Copy Number Variation in Gluthatione S-Transferase Variants using Multiplex Ligation-Dependent Probe Amplification in a Health Population in Goiânia – Go

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to a lot of diseases. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine GST variants in 100 healthy individuals from Goiânia – GO with a new methodology. GSTM1, GSTT1 and GSTP1 variants were analyzed by a MPLA (Multiplex Ligation by Probe Amplification) approach. The results obtained for the GSTM1 gene in exon 3 we found 43 deletions and 24 duplications, for exon 5 we found 45 deletions and 11 duplications, for the GSTT1 gene in exon 1 we found 14 deletions, and 18 duplications, for exon 5 we found 23 deletions and 28 duplications. For the GSTP1 gene in exon 3, we found only 1 deletion and for exon 4 we do not found any alteration. These findings in healthy population, give us such more information for the future epidemiological and clinical studies. Using to examine the effect of these combinations in drugs metabolism and cancer predisposition, further largest group would be needed, since their frequencies are quite low. To our of GSTs variations, this is the first study with this methodology indicating the frequencies of genetic variants of GST superfamily in a health population in a Goiânia population, and this study gives us new possibilities and data for new research using this same methodology.


Introduction
Individual inherited genetic differences related to polymorphism in detoxification enzymes could be an important factor not only in carcinogen metabolism but also in cancer susceptibility [1]. Functional genetic polymorphisms have been described for Glutathione-S-transferase (GSTs) genes, a superfamily of phase II metabolizing enzymes. GSTs catalyze the conjugation of reduced glutathione (GSH) to a wide variety of electrophilic compounds in order to make them more soluble enabling their elimination [2].
As a result of this detoxification activity, GSTs protect the cell from DNA damage, genomic instability and cancer development. In addition, as nonenzymatic proteins, GSTs can modulate signaling pathways that control cell proliferation, cell differentiation and apoptosis, among other processes [3,4].
Differences in GSTs activity may modify the risk of cancer development and also may impact on the heterogeneous responses to toxic substances or specific therapies [2]. Moreover, GST mutations are known to contribute to in//ter-individual and ethnic variability in the susceptibility to environmental risk factors, cancer predisposition and drug responsiveness.
Several epidemiological studies evaluated the role of GST polymorphisms on CML susceptibility, but conflicting results have been achieved [5,6]. CNVs are genomic that differ in copy number (CN) in compared genomes. CNVs include deletions, duplications, multiple duplications or more complex rearrangements. Common CNVs, also known as copy number polymorphisms (CNPs), account for approximately 10% of the human genome.
Although CNVs are more frequent in intergenic regions, they overlap hundreds of protein-coding genes, regulatory sequences, and other functional genetic elements. Although the majority of CNVs are probably neutral, increasing numbers of CNVs are being associated with various human phenotypes, including diseases [7]. With the widespread use of substances (supplements, medicines, among others) and interaction with the environment and life style, there is a need to trace the metabolic genetic profile of people who practice physical activity to power end know and elucidate the that as changes found could affect the metabolism of these people. Therefore, the aim of the present study was investigate, for the first time, the importance of GST genetic mutations by CNV analysis in the health population in Goiânia -GO.

Subjects
This study was carried out in the Genetic Molecular and Citogenetic

Sample collection and DNA analyzes
Peripheral blood (5mL) was collected in EDTA vacutainer tubes from all participating individuals after obtaining their written consent. Genomic DNA extraction was performed from whole blood using Purelink DNA Kit (Invitrogen ® ).   For the GSTP1 gene in exon 3, we found only 1 deletion in homozygous (1 woman). For exon 4 we do not found any alteration.

Discussion
Deletion in the GSTs genes apparently results from unequal homologous exchanges involving flanking regions containing sequences with high identity that were identified as regions of deletion / addition of the null genotype [8,9,10,11]. The homozygous deletion of the GSTM1 gene is observed at frequencies ranging from 20 to 70% in different populations, while for gene GSTT1 this variation is 11 to 38% [12,13,14].
The functional allele of GSTM1 are active in detoxification of various substances, such as polycyclic aromatic hydrocarbons and other products of combustion, which are potentially genotoxic oxidizing agents [15], while GSTT1 presents important activity in detoxification of peroxidized lipids and DNA oxidation products [16]. So that deletion of these isoforms has been associated with several types of diseases correlated with smoking, with emphasis on susceptibility studies to cancer [17,18,19,20].
GSTP1 is a major enzyme metabolizing anticancer drugs like oxiplatin, cyclophosphamide which are used in the treatment of breast and colorectal cancer. An over expression of this enzyme causes resistance to drugs like cisplatin [21]. Therefore, investigation of these mutations will provide a clue to the investigation of responders to cancer therapy with certain chemotherapeutic drugs.
It appears that the absence of one or more forms of GST can become more susceptible to chemical and oxidative stress cell, which can lead to cell dysfunction, being of great importance to characterize the influence of deletion polymorphism GSTM1 and GSTT1 risk disease [3,4].
In addition, pharmacogenetic has recently taken an important role showing significant differences, inter and intra population in metabolism, efficacy and toxicity of drugs, and this fact varies between regions studied. The Brazilian population, especially in the central-west region of Brazil offers a great research potential because it presents a unique admixture and diversity of variant combinations in different loci enabling gene interaction studies [22].
Deletion in the GSTs genes apparently results from unequal homologous exchanges involving flanking regions containing sequences with high identity that were identified as regions of deletion / addition of the null genotype [8,9,10,11]. The homozygous deletion of the GSTM1 gene is observed at frequencies ranging from 20 to 70% in different populations, while for gene GSTT1 this variation is 11 to 38% [12,13,14].

Conclusion
This study provides de first results of alterations distribution of GSTs M1, T1 and P1 mutations in a health population in Goiânia-Go, using the MLPa technic. Hence, it opens up new avenues for further investigations by epidemiologists in determining inter individual variation in genetic susceptibility to various diseases caused due to gene-environment interaction and the application of MLPA-based genotyping to routine clinical analysis will enable patients to be assigned to more accurate genotypes at a reasonable cost in a large number of individuals at the majority of locations.