PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus

Charnaud, Sarah C.; Munro, Jacob E.; Semenec, Lucie; Mazhari, Ramin; Brewster, Jessica; Bourke, Caitlin; Ruybal‐Pesántez, Shazia; James, Rob S.; Lautu-Gumal, Dulcie; Karunajeewa, Harin; Müeller, Ivo; Bahlo, Melanie

doi:10.1038/s42003-022-03102-8

Cited by 15 publications

(17 citation statements)

References 46 publications

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“…However, this is speculative and has yet to be tested. Other reports, in Micronesian populations, have described allele frequencies of 0.009 in a Ni-Vanuatu sample (Gutiérrez Rico et al, 2020), significantly less than our 0.092 (P = 3.9×10 -10 ) and 0.058 in a large sample of Solomon Islanders (Charnaud et al, 2022), comparable but significantly less than our 0.092 (P=0.038).…”

Section: Discussioncontrasting

confidence: 74%

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Māori and Pacific peoples

Hitchman

Faatoese

Merriman

et al. 2022

Preprint

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The enzyme cytochrome P450 2D6 (CYP2D6) metabolises approximately 25% of commonly prescribed drugs, including analgesics, anti-hypertensives, and anti-depressants, among many others. Genetic variation in drug metabolising genes can alter how an individual responds to prescribed drugs, including predisposing to adverse drug reactions. The majority of research on the CYP2D6 gene has been carried out in European and East Asian populations, with Indigenous and minority populations greatly underrepresented. However, genetic variation is often population specific and analysis of diverse ethnic groups can reveal differences in alleles that may be of clinical significance. For this reason, we set out to examine the range and frequency of CYP2D6 variants in a sample of 202 Māori and Pacific people living in Aotearoa (New Zealand). We carried out a long PCR to isolate the CYP2D6 region before performing nanopore sequencing to identify all variants and alleles in these samples. We identified eleven novel variants, three of which were exonic missense variations. Six of these occurred in single samples and one was found in 19 samples (9.4% of the cohort). The remaining four novel variants were identified in two samples each. In addition, five new suballeles of CYP2D6 were identified. One striking finding was that CYP2D6*71, an allele of unknown functional status which has been rarely observed in previous studies, occurs at a relatively high frequency (9.2%) within this cohort. These data will help to ensure that CYP2D6 genetic analysis for pharmacogenetic purposes can be carried out accurately and effectively in this population group.

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Section: Discussioncontrasting

confidence: 74%

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Māori and Pacific peoples

Hitchman

Faatoese

Merriman

et al. 2022

Preprint

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“… a Reference sequence NG_008376.4 (1 = sequence start). b Variant also identified by Charnaud et al (2022) . Italic values are Star allele designations.…”

Section: Resultsmentioning

confidence: 99%

“…We identified nine known star alleles in this cohort, including CYP2D6*71, a previously identified rare allele of uncertain function, which comprised ~9% of the alleles detected. The frequency of this allele prevented us from inferring metabolic b Variant also identified by Charnaud et al (2022).…”

Section: Discussionmentioning

confidence: 99%

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Māori and Pacific peoples

et al. 2022

View full text Add to dashboard Cite

The enzyme cytochrome P450 2D6 (CYP2D6) metabolises approximately 25% of commonly prescribed drugs, including analgesics, anti-hypertensives, and anti-depressants, among many others. Genetic variation in drug metabolising genes can alter how an individual responds to prescribed drugs, including predisposing to adverse drug reactions. The majority of research on the CYP2D6 gene has been carried out in European and East Asian populations, with many Indigenous and minority populations, such as those from Oceania, greatly underrepresented. However, genetic variation is often population specific and analysis of diverse ethnic groups can reveal differences in alleles that may be of clinical significance. For this reason, we set out to examine the range and frequency of CYP2D6 variants in a sample of 202 Māori and Pacific people living in Aotearoa (New Zealand). We carried out long PCR to isolate the CYP2D6 region before performing nanopore sequencing to identify all variants and alleles in these samples. We identified twelve variants which have previously not been reported in the PharmVar CYP2D6 database, three of which were exonic missense variations. Six of these occurred in single samples and one was found in 19 samples (9.4% of the cohort). The remaining five variants were identified in two samples each. Identified variants formed twelve new CYP2D6 suballeles and four new star alleles, now recorded in the PharmVar database. One striking finding was that CYP2D6*71, an allele of uncertain functional status which has been rarely observed in previous studies, occurs at a relatively high frequency (8.9%) within this cohort. These data will help to ensure that CYP2D6 genetic analysis for pharmacogenetic purposes can be carried out accurately and effectively in this population group.

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“…Data were crossvalidated versus previous data from multiple technologies (Carvalho Henriques et al, 2021b), and many of the resulting percentage alignments were above 99%. This method would therefore appear to be more accurate and efficient than any of the other SMRT HiFi methodologies reported to date (Fukunaga et al, 2021;van der Lee et al, 2021;Charnaud et al, 2022) for the detection and characterization of CYP2D6 fusion genes. In addition, we have since used the forward primer for the E amplicon and the reverse primer for the G amplicon to generate an L-PCR product for non-characterization apart from a minority of CYP2D7-2D6 hybrids for which the H amplicon appears to be required.…”

Section: Discussionmentioning

confidence: 98%

“…In the tamoxifen-treated dataset, only four individuals with hybrids were identified by the SMRT, and the hybrid haplotype was not specified (Supplementary Table S2; van der Lee et al, 2021). The latest relevant SMRT paper used a long-range polymerase chain reaction (L-PCR) technique that amplified a 6.1 kb region spanning from 712 bp upstream to 1176 bp downstream of the NB_008376.4 CYP2D6 RefSeq coding sequence, and, in addition (by design), amplified the corresponding region from CYP2D7 , generating a 7.6 kb amplicon in an analysis of 377 Solomon Islanders (Charnaud et al, 2022). From the SMRT data, 27/365 (7.6%) samples appeared to have a CYP2D6-2D7 fusion haplotype with breakpoints in exon 8 (consistent with a CYP2D6*63) , and 7/365 (2%) samples appeared to have CYP2D7-2D6 fusions (CYP2D6*13) However, there was a degree of discrepancy between the above and TaqMan CNV intron 9 and exon 9 data, with not all of the samples with a CYP2D6-2D7 predicted fusion haplotype having a higher intron 2 than exon 9 CNV count, and 1/7 of the predicted CYP2D7-2D6 fusions not having a high exon 9 count.…”

Section: Introductionmentioning

confidence: 99%

Single molecule long-read real-time amplicon-based sequencing ofCYP2D6: a proof-of-concept with hybrid haplotypes

Dong

Thamilselvan

et al. 2022

Preprint

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CYP2D6 is a widely expressed human xenobiotic metabolizing enzyme, best known for its role in the hepatic phase I metabolism of up to 25% of prescribed medications, which is also expressed in other organs including the brain, where its potential role in physiology and mental health traits and disorders is under further investigation. Owing to the presence of homologous pseudogenes in the CYP2D locus and transposable repeat elements in the intergenic regions, the gene encoding the CYP2D6 enzyme, CYP2D6, is one of the most hypervariable known human genes - with more than 140 core haplotypes. Haplotypes include structural variants, with a subtype of these known as fusion genes comprising part of CYP2D6 and part of its adjacent pseudogene, CYP2D7. The fusion genes are particularly challenging to identify. The CYP2D6 enzyme activity corresponding to some of these fusion genes is known, while for others it is unknown. The most recent (high fidelity, or HiFi) version of single molecule real-time (SMRT) long-read sequencing can cover whole CYP2D6 haplotypes in a single continuous sequence read, ideal for structural variant detection. In addition, the accuracy of base calling has increased to a level sufficient for accurate characterization of single nucleotide variants. As new CYP2D6 haplotypes are continuously being discovered, and likely many more remain to be identified in populations that are relatively understudied to date, a method of characterization that employs sequencing with at least this degree of accuracy is required. The aim of the work reported herein was to develop an efficient and accurate HiFi SMRT amplicon-based method capable of detecting the full range of CYP2D6 haplotypes including fusion genes. We report proof-of-concept for 20 amplicons, aligned to fusion gene haplotypes, with prior cross-validation data. Amplicons with CYP2D6-D7 fusion genes aligned to *36, *63, *68, and *4 (*4-like; *4N, or *4.013) hybrid haplotypes. Amplicons with CYP2D7-D6 fusion genes aligned to the *13 subhaplotypes predicted (e.g., *13F, *13A2). Data analysis was efficient, and further method development indicates that this technique could suffice for the characterization of the full range of CYP2D6 haplotypes. Although included in drug labelling by regulatory bodies (the U.S. Food and Drug Administration, the European Medicines Agency, the Pharmaceuticals and Medical Devices Agency) and prescribing recommendations by consortia (Clinical Pharmacogenetics Implementation Consortium and the Dutch Pharmacogenetics Working Group), the identification of CYP2D6 variants is not yet routine in clinical practice. The HiFi sequencing method reported herein is suitable for high throughput, efficient, identification of the full range of known CYP2D6 haplotypes and novel haplotypes, and can be completed in a week or less. Moreover, the method that we have developed could be extended to other complex loci and to other species in a multiplexed high throughput assay.

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PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus

Cited by 15 publications

References 46 publications

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Māori and Pacific peoples

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Māori and Pacific peoples

Allelic diversity of the pharmacogene CYP2D6 in New Zealand Māori and Pacific peoples

Single molecule long-read real-time amplicon-based sequencing ofCYP2D6: a proof-of-concept with hybrid haplotypes

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