High-Resolution Melt Analysis to Detect Sequence Variations in Highly Homologous Gene Regions: Application to CYP2B6

Abstract

High-resolution melt (HRM) analysis using ‘release-on-demand‘ dyes, such as EvaGreen^® has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African–Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.

KEYWORDS::

• CYP2B6
• CYP2B7
• genotyping
• high-resolution melt analysis
• HRM
• SNPs

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The content of this article does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the NIH.

Financial & competing interests disclosure

This project was supported in part by a grant from the Tom Keaveny Endowed Fund for Pediatric Cancer Research. The Liver Tissue Cell Distribution System was funded by NIH Contract #N01-DK-7-0004/HHSN267200700004C. The project entitled ‘Laboratory of Developmental Biology‘ was supported by NIH Award Number 5R24HD0008836 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. The authors are also grateful to Xenotech, LLC for their generous gift of tissue samples. Furthermore, the authors would like to thank L Ndjountché and A Bagherian for technical assistance. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

This project was supported in part by a grant from the Tom Keaveny Endowed Fund for Pediatric Cancer Research. The Liver Tissue Cell Distribution System was funded by NIH Contract #N01-DK-7-0004/HHSN267200700004C. The project entitled ‘Laboratory of Developmental Biology‘ was supported by NIH Award Number 5R24HD0008836 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. The authors are also grateful to Xenotech, LLC for their generous gift of tissue samples. Furthermore, the authors would like to thank L Ndjountché and A Bagherian for technical assistance. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.