, and biallelic BRIP1 mutations cause Fanconi anemia subtype FA-J11,12. However, FA-J is associated with the classical Fanconi anemia phenotype, and there have not been any reports of individuals with FA-J with a childhood solid tumor11,12.
It is plausible that heterozygosity for mutations in other Fanconi anemia genes may also be involved in breast cancer susceptibility. However, epidemiological studies of relatives of individuals with Fanconi anemia have not demonstrated this, suggesting that breast cancer susceptibility is associated with only a subset of Fanconi anemia genes. This is consistent with the negative results of mutational screens of other Fanconi anemia genes in familial breast cancer cases13. The biological features that determine whether a Fanconi anemia gene is also a breast cancer predisposition gene are unknown. However, it is notable that the three Fanconi anemia genes currently associated with breast cancer susceptibility (BRCA2, PALB2 and BRIP1) are not part of the Fanconi anemia core complex and are the only known Fanconi anemia genes that act downstream of FANCD2 (Fig. 1b).
We estimate that PALB2 mutations are associated with an approximately twofold higher risk of female breast cancer. Therefore, despite the fact that PALB2 is functionally associated with BRCA2 and that biallelic mutations in both genes cause similar phenotypes, the increase in breast cancer risk associated with PALB2 monoallelic mutations is clearly more modest than that conferred by BRCA2 monoallelic mutations, which result in approximately a tenfold increase in risk. These differences in risk are reminiscent of those previously reported between BRCA1 mutations, which also confer a greater than tenfold increase in risk of breast cancer, and mutations in BRIP1, which confer only a twofold increase in risk6. The explanations for the apparent differences in risk associated with mutations in these genes, despite the close functional interactions between the proteins they encode, are currently unknown. Thus, our data provide further evidence of the close link between breast cancer susceptibility and the Fanconi anemia-DNA repair pathway, but they also demonstrate that the relationship is complex at both the phenotypic and molecular levels.
With the identification of PALB2 as a new breast cancer predisposition gene, a clearer picture of the genetic architecture of breast cancer susceptibility is emerging. BRCA1 and BRCA2 are likely to be the only major high-penetrance breast cancer susceptibility genes (leading to more than a tenfold higher risk). Mutations in TP53 also confer high risks of breast cancer but are much rarer14. These genes are characterized by multiple, rare, inactivating mutations that together account for approximately 15%–20% of the familial risk of the disease14. A similar mutation spectrum has now been identified in four additional genes that encode proteins that interact biologically with BRCA1, BRCA2 and/or p53. Three of these proteins, CHK2, ATM and BRIP1, interact with BRCA1, p53 or both (refs. 8,15). PALB2 is the first that interacts with BRCA2. However, compared with risks associated with mutations in BRCA1, BRCA2 and TP53, the risks associated with mutations in CHEK2, ATM, BRIP1 and PALB2 are much lower4-6. Moreover, inactivating mutations in each of these genes are rare, with fewer than 1% of the population being heterozygotes. As such, the contribution of each gene to the familial risk of breast cancer is small. Collectively, however, they already account for ~2.3% of the overall familial relative risk. Thus, this class of susceptibility gene may make an appreciable contribution to breast cancer predisposition.
We thank the participating families who were recruited to the study by the Familial Breast Cancer Collaboration (UK) which includes the following contributors: A. Ardern-Jones, J. Berg, A. Brady, N. Bradshaw, C. Brewer, G. Brice, B. Bullman, J. Campbell, B. Castle, R. Cetnarsryj, C. Chapman, C. Chu, N. Coates, T. Cole, R. Davidson, A. Donaldson, H. Dorkins, F. Douglas, D. Eccles, R. Eeles, F. Elmslie, D.G. Evans, S. Goff, S. Goodman, D. Goudie, J. Gray, L. Greenhalgh, H. Gregory, N. Haites, S.V. Hodgson, T. Homfray, R.S. Houlston, L. Izatt, L. Jeffers, V. Johnson-Roffey, F. Kavalier, C. Kirk, F. Lalloo, I. Locke, M. Longmuir, J. Mackay, A. Magee, S. Mansour, Z. Miedzybrodzka, J. Miller, P. Morrison, V. Murday, J. Paterson, M. Porteous, N. Rahman, M. Rogers, S. Rowe, S. Shanley, A. Saggar, G. Scott, L. Side, L. Snadden, M. Steel, M. Thomas and S. Thomas. We thank A. Hall and E. Mackie for coordination of sample collection. We acknowledge use of DNA from the British 1958 Birth Cohort collection, funded by the Medical Research Council grant G0000934 and the Wellcome Trust grant 068545/Z/02. P.K. is supported by a grant from the Breast Cancer Campaign. A.E. is supported by US Army Medical Research and Material Command grant W81XWH-05-1-0204. This research was supported by donations from the Geoffrey Berger and Daniel Falkner Charitable Trusts, by the Institute of Cancer Research and by Cancer Research UK.
Note: Supplementary information is available on the Nature Genetics website.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
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