LEIDEN, THE NETHERLANDS

Transcription

1 Full-length CYP2D6 diplotyping for better drug dosage and response management Henk Buermans, PhD Leiden University Medical Center Human Genetics, LGTC LEIDEN, THE NETHERLANDS

2 CYP2D6 Function Metabolism of xenobiotics (drugs) ~25% of prescription drugs are processed by CYP2D6 High expression in the Liver High variation between individuals in efficiency and amount of CYP2D6 enzyme If a drug is metabolized too quickly, it may decrease the drug's efficacy while if the drug is metabolized too slowly, toxicity may result Inactive CYP2D6 Tamoxifen 2 active Endoxifen

3 CYP2D6 Variants and protein activity Specific combinations of variants predict the phenotype 3 Poor Metabolizer Little or no CYP2D6 function *3, *4, *5, *6, *11. Intermediate Metabolizers Metabolize drugs at a rate somewhere between the poor and extensive metabolizers *9, *10, *17, *29... Extensive Metabolizer Normal CYP2D6 function *1, *2, *35... Ultrarapid Metabolizer Multiple copies of the CYP2D6 gene are expressed, and therefore greater-than-normal CYP2D6 function *1xN, *2xN...

4 CYP2D6 Variants and protein activity database ( ) 193 variants & 163 haplogroups

5 CYP2D6 Variants and protein activity CYP2D6 variant assay Haplotype per gene copy with variants Diplotype Lookup table for metabolic activity for a specific drug Tailored drug regimen per individual

6 Classical CYP2D6 genotyping methods are sub optimal Fixed features for Array platforms Limited number of variants included Limited haplogroups represented Gaedigk A, International Review of Psychiatry, October 2013; 25(5): No phasing information Most platforms only test whether a duplication is present, but do not: discriminate among duplications quantitatively determine copy number High sequence homology between CYP genes (CYP2D7 pseudogene) Costly (reagents + time) 6

7 Different technologies for CYP2D6 profiling x 5 Entire gene locus: 6,537 bases PharmGKB Roche DMET % 0.45% 0.36% Profile variants with well described association with specific haplotypes

8 PacBio RSII Long read SMRT sequencing Advantages for CYP2D6 genotyping: High Multiplexing Capacity No GC bias (at sequencing level) Long read technology spanning kilobases Sequence the complete CYP2D6 gene High accuracy consensus sequences Variant Phasing Short time to DNA sequence data 8

9 PacBio CYP2D6 Amplicon Setup Two-step Barcoding Scheme 1: locus specific amplification with M13-F and M13-R sequence tails Patient 1 Patient 2 M13-F M13-F locus1 M13-R M13-R 2: Barcoding with re-usable M13 barcode primers index1 Patient 1 M13-F index2 Patient 2 M13-F M13-R locus1 index1 Equimolar pool PacBio RSII M13-R index2

10 PacBio Data Analysis Barcoded samples (n=12) per SMRT cell Sequenced on RSII with P6/C4 Long Amplicon analysis v2 For each individual one or more Haplogroup sequences Re-orient sequences to the plus strand sequence Removal of PCR primer sequences 10

11 PacBio Data Analysis Barcoded samples (n=12) per SMRT cell Align to chr22 Hg38 with BWA MEM Sequenced on RSII with P6/C4 Variant calling with bcftools (v1.2) Long Amplicon analysis v2 LeftAlignAndTrimVariants with GATK For each individual one or more Haplogroup sequences Merge all vcf files Re-orient sequences to the plus strand sequence Removal of PCR primer sequences 11 Describe all variants in HGVS format (Ref = Hg38 genomic positions, plus strand) Call haplotypes. Use the Translation table ( feb 2016) 196 variants & 163 genotypes / haplogroups

12 PacBio CYP2D6 Haplotyping results Two haplogroup sequences Hg-1: 19 variants: 18 snp + 1 ins + 0 del Hg-2: 25 variants: 23 snp + 1 ins + 1 del Hg-1 Hg HOM HET variants for CYP2D6 *4A no activity 14 variants for CYP2D6 *41 decreased activity diplotype: CYP2D6 *4A/*41

13 PacBio CYP2D6 Haplotyping results

14 PacBio CYP2D6 Haplotyping results Gene duplication Increased subread coverage ratio PCR for deletion: Negative PCR for duplication: Positive 14 Hg1 *1A Hg2 *2A 1x product for Hg1 *2A Double yield for Hg2

15 PacBio CYP2D6 Haplotyping results One Haplogroup sequence PCR for deletion: Negative PCR for duplication: Positive Hg1 Gene duplication 15 Hg2 *2A *2A *2A

16 PacBio CYP2D6 Haplotyping results Gene Deletion events One Haplogroup sequence Hg1 Hg2 16 *2A PCR for deletion: Positive PCR for duplication: Negative Only LR-PCR product for Hg1 *5 gene deletion

17 CYP2D6 Variant Effects Total 40 samples 71 unique variants Ensembl VEP Consequences upstream_gene_variant: 48% intron_variant: 24% non_coding_transcript_variant: 13% downstream_gene_variant: 4% missense_variant: 4% synonymous_variant: 2% frameshift_variant: 2% splice_region_variant: 1% non_coding_transcript_exon_variant: 1% Others Coding Consequences missense_variant: 51% synonymous_variant: 24% frameshift_variant: 19% inframe_deletion: 5%

18 CYP2D6 Variant Effects Total 40 samples 71 unique variants NOT listed in PharmGKB database: with rsid downstream_gene_variant 2 intron_variant 4 upstream_gene_variant 4 frameshift_variant 1 missense_variant 2 Individual also had a CYP2D6 *5 gene deletion 7x G 6x G Needs re-analysis 26 No rsid PolyPhen & Sift Prediction 22:g C>T benign(0.041) & tolerated(0.22) PolyPhen & Sift Prediction 22:g C>T [rs ] benign(0.003) & tolerated(0.22) 22:g C>T [rs ] benign(0.003) % deleterious(0.05)

19 Novel CYP2D6 variants confirmed 22:g _ delTGT 22:g G>C * Reference deltgt Present in 80% of all individuals in this study Present in one individual in this study

20 Conclusion & Discussion PacBio Long Read CYP2D6 haplotyping outperforms existing technologies High multiplexing capabilities Full length vs selected variants Variant Phasing Accurate variant calls haplotype calls Gene duplications can be detected Run in parallel with PCR for Gene deletions, duplications and CYP2D6-7 fusion eventsplus Found many additional variants that had not previously been associated with the defined haplogroups Novel Variants detected & Confirmed 20

21 Acknowledgements Dept for Human Genetics / LGTC Johan den Dunnen Stefan White Guy Allard Yahya Anvar Rolf Vossen Dept. Clinical Pharmacy and Toxicology Tahar van der Straaten Paul Bank Henk-Jan Guchelaar Jesse Swen Contact: info@lgtc.nl