Mol. Hum. Reprod. Advance Access originally published online on July 23, 2004
Molecular Human Reproduction 2004 10(9):651-654; doi:10.1093/molehr/gah093
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Analysis of the codon 72 polymorphism of the TP53 gene in patients with endometriosis
1II Department of Obstetrics and Gynecology, University of Milano, Milano, Italy and 2Molecular Biology Laboratory, Istituto Auxologico Italiano, Milano, Italy
3 To whom correspondence should be addressed at: Molecular Biology Laboratory, Istituto Auxologico Italiano, Via Zucchi, 18 - 20095 Cusano Milanino, Milano, Italy. Email: a.diblasio{at}auxologico.it
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Abstract |
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Endometriosis is a benign gynaecologic disease that is associated with a certain risk for malignant degeneration. The disease has a genetic background, but the locations of possible genomic aberrations are still poorly clarified. In this context, the proline form of TP53 codon 72 polymorphism has been recently associated with the risk of developing endometriosis. In this case-control study, we aimed to investigate further the potential association between endometriosis and this polymorphism in order to evaluate whether this genetic variant may influence the susceptibility to the disease. Genomic DNA was obtained from a consecutive series of 303 Italian Caucasian women of reproductive age who underwent laparoscopy for benign gynaecological pathologies. Endometriosis was defined according to the criteria of Holt and Weiss [Holt V and Weiss NS (2000) Recommendations for the design of epidemiologic studies of endometriosis. Epidemiol 11,654–659] for the definite disease. Subjects of similar age without laparoscopic evidence of the disease served as control group. Molecular analysis of TP53 codon 72 polymorphism was performed by PCR amplification. Endometriosis was documented in 151 women. We found no statistically significant difference in the distribution of TP53 codon 72 polymorphism genotypes between patients with and without endometriosis. The respective proportions of arginine homozygotes, heterozygotes and proline homozygotes were 55.6, 39.7 and 4.6% in the group with endometriosis and 59.9, 30.9 and 9.2% in the control group. Moreover, no statistically significant association was demonstrated between TP53 codon 72 polymorphism and the various clinical manifestations of the disease, although a non-significant tendency towards an increased frequency of the proline allele was observed in association with specific manifestations of the disease reflecting a more severe form. Our results suggest that the TP53 codon 72 polymorphism does not confer genetic susceptibility to endometriosis in the Italian population. However, a possible susceptibility role of this polymorphism in endometriosis development towards very severe forms cannot be ruled out.
Key words: endometriosis/p53/codon 72 polymorphism
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Introduction |
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The TP53 gene, located on chromosome 17p13, is one of the most important tumour suppressor genes controlling DNA transcription and cell cycle regulation. The p53 protein induces apoptosis or cell cycle arrest in response to DNA damage, allowing the injured cells to be destroyed or repaired before reinitiating replicative DNA synthesis (Robles et al., 2002
). Functional inactivation of p53 pathways through genetic and epigenetic events, affecting the TP53 gene itself and/or its interacting partners, occurs with a high frequency in cancers. Indeed, mutations of this gene are associated with instability in cell development and cycle progression (Robles et al., 2002). Numerous cancers are related to somatic abnormalities of the TP53 gene, including cancer of the cervix, ovary, bladder, prostate, liver, intestine, lung, brain, mouth, nose and throat, oesophagus, breast and lymphoma (Hollstein et al., 1991). In contrast, germline variants of the TP53 gene are less studied and there is controversy about their importance in various tumours (Storey et al., 1998; Zehbe et al., 2001; Koushik et al., 2004; Zhang et al., 2004).
More specifically, two common polymorphic variants have been described at codon 72 of the TP53 gene (Storey et al., 1998; Zehbe et al., 2001; Robles et al., 2002; Koushik et al., 2004; Zhang et al., 2004). This polymorphism derives from a single-nucleotide substitution at codon 72 that results in the presence of either proline (p53pro) or arginine (p53arg) in the amino-acid sequence. This non-conservative amino-acid change involves one of the five PXXP SH3-binding motifs found in the p53 proline-rich domain and affects certain p53 biochemical and functional properties. The proline form of p53 is a stronger transcriptional activator, but a poorer inducer of apoptosis than the arginine form (Robles et al., 2002). In addition, the arginine form is more susceptible to degradation by the human papillomavirus E6 protein (Koushik et al., 2004). While some studies reported that p53arg homozygosity is associated with some forms of cancer, other studies revealed higher risks in p53pro homozygotes (Storey et al., 1998; Zehbe et al., 2001; Inserra et al., 2003; Koushik et al., 2004; Zhang et al., 2004).
Recently, Chang et al. (2002) have demonstrated an association between the proline form of TP53 codon 72 polymorphism and endometriosis. Indeed, although the specific aetiology of this disease remains still unclear, a genetic basis for its development has been proposed on the basis of about 7% concordance in identical twins and an increased risk in siblings, compared with the general population (Simpson et al., 2003). This risk suggests that, as with most adult-onset conditions, the disease is characterized by a polygenic and multifactorial aetiology. Thus, one of the current tasks is to determine the number and locations of genes responsible for inherited susceptibility to the disease. Candidate genes specifically studied for association or linkage with endometriosis include genes coding for detoxification enzymes (Simpson et al., 2003), for steroid receptors (Wieser et al., 2002) and for inflammatory cytokines and adhesion molecules (Simpson et al., 2003; Vigano et al., 2003). However, association studies applied to endometriosis are probably complicated by its multiple manifestations, by the problematical diagnosis of a disease with very different degrees of severity and by the complexity in selecting an adequate control population (Holt and Weiss, 2000). Consequently, an unequivocal consensus regarding the association of any potential candidate gene and this disease is lacking (Simpson et al., 2003).
To further evaluate the relationship between the TP53 codon 72 polymorphism and endometriosis, and investigate the possibility that this association may be of help in the identification of patients at risk for a more aggressive disease, we have compared the frequency of the proline/arginine forms of TP53 codon 72 polymorphism between subjects with and without endometriosis in a cohort of Italian Caucasian women.
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Materials and methods |
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Subjects
The TP53 codon 72 polymorphism was evaluated in women who attended the endoscopic surgical service of the II Department of Obstetrics and Gynecology of the University of Milan to undergo gynaecological laparoscopy. All studied individuals were of Italian Caucasian origin. From January 2001 to June 2003, 303 women were enrolled in the study. All the women underwent complete pre-surgery clinical examination before the diagnostic-operative laparoscopy. Indications to laparoscopy included chronic pelvic pain, infertility, ovarian cysts and myomas. None of the women was taking any medication except for non-steroidal anti-inflammatory drugs. Endometriosis was always diagnosed during the laparoscopic intervention and criteria for inclusion in the study were those proposed by Holt and Weiss (2000)
. Specifically, these criteria include patients with ovarian endometriomas of any size or with pelvic endometriotic implants of any size >5 mm deep or with pelvic endometriotic implants of any size with adhesion not attributable to another cause. Diagnosis of ovarian endometriotic cysts and deep peritoneal lesions was always confirmed histologically. Diagnosis of superficial peritoneal lesions was based on direct visualization when endometriotic implants presented as typical lesions. Lesions whose aspect was dubious were always removed in order to confirm their endometriotic nature by histologic examination. Three physicians active in the evaluation and treatment of endometriosis staged patients according to the revised American Society for Reproductive Medicine (rASRM) classification (ASRM, 1997). Endometriosis was documented in 151 women (49%). Stage of disease was found to be minimal or mild (stages I–II) in 26 cases (17%), moderate (stage III) in 69 cases (45%) and severe (stage IV) in 56 cases (37%). Other gynaecological benign pathologies were present in 5 of these 151 women with endometriosis (5 cases of uterine myomas). The presence of endometriotic cysts (endometriomas) was observed in 114 (75%) of these 151 women. Deep endometriosis, defined as lesions infiltrating to a depth of at least 5 mm beneath the peritoneal surface, was observed in 34 (22.5%) women. One hundred and fifty-two women who underwent laparoscopy and in whom no endometriosis was found served as control group. Specifically, they included 29 cases with a regular pelvis, 46 cases of serous or mucinous cysts, 23 cases of dermoid cysts, 27 cases of uterine myomas, 1 case of ovarian fibroid, 12 cases of uterine malformations, 7 cases of paraovarian cysts, 19 cases of pelvic inflammatory disease and 1 case of appendicitis. The mean 
of patients with endometriosis and controls was 
years and 
years, respectively. Patients were informed that blood would be used for research purposes and gave written consent. Approval for this study was granted by the local Human Institutional Investigation Committee.
Molecular analysis of the TP53 codon 72 polymorphism
Genomic DNA was isolated as previously described (Vigano et al., 2003). Genomic DNA (150 ng) was amplified by PCR using primers that detect TP53 codon 72 in proline form (5'-GCCAGAGGCTGCTCCCCC-3'; 5'-CGTGCAAGTCACAGACTT-3') and arginine form (5'-TCCCCCTTGCCGTCCCAA-3'; 5'-CTGGTGCAGGGGCCACGC3') according to the procedure described by Storey et al. (1998). PCR was performed in a total volume of 25 µl containing genomic DNA, 5 pmol of each primer, 1x Taq polymerase buffer and 0.5 units of Red Taq (Sigma). Cycling conditions for p53 codon 72 in proline form was set as follows: one cycle at 94°C for 5 min, 35 cycles at 94°C for 15 s, 52°C for 20 s, and 72°C for 30 s, and one final cycle of extension at 72°C for 5 min. Conditions for TP53 codon 72 in arginine form were the same as for the proline form except that the annealing was done at 50°C. The PCR products derived from both the reactions examining the TP53 codon 72 in proline form and arginine form from the same patient were mixed together, and 10 µl of this solution was loaded into 3% agarose gel containing ethidium bromide for electrophoresis. Alleles were analysed examining the distributions of TP53 polymorphisms in both groups and evaluating the correlation between the TP53 genotypes and endometriosis (Figure 1).
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Statistical analysis
Considering the reported 25–30% prevalence of the mutated allele (Inserra et al., 2003
), the sample size used in this study allowed us to detect at the usual level of study power (80% statistical power of avoiding a type II error and 5% level of significance) at least a twofold increase in the risk to develop endometriosis. Statistical analysis was done using the SPSS statistical package (SPSS, Chicago, IL). Allele and genotype frequencies were compared between groups using the 
2 test or Fisher's exact test, as appropriate. The odds ratio (OR) was used to measure the strength of the association between allele frequencies and endometriosis. |
Results |
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In this study we have evaluated the genotype data of 151 endometriosis patients and 153 controls. Patients and controls were examined before the results of genetic analysis were known and laparoscopic surgeons were therefore masked to the TP53 codon 72 polymorphism of the individuals. Since diagnosis of endometriosis can be definitively proven by laparoscopy and the disease is often asymptomatic, controls consisted of women in which the disease was laparoscopically ruled out. The frequencies of TP53 codon 72 polymorphism alleles in our control population are in line with those reported for a group of Italian women and a white non-Hispanic population (Zehbe et al., 2001
; Zhang et al., 2004). In both patients with endometriosis and controls, the genotype frequency pattern showed dominance of the arginine allele. The frequency of the proline allele was not significantly different between the study and control groups (24.5% versus 24.7%, respectively) (Table I). The genotype frequencies did not significantly differ between the study and control groups as well (Table I). We have also reanalysed the data considering only the cases with histologically proven disease, but genotype and allele frequencies between patients and controls were still very similar (data not shown).
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We then analysed the endometriosis group according to the presence or absence of the different forms and features of the disease (superficial and deep peritoneal endometriosis, extent of adhesions and presence of endometriotic cysts). As shown in Table II, the proline/arginine distribution of the TP53 codon 72 polymorphism did not appear to be associated with the presence of deep endometriosis, although a modest but not statistically significant increase of the proline form frequency could be observed in patients with two or more cysts, with a high rASRM adhesion score and with a stage IV disease.
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The proline/arginine distribution of the TP53 codon 72 polymorphism has also been evaluated as a function of the diagnosis of the controls, but no significant associations could be found (Table III).
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Discussion |
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The results presented allow us to exclude, in the Italian population, a two- or more-fold increased risk of endometriosis when harbouring the variant of the TP53 codon 72 polymorphism. This is in contrast to data from a recent study by Chang et al. (2002)
reporting that the proline/arginine heterozygotes and proline homozygotes had a 3.9-fold increased risk to develop endometriosis when compared to the arginine homozygotes.
Researchers recognize that few published reports of significant associations between genetic variants and phenotypic manifestations can be unequivocally replicated (Ioannidis et al., 2003). Indeed, it has been reported that only 16% of these associations are subsequently replicated with formal statistical significance (Ioannidis et al., 2003). Reasons for heterogeneity in the size of the effect of a disease gene between study settings are unclear. This aspect has been recently discussed in detail (Ioannidis et al., 2003). Inability to replicate genetic associations include false positive results obtained by chance in the initial studies, the possibility that the disease-causing allele is in linkage disequilibrium with the locus under study, selection biases, and heterogeneity between ethnic groups. In this context, replication of associations, before declaration of evidence as convincing, is gaining increasing acceptance. Replication studies have the advantage of avoiding the same biases as the first study and require a less stringent P value, because a more specific hypothesis is being tested. They are more likely to give effect sizes smaller and closer to the true effect size than initial published reports (Ioannidis et al., 2003). These considerations could explain the discrepancy between our findings and those reported by Chang et al. (2002) and, in this view, our study acquires a specific significance.
One major reason for controversy with the study by Chang et al. (2002) may be the different ethnicity. Indeed, while allele frequencies detected in this study are in line with those reported for other Caucasian populations (Zehbe et al., 2001; Zhang et al., 2004), they differ considerably from those found in the Taiwanese study (Chang et al., 2002). Race-specific variation in the distribution of genotypes in the TP53 codon 72 polymorphism has been already demonstrated. Indeed, also in other studies considering other Asian populations, the frequency of the arginine allele is lower than in the Caucasian group (61% versus 70%, respectively) (Inserra et al., 2003).
Moreover, it has to be noted that a few studies have suggested that Asian women are at higher risk of endometriosis compared to women of other races (Missmer and Cramer, 2003). This variation in the incidence in endometriosis in the various ethnic groups might be another explanation for the differences observed between the two studies. The TP53 codon 72 polymorphism might differently influence the manifestation of endometriosis in different ethnic groups.
We also cannot rule out the presence of small effects since this study does not have enough statistical power to detect a risk lower than two. Nor does this study have adequate power to detect small increases in the risk to develop specific forms of the disease. This concept has to be taken into particular consideration since a trend towards an increased frequency of the proline allele was observed in association with specific manifestations of the disease reflecting a more severe form. Thus, a possible susceptibility role of this polymorphism at a late stage of endometriosis development cannot be excluded.
The variants of TP53 polymorphism at codon 72 are quite different in their ability to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis and to repress the transformation of primary cells (Robles et al., 2002). They may serve as risk factors for major human neoplasms and may play a role in modulating environmental risk factors for cancer (Storey et al., 1998; Zehbe et al., 2001; Robles et al., 2002; Koushik et al., 2004; Zhang et al., 2004). The possibility of an association between the TP53 codon 72 polymorphism and endometriosis would be particularly interesting in relation to the identification of genetic markers of potential malignant transformation of the disease. Several studies have indeed investigated whether transformation from endometriosis to endometrioid and clear cell ovarian carcinoma could involve mutations in this gene and/or protein alterations (Campbell and Thomas, 2001; Vignali et al., 2002). Evaluation of chromosome 17 aberrations by fluorescence in-situ hybridization (FISH) and allelotyping have revealed controversial findings (Jiang et al., 1998; Bishoff et al., 2002) and most studies failed to detect mutations in exons 5–9 of the TP53 gene (Vercellini et al., 1994; Jiang et al., 1996). p53 immunoreactivity was undetectable in samples of endometriosis not associated with carcinomas (Nezhat and Kalir, 2002) while the expression of p53 in endometriotic lesions next to carcinomas as a potential sign of a continuum from endometriosis to cancer is still debated (Mhawech et al., 2002; Nezhat et al., 2002). Therefore, further studies are needed to clarify the exact role of p53 in endometriosis degeneration towards malignancy and the potential association of the codon 72 polymorphism with very severe forms of the disease.
In conclusion, with the power to detect at least a double risk, this study failed to demonstrate any association between TP53 codon 72 polymorphism and endometriosis development in an Italian population. However, from this study it cannot be excluded that variations in ethnicity and/or statistical power limitation may mask smaller effects.
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Submitted on May 13, 2004; resubmitted on July 1, 2004; accepted on July 6, 2004.
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