1 Department of Surgery, University of Western Australia, Nedlands 6907, Australia and
2 Division of Endocrine Oncology, Department of Medical Oncology, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands
Abstract
The tumour suppressor gene TP53 and its downstream effector p21 are thought to play major roles in the development of breast cancer. We investigated three common sequence variants in TP53 and p21 for possible associations with the risk of breast cancer and with various phenotypic features of this disease. A total of 351 cases were available for study. Germline DNA obtained from female subjects of similar age but without cancer was used to estimate the TP53 and p21 genotype frequencies in a control population. A single nucleotide polymorphism in intron 2 of p21 was associated with slightly increased breast cancer risk (RR = 1.4, P = 0.011) and with well/moderately differentiated tumour histology (P = 0.029). The 16 bp insertion polymorphism in intron 3 of TP53 was associated with poor histological grade (OR = 2.3, P = 0.013) independently of other pathological features. The codon 31 polymorphism in p21 was strongly linked to negative progesterone receptor status (OR = 3.4, P = 0.0001), suggesting this variant may have functional significance for the progesterone signalling pathway in breast cancer. These results add to the growing body of evidence that genetic variants can influence not only the risk of breast cancer but also the disease phenotype.
Abbreviations: F-SSCP, fluorescent single strand conformation polymorphism.
Introduction
Known risk factors for breast cancer include family history of the disease, age over 50 years and high oestrogen exposure (reviewed in ref. 1). First-degree relatives of breast cancer patients have a 2-fold increased risk compared with the general population; however, only a small proportion of this is attributable to highly penetrant but rare BRCA1/2 germline mutations (2). Apart from shared environmental factors, the remaining familial risk may be due to common, low-penetrance genetic variants that are also referred to as modifier genes. Because of their higher frequencies in the general population, these polymorphisms may be responsible for a greater overall risk than BRCA1/2 mutations (3). Candidate modifier genes are involved in carcinogen metabolism, DNA damage repair, steroid metabolism and steroid receptor activation pathways. A recent review of published case-control studies found evidence for an increased risk of breast cancer associated with variants of the CYP19, GSTP1 and TP53 genes (4). As stated by the authors, more precise estimates of the risks associated with these and other yet to be identified candidate genes await further study.
Polymorphisms in TP53 are considered candidate risk factors because of the crucial role played by this gene in the maintenance of genomic integrity following genotoxic insult (5). Highly penetrant germline mutations in TP53 are very rare, however polymorphisms are quite common and at least 10 different variants have been described (6). Those investigated for associations with breast cancer include a 16 bp insertion in intron 3 (79), an Arg72Pro polymorphism in exon 4 (8,9) and a single nucleotide polymorphism in intron 6 (811). Only the codon 72 polymorphism appears to be significantly associated with the risk of breast cancer (4).
A major downstream component of the TP53 tumour suppressor pathway is the p21 cyclin dependent kinase inhibitor, also known as WAF1 or CIP1 (12). It was initially thought that somatic mutations in this gene might be involved in tumour formation, particularly for cases having wild-type TP53; however, p21 mutations proved to be extremely rare in a variety of cancer types investigated (1315). Polymorphisms in p21 have been described, with the two most common being Ser31Arg in exon 2 and a single nucleotide polymorphism in the 3' untranslated region of exon 3, 20 bp downstream from the stop codon (13). Another polymorphism, Asp149Gly, has also been reported in an Indian population (16). Interestingly, both codons 31 and 149 polymorphisms appear to occur more frequently in patients whose tumours contain wild-type TP53 (16,17). Another suspected p21 polymorphism occurs in the 5' region of intron 2 but this remains to be confirmed (18). To our knowledge, only two studies have examined p21 polymorphisms and the risk of breast cancer and both have been on the codon 31 polymorphism (14,19).
Several groups have reported interesting associations between various genetic polymorphisms and phenotypic features of breast cancer. These include TP53 codon 72 and low-grade histology (8), CYP1B1 codon 432 and steroid receptor status (20), VDR and nodal status (21), GSTP1 codon 105 and patient survival (22) and SRD5A2 codon 89 with both survival and grade (23). In the present study we investigated a large, population-based series of breast cancers for the occurrence of a polymorphism in TP53 (16 bp insert in intron 3, TP53PIN3) and two polymorphisms in p21 (codon 31, p21c31; intron 2 snp, p21int2). The major aim of this work was to evaluate possible associations between these polymorphisms and clinico-pathological features of breast cancer.
Materials and methods
Breast cancers
Eligible cases were women with primary breast cancer (n = 351) treated by mastectomy or breast conserving surgery between 1990 and 1993 at either the Sir Charles Gairdner or Royal Perth Hospitals in Perth, Australia. Genomic DNA was extracted from surgically resected tumour samples using standard procedures. The median age at surgery was 58 years (range, 1892 years) and the median follow-up time was 87 months (range, 2116 months). Clinical and pathological features of this tumour series were described earlier (24). The majority of subjects (>95%) were Caucasian of European descent. Information on family history of breast cancer was not available. DNA obtained from individuals of similar age but without cancer was used to estimate p21c31 and p21int2 genotype frequencies in a control population.
Genotyping for TP53PIN3, p21c31 and p21int2
The 16 bp duplication in intron 3 of TP53 was amplified by PCR and visualized on agarose gels essentially as described earlier (9). Primers used were forward: 5'-CTGAAAACAACGTTCTGGTA-3' and reverse: 5'-AAGGGGGACTGTAGATGGGTG-3'. DNA template (200 ng) was added to 25 µl of PCR reaction mix consisting of 1x reaction buffer, 0.2 mM dioxynucleotide triphosphates, 1.5 mM MgCl2, 1 µM of each primer (Life Technologies, The Netherlands) and 0.4 U Taq polymerase (Qiagen, The Netherlands). Negative controls comprising of no added DNA were included in each run. Amplification was performed in a Peltier Thermal Cycler starting with a 4 min denaturation step at 94°C and followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 60°C, 1 min extension at 72°C and ending with a final extension of 3 min at 72°C. TP53 intron 3 PCR product was visualized on 3.5% metaphor agarose gels. Depending on the presence or absence of the duplication, the size of the TP53PIN3 PCR product was 135 or 119 bp, respectively. The TP53 gene is located on chromosomal arm 17p, a region that is frequently the target of allelic loss in breast cancer. However, because the primary breast tumour specimens from which the DNA was obtained for genotyping always contain a relatively high proportion (>50%) of non-tumour tissue (stroma, lymphoid, non-tumour areas), this contribution of germline DNA ensures accurate genotyping of the TP53 gene.
PCR amplifications using HEX-labelled fluorescent primers (Geneworks, South Australia) were carried out for the p21 gene regions spanning polymorphisms in codon 31 (p21c31) and a previously reported (18) CG variant located 17 bp from the 5' end of intron 2 (p21int2). The forward primer sequence for p21c31 was 5'-CGCCATGTCAGAACCGGCT-3' and the reverse was 5'-TTCCATCGCTCACGGGCC-3', giving a PCR product size of 153 bp. The forward primer sequence for p21int2 was 5'-TCGCTCAGGGGAGCAGGCTGAA-3' and the reverse was 5'-GAGAATCCTGGTCCCTTAC-3', giving a PCR product size of 129 bp. Annealing temperatures were 60 and 62°C, respectively. PCR was carried out in a volume of 15 µl comprising a mix of 1x reaction buffer, 0.2 mM dioxynucleotide triphosphates, 2.5 mM MgCl2, 0.4 µM of each primer and 0.3 U Taq polymerase (Qiagen, Australia). Samples were heated to 94°C before addition of DNA template (200 ng). PCR comprised 10 min of denaturation at 94°C followed by 32 cycles of 45 s at 94°C, 45 s at the appropriate annealing temperature and 45 s at 72°C. Final extension was for 5 min at 72°C. PCR product was screened for polymorphisms using fluorescent single strand conformation polymorphism (F-SSCP) essentially as described previously (25). Briefly, 2 µl of PCR product was mixed with 4 µl of deionized formamide loading buffer and denatured at 94°C for 3 min. One microlitre of this mix was then loaded onto a non-denaturing, 8% polyacrylamide/2% glycerol gel and run on the Gel-Scan 2000 DNA fragment analyser (Corbett Research, Sydney, Australia). The sample was pulse loaded for 20 s at 1200 V, the wells rinsed and the gel run for 90 min at 1200 V in 0.8x TBE buffer at a constant temperature of 22°C. ONE-D scan 1.3 software (Scanalytics, Billerica, MA, USA) was used to analyse the electrophoretogram. DNA sequencing of wild-type and variant homozygote samples was carried out for the two p21 polymorphisms in order to confirm the identity of banding patterns observed on F-SSCP gels. Limitations on resources meant that ~80% of the available breast cancer cases were genotyped for the two p21 polymorphisms.
Statistical analyses
The 2 test (Pearson statistic) was used to determine associations between presence of the TP53PIN3, p21c31 and p21int2 polymorphisms and various clinico-pathological features of the breast tumours, as well as for independence of the alleles (HardyWeinberg equilibrium). KaplanMeier analysis was used to assess 5 year cumulative survival probability and differences were evaluated using the log-rank test. Cox's proportional hazards univariate and multivariate analyses were used to calculate hazard ratios and 95% confidence intervals. Wald's test was used to calculate 95% confidence intervals for odds ratios. All P values are derived from two-tailed statistical tests. Analyses were carried out using the SPSS software package (Chicago, IL, USA).
Results
TP53PIN3 polymorphism
The 16 bp duplication in TP53 intron 3 (TP53PIN3) was found in 74 (21%) of 351 breast cancer cases analysed, with 73 being heterozygous and one homozygous. The wild-type and variant alleles were in HardyWeinberg equilibrium. Although the frequency of this polymorphism in a control population was not examined here, the combined results from six previous studies (6,7,2629) on 1525 controls of Caucasian origin revealed a similar frequency (24 versus 21%, P = NS). TP53PIN3 was significantly associated with poor histological grade [OR = 2.4, 95% CI (1.34.2), P = 0.004] but not with the clinico-pathological features of nodal involvement, tumour size or steroid receptor status (Table I). Trends were apparent for associations between TP53PIN3 and younger patient age as well as with mutant TP53 status. Multivariate logistic regression analysis showed that TP53PIN3 was independently predictive of poor grade [OR = 2.3, 95% CI (1.24.6), P = 0.013], together with the factors of young age, large tumour size and mutant TP53 (results not shown). No difference was apparent for the survival of patients with the TP53PIN3 polymorphism compared with those with wild-type allele [HR = 1.3, 95% CI (0.72.3), P = 0.43].
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Breast cancer is a clinically heterogenous disease, as evidenced by widely variable morphological appearance and distinctive gene expression profiles (30). Tumour heterogeneity is also reflected in the different responses that morphologically similar tumours can show towards the same adjuvant therapies. Because of possible effects on protein function or expression, it is reasonable to suspect that polymorphisms in genes involved in carcinogen metabolism, oestrogen production, DNA repair and cell-cycle control could predispose individuals to the development of breast cancer, as well as influencing the clinical phenotype of the tumour. Genetic variants associated with an amino acid change can obviously have consequences for protein function, while those that occur in promoter or intronic regions could alter the level of gene expression. Alternately, the genetic variant may have no direct functional implications but could be linked to other polymorphisms that have altered functions relative to the wild-type sequence.
Because of the putative roles of TP53 and p21 in breast cancer development, we chose in the present study to investigate polymorphisms in these genes. Breast cancer is commonly found in individuals with germline TP53 mutations, while somatic mutations in this gene are associated with aggressive tumour features including poorly differentiated grade and absence of steroid receptors (24). Together, these observations suggest that changes to the normal function of TP53 can not only increase the risk of breast cancer but also directly or indirectly influence the resulting phenotype. Similar to three previous studies (79), we report here that a relatively common intronic variant of TP53 does not increase the risk of breast cancer (Table I). Interestingly, this polymorphism was twice as frequent in patients whose tumours were poorly differentiated compared with those with lower grade tumours (Table I
). Furthermore, this association was independent of other tumour-specific factors including nodal involvement, tumour size and steroid receptor status. An earlier study reported lack of association between TP53PIN3 and tumour grade but found instead that the codon 72 polymorphism in this gene was more frequent in patients with well-differentiated breast cancer (8). TP53PIN3 has been reported to be in strong linkage disequilibrium (>90%) with a polymorphism in intron 6, but not with that in codon 72 (9). It would be of interest to determine the prevalence of the codon 72 polymorphism in the current breast tumour series so that linkage with the TP53PIN3 and intron 6 variants could be evaluated as well as possible associations with histological grade. Although the functional relevance of TP53PIN3 is unknown, intronic sequences in this gene have been implicated in the regulation of gene expression and in DNAprotein interactions (31,32). TP53PIN3 may also be in linkage disequilibria with other as yet unidentified genes, thus explaining its association with a distinctive phenotype.
We also investigated known and suspected polymorphisms in the p21 cell-cycle control gene, a downstream component of the TP53 pathway. The p21c31 variant has been linked to an increased risk of cancer in some (17,29,33,34) but not all (19,35) previous studies. In agreement with an earlier investigation of 53 invasive breast carcinomas (19), the present study found no evidence to link p21c31 with an increased risk of this disease (Table II). Interestingly, however, this variant was three times more frequent in patients whose tumours lacked progesterone receptors compared with those with positive status. To our knowledge associations between the p21c31 variant and breast tumour phenotype have not so far been reported. In vitro transfection studies suggest the arginine allele of this variant has similar functional activity to the wild-type serine allele (36). Furthermore, no correlation was observed between the variant and p21 mRNA expression levels in gastric tissues (37) or between the variant and p21 protein expression in breast carcinomas (19). These findings suggest the p21c31 variant does not alter p21 function or expression compared with the wild-type sequence. However, progesterone is known to regulate the transcription of p21 in breast cancer cells (38,39) and it is tempting to speculate this could explain the observed association between absence of progesterone receptors and the presence of the p21c31 variant. The p21c31 genotype, through linkage to one or more progesterone-sensitive polymorphisms in the promoter region, may provide selective pressures for the development of breast tumours having low progesterone receptor levels.
This study confirmed the presence of a suspected p21 intronic polymorphism near the 5' end of intron 2 (18). The p21int2 variant was associated with a small but significant increased risk for breast cancer [RR = 1.37, 95% CI (1.061.76), P = 0.011]. Similar to the TP53PIN3 polymorphism, p21int2 showed an association with histological grade, being more frequent in patients with well/moderately differentiated tumours compared with those with poorly differentiated tumours (Table III). The close proximity of this CG polymorphism to the 5' splice site of intron 2 (17 bp downstream) suggests it may influence mRNA splicing. In eukaryotes the splice recognition site is thought to be highly conserved, consisting of the nucleotides GT at the 5' end of the intron and AG at the 3' end (40). Intron 2 of p21 does not conform to these consensus sequences, having an extra G nucleotide at the 5' end and a CA at the 3' end (NCBI accession no. Z85996). Further work is required to determine whether the p21int2 variant described here is associated with aberrant mRNA splicing and whether this has any phenotypic effects.
In conclusion, the results of this study suggest two common intronic variants in the TP53 and p21 genes are associated with opposite phenotypic features of histological grade in breast cancer. Further studies are needed to ascertain whether these associations are due to effects on gene expression that in turn have phenotypic consequences, or whether they are due to linkage with other, as yet unidentified variants that are themselves associated with functional differences. The p21c31 coding variant showed a strong association with the progesterone receptor status in this breast tumour series, suggesting a possible link between the progesterone and p21 signalling pathways. Finally, additional studies on large and diverse populations are needed to confirm the increased risk of breast cancer associated with the p21int2 variant as well as the observed genotype/phenotype associations.
Notes
3 To whom correspondence should be addressedEmail: bjiac{at}cyllene.uwa.edu.au
4 These authors contributed equally to the work
References