The TP53 Arg72Pro and MDM2 309G>T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance.
METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework.
RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93-1.10, P(trend)=0.77; MDM2: HR=0.96, 95%CI: 0.84-1.09, P(trend)=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87-1.12, P(trend)=0.83; MDM2: HR=0.98, 95%CI: 0.80-1.21, P(trend)=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association.
CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.

The TP53 pathway is crucial for tumour suppression, acting through regulation of cell-cycle control, apoptosis, senescence and DNA repair. The TP53 gene and its negative regulator MDM2 are central to this pathway, promoting polyubiquitination and degradation of TP53, and also controlling the TP53 synthesis (Toledo and Wahl, 2006; Candeias ). Inactivation of the TP53 pathway has an important role in BRCA1- and BRCA2-associated tumourigenesis. BRCA1 and BRCA2 mutations are associated with genomic instability caused by defective cell-cycle checkpoint and DNA damage repair (Deng, 2006). Mouse model studies have highlighted functional links between these genes. Biallelic inactivation of BRCA1 and BRCA2 in mice have shown that embryonic lethality because of growth retardation can be partially rescued in a Trp53 null background (Evers and Jonkers, 2006). The development of mammary tumours in conditional BRCA1 and BRCA2 knockout mice was considerably accelerated in a Trp53 knockout background (Evers and Jonkers, 2006). In addition, a high incidence of TP53 mutations has been found in breast tumours of human BRCA1 and BRCA2 mutation carriers (Greenblatt ; Manie ). The observed interactions between TP53 and BRCA pathways are integral to the progression of tumourigenesis in breast cancer.

A TP53 polymorphism (rs1042522) has been found to be of functional significance, with the Pro72 allele being less efficient than Arg72 at inducing apoptosis, mainly due to weaker binding and ubiquitination by MDM2 of the Pro72 variant protein (Dumont ; Osorio ). An SNP in the promoter region of MDM2 (309T>G, rs2279744) has been shown to increase MDM2 transcriptional activity, thus attenuating the TP53 pathway (Bond ). This latter SNP was associated with an earlier onset of breast cancer in Li–Fraumeni patients carrying TP53 mutations (Bougeard ; Ruijs ). The effect on breast cancer risk of the TP53 Arg72Pro and the MDM2 309T>G polymorphisms, separately and in combination, was investigated in a large case–control study by the Breast Cancer Association Consortium (BCAC), but no association was detected (Schmidt ). However, several smaller studies examined these polymorphisms in BRCA1 and BRCA2 mutation carriers (Martin ; Tommiska ; Copson ; Osorio ; Wasielewski ; Yarden ), and some suggested an association between the TP53 Pro72 and the MDM2 309G alleles with an earlier age at breast cancer diagnosis (Martin ; Tommiska ; Osorio ; Yarden ). We therefore investigated the associations between breast cancer risk and these TP53 and MDM2 polymorphisms in a large series of BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) (Chenevix-Trench ).

Materials and methods

Study sample

Eligibility was restricted to female carriers with pathogenic mutations in BRCA1 or BRCA2 who were ⩾18 years. Data were obtained from 13 CIMBA studies (Table 1). The majority of carriers were recruited through cancer genetics clinics offering genetic testing, and enrolled into national or regional studies. Information collected included the year of birth; mutation description; age at last followup; ages at breast and ovarian cancer diagnosis; and age at bilateral prophylactic mastectomy. Information was also available on the country of residence, which was defined to be the country of the clinic at which the carrier family was recruited for the study. Related individuals were identified through a unique family identifier. Further details of the information collected on the BRCA1 and BRCA2 mutation carriers and other details of the CIMBA initiative can be found elsewhere. Additional specific acknowledgements to the CIMBA collaborating centres are included in the Supplementary Appendix. (http://www.srl.cam.ac.uk/consortia/cimba/index.html) (Chenevix-Trench ). All carriers participated in clinical and research studies at the host institutions under IRB-approved protocols.

Table 1

Number of BRCA1 and BRCA2 mutation carriers by study and by single nucleotide polymorphism (SNP)

Study	Country	TP53 Arg72Pro (rs1042522)	MDM2 309T>G (rs2279744)	Genotyping platform
Spanish National Cancer Centre (CNIO)	Spain	788	0	Restriction enzyme digestion
Deutsches Krebsforschungszentrum (DKFZ)	Germany	170	0	PCR-based RFLP
Epidemiological study of BRCA1 and BRCA2 mutation carriers (EMBRACE)	U.K. and Eire	1131	0	iPLEX
Genetic Modifiers of cancer risk in BRCA1/2 mutation carriers (GEMO)	France and U.S.A.	1405	1357	Taqman
German Consortium of Hereditary Breast and Ovarian Cancer (GC-HBOC)	Germany	815	0	Taqman
Helsinki Breast Cancer Study (HEBCS)	Finland	188	187	rs1042522: Amplifluor(tm) fluorescent genotyping (Kbiosciences); rs2279744: RFLP
HEreditary Breast and Ovarian study Netherlands (HEBON)	The Netherlands	438	432	Taqman
INterdisciplinary HEalth Research International Team BReast CAncer susceptibility (INHERIT BRCAs)	Quebec-Canada	146	155	Taqman
kConFab	Australia	790	0	iPLEX
National Cancer Institute (NCI)	USA	190	0	Taqman
National Israeli Cancer Control Center (NICCC)	Israel	470	0	Taqman
Ontario Cancer Genetics Network (OCGN)	Canada	84	91	Taqman
University of Pennsylvania (UPENN)	USA	396	0	iPLEX
Total		7011	2222

Genotyping

We pooled genotype data from studies within CIMBA that had previously genotyped polymorphisms rs1042522 and rs2279744 (see Table 1). Deviation from Hardy–Weinberg equilibrium among unrelated subjects was evaluated separately for each SNP and study. There was evidence for deviation for only one study (P=0.03), but cluster plot examination did not show any unusual pattern and the study was included in the analysis. Where available study specific genotyping quality control data were examined and data were included if the call rate was over 95% and the concordance among duplicates was over 98%.

Statistical analysis

Mutation carriers were classified according to their age at diagnosis of breast cancer or their age at last follow up. For this purpose, individuals were censored at the age of first breast cancer diagnosis, ovarian cancer diagnosis, bilateral prophylactic mastectomy or the age at last observation. Only individuals censored at breast cancer diagnosis were assumed to be affected (Table 2).

Table 2

Summary characteristics for the 7109 eligible BRCA1 and BRCA2 carriers used in the analysis and typed for either single nucleotide polymorphism (SNP)

	BRCA1		BRCA2
Characteristic	Unaffected	Breast cancer	Unaffected	Breast cancer
Number	2055	2567	1051	1436
Person-years follow-up	87 571	104 679	46 315	63 080
Median age at censure (IQR)	41 (33–51)	40 (34–46)	42 (34–52)	43 (37–50)
Age at censure (years), N (%)
<30	327 (15.9)	225 (8.8)	139 (13.2)	78 (5.4)
30–39	584 (28.4)	1052 (41.0)	296 (28.1)	462 (32.2)
40–49	574 (27.9)	880 (34.3)	286 (27.2)	511 (35.6)
50–59	364 (17.7)	296 (11.5)	196 (18.7)	278 (19.4)
60–69	134 (6.5)	87 (3.4)	82 (7.8)	81 (5.6)
70+	72 (3.5)	27 (1.0)	52 (4.9)	26 (1.8)
Year of birth, N (%)
<1920	18 (0.9)	32 (1.3)	12 (1.1)	10 (0.7)
1920–29	63 (3.1)	117 (4.6)	39 (3.7)	83 (5.8)
1930–39	171 (8.3)	267 (10.4)	96 (9.1)	196 (13.7)
1940–49	326 (15.9)	657 (25.6)	143 (13.6)	358 (24.9)
1950–59	481 (23.4)	820 (31.9)	241 (22.9)	459 (32.0)
1960+	996 (48.5)	674 (26.3)	520 (49.5)	330 (23.0)

Abbreviation: IQR=interquartile range.

To correct for a potential bias related to the fact that BRCA1 and BRCA2 mutation carriers are not randomly sampled with respect to their disease status, the data were analysed within a survival analysis framework, by modelling the retrospective likelihood of the observed genotypes conditional on the disease phenotypes. A detailed description of the retrospective likelihood approach has been published (Antoniou ). We used a Cox proportional hazards model, where the effect of each SNP was modelled either as a per-allele hazard ratio (HR) or using separate HRs for heterozygotes and homozygotes. To assess the combined effects of the SNPs, we fitted a model in which a separate HR parameter was estimated for each multilocus genotype. More details of the statistical analysis can be found elsewhere (Antoniou ).

Results

In total, 7011 BRCA1 and BRCA2 mutation carriers were genotyped for TP53 Arg72Pro and 2222 mutation carriers were genotyped for MDM2 309T>G (Table 1). Table 2 shows summary statistics for the cohort of BRCA1 and BRCA2 mutation carriers with an observed genotype for either the TP53 or MDM2 polymorphism. There was no evidence of an association between either SNP and breast cancer risk in BRCA1 or BRCA2 mutation carriers combined or analysed separately (TP53 Arg72Pro: Ptrend=0.89, 0.77 and 0.83, respectively; MDM2 309T>G: Ptrend=0.60, 0.54 and 0.88, respectively) (Table 3). There was no evidence for heterogeneity in the HRs between studies (TP53 Arg72Pro: P=0.22 and 0.93, MDM2 309T>G: P=0.11 and 0.82 for BRCA1 or BRCA2 mutation carriers respectively). The HRs for the 9 TP53–MDM2 combined genotypes, estimated separately in BRCA1 and BRCA2 mutation carriers, ranged between 0.72 and 1.31, but none of them were significant.

Table 3

Genotype frequencies by mutant gene and breast cancer status with hazard ratio (HR) estimates

	Unaffected (%)	Affected (%)	HR	95% CI	P-value
TP53 Arg72Pro (rs1042522)
BRCA1/2
GG	1660 (54.4)	2164 (54.7)	1.00
GC	1178 (38.6)	1508 (38.1)	1.00	0.92–1.10
CC	214 (7.0)	287 (7.3)	1.01	0.85–1.20
2-df test					0.99
Per allele			1.01	0.94–1.08	0.89
BRCA1
GG	1127 (56.0)	1399 (55.2)	1.00
GC	748 (37.2)	947 (37.4)	1.01	0.90–1.13
CC	138 (6.9)	188 (7.4)	1.03	0.84–1.27
2-df test					0.96
Per allele			1.01	0.93–1.10	0.77
BRCA2
GG	533 (51.3)	765 (53.7)	1.00
GC	430 (41.4)	561 (39.4)	0.98	0.84–1.14
CC	76 (7.3)	99 (6.9)	0.99	0.72–1.36
2-df test					0.95
Per allele			0.99	0.87–1.12	0.83
MDM2 309T>G (rs2279744)
BRCA1/2
TT	358 (40.3)	530 (39.8)	1.00
TG	405 (45.6)	615 (46.1)	0.99	0.84–1.18
GG	126 (14.2)	188 (14.1)	0.93	0.73–1.17
2-df test					0.79
Per allele			0.97	0.87–1.08	0.60
BRCA1
TT	275 (39.7)	369 (39.5)	1.00
TG	323 (46.6)	443 (47.4)	0.98	0.81–1.19
GG	95 (13.7)	123 (13.2)	0.91	0.67–1.19
2-df test					0.78
Per allele			0.96	0.84–1.09	0.54
BRCA2
TT	83 (42.4)	161 (40.5)	1.00
TG	82 (41.8)	172 (43.2)	1.07	0.77–1.50
GG	31 (15.8)	65 (16.3)	0.93	0.60–1.44
2-df test					0.83
Per allele			0.98	0.80–1.21	0.88

Discussion

To our knowledge, this is the largest study to investigate the hypothesis that TP53 Arg72Pro and MDM2 309T>G influence breast cancer risk in BRCA1 and BRCA2 mutation carriers individually or in combination. Our findings of no association for these SNPs suggest that they have little or no effect on BRCA-related breast cancer risk. These results are consistent with the absence of risk association in the recent TP53 haplotype analysis, involving Arg72Pro and an intronic polymorphism c.97-147ins16 bp, in a series of 2932 BRCA1 and BRCA2 carriers from CIMBA (Osorio ). Our sample of mutation carriers had power of approximately 75% for TP53 and 40% for MDM2 to detect significant associations (P<0.05) for a per-allele HR of 1.1 and power of 100 and 90% respectively for a HR of 1.2, suggesting that we can reliably dismiss previously suggested associations (Martin ; Osorio ; Yarden ).

Yarden et al showed that the MDM2 GG genotype among Ashkenazi BRCA1/2 mutations carriers was significantly associated with breast cancer diagnosed

The BCAC study of 5191 cases and 3834 controls found no evidence of an association of TP53 Arg72Pro and MDM2 309T>G either with breast cancer overall or with oestrogen receptor (ER) status of tumours (Schmidt ). As the majority of BRCA1 mutation-associated breast tumours are ER-negative (Lakhani ), the absence of an association in our study of breast cancer with the TP53 and MDM2 SNPs in BRCA1 mutation carriers is consistent with the lack of an association with ER-negative cancers in the general population.