Characterisation of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing

The main aim of this study was to improve the characterisation of complex structural variations (SVs) and copy number variations (CNVs) in the CYP2D6-CYP2D7-CYP2D8 loci, which play a critical role in drug metabolism. These loci, particularly the highly polymorphic CYP2D6 gene, are challenging to accurately genotype due to the presence of pseudogenes (CYP2D7 and CYP2D8) and complex SVs. The study focused on using a CRISPR-Cas9-based enrichment method combined with Oxford Nanopore sequencing to resolve these complexities and improve the accuracy of CYP2D6 genotyping, which is crucial for assigning accurate drug metaboliser status.

The MinION sequencer (Oxford Nanopore Technologies) was used to perform single-molecule sequencing, which, thanks to the long reads, allowed for the continuous capture of the entire 52 kb CYP2D6-CYP2D7-CYP2D8 loci. This method enabled full haplotype phasing and precise characterisation of SVs without the need for PCR amplification, overcoming limitations seen with short-read sequencing technologies.

The major findings of the study demonstrated that the CRISPR-Cas9 enrichment combined with nanopore sequencing successfully identified novel CYP2D6 suballeles and resolved complex diplotypes in multiple samples. The method also uncovered 17 unique CYP2D7 haplotypes and 18 CYP2D8 haplotypes. This approach showed high accuracy in detecting SVs and CNVs, providing a more comprehensive analysis compared to traditional genotyping methods. The study highlights the clinical potential of this method for improving pharmacogenetic testing and drug therapy personalisation by enabling precise CYP2D6 genotyping.