Original paper
Association of methylenetetrahydrofolate reductase gene polymorphisms with basal cell carcinoma development

Introduction

Non-melanocytic skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common malignancies in the Caucasian race. Basal cell carcinoma accounts for about 80% of all skin cancers. Although it is characterized by slow growth and local invasiveness, it may lead to the destruction of vast areas of the skin, posing a therapeutic problem [1, 2]. In the pathogenesis of BCC one should take into account the interaction between genetic and environmental factors, especially exposure to solar radiation.

Ultraviolet radiation (UVR), through DNA damage, generating photoproducts, and development of mutations in genes which regulate the cell cycle, contributes to the initiation of carcinogenesis [3]. Ultraviolet radiation can also cause a local state of immunosuppression, including by reducing the activity of Langerhans cells, which also pro­mote this phenomenon [4, 5]. The gene encoding methy­lenetetrahydrofolate reductase (MTHFR) is located on chromosome 1 (1p36.3), consists of 11 exons and has 9 alternative transcripts. The MTHFR gene encodes an NADPH reductase, which catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate [6], which plays a crucial role in DNA synthesis and repair.

Repair of DNA damage is a key phenomenon that protects the organism against development of cancers. This was a prerequisite to search for association of polymorphisms of genes involved in DNA repair with risk of cancer [7, 8]. Literature data also point to the participation of MTHFR gene polymorphisms in oncogenesis [7]. The most important functional polymorphism of the MTHFR gene is transition C/T at nucleotide 665 and A/C at nucleotide 1286 from the transcription start site [9-11].

Polymorphism 665C/T (exon 4) causes an alanine to valine substitution at codon 222 and polymorphism 1286A/C (exon 7) causes conversion of glutamic acid to alanine at codon 429 [7, 12], which leads to generation of a thermolabile form of MTHFR and a decrease of its enzymatic activity.

To date, there have been demonstrated relationships between selected polymorphisms of the MTHFR gene and increased risk of developing breast [13], colorectal [14] and gastric cancer [15], and squamous cell carcinoma [7, 12, 16, 17].

Because of the relatively sparse literature data concerning the role of MTHFR polymorphisms in the development of basal cell carcinoma [7, 12], the aim of our study was to determine the relationship between polymorphisms 665C/T and 1286A/C in the MTHFR gene and risk of BCC in the Polish population.

Material and methods

The study group consisted of 142 patients with histologically confirmed BCC and 142 healthy individuals, as a control group, divided according to sex and age. Clinical characteristics are shown in table 1.

All patients gave written consent to participate in the study. The volunteers were generally healthy with a negative medical history of patient and family history for occurrence of skin cancer. The study was approved by the Local Ethics Committee at the Medical University of Lodz (No. RNN/171/06/KE).

Genotyping of the 665C/T MTHFR gene variant

Genomic DNA containing the polymorphism 665C/T was amplified by polymerase chain reaction, using 665CT1 primer in intron 5: 5´-AGGACTCTCTCTGCCCAG-3` (forward) and 665CT2 in intron 6: 5´-TCA CAA AGC GGA AGA ATG-3 (reverse) using the AccuPrime™ Taq polymerase system (Invitrogen, California, USA) and 50 ng of genomic DNA. This PCR reaction resulted in the synthesis of a 227 bp fragment (665CC). The MTHFR gene contains a C to T substitution at nucleotide 665; the alteration created a HinfI restriction site. Obtained PCR products were then digested with the restriction enzyme FastDigest® HinfI (Fermentas, Ontario, Canada), at 37°C for 30 min. FastDigest® HinfI did not digest the fragment derived from the C allele, whereas it digested the fragment of the same length from the T allele into 169 bp and 58 bp fragments. These fragments were then electrophoresed using a 2% agarose gel with ethidium bromide and visualized under UV light. Homozygous CC results in one fragment of 227 bp, while homozygous TT results in two fragments of 169 bp and 58 bp. Heterozygotes (CT) show all three bands of 227 bp, 169 bp and 58 bp (fig. 1).

Genotyping of the 1286A/C MTHFR gene variant

Genomic DNA containing the polymorphism 1286A/C was amplified by PCR, using the forward primer 1286AC1 in intron 8 (5´-TGA AGA GCA AGT CCC CCA AG-3`) and the reverse primer 1286AC2 in intron 9 (5´-CAA CAA AGA CCC AGC CTG TC-3`), using the same polymerase system. This reaction resulted in the synthesis of a 325 bp fragment (1286CC). In exon 8 of the MTHFR gene, an A to C substitution leads to an amino acid change from glutaminic acid to alanine at codon 429 of the protein. This Glu429Ala polymorphism is detectable with a restriction enzyme, FastDigest® MboII. Obtained fragments were then electrophoresed by using a 2% agarose gel with ethidium bromide and visualized under UV light. The MTHFR CC genotype was represented by a single fragment of 325 bp, AA genotype by two fragments of 253 bp and 72 bp, while heterozygotes (AC) showed all three bands of 325 bp, 253 bp and 72 bp (fig. 2).

Additionally, we analysed the association of these polymorphisms with phenotypic features, such as eye colour, hair and skin phototype according to Fitzpatrick [18].

Statistical analysis

To assess the relationships between dependent or independent variables, logistic regression was used. Frequencies of genotypes and alleles in the studied population were analysed for deviation from Hardy-Weinberg equilibrium and tests for association were performed (Institute of Human Genetics, Technical University Munich, Germany, http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/ hwa1.pl). Risk assessment of individual genotypes coexisting with the disease and other characteristics was performed using the odds ratio (OR). Results were considered statistically significant at the significance level p < 0.05. Statistical analysis was performed using STATISTICA software.

Results

The distribution of genotypes of the polymorphisms 665C/T and 1286 A/C in the MTHFR gene in both groups was consistent with Hardy-Weinberg equilibrium.

Analysing the distribution of genotypes for polymorphism 665C/ T in the control group showed that the CC genotype occurs with frequency of 80.3%, CT – 12.0%, TT – 7.7%.

In patients with BCC, compared with the control group, the heterozygous genotype CT occurred statistically significantly more often (30.3% vs. 11.97%, OR = 3.392, p = 0.00008). Moreover, in patients with BCC (39.6%), allele T (genotype CT and TT) is more frequent compared with the control group (19.7%). In the case of carriers of this allele, there was observed increased risk of developing BCC (OR = 2.094, p = 0.00068), compared with the control group (tab. 2, fig. 3).

Analysing the distribution of genotypes 1286A/C MTHFR in the control group showed that genotype AA occurs with frequency 52.1%, AC – 45.8% , CC – 2.11%. In patients with BCC the presence of CC genotype was statistically significantly more frequent (7.7% vs. 2.1%, OR = 4.24, p = 0.032). Moreover, in patients with BCC, compared with the control group, allele C was more frequently present (genotype AC and CC, 54.9% vs. 47.9%, respectively), but this association showed no statistical significance (p > 0.05) (tab. 2, fig. 4).

There were no relationships between investigated polymorphisms and eye colour, hair and skin phototype (p < 0.05 for all comparisons).

Discussion

Normal serum folate level is essential, in physiological conditions, for repair of DNA damaged by ultraviolet radiation. Hence, folates, and their metabolites, are necessary for normal cell proliferation and DNA repair in rapidly dividing cells, such as keratinocytes. In the metabolism of folates, a crucial role is played by methylenetetrahydrofolate reductase, which is involved in the folate pathway and catalyses a reduction reaction from 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, a substrate for the remethylation of homocysteine to methionine [19-23].

The most common genetic phenomenon in the process of carcinogenesis is incorrect DNA methylation [24, 25]. Polymorphisms in the MTHFR gene give rise to reductase with decreased activity and thus affect the folic acid metabolic pathway, which leads to disorders in methylation and initiation of carcinogenesis. In spite of numerous studies, the relationship between two common polymorphisms of the MTHFR gene with diminished DNA methylation in the development of BCC has not been analysed [17]. Festa et al. [7] in studies of patients with BCC, among the Scandinavian population, have shown that the presence of a combination of genotypes of TT/AA, both polymorphisms 665 C/T and 1286 A/C in the MTHFR gene, was associated with an increased risk of developing this cancer. However, this relationship has not been confirmed in other studies, in patients from the American population [12].

In this study we confirmed the association between the occurrence of some genotypes and the analysed polymorphisms in the MTHFR gene and the development of BCC. Based on the obtained results we found that the presence of the CC or CT genotype is associated with an increased risk of cancer development. Genotype 665 CT causes more than three-fold increased risk of BCC, while the presence of CC genotype in the polymorphism 1268 A/C augments the risk more than four times. Hence, these two genotypes might be considered as a factor predisposing to the development of BCC in the Polish population. The literature data suggest that the 665T allele is associated with reduced enzyme activity, which leads to elevated concentration of homocysteine and lower folate level in plasma, DNA repair abnormalities and consequently to carcinogenesis [9]. In the present study we also found a relationship between presence of the T allele and the development of BCC, which confirms the above hypothesis.

The presence of polymorphism 1286 A/C also inhibits the functional activity of the enzyme, although it does not directly affect the level of homocysteine and folic acid in the plasma. The study showed, however, that the presence of allele 1286C in patients with low folate concentration can cause impairment of their metabolic pathway [10]. In the present study we also found an association of CC genotype with risk of developing basal cell carcinoma, but this dependence was not analysed for folate concentration in serum of patients with BCC.

In the process of skin carcinogenesis, ultraviolet radiation plays a key role through the possibility of DNA damage. In addition, UVR causes degradation of folate in the photolysis process, which causes a deficiency in the serum and consequently leads to abnormal repair processes damaged by UVR genomic DNA [26].

On the basis of these results, we conclude that the interaction between recognized pathogenetic environmental (excessive exposure to ultraviolet radiation) and genetic (polymorphisms in the gene MTHFR) factors predisposes to non-melanocytic skin cancers, including BCC.

Acknowledgements

The project was funded by Medical University Research Grant nr 503-1152-1 and Polish Scientifique Committee nr NN402474731.

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