Characterization of NAT2 Using Long-Read Sequencing: Allele, Diplotype, and Phenotype Call Accuracy Compared to Other Testing Strategies.

NAT2 variants determine acetylation capacity for aromatic amines and hydrazines; variant phasing, beyond the reach of targeted genotyping and short-read sequencing, governs diplotype and then phenotype assignment.

NAT2 typing, which classifies individuals as rapid, intermediate or slow acetylators, is limited by the inability of targeted genotyping and short-read sequencing to phase variants, a source of ambiguous diplotype and phenotype calls. The authors compared long-read sequencing, short-read sequencing and simulated SNP panels across 1,828 long-read and 662 paired short-read patient samples from the Genomic Answers for Kids program, using a custom tool, staR-NAT2, which identified 11 new star alleles and four new suballeles. Long-read data achieved over 99% diplotype accuracy with pb-StarPhase and Aldy, excluding novel haplotypes, against 64% diplotype concordance for short-reads, a gap that nonetheless altered only 4% of phenotype calls. The 4-SNP and 5-SNP panels predicted phenotype correctly in more than 95% of subjects, but yielded discordant or ambiguous phenotypes in 4.5% and 25% of non-white individuals respectively. Among 1,144 unrelated individuals, population-specific alleles were identified, including alleles 14 (8.9%) and 43 (3.57%) in Black individuals and allele 7 (7.35%) in Hispanic individuals.

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The paper's own data partly undercut its conclusion: if a 4-SNP panel already predicts the correct phenotype in more than 95% of cases, the superiority of long-read sequencing lies in diplotype resolution, not in the treatment decision. The argument that holds is equity, with 25% discordant or ambiguous phenotypes in non-white individuals using the 5-SNP panel, a bias long-read sequencing genuinely corrects. That said, NAT2 carries no level A dosing recommendation: what is being refined here is a typing method whose clinical use remains to be demonstrated, precisely the criticism levelled this week at dolutegravir pharmacogenomics.

Analysis by Dr Thibaut Benquey