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Genotyping, characterization, and imputation of known and novel CYP2A6 structural variants using SNP array data

Journal of Human Genetics volume 68, pages 533–541 (2023)Cite this article

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Abstract

CYP2A6 metabolically inactivates nicotine. Faster CYP2A6 activity is associated with heavier smoking and higher lung cancer risk. The CYP2A6 gene is polymorphic, including functional structural variants (SV) such as gene deletions (CYP2A6*4), duplications (CYP2A6*1 × 2), and hybrids with the CYP2A7 pseudogene (CYP2A6*12, CYP2A6*34). SVs are challenging to genotype due to their complex genetic architecture. Our aims were to develop a reliable protocol for SV genotyping, functionally phenotype known and novel SVs, and investigate the feasibility of CYP2A6 SV imputation from SNP array data in two ancestry populations. European- (EUR; n = 935) and African- (AFR; n = 964) ancestry individuals from smoking cessation trials were genotyped for SNPs using an Illumina array and for CYP2A6 SVs using Taqman copy number (CN) assays. SV-specific PCR amplification and Sanger sequencing was used to characterize a novel SV. Individuals with SVs were phenotyped using the nicotine metabolite ratio, a biomarker of CYP2A6 activity. SV diplotype and SNP array data were integrated and phased to generate ancestry-specific SV reference panels. Leave-one-out cross-validation was used to investigate the feasibility of CYP2A6 SV imputation. A minimal protocol requiring three Taqman CN assays for CYP2A6 SV genotyping was developed and known SV associations with activity were replicated. The first domain swap CYP2A6-CYP2A7 hybrid SV, CYP2A6*53, was identified, sequenced, and associated with lower CYP2A6 activity. In both EURs and AFRs, most SV alleles were identified using imputation (>70% and >60%, respectively); importantly, false positive rates were <1%. These results confirm that CYP2A6 SV imputation can identify most SV alleles, including a novel SV.

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Data availability

Full exon and breakpoint DNA sequences for the CYP2A6*53 SV are available in Supplementary Fig. 1.

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Acknowledgements

We acknowledge the contributions of Dr. Caryn Lerman in the design and administration of the Pharmacogenetics of Nicotine Addiction Treatment 2 clinical trial (NCT01314001), of Dr. Michiaki Kubo in contributing to the deep exon sequencing and initial Taqman CNV assay genotyping of participants, and of Dr. Andrea Gaedigk and the CYP2A6 Pharmvar expert panel in naming the novel structural variant, CYP2A6*53, and inspiring the CYP2A6 structural variant schematics in Fig. 1. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (Tyndale, the Canada Research Chair in Pharmacogenomics), Canadian Institutes of Health Research (Tyndale, FDN-154294; Tyndale, Knight, and Chenoweth, PJY-159710); National Institute on Drug Abuse (Tyndale, PGRN U01-DA20830), and National Institutes of Cancer (Cox, NCI CA091912) as well as the Centre for Addiction and Mental Health and the CAMH Foundation. JSA funded in part by P20GM130414, a NIH funded Center of Biomedical Research Excellence (COBRE).

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, University of Toronto, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Alec W. R. Langlois, Ahmed El-Boraie, Meghan J. Chenoweth & Rachel F. Tyndale

  2. Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, 100 Stokes Street, Toronto, ON, M6J 1H4, Canada

    Alec W. R. Langlois, Ahmed El-Boraie, Jennie G. Pouget, Meghan J. Chenoweth & Rachel F. Tyndale

  3. Department of Psychiatry, University of Toronto, 250 College Street, Toronto, ON, M5T 1R8, Canada

    Jennie G. Pouget, Meghan J. Chenoweth & Rachel F. Tyndale

  4. Department of Population Health, University of Kansas School of Medicine, Kansas City, KS, 66160, USA

    Lisa Sanderson Cox

  5. Departments of Behavioral and Social Sciences and Medicine, Brown University School of Public Health, Providence, RI, 02912, USA

    Jasjit S. Ahluwalia

  6. Center for Integrative Medical Sciences, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

    Koya Fukunaga & Taisei Mushiroda

  7. Data Science Institute and Medical School, Lancaster University, Lancaster, UK

    Jo Knight

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Contributions

AL, JP, JK, MC, and RT designed the research; AE, LC, JA, KF, and TM provided resources, AL performed the research; AL analyzed the data; AL and RT wrote the manuscript; all authors contributed to editing the manuscript.

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Correspondence to Rachel F. Tyndale.

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Some of the authors declared financial and non-financial relationships and activities, and conflicts of interest regarding this manuscript as indicated in the Supplementary Materials.

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Ethical approval for these analyses was obtained from the University of Toronto (PNAT2 REB number: 25510; KIS3 REB number: 38361) and at all original participating sites of the trials.

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Langlois, A.W.R., El-Boraie, A., Pouget, J.G. et al. Genotyping, characterization, and imputation of known and novel CYP2A6 structural variants using SNP array data. J Hum Genet 68, 533–541 (2023). https://doi.org/10.1038/s10038-023-01148-y

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