Dataset Information

A novel simple method for determining CYP2D6 gene copy number and identifying allele(s) with duplication/multiplication.

ABSTRACT:

Background

Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.

Methods

We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.

Results

The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.

Conclusions

Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.

SUBMITTER: Langaee T

PROVIDER: S-EPMC4308104 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A novel simple method for determining CYP2D6 gene copy number and identifying allele(s) with duplication/multiplication.

Langaee Taimour T Hamadeh Issam I Chapman Arlene B AB Gums John G JG Johnson Julie A JA

PloS one 20150127 1

<h4>Background</h4>Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.<h4>Methods</h4>We developed a simple allele quantification-based Pyro ...[more]

PMID: 25625348

Similar Datasets

Project description:BackgroundCYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the "One-pot" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5'UTR, intron 6, and exon 9 regions.MethodsGenomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA.ResultsOverall, results between the two platforms and digestions methods were consistent. The "One-pot" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference.ConclusionThe Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

Dataset Information

A novel simple method for determining CYP2D6 gene copy number and identifying allele(s) with duplication/multiplication.

Background

Methods

Results

Conclusions

Publications

A novel simple method for determining CYP2D6 gene copy number and identifying allele(s) with duplication/multiplication.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets