Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe.

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.