An evaluation of the polymorphisms Ins16bp and Arg72Pro in p53 as breast cancer risk modifiers in BRCA1 and BRCA2 mutation carriers

doi:10.1038/sj.bjc.6604624

Br J Cancer. 2008 September 16; 99(6): 974–977.

Published online 2008 September 9. doi: 10.1038/sj.bjc.6604624

PMCID: PMC2538750

An evaluation of the polymorphisms Ins16bp and Arg72Pro in p53 as breast cancer risk modifiers in BRCA1 and BRCA2 mutation carriers

A Osorio,¹^* M Pollán,² G Pita,³ R K Schmutzler,⁴ B Versmold,⁴ C Engel,⁵ A Meindl,⁶ N Arnold,⁷ S Preisler-Adams,⁸ D Niederacher,⁹ W Hofmann,¹⁰ D Gadzicki,¹¹ A Jakubowska,¹² U Hamann,¹³ J Lubinski,¹² A Toloczko-Grabarek,¹² C Cybulski,¹² T Debniak,¹² G Llort,¹⁴ D Yannoukakos,¹⁵ O Díez,^16,¹⁷ B Peissel,¹⁸ P Peterlongo,^19,²⁰ P Radice,^19,²⁰ T Heikkinen,²¹ H Nevanlinna,²¹ P L Mai,²² J T Loud,²² L McGuffog,²³ A C Antoniou,²³ and J Benitez^1,³

¹Human Cancer Genetics Programme, Human Genetics Group, Spanish National Cancer Centre (CNIO), Madrid, Spain

²National Epidemiology Centre, Instituto de Salud Carlos III & CIBERESP, Madrid, Spain

³Human Cancer Genetics Programme, Genotyping Unit, Spanish National Cancer Centre (CNIO), Madrid, Spain

⁴Division of Molecular Gynaeco-Oncology, Department of Obstetrics and Gynaecology, University of Cologne, Cologne, Germany

⁵Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany

⁶Department of Obstetrics and Gynaecology, Technical University, Munich, Germany

⁷Department of Obstetrics and Gynaecology, University of Schleswig-Holstein, Campus Kiel, Germany

⁸Institute of Human Genetics, University of Münster, Münster, Germany

⁹Department of Obstetrics and Gynaecology, Molecular Genetics Laboratory, University of Düsseldorf, Düsseldorf, Germany

¹⁰Institute of Human Genetics, Charite-University Medical Centre, Berlin, Germany

¹¹Institute of Cellular and Molecular Pathology, Medical University, Hannover, Germany

¹²Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland

¹³German Cancer Research Center, Molecular Genetics of Breast Cancer, Heidelberg, Germany

¹⁴Cancer Genetic Counselling Progam, Catalan Institute of Oncology (ICO), Barcelona, Spain

¹⁵Molecular Diagnostics Laboratory IRRP, NCSR Demokritos, Athens, Greece

¹⁶Genetics Service, Hospital de Sant Pau, Barcelona, Spain

¹⁷Oncogenetics Laboratory, Hospital Vall d'Hebron, Barcelona, Spain

¹⁸Medical Genetics Service, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

¹⁹Unit of Genetic Susceptibility to Cancer, Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

²⁰IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy

²¹Department of Obstetrics and Gynecology, Helsinki University Central Hospital (HUCH), Helsinki, Finland

²²Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, MD, USA

²³Cancer Research UK Genetic Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK

^*Author for correspondence: Email: aosorio/at/cnio.es

Received July 4, 2007; Accepted July 30, 2008.

This article has been cited by other articles in PMC.

Abstract

The close functional relationship between p53 and the breast cancer susceptibility genes BRCA1 and BRCA2 has promoted the investigation of various polymorphisms in the p53 gene as possible risk modifiers in BRCA1/2 mutation carriers. Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, and it has been recently reported that a p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at the onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population. In this study, we have evaluated this association in a series of 2932 BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2.

Keywords: BRCA1, BRCA2, p53, breast cancer

Given the involvement of p53 in cell cycle control, DNA repair and apoptosis, the role of this gene in cancer susceptibility has been extensively studied. Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, although not all studies have yielded consistent results (Weston et al, 1997; Wang-Gohrke et al, 1998; Suspitsin et al, 2003; Damin et al, 2006; Baynes et al, 2007; Costa et al, 2008). The Arg72Pro single-nucleotide polymorphism (SNP) has gained special attention, as there is consistent evidence of functional differences in apoptotic rates between the Arg and Pro variants (Biros et al, 2002; Wu et al, 2002; Dumont et al, 2003). In addition, the close functional relationship between p53 and the breast cancer susceptibility genes BRCA1 and BRCA2 (Jonkers et al, 2001; Ongusaha et al, 2003; Liu et al, 2007) has promoted the investigation of the Arg72Pro SNP as a possible risk modifier in BRCA1/2 mutation carriers (Martin et al, 2003). Indeed, it was recently reported that a p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population (Osorio et al, 2006). In this study, we have evaluated this association in a series of 2932 BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA) (Chenevix-Trench et al, 2007).

Materials and Methods

Patients

A total of 2088 BRCA1 mutation carriers, 841 BRCA2 mutation carriers and 3 carriers of mutations in both genes ascertained from eight centres participating in CIMBA were included in this study (Table 1). The inclusion criteria for subjects is described elsewhere (Chenevix-Trench et al, 2007).

Table 1

Genotype distribution of the two p53 polymorphisms in the BRCA1 and BRCA2 mutation carriers by participating study

Genotyping

Genotypes for the two polymorphisms – Ins16bp and Arg72Pro – were determined for each sample using previously described methodology (Osorio et al, 2006). In some cases, the Ins16bp SNP was genotyped by DHPLC on the WAVE HT system (Transgenomic, Omaha, NE, USA) using an acetonitrile gradient and profiles analysed with the Navigator™ software (Transgenomic), and the Arg72Pro SNP was genotyped by Taqman (Applied Biosystems, Foster City, CA, USA). Hardy–Weinberg equilibrium (HWE) for each polymorphism was tested using the likelihood ratio test among unrelated individuals. The German Consortium of Hereditary Breast and Ovarian Cancer (GCHBOC) study gave HWE P-values of 0.005 and 0.043 for Ins16bp and Arg72Pro, respectively; therefore, all genotypes from that patient subset were confirmed by an alternative technique (DHPLC). In addition, individuals homozygous for Ins16bp were directly sequenced. The concordance rate was 100% in both instances; accordingly, the GCHBOC mutation carriers were included in all subsequent analyses. Call rates ranged between 92 and 100% across studies.

Statistical analysis

Haplotypes were imputed using the R-package ‘hapassoc' (Burkett and McNeney, 2006). Associations of individual haplotypes with time to breast cancer or ovarian cancer diagnosis were evaluated using weighted Cox proportional hazards models, using age as the time variable (Antoniou et al, 2005). Carriers were censored at the first occurrence of breast or ovarian cancer or bilateral prophylactic mastectomy. To allow for correlations between members of the same family, Huber and White robust estimators of variance were used, considering women clustered within families (Huber, 1967). The most frequent haplotype was taken as the reference and all other haplotypes were included in the multivariate model considering the number of copies of that particular variant. Models were adjusted for ethnicity, birth cohort and centre of recruitment. The analysis considered BRCA1 and BRCA2 mutation carriers separately (Table 2) and all carriers combined (data not shown).

Table 2

Haplotype frequencies^a by mutation and disease status and HR estimates for breast cancer

Results and Discussion

Genotype distributions and frequencies for the Ins16bp and Arg72Pro polymorphisms are shown in Table 1. Allele frequencies were similar to those previously published (Osorio et al, 2006), and genotype frequencies were consistent with HWE, except for the carriers from GCHBOC (see Materials and Methods). Haplotypes were inferred, and haplotype- and genotype-specific hazard ratios were estimated separately for each of breast (Table 2) and ovarian cancer (data not shown), among BRCA1 and BRCA2 mutation carriers. No evidence of association was found for any of the genotypes or haplotypes analysed with either breast or ovarian cancer risk, including the No Ins-72Pro haplotype, previously reported to be associated with an increased risk to develop a first primary tumour before 35 years of age in BRCA2 mutation carriers (Osorio et al, 2006).

To confirm that this negative result was not due to the different analytic approach performed in this study, we carried out a logistic regression analysis, as was done in the original study (Osorio et al, 2006), considering those with age at diagnosis younger than 35 as cases, and did not find a positive association between early diagnosis and this haplotype. In the original study, the result was corroborated by a functional assay (Osorio et al, 2006), in which a decrease in apoptotic rate was found to be associated with the No Ins-72Pro haplotype. However, although concordant, both the genetic and the functional studies were limited by the small sample size (265 and 24 individuals, respectively), as reflected in the marginal statistically significant results described in that report.

In summary, the previously reported association of the No Ins-72Pro haplotype in p53 with an increased cancer risk in BRCA2 mutation carriers (Osorio et al, 2006) has not been validated in a larger series proceeding from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA). In this series of 2932 BRCA1/2 mutation carriers, no evidence of modification of breast or ovarian cancer risk by any of the two polymorphisms, Ins16bp and Arg72Pro, or their haplotype combinations has been detected. The lack of confirmation of a previously reported association found in a much smaller series highlights the necessity of international collaborative efforts aimed at achieving the statistical power required to reach reliable definitive conclusions in genetic association studies.

Acknowledgments

CNIO study: we thank RM Alonso and RL Milne for their assistance. This study was partially supported by Mutua Madrileña, Genome Spain and Marato TV Foundations. The Milan study is supported by the Fondazione Italiana per la Ricerca sul Cancro. DKFZ study: we thank D Torres and MU Rashid for providing DNA samples and supplying data. AC Antoniou, L McGuffog and the CIMBA data management are funded by Cancer Research UK.

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