Asia Pac Allergy.  2013 Oct;3(4):231-240. 10.5415/apallergy.2013.3.4.231.

CYP1A2 polymorphism and theophylline clearance in Korean non-smoking asthmatics

• Yim EY
• Kang HR
• Jung JW
• Sohn SW
• Cho SH

• KMID: 2397300
• DOI: http://doi.org/10.5415/apallergy.2013.3.4.231

Abstract

BACKGROUND
Theophylline is mainly metabolized by cytochrome P450 (CYP) 1A2 and CYP2E1 which show inter-individual variations. However, the underlying mechanism remains unknown in humans. We investigated the relationship between differences in theophylline clearance and genetic polymorphisms in the CYP1A2 and CYP2E1 gene in 89 Korean asthmatic patients.
METHODS
Polymerase chain reaction (PCR) was performed on the 5'-flanking region of those genes. PCR products were directly sequenced and confirmed using the SNaP shot method. We determined whether the detected SNPs affected gene transcription using electrophoretic mobility shift assay (EMSA). Theophylline clearance (mL/kg/h) was assessed by using a Bayesian approach.
RESULTS
Genetic polymorphisms were identified at 7 sites in the CYP1A2 gene and at 10 sites in the CYP2E1. Among them, subjects with genotypes (GA+AA) of the -3860G>A polymorphism were found to show higher theophylline clearance than those with genotypes GG (29.11 ± 0.91 mL/kg/h vs. 26.12 ± 0.80 mL/kg/h, p = 0.014). This polymorphic site was revealed to be a protein binding site by conducting EMSA on nuclear hepatocyte extracts.
CONCLUSION
In conclusion, increased theophylline clearance was significantly related to the -3860G>A polymorphism, which could be associated with increased CYP1A2 inducibility in Korean non-smoking asthmatics.

Keyword

Theophylline; Cytochrome P450; CYP1A2; Polymorphism; Clearance; Electrophoretic mobility shift assay

MeSH Terms

Bayes Theorem
Cytochrome P-450 CYP1A2*
Cytochrome P-450 CYP2E1
Cytochrome P-450 Enzyme System
Electrophoretic Mobility Shift Assay
Genotype
Hepatocytes
Humans
Methods
Polymerase Chain Reaction
Polymorphism, Genetic
Polymorphism, Single Nucleotide
Protein Binding
Theophylline*
Cytochrome P-450 CYP1A2
Cytochrome P-450 CYP2E1
Cytochrome P-450 Enzyme System
Theophylline

Figure

• 

  Fig. 1 Genotyping of the CYP1A2 and CYP2E1 gene polymorphisms. Direct sequencing was carried out and seven polymorphic sites in the promoter regions of the (A) CYP1A2 gene and ten polymorphic sites in the promoter regions of the (B) CYP2E1 gene were detected.

• 

  Fig. 2 Theophylline clearance and genetic polymorphisms at -3860 (G>A) of CYP1A2. A positive association was found between theophylline clearance and genotypes including A allele (GA or AA) vs. the GG genotype at -3860 (G>A) of CYP1A2.

• 

  Fig. 3 Electrophoretic Mobility Shift Assay (EMSA) with the polymorphic site of DNAs of 3860G>A polymorphism. Protein DNA-binding affinities of the GG and AA alleles at the -3860 (G>A) region were determined by EMSA. The formation of a complex with the wild or mutant type oligomer was inhibited by the presence of a 50-and 100-fold molar excess of the unlabeled wild type or mutant type oligomer competitor. However, the binding affinity of the oligomer with the A allele was higher than that to the G allele at the -3860G>A point mutation.

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