P113

Abstract

Background

Heparansulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. d-glucuronyl C5-epimerase (GLCE) is a crucial enzyme in HS synthesis, converting d-glucuronic acid (GlcA) to l-iduronic acid (IdoA) to increase HS flexibility. Aberrant modification may result in wrong structure of polysaccharide chains of HS and defects of microenvironment associated with malignant transformation.We previously experimentally identified the GLCE polymorphism Ile597Val. Its localization close to the activity center of the enzyme and different physical parameters of the involved amino acids suggest that the polymorphism is functional. So, three different variants of GLCE dimers with different enzymatic activity may exist in heterozygous carriers. Bioinformatics’ search (PubMed resource) revealed interracial variations in allele frequency distribution. Unusual high frequency of allele G was shown for black race (45%) compared with white race (17%). Taking into account the increased resistance of negroid race to breast cancer, we assume a potential involvement of the GLCE polymorphism in breast cancer.

Aim

The estimation of effects of GLCE functional polymorphism A2017G (Ile597Val) on the gene expression levels in normal and breast cancer cells and LOH in breast tumors.

Materials and methods

Breast cancer patients (n = 144.) had histologically verified diagnoses. Blood and breast cancer tissue samples as well as matched control tissues were collected from each patient during surgery. Genomic DNA was isolated by phenol extraction. Total RNA was isolated by TRIZol, RNA quantity was accessed by Qubit instrument with appropriate reagents and cDNA was obtained using First Strand cDNA Synthesis kit. SNP A2017G (rs3865014) was analyzed by Custom Real-Time SNP Array and GLCE expression levels were determined using Taq-Man-based Real-Time PCR (Applied Biosystems). Statistical analysis was carried out using a Statistika 9.0 software.

Results

AA genotype carriers had a 2-fold increase in GLCE mRNA levels in tumors compared with control surrounding tissues (0.37 ± 0.77 versus 0.17 ± 0.16, respectively, p < 0.05). Oppositely, AG genotype carriers had a 1.5-fold decrease in GLCE mRNA levels in tumors compared with control surrounding tissues (0.39 ± 0.29 versus 0.58 ± 0.33, respectively, p < 0.05 ). However, in any case,the GLCE expression in both normal tissues and breast tumors was more active in AG genotype carriers than in AA carriers. It is known that LOH is often associated with molecular mechanisms of carcinogenesis, we studied this process for the same patients. According our results, LOH was detected in about 10% of cases (5/52 patients), among which G was lost in 3 patients and A was lost in 2 patients.

Conclusion

The obtained data show a possible association between GLCE Ile597Val polymorphism and breast cancer, although the nature of the association remains ambiguous.