GSTM1 and GSTT1 Allele Frequencies among Various Indian and non-Indian Ethnic Groups

Drug/xenobiotic compound metabolizing enzymes (XMEs) are essential for various disease-free state of an individual. They are capable of converting the active carcinogens/toxic compounds to inactive/non toxic compounds and vice versa (Lacko et al., 2009). The combination of Phase I and Phase II biotransforming enzymes along with the receptors associated to them act inevitably accurate to maintain an individual’s safety from environmental pollutants and other xenobiotics. One of the Phase II enzyme system, Glutathione-S-Transferase (GST) constitutes a family of multifunctional enzymes that has the ability to conjugate electrophiles and detoxify the agents of environmental concern including pesticides, therapeutic drugs, dietary components and a wide range of epoxides (Reszka, 2006). Knowledge of GST has lead to hypotheses about the role of the GSTM1 and GSTT1 genes in cancer etiology (Rebbeck, 1997). High activity of Phase I enzymes or high exposure to environmental agents and null activity of Phase II enzymes increases the individual risk association to various disease or cancer, as the enzymes majorly inactivate the toxic and/or active pro-carcinogenic


Introduction
Drug/xenobiotic compound metabolizing enzymes (XMEs) are essential for various disease-free state of an individual. They are capable of converting the active carcinogens/toxic compounds to inactive/non toxic compounds and vice versa (Lacko et al., 2009). The combination of Phase I and Phase II biotransforming enzymes along with the receptors associated to them act inevitably accurate to maintain an individual's safety from environmental pollutants and other xenobiotics. One of the Phase II enzyme system, Glutathione-S-Transferase (GST) constitutes a family of multifunctional enzymes that has the ability to conjugate electrophiles and detoxify the agents of environmental concern including pesticides, therapeutic drugs, dietary components and a wide range of epoxides (Reszka, 2006).
Knowledge of GST has lead to hypotheses about the role of the GSTM1 and GSTT1 genes in cancer etiology (Rebbeck, 1997). High activity of Phase I enzymes or high exposure to environmental agents and null activity of Phase II enzymes increases the individual risk association to various disease or cancer, as the enzymes majorly inactivate the toxic and/or active pro-carcinogenic RESEARCH ARTICLE

GSTM1 and GSTT1 Allele Frequencies among Various Indian and non-Indian Ethnic Groups
KP Senthilkumar 1,2 , R Thirumurugan 1,3 * intermediates of Phase I enzymes (Hirvonen, 1999). The null allele genotype with deletion mutation in GST has complete loss of gene function. The homozygous null types were associated with increased susceptibility to various genetic and metabolic disorders (Habdous et al., 2004;Anantharaman et al., 2007;Nosheen et al., 2010;Amer et al., 2011;Masood et al., 2011;Lordelo et al., 2012;Jiang et al., 2012). The variations in frequency distributions of the alleles are ethnic dependent and are even responsible for the efficacy and toxicity with various drugs (Kurose et al., 2012).
Growing literature on genetic polymorphism of GSTM1 and T1 null alleles with different ethnic groups or population affiliation were observed recently as they differ significantly from each other. Even though few studies have been reported in Indian ethnics for GST polymorphism (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010) all were not able to show the exiting deviation among the intra ethnics of Indians significantly and none of them have the reports from Gujarat, which is the highly polluted state of India (Kathuria, 2007;Singh and Kohli, 2012). Studies from other non Indian ethnics also claims the existing inter ethnic differences 6264 among the world population and Indian ethnics (Oke et al.,1998;Rossini et al., 2002;Hamdy et al., 2003;Magno et al., 2009;Ebeshi et al., 2011;Kurose et al., 2012). In this study, we investigated the hypothesised intra-Indian ethnic's and inter-Indian ethnic's differences among different populations with reference to our population based subjects from Gujarat for GSTM1 and GSTT1 null type allele frequency. The data acquainted from the study could be useful for understanding the significant deviation among XMEs in different populations and also useful for predicting the possible GSTM1 and T1 dependent drug tolerance, toxicity and/or susceptibility to various cancers among Gujaratians.

Subjects
The study was approved by the institutional ethical committee of Sh.NP Cancer Institute, Rajkot Cancer Society; India. Two mL of blood samples were collected from 504 healthy unrelated (2 male: 1 female) volunteers of Gujarat origin, after signing the informed consent to participate in the study. The age of subjects ranged from 40-80 with the mean age of 60 years.

DNA isolation and Genotyping
Genomic DNA from whole blood was isolated by salting out method of Lahiri and Nurnberger (1991). Multiplex polymerase chain reaction by Kui et al. (2006) method was performed for identification of GSTM1 and T1 null types with albumin gene as internal control. Primers used were GSTM1-F: 5' GAA CTC CCT GAA AAG CTA AAG C 3', GSTM1-R: 5' GTT GGG CTC AAA TAT ACG GTG G 3', GSTT1-F: 5' TTC CTT ACT GGT CCT CAC ATC TC 3', GSTT1-R: 5' TCA CCG GAT CAT GGC CAG CA 3', Albumin F: 5' GCC CTC TGC TAA CAA GTC CTA 3' and Albumin R: 5' GCC CTA AAA AGA AAA TCG CCA ATC 3'. The amplified 215 bp, 480 bp, and 350 bp were analyzed for GSTM1, GSTT1, and albumin presence respectively. Homozygous null types were identified by the absence of respective bands and the efficiency of the reaction was confirmed by the presence of the albumin band in all the samples. 25 µl reaction was used to amplify the template DNA with 10 µl of 1:10 diluted DNA sample; 200 µM of all dNTPs; Forward and reverse primers were used in 5 pM for GSTM1, T1 and albumin; 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 and 2.5U of Taq DNA polymerase. (All molecular biology grade chemicals used were from Merkgenei private Limited, Mumbai -India).

Different ethnics used in the study
The GST null allele frequency comparative analysis included the previous reports from Indian ethnics ( Table  2 and 4) and other non Indian ethnics (Table 3 and 5). Among the intra Indian ethnics, reports from South India (Naveen et al., 2004;Vetriselvi et al., 2006), Lucknow (Konwar et al., 2010), North India (Mishra et al., 2004), Western central India (Buch et al., 2001), Orissa (Roy et al., 1998) and other stratified report of Tamilnadu-Pondicherry, Andhra Pradesh, Kerala and Karnataka (Naveen et al., 2004) were comparatively studied with reference to Gujaratians. Combined review report of GST null allele frequency from ethnics of Eastern Asians and Europeans countries (Kurose et al., 2012) and reports from various investigators of Egyptian (Hamdy et al., 2003), Brazilian (Rossini et al., 2002;Magno et al., 2009) Nigerian (Ebeshi et al., 2011) and Turkish (Oke et al., 1998) were used for comparative study as the inter Indian ethnic groups. Asian and European countries were grouped under population affiliation of i) Eastern Asians countries -Japan, Korea, China, Taiwan and Mongolia ii) South Eastern Asia countries -Vietnam, Thailand, Philippines, Indonesia, Malaysia and Singapore iii) Northern European countries -Norway, Sweden, Denmark, Finland and UK iv) Southern European countries -Italy, Spain, Portugal, Slovenia and Greece v) Western European countries -Netherland, Germany and France vi) Eastern European countries -Checz, Bulgaria, Poland, Slovakia and Russia and other ethnics as vii) Caucasian includes Caucasian American and Caucasian Canadians and viii) African includes African Americans and South African Xhosa (Kurose et al., 2012).

Statistics
Null Allele frequency of GSTM1 and GSTT1 was calculated using Statistical Package for Social Studies (SPSS 16) software for Windows. Two sided Fisher's exact test was done with statistical significance set at p<0.05 to compare frequency variation of null alleles among other inter-Indian and intra-Indian ethnics with reference to ethnics of Gujarat.

Discussion
XMEs were vital to consider for any drug before its practice in an individual. The null/defective allelic frequency estimate for a drug to get metabolized in a given population is the deciding factor for the effectiveness of the drug. Clinical trials for lethal/effective curable dose of a drug among the subjects were critical in approval of the drug (Deisseroth et al., 2012;Poste et al., 2012). In addition the difference in the ethnicity of the subjects used for clinical trials and the population used for treatment were crucial for the main effect and side effect of the drug designed (Andres et al., 2012;Kappert et al., 2012). On the other hand, XMEs defective or null type alleles were reported as the risk factors associated with various cancers and diseases (Habdous et al., 2004;Anantharaman et al., 2007;Nosheen et al., 2010;Amer et al., 2011;Masood et al., 2011;Lordelo et al., 2012;Jiang et al., 2012). In this population based basic study, one of the phase II XMEs, GST has been investigated in the suspected population of highly polluted Gujarat for the first time. GSTM1 null allele frequency of Gujarat (20%) was significantly similar to most of known reports by various other authors from ethnics of Orissa, South India, North India and Western Central India. But was significantly different and low to (36.4%) Karnataka and (36.5%) Lucknow population reported earlier by Naveen et al. 2004 andKonwar et al. 2010 respectively among Indian ethnics at p<0.5 level. This observation of intra ethnic difference shows the diversity of GSTM1 null type among some Indian population if not all. Therefore any drug that requires GSTM1-XMEs could be used among majority of Indian ethnics, as Intra ethnic difference for GSTM1 among Indians was less. At the same time the existence of Inter ethnics difference among Indians and non Indians ethnics for GSTM1 null allele have to be considered significantly for GSTM1-XMEs dependent drug's uptake or dosage, as the difference was more than two and half fold high in South Eastern Asian and Egyptian at P<0.000001 level ( Table 3). The existence of significant GSTM1 null allele frequency's similarity among majority ethnics of Indians was unexpectedly deviating from other reports of various authors (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006; Konwar et al., 2010;Kurose et al., 2012) while, non-Indians ethnics significant deviation was in agreement to the previous reports from various regions (Rossini et al., 2002;Hamdy et al., 2003;Magno et al., 2009;Ebeshi et al., 2011;Kurose et al., 2012). GSTT1 null frequency of Gujarat (35.5%) was significantly higher than all other reported Indian populations (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010) of 13-19.1% and also significantly different to Western Central India, Tamilnadu-Pondicherry and Lucknow at P<0.001 (Table 4). This significant deviation in null allele frequency for GSTT1 was similar to the expectations of the existence of intra ethnic variation by other investigators from India (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010). Among the world populations, (Table 5) GSTT1 null allele frequency of European, Caucasian and Turkish populations were low to Gujaratians and significantly different at P<0.05 level was as per the expectations of various investigators (Oke et al., 1998;Kurose et al., 2012). This observation of significant intra and inter ethnic difference among majority of Indian and non Indian ethnics for GSTT1 and GSTM1 null allele frequency has to be considered critically for the GST-XMEs dependent drug's uptake or dosage of inter-individuality response was in agreement to Kunak et al. (2012). The existing intra ethnic differences among various Indian ethnics were in accordance to various investigators' for GSTM1 but with unexpected significant similarity for GSTT1 (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010). Gujarat was known for its highly polluted environment and the null allele frequency of GSTT1 observed was to be high among the populations. This combined interaction of high pollution and high GSTT1 null frequency among the population, insists the probable susceptible status of Gujaratians for various diseases or disorders and ethnic-variable drug responses. Therefore the population under risk has to be considered for ethnic specific drugs treatments, as recommended earlier by Laing et al. (2011) with precaution to the environmental and genetic risks involved in them.
In conclusion, the distribution pattern of GSTM1 null alleles in Gujarat was not significantly different to other Indian ethnics but differ from most of non Indian ethnics. GSTT1 null allele's frequency of Gujarat population varies among intra ethnics of India and non Indian ethnics of Europe. The data acquainted from this population study adds basic information about the defective null alleles of phase II XMEs such as GSTT1 and GSTM1 in Gujaratians. This information of Gujaratians GSTM1 and GSTT1 null allele's comparisons with other populations suggests the existence of significant deviation in frequency of inter-Indian ethnics and intra-Indian ethnics respectively. The study also reports for the first time the presence of possible GSTT1 dependent drug tolerance/toxicity, variability in drug response among the Gujaratians in India. And the population might be at high risk for susceptibility to the diseases and various cancers associated with null alleles of GSTT1, as the environmental pollution rate was also alarming in the region. On the other hand, the physicians' too have difficulties in treating the ethnics with GSTT1 dependent drugs for various diseases. Further studies in clinical molecular biology are required for eliminating the alarming susceptible status of Gujaratians and their cure without GSTT1 dependent drugs for various cancers or diseases, accustomed to gene-environment interactions and ethnicity.