A Nanopore sequencing-based pharmacogenomic panel to personalize tuberculosis drug dosing

Abstract

Rationale: Standardized dosing of anti-tubercular (TB) drugs leads to variable plasma drug levels, which are associated with adverse drug reactions, delayed treatment response, and relapse. Mutations in genes affecting drug metabolism explain considerable interindividual pharmacokinetic variability; however, pharmacogenomic (PGx) assays that predict metabolism of anti-TB drugs have been lacking.

Objectives: To develop a Nanopore sequencing panel and validate its performance in active TB patients to personalize treatment dosing.

Measurements and main results: We developed a Nanopore sequencing panel targeting 15 single nucleotide polymorphisms (SNP) in 5 genes affecting the metabolism of isoniazid (INH), rifampin (RIF), linezolid and bedaquiline. For validation, we sequenced DNA samples (n=48) from the 1000 genomes project and compared variant calling accuracy with Illumina genome sequencing. We then sequenced DNA samples from patients with active TB (n=100) from South Africa on a MinION Mk1C and evaluated the relationship between genotypes and pharmacokinetic parameters for INH and RIF.

Results: The PGx panel achieved 100% concordance with Illumina sequencing in variant identification for the samples from the 1000 Genomes Project. In the clinical cohort, coverage was >100x for 1498/1500 (99.8%) amplicons across the 100 samples. One third (33%) of participants were identified as slow, 47% were intermediate and 20% were rapid isoniazid acetylators. Isoniazid clearance was significantly impacted by acetylator status (p<0.0001) with median (IQR) clearances of 11.2 L/h (9.3-13.4), 27.2 L/h (22.0-31.7), and 45.1 L/h (34.1-51.1) in slow, intermediate, and rapid acetylators. Rifampin clearance was 17.3% (2.50-29.9) lower in individuals with homozygous AADAC rs1803155 G>A substitutions (p=0.0015).

Conclusion: Targeted sequencing can enable detection of polymorphisms influencing TB drug metabolism on a low-cost, portable instrument to personalize dosing for TB treatment or prevention.

Keywords: NAT2; Nanopore; isoniazid; pharmacogenomics; targeted sequencing; tuberculosis.

Figure 1.

(A) Nanopore PGx panel. The top row contains the anti-TB drugs for which pharmacogenomic associations were identified from published studies. Genes and their location on chromosome are listed in rows two and three. Based on the position of targeted SNPs, targets were divided into eight amplicons corresponding to five genes (red). The amplicons are not scaled to their product length. The last row contains information on the positions in pharmacogenes which were included in the PGx panel. (B) Amplicon coverage in PGx panel: Sequencing coverage (log₁₀ scale) per amplicon in 100 samples from the PK cohort, sequenced on a MinION Mk1C sequencer in a single-tube reaction.

Figure 2.

(A) Distribution of homozygous wildtype (purple), homozygous alternate (blue) and heterozygous alleles (yellow) at 15 polymorphic sites in active TB patients (n=100) from PK cohort sequenced on MinION sequencer. (B) NAT2 haplotypes in red are slow acetylator types, those in green are rapid acetylator haplotypes. Connections in red indicate two slow acetylator haplotypes, those in green indicate two rapid haplotypes, and those in yellow indicate one rapid and one slow haplotype (intermediate acetylation)

Figure 3.

Predicted phenotype and INH clearance (A) Predicted NAT2 phenotype and INH clearance in active TB patients (n=100) rapid acetylators (purple) retained plasma drug levels for a shorter period than intermediate (blue) and slow (light blue) predicted phenotypes (p-value= <0.0001). (B) The area under the plasma drug concentration-time curve (AUC) for 0–24hrs was lowest for rapid acetylators (5.80 (4.38–9.48) mg*h/L), moderate for intermediate acetylators (10.7 (7.94–14.6) mg*h/L) and highest in slow acetylators (23.2 (18.3–30.9) mg*h/L)

Figure 4.

(A) AADAC rs1803155 G>T intron mutation and effect on rifampicin clearance: RIF clearance was 16.5% (1.30–29.3) lower in individuals who were homozygous alternate (purple) for AADAC rs1803155 G>A substitutions (p=0.0015) than heterozygous (blue) and wild type (light blue) (B) The area under the plasma drug concentration-time curve (AUC) for 0–24hrs in RIF: Homozygous alternate (purple) for AADAC rs1803155 G>A substitutions heterozygous (blue) and wild type (light blue)