Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule...

Area of Science:

• Genomics
• Pharmacogenomics
• Molecular Biology

Background:

• Complex genomic regions, including pseudogenes and structural variations (SVs), challenge accurate genetic analysis with short-read sequencing.
• The CYP2D6 locus is critical for drug metabolism but contains highly similar pseudogenes (CYP2D7, CYP2D8) and complex SVs, hindering accurate genotyping.
• Inaccurate CYP2D6 genotyping can lead to incorrect enzyme activity assignments, impacting drug dosing and disproportionately affecting certain populations.

Purpose of the Study:

• To develop a novel method for accurate and comprehensive genotyping of the entire CYP2D6-CYP2D7-CYP2D8 loci.
• To overcome the limitations of existing sequencing technologies in resolving complex structural variations within this pharmacogenomic region.
• To enable precise determination of CYP2D6 diplotypes for improved clinical phenotyping and drug therapy guidance.

Main Methods:

• Developed a PCR-free CRISPR-Cas9 enrichment strategy for targeted long-read sequencing.
• Applied the method to clinically relevant samples (blood, saliva, liver tissue).
• Generated high-coverage, continuous long reads (up to 52 kb) to span the entire loci, including structural variations.

Main Results:

• Successfully generated continuous long reads that fully characterized the CYP2D6-CYP2D7-CYP2D8 loci, irrespective of SVs.
• Enabled phased dissection of the loci structure, accurately resolving complex CYP2D6 diplotypes with a single assay.
• Identified three novel CYP2D6 suballeles and fully characterized 17 CYP2D7 and 18 CYP2D8 unique haplotypes.

Conclusions:

• The developed CRISPR-Cas9 enrichment and long-read sequencing method provides accurate and comprehensive CYP2D6 genotyping.
• This approach significantly improves the ability to resolve complex genetic variations within challenging genomic regions.
• The method holds potential for enhancing clinical phenotyping, informing drug therapy, and can be adapted for other difficult-to-analyze genomic loci.