Functional effects of genetic polymorphisms in the N-acetyltransferase 1 coding and 3' untranslated regions

Abstract

Background: The functional effects of N-acetyltransferase 1 (NAT1) polymorphisms and haplotypes are poorly understood, compromising the validity of associations reported with diseases, including birth defects and numerous cancers.

Methods: We investigated the effects of genetic polymorphisms within the NAT1 coding region and the 3'-untranslated region (3'-UTR) and their associated haplotypes on N- and O-acetyltransferase catalytic activities, and NAT1 mRNA and protein levels following recombinant expression in COS-1 cells.

Results: 1088T>A (rs1057126; 3'-UTR) and 1095C>A (rs15561; 3'-UTR) each slightly reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. A 9-bp (TAATAATAA) deletion between nucleotides 1065 and 1090 (3'-UTR) reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. In contrast, a 445G>A (rs4987076; V149I), 459G>A (rs4986990; T153T), and 640T>G (rs4986783; S214A) coding region haplotype present in NAT1*11 increased NAT1 catalytic activity and NAT1 protein, but not NAT1 mRNA levels. A combination of the 9-bp (TAATAATAA) deletion and the 445G>A, 459G>A, and 640T>G coding region haplotypes, both present in NAT1*11, appeared to neutralize the opposing effects on NAT1 protein and catalytic activity, resulting in levels of NAT1 protein and catalytic activity that did not differ significantly from the NAT1*4 reference.

Conclusions: Because 1095C>A (3'-UTR) is the sole polymorphism present in NAT1*3, our data suggest that NAT1*3 is not functionally equivalent to the NAT1*4 reference. Furthermore, our findings provide biologic support for reported associations of 1088T>A and 1095C>A polymorphisms with birth defects.

Figure 1

Vector structure of the cloned NAT1 fragments containing the 873-bp NAT1 open reading frame (ORF) and 302 bp of the NAT1 3′-UTR. NAT1 mRNA transcription was driven by cytomegalovirus (CMV) promoter, whereas RNA processing at the 3-end was facilitated by the NAT1 putative polyA signals since the BGH polyA signal in the vector was deleted.

Figure 2

Effects of 1088T>A and 1095C>A on NAT1 activities towards PABA and ABP N-acetyltransferase, and N-OH-PhIP O-acetyltransferase activities and NAT1 mRNA. Each bar represents Mean ± SEM for three transfections. a) p<0.05; b) p<0.01; c) p<0.001 significantly different from the NAT1*4 reference.

Figure 3

Effects of NAT1 polymorphisms on protein expression. Ratios of NAT1 protein to beta-galactosidase protein are shown following densitometric scanning of the representative Western blots shown above the graph. Top Western blot: A: Positive control (recombinant NAT1 from yeast); B: Negative control (mock transfection); C: NAT1*4 reference; D: 1095C>A; E: 1088T>A. Bottom Western blot: A: 9-bp deletion haplotype; B: 445G>A, 459G>A, 640T>G haplotype; C: NAT1*4 reference; D: Combination 445G>A, 459G>A, 640T>G, 9-bp deletion haplotype; E: Negative control (mock transfection); F: Positive control (recombinant NAT1 from yeast). Each bar represents Mean ± SEM for three transfections. a) p<0.001 from NAT1*4 reference; b) p<0.01 from 445G>A, 459G>A, 640T>G haplotype; c) p<0.05 from combination 445G>A, 459G>A, 640T>G, 9-bp deletion haplotype.

Figure 4

Effects of combination 445G>A, 459G>A, and 640T>G and 9-bp (TAATAATAA) deletion between 1065-1090 in the 3′-UTR on PABA and ABP N-acetyltransferase and N-OH-PhIP O-acetyltransferase activities and NAT1 mRNA. Each bar represents Mean ± SEM for three transfections. a) p<0.001 from 9-bp deletion; b) p<0.05 from NAT1*4 (REF); c) p<0.01 from 9-bp deletion; d) p<0.01 from NAT1*4 (REF); e) p<0.01 from combination 445G>A, 459G>A, and 640T>G and 9-bp deletion haplotype; f) p<0.05 from 9-bp deletion.