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 bee n reported, their relationship to phenotypic variations in NAT1 functi on remains unclear. We have used in-vivo and in-vitro phenotyping test s, along with PCR-based cloning and heterologous expression, to invest igate the extent of variation in NAT1 function and to characterize nov el allelic variants at the NAT1 gene locus. The NAT1-selective substra te 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 o ral ingestion of Nemasol Sodium(R). Excluding outliers, a 65-fold vari ation 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 moderate ly (r = 0.74, p < 0.0005). One individual (case 244) had a marked impa irment of PAS N-acetylation, with 10-fold lower urinary and plasma AcP AS: PAS ratios compared with other subjects. Biochemical investigation s 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 decrease d V-max for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from sev en other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT114 and NAT1*15, in case 2 44, as well as one variant, NAT111, which has been observed in previo us investigations. NAT114 contained a missense mutation (G(560)-->A) that is predicted to change a single amino acid (Arg(187)-->Gln), as w ell as two 3' non-coding region mutations (T-1088-->A and C-1095-->A) that have previously been observed in the NAT110 allelic variant. NAT 115 had a single nonsense mutation (C-559-->T; Arg(187)-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT114 mirrored the defective enzyme function in whole blood lysates from ca se 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on t he other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functi onally silent. The frequencies of NAT111, NAT1*14 and NAT1*15 were 0. 021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in sample. (C) 1998 Chapman & Hall Ltd.