Identification and characterization of CYP3A420, a novel rare CYP3A4 allele without functional activity

Background The major drug‐metabolizing enzyme cytochrome P450 (CYP) 3A4 is genetically conserved. One outlier of Brazilian descent was found in a clinical pharmacokinetic trial exhibiting a 6‐fold higher exposure than expected to an investigational drug, shown to be a CYP3A4 substrate. We aimed to investigate the genetic background of this finding. Methods The allelic variant of the CYP3A4 gene present in the outlier was sequenced, and the corresponding complementary deoxyribonucleic acid was expressed in yeast and human embryonic kidney cells. The outlier was phenotyped by use of intravenous administration of 1 mg midazolam. Analysis of phenotype and genotype correlation was carried out. The prevalence of the new allele was screened for in a white population. Results We identified a subject who heterozygously carried a novel CYP3A4 allele, named CYP3A4*20, with a premature stop codon yielding a truncated protein. Heterologous expression revealed that the CYP3A4.20 enzyme does not incorporate heme and thus is devoid of catalytic activity. CYP3A phenotyping in vivo showed that CYP3A4*20 exhibits a clear genotype‐phenotype correlation, demonstrated by the subject's low systemic midazolam clearance (2.99 mL · min−1 · kg−1). Genotyping of a white German population (n=428) and relatives of the subject, as well as a review of published CYP3A4 sequencing data, suggests that CYP3A4*20 is a rare variant allele (