Analysis of R213R and 13494 g→a polymorphisms of the p53 gene in individuals with esophagitis, intestinal metaplasia of the cardia and Barrett’s Esophagus compared with a control group

R213R (exon 6) polymorphism: to identify the R213R polymorphism, digestion of the long PCR product was carried out using TaqI (New England BioLabs™) enzyme to cleave the T′CGA site, according to the manufacturer’s instructions. The digestion used 10 μl of the PCR product and 2 U of the enzyme in a total final volume of 30 μl. The reaction was kept at 65°C in a thermocycler for 2 h and 30 min. Fragments resulting from cleavage of the PCR product with the TaqI enzyme are 385, 309 and 925 bp in the presence of the CGA sequence, and 694 and 925 bp in the presence of the CGG sequence.

13494 g→a (intron 6) polymorphism: to identify the 13494 g→a polymorphism, digestion on the long PCR product was carried out using the MspI (New England BioLabs™) enzyme to cleave the C′CGG site, according to the manufacturer’s instructions. The digestion used 10 μl of the PCR product and 2 U of the enzyme in a total final volume of 30 μl. The reaction was incubated at 37°C in dry bath. Fragments resulting from cleavage of the PCR product with the MspI enzyme are 124, 356, 276, 299, 168, 276 and 120 bp in the presence of the CGG sequence, and 124, 632, 299, 168, 276 and 120 bp in the presence of the CAG sequence.

Both digestion products were subject to electrophoresis in 3% agarose gel. The band pattern found is visualized in Fig. 1.

The statistical analysis of results was performed using a Pearson’s chi-square test in the MedCalc™ software.

For this study, the DNA extracted from blood and affected tissue of the same patient was analyzed. Only individuals who presented a heterozygous pattern in their blood were considered (n = 2 for the R213R polymorphism and n = 12 for the 13494 g→a polymorphism), respectively 8% and 33% of the sample for each polymorphism.

The loss of cell cycle control and the progression to cancer in TP53 events in exons 5–8 are already well established. The functional significance and the frequency of polymorphisms in these regions still remain unknown. It is believed that they may contribute to a higher susceptibility (or resistance) to developing cancer (Vos et al. 2003).

A consistent association between these polymorphisms and several types of human neoplasias may serve as argument supporting such sites as susceptibility determiners (IARC). Furthermore, mutations in flanking regions of these exons are probably rare in spite of the biological significance they present.

The genes involved with tumor genesis are potential molecular markers associated to susceptibility to cancer. We thus investigated the relationship linking the R213R and 13494 g→a polymorphisms of the p53 gene and the premalignant esophageal alterations (esophagitis, IMC and BE). Also, countless studies indicate an association between the R72P polymorphism at exon 4 of TP53 and the increased risk of developing neoplasias (Malcolm et al. 2000; Gottschlich et al. 2000; Nakano et al. 2000; Lacerda et al. 2005). Hence, we also carried out a joint analysis of the three polymorphisms described by means of a haplotype.

In the present study the genotypic distribution of the R213R and 13494 g→a polymorphisms are in agreement with the Hardy-Weinberg equilibrium in the patient group as well as in the control group. The genotypic analysis of both polymorphisms did not present a significantly different allele distribution compared to the control samples, which leads us to conclude that there is no correlation, at these stages, between such molecular findings and the histological types, although a strong ethnic variation with regard to the prevalence of these two polymorphisms (Leistner-Segal et al. 2006, Segal et al. 2004) is known to exist.

While analyzing the R213R polymorphism, Mazars et al. 1992 obtained results similar to ours for ovarian carcinoma and blood bank donor controls with prevalence of 3% and 2.6% for the polymorphic allele, respectively. In the analysis of the 13494 g→a polymorphism for gastrointestinal and breast tumors, Peller et al. 1995 also found results similar to ours, with frequencies of the heterozygous AG genotype of 32% and 68% for homozygous GG genotypes, for the patient group. These results match the allele frequency described by Chumakov and Jenkins 1991, which was 31% for the AG genotype, and 69% for the GG genotype.

It was anticipated that there would be a smaller proportion of the polymorphism in the exon 6 since the coding sequence is more conserved than the intronic region, mainly because the region in which this polymorphism is located carries great importance to the activity of the p53 protein. Eventual differences detected in several studies may be explained by ethnic differences between the groups analyzed (Brenna et al. 2004). Different genetic attributes may bestow protection against or risk for developing cancer in populations of diverse ancestries (Rocha 2005). However, other reasons such as sample size, DNA source, and techniques employed should also be taken into consideration (Brenna et al. 2004).

In the combined analysis with the R72P polymorphism previously studied by our group, a similar frequency was observed in both controls and patients. According to Table 3, the most frequent haplotype was [AA, GG, RR] which occurred in 34.6% of analyzed patients. Since the most frequent haplotype is the same in patient and control groups, we may think again that the absence of association between these three polymorphisms and the esophageal alterations stems from the fact that molecular alterations can only be identified in more advanced stages of the normal tissue-metaplasia-adenocarcinoma progression.

The LOH findings are in agreement with the occurrence of genomic instability seen in various types of cancer, and serve as encouragement to the understanding of DNA repair processes in replication and recombination events (Thiagalingam et al. 2002). All tissue samples presented the wild allele indicating that cells carrying the analyzed polymorphisms were heterozygotic or that many normal cells (lacking polymorphism) were present in the tissue from which DNA was extracted (Campomenosi et al. 1996, Doak et al. 2003). This fact may have minimized the LOH findings in our study.

The allelic loss associated to esophageal carcinogenesis was found by Kawaguchi for the codon 72 polymorphism (Kawaguchi et al. 2000). Abnormalities involving the TP53 are common with esophageal adenocarcinoma and have been detected in pre-malignant tissues surrounding the esophageal cancer (Prevo et al. 1999; Reid et al. 2001). Neshat et al. 1994 reported molecular and genomic alterations identifying LOH in 94% of Barrett cancers and 88% of mutations in the TP53. Allelic loss of 17p has been detected in initial stages of high-degree metaplasia and dysplasia, leading to aneuploidy and suggesting that inactivation of the TP53 is a first step in carcinogenesis. Both cases of LOH located in the intron 6 reinforce the data in literature for these events being found in altered tissues and also highlight the presence of alterations within the non-coding region. Such alterations may alter the phenotype of the protein being coded and are of considerable value in understanding the genic expression.

According to Campomenosi et al. 1996 the association of the molecular findings with the histological type occurs upon development of the BE since the type I metaplasia (common in BE) would be more responsive to damage caused by GERD and therefore more prone to developing aneuploidies than the tissues analyzed in our study (Li et al. 2005). Without doubt, this could provide an explanation to the fact that our results do not differ from those in the control population, since the tissues assayed were in early stages of BE development.

There is increasing evidence that new mechanisms including mutation in splicing sites, introns and promoter regions are involved in the genic expression. Identification of these alterations may help select patients with a higher risk of developing cancer, allowing optimization of treatments (Lacerda et al. 2005).

Considering our data regarding the incidence of alleles, genotypes and haplotypes of the R213R, 13494 g→a and R72P polymorphisms of the p53 gene in patients and controls, we can suggest that these three sites do not represent genetic susceptibility determiners to esophagitis, intestinal metaplasia of the cardia and BE. Additional studies are required in order to investigate these three polymorphisms in advanced stages of carcinogenesis.