Quantitative trait locus analysis reveals two intragenic sites that influence O6 -alkylguanine-DNA alkyltransferase activity in peripheral blood mononuclear cells

Abstract

The repair of specific types of DNA alkylation damage by O6-alkylguanine-DNA alkyltransferase (MGMT) is a major mechanism of resistance to the carcinogenic and chemotherapeutic effects of certain alkylating agents. MGMT expression levels vary widely between individuals but the underlying causes of this variability are not known. To address this, we used an expressed single nucleotide polymorphism (SNP) and demonstrated that the MGMT alleles are frequently expressed at different levels in peripheral blood mononuclear cells (PBMC). This suggests that there is a genetic component of inter-allelic variation of MGMT levels that maps close to or within the MGMT locus. We then used quantitative trait locus (QTL) analysis using intragenic SNPs and found that there are at least two sites influencing inter-individual variation in PBMC MGMT activity. One is characterized by an SNP at the 3' end of the first intron and the second by two SNPs in the last exon. The latter are in perfect disequilibrium and both result in amino acid substitutions-one of them, Ile143Val, affecting an amino acid close to the Cys145 residue at the active site of MGMT. Using in vitro assays, we further showed that while the Val143 variant did not affect the activity of the protein on methylated DNA substrate, it was more resistant to inactivation by the MGMT pseudosubstrate, O6-(4-bromothenyl)guanine. These findings suggest that further investigations of the potential epidemiological and clinical significance of inherited differences in MGMT expression and activity are warranted.

Differential inactivation of polymorphic variants of human O6-alkylguanine-DNA alkyltransferase

“…The alteration of Val for Ile143 in the binding pocket may cause subtle changes that affect these interactions. Our results are in agreement with a recent report that there was a small difference in the sensitivity to inactivation by PaTrin-2 when I143V/K178R and I143V variants were compared to wild type [24]. Although the increase in ED 50 values were not calculated in these experiments there was 76% loss of activity with wild type and only 67% loss with the I143V alteration with 10 μM Patrin-2 suggesting that the alteration is similar in magnitude to that seen with BG.…”

“…The Ile143 is located in the active site pocket and is very close to the Cys145 acceptor site. However, this protein appears not to differ from wild type in the repair of methylated DNA [19,24,39]. This is not surprising since the position equivalent to Ile143 in hAGT is variable when sequences from a wide range of organisms are compared with the most common alteration being the presence of Val rather than Ile.…”

“…The SNPS leading to the I143V and K178R changes are in almost perfect disequilibrium [17,18,24,30] and it is highly likely that both changes occur in the protein derived from this gene. In order to examine the extent to which two linked alterations contribute to the resistance to inhibitors observed, N-terminal His 6 -tagged hAGT proteins with the individual mutations were produced separately and tested (Table 3).…”

“…Variants W65C [15,16], L84F [15][16][17][18][19][20][21][22][23][24][25][26][27][28], I143V/ K178R [17][18][19][20][21][22][23][24]27,[29][30][31] and G160R [29,32] have been reported by multiple laboratories. These reports raise the possibility that there may be individual variation in response to hAGT inactivators.…”

“…However, subsequent studies have shown that the frequency of the G160R variant is very low ( >1%) [17,29,30,34,35]. Recently, it was reported that the I143V/K178R variant, which is much more common with a frequency of c. 24%, may be resistant to PaTrin-2 [24].…”