Diverse point mutations in the human gene for polymorphic N-acetyltransferase.

Abstract

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variation. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetylCoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT] and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Hepatic cytosolic N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5) participates in the detoxification of a plethora of hydrazine and arylamine drugs, as well as in activation pathways of occupational carcinogens (1, 2). A reactive cysteinyl thiolate is part of the active site of NAT (3), which displays a ping-pong reaction mechanism with a covalent acetyl-cysteinyl-NAT species as the catalytic intermediate (3, 4). Hereditary differences in N-acetylation activity among individuals and in populations of diverse raciogeographic origin have led to a phenotypic classification of humans as rapid or slow acetylators. This genetic heterogeneity in NAT activity, widely referred to as the N-acetylation polymorphism, is determined in people and several animal species by a single autosomal gene with two major alleles expressed codominantly (2, 5). On the other hand, rates of elimination in vivo and acetylation in vitro of certain drugs (e.g., p-aminosalicylic acid) do not differ appreciably among rapid and slow acetylators, indicating that metabolism of these substrates takes place without genetic variation. The term monomorphic has been coined for genetically invariant N-acetylation activity (ref. 2, pp. 137-138). The molecular mechanisms for monomorphic and polymorphic N-acetylation in humans are not yet fully understood.The amino acid composition reported for several tryptic peptides of electrophoretically homogeneous liver NAT from homozygous rapid acetylator rabbits (4) proved pivotal in the design of oligonucleotide screening probes and isolation of rabbit NAT cDNAs (6, 7), which eventually enabled identification of human NAT cDNA and genomic clones (7,8). The libraries screened for human NAT cDNAs were made with mRNA from two livers of undetermined acetylator phenotype (7). It was inferred from the substrate selectivity profiles of the cDNA products expressed in Chinese hamster ovary cells that two of the isolated cDNA clones coded for the genetically variant NAT and one coded for the monomorphic protein (7). Three NAT gene loci, corresponding to EcoRI fragments of 1.3 kilobases (kb) (NATJ), 1.9 kb (NAT2), and 4.7 kb (NA TP), were identified upon screening of a library constructed with leukocyte DNA from a heterozygous person. Indirect evidence f...

An improved method for genotyping ofN-acetyltransferase polymorphism by polymerase chain reaction

“…Subsequent study revealed that this allele has the same three nucleotide substitutions i.e. nt (nucleotide position according to Ohsako and Deguchi (1990)) 341,481, and 803 as ra reported recently by Vatsis et al (1991) (manuscript in preparation). Allele 4 is referred to as a 'slow' type allele (Vatsis et al, 1991).…”

“…nt (nucleotide position according to Ohsako and Deguchi (1990)) 341,481, and 803 as ra reported recently by Vatsis et al (1991) (manuscript in preparation). Allele 4 is referred to as a 'slow' type allele (Vatsis et al, 1991). Consequently Nacetylator phenotypes can be predicted by determining which combination of the 4 known alleles an individual has.…”

“…slow, intermediate, and rapid acetylators were correlated with five genotypes generated by combinations of the 3 alleles . Recently we showed that another allele which has no KpnI site (Hickman and Sire, 1991;Blum et al, 1991;Vatsis et al, 1991) also exists among the Japanese, and is designated as allele 4 (Mashimo et al, 1992). Subsequent study revealed that this allele has the same three nucleotide substitutions i.e.…”

N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplotype on urinary bladder cancer risk

“…NAT1 and NAT2 are products of single, intronless exons containing single 870 bp open-reading frames encoding 290 amino acids (Blum et al, 1991;Vatsis et al, 1991). NAT1, NAT2 and a pseudogene NATP are located on the short arm of human chromosome 8 NAT2 polymorphism and urinary bladder cancer risk DW Hein (Blum et al, 1990;Hickman et al, 1994) in the orientation NAT1:NATP:NAT2 (Matas et al, 1997).…”

Chromosomal localization of human genes for arylamine N-acetyltransferase

“…One of the alleles confers the fastacetylator type, whereas the other alleles are associated with slow acetylation (Vatsis et al, 1991;Hickman et al, 1992). The AAC-I locus encodes what has been considered as monomorphic N-acetyltransferase (NAT).…”