TP53-binding protein variants and breast cancer risk: a case-control study
© Frank et al.; licensee BioMed Central Ltd. 2005
Received: 25 February 2005
Accepted: 12 April 2005
Published: 6 May 2005
The TP53-binding protein (53BP1) has been shown to influence TP53-mediated transcriptional activation, thus playing a pivotal role in DNA damage signalling. Genetic aberrations in TP53 and in ATM and CHEK2 predispose to cancer. We have therefore examined the effects of 53BP1 single nucleotide polymorphisms (D353E, G412S, and K1136Q) and the novel 53BP1 6bp deletion (1347_1352delTATCCC) on breast cancer risk.
Allelic discrimination was performed to investigate the frequencies of 53BP1 D353E, G412S, and K1136Q and of 1347_1352delTATCCC in 353 patients with breast cancer and 960 control individuals.
No significant association of 53BP1 D353E, G412S, or K1136Q with breast cancer risk was detected. 53BP1 1347_1352delTATCCC, leading to the loss of an isoleucine and a proline residue, showed a nonsignificant inverse association with breast cancer risk (odds ratio = 0.61, 95% confidence interval = 0.22 to 1.68, P = 0.34).
The lack of association casts doubt on the putative effects of D353E, G412S, and K1136Q on breast cancer risk. Investigating a larger study cohort might elucidate the influence of the 6bp deletion 1347_1352delTATCCC. Studying the functional effect and the impact of this variant on the risk of other cancers may be revealing.
The TP53-binding protein (53BP1), a conserved nuclear protein, was initially identified to interact with the DNA-binding domain of TP53, thus enhancing TP53-mediated transcriptional activation [1, 2]. In response to exogenous exposure to ionising radiation, 53BP1 becomes hyperphosphorylated and rapidly localises to sites of DNA double-strand breaks, demonstrating its determining role in DNA damage signalling [3, 4]. 53BP1-deficient mice exhibit growth retardation, high radiation sensitivity, and tumour development – features that are indicative of a defective DNA damage response . 53BP1 is involved in the phosphorylation of various ataxia telangiectasia mutated protein (ATM) substrates such as cell cycle checkpoint kinase 2 (CHEK2) [3, 6]. Mutations in ATM, CKEK2, and its substrate, TP53, have been shown to predispose to cancer [6–9]. Therefore, we selected 53BP1 as an attractive candidate gene for breast cancer susceptibility.
This is the first study to investigate the effects of the 53BP1 single nucleotide polymorphisms (SNPs) D353E (1059C>G), G412S (1234G>A), and K1136Q (3406A>C) on breast cancer risk, analysing 353 German patients with breast cancer and 960 controls. 53BP1 D353E, G412S, and K1136Q showed no association with breast cancer risk. In addition, we detected a novel, very rare 53BP1 6bp deletion (1347_1352delTATCCC) showing an inverse association with breast cancer risk (age-adjusted odds ratio (OR) = 0.61, 95% confidence interval (CI) = 0.22 to 1.68), lacking significance (P = 0.34).
Materials and methods
A randomly chosen set of 23 German patients with familial breast cancer was initially screened for annotated 53BP1 SNPs (dbSNP database; NCBI (National Center for Biotechnology Information)) by DNA sequencing. Sequencing primers are available upon request. The initial analysis included 53BP1 exons 9, 11, and 17, harbouring three reported nonsynonymous polymorphisms (D353E: rs560191; G412S: rs689647; and K1136Q: rs2602141). When sequencing exon 11, we additionally detected the 6bp deletion 1347_1352delTATCCC. All validated variants were chosen for further analyses using a large cohort of breast cancer patients.
The breast cancer patients were 353 unrelated German women (mean age 44.8 years, range 21 to 80 years) who were negative for BRCA1 and BRCA2 mutations. In accordance with the German Consortium for Hereditary Breast and Ovarian Cancer, they were classified into six categories based on family history: (A1) families with two or more breast cancer cases including two cases with onset below the age of 50 (39.3% of analysed cases); (A2) families with at least one male breast cancer case (0.9%); (B) families with at least one breast cancer and one ovarian cancer case (16.2%); (C) families with at least two breast cancer cases including one case diagnosed before the age of 50 (33.5%); (D) families with at least two breast cancer cases comprising two cases diagnosed after the age of 50 (5.5%); (E) single cases of breast cancer diagnosed before the age of 35 (4.6%) . They were collected during the years 1997 to 2004 through the Institute of Human Genetics (Heidelberg, Germany) and the Department of Gynaecology and Obstetrics (Cologne, Germany). The control series included 960 blood donors (mean age 30.5 years, range 18 to 67 years) collected by the Institute of Transfusion Medicine and Immunology (Mannheim, Germany) having the same ethnic background as the breast cancer patients. Both study populations have been described earlier . The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany).
PCR amplification and sequencing were performed as previously described . Conditions are available on request.
53BP1 polymorphisms D353E, G412S, and K1136Q were analysed using TaqMan allelic discrimination. TaqMan assays were performed in a reaction volume of 10 μl comprising 5ng of genomic DNA, each probe at 50 nM, each primer at 225 nM, and 1× Universal Master Mix with the following amplification conditions: 2 min at 50°C, 10 min at 95°C and 35 to 45 cycles at 92°C for 15 s and 60°C for 1 min. Amplification products were measured and analysed with the ABI Prism 7900 HT sequence detection system and the SDS software (version 1.2; Applied Biosystems, Foster City, CA, USA). TaqMan probes and primers were provided by the assay-on-demand and assay-by-design services, respectively (Applied Biosystems). 53BP1 1347_1352delTATCCC was analysed using the MGB Eclipse™ Probe System by Epoch Biosciences (Bothell, WA, USA). Allelic discrimination was carried out as recommended by the manufacturers using the following probes: D353E: assay-on-demand C_2944794_10; G412S: VIC-ACTTCAAAGTGGTGAACC, FAM-AACTTCAAAGTAGTGAACC; K1136Q: VIC-GGAGTACTAATAAGGAAA, FAM-CGGAGTACTAATCAGGAAA; 1347_1352delTATCCC: FAM-CACTTCATCCCAT; TET-CACTTCCTATCCCATC. Primers and probes were designed based on GenBank NM_005657 (NCBI) and are available on request. More than 5% of the genotyping results were confirmed by sequencing, and genotype distributions were consistent with Hardy–Weinberg equilibrium.
Calculations of Hardy–Weinberg equilibrium, genotype-specific OR, and 95% CI were carried out using a tool offered by the Institute of Human Genetics, Technical University Munich, Munich, Germany . Age-adjusted ORs and corresponding 95% CIs were computed by means of unconditional logistic regression using SAS (Version 8.2; SAS Institute Inc, Cary, NC, USA). Haplotypes were assigned to subjects using the SNPHAP software (see ), which also reports the posterior probability of the most likely assignment [14, 15].
Results and discussion
Genotype frequencies of 53BP1 polymorphisms in breast cancer patients and controls
No. (%) of cases
No. (%) of controls
AOR (95% CI)
CC vs GC+GG
AA+AG vs GG
delTATCCC/wt vs wt/wt
CC+CA vs AA
Haplotype distribution of 53BP1 polymorphismsa in breast cancer patients and control individuals
95 % CI
Mean posterior probability
Mean posterior probability
The three known 53BP1 SNPs – D353E, G412S, and K1136Q – lacked association with breast cancer risk. However, we detected a novel, very rare 6bp deletion, 1347_1352delTATCCC, that showed a statistically nonsignificant inverse association with breast cancer risk. Concerning the latter, a much larger study cohort is required to verify any putative significant effect. Additionally, it would be valuable to investigate a possible functional effect of this 53BP1 deletion and its impact on other cancers.
TP53 binding protein
ataxia telangiectasia mutated protein
cell cycle checkpoint kinase 2
polymerase chain reaction
single nucleotide polymorphism.
The authors wish to thank Kerstin Wagner for critical comments on the manuscript and are grateful to Prof CR Bartram (Heidelberg) and Prof RK Schmutzler (Cologne) for providing the German breast cancer samples, which were collected within a project funded by the Deutsche Krebshilfe.
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