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

Area of Science:

• Pharmacogenomics
• Genetics
• Drug Metabolism

Background:

• The N-acetyltransferase 2 (NAT2) gene is crucial for metabolizing drugs like aromatic amines and hydrazines.
• Genetic variations in NAT2 determine individual acetylation capacity, classifying people as rapid, intermediate, or poor metabolizers for drug dosing.
• Current short-read sequencing methods struggle with variant phasing, leading to ambiguous NAT2 diplotype and phenotype calls.

Purpose of the Study:

• To evaluate the accuracy of long-read sequencing for NAT2 variant phasing and diplotype determination compared to short-read sequencing.
• To identify novel NAT2 alleles and assess the performance of different genotyping strategies in a large patient cohort.
• To highlight the impact of sequencing technology on the accuracy and equity of pharmacogenetic testing.

Main Methods:

• Utilized long-read sequencing on 1828 samples and paired short-read sequencing on 662 samples from the Genomic Answers for Kids program.
• Employed custom tool staR-NAT2 for variant identification and phasing tools (pb-StarPhase, Aldy) for diplotype accuracy assessment.
• Compared long-read results with short-read data and simulated SNP panels, analyzing phenotype prediction accuracy and population-specific allele frequencies.

Main Results:

• Long-read sequencing achieved >99% diplotype accuracy, unequivocally establishing genotypes.
• Short-read sequencing showed significantly lower diplotype concordance (64%) but still predicted phenotype accurately for most individuals (>95%).
• Short-read based SNP panels exhibited significant discordance and ambiguity for non-white populations, revealing population-specific NAT2 alleles.

Conclusions:

• Long-read sequencing offers superior accuracy and phasing capabilities for comprehensive NAT2 pharmacogenetic testing.
• Short-read sequencing and SNP panels have limitations in resolving complex genotypes and ensuring equitable testing across diverse populations.
• Accurate NAT2 genotyping via long-read sequencing is essential for precise, individualized drug therapy and reducing health disparities.