Identification and phenotype characterization of two CYP3A haplotypes causing different enzymatic capacity in fetal livers

Abstract

BACKGROUND: The fetal liver cytochrome P450 (CYP) 3A enzymes metabolize potentially toxic and teratogenic substrates and drugs in addition to endogenous hormones and differentiation factors. CYP3A7 is the most abundant CYP in the human liver during fetal stages and the first months of postnatal age and shows a large interindividual variability of unknown molecular basis. METHODS: A new variant gene (CUpsilonP3A7*2), which carries a mutation in exon 11 of CUpsilonP3A7 causing a T409R substitution, was identified by direct sequencing. Genotype analysis was performed by use of polymerase chain reaction followed by restriction enzyme analysis. CYP3A7.2 activity was assessed in heterologous expression systems and human fetal liver microsomes. RESULTS: The frequency of CUpsilonP3A7*2 was 8%, 17%, 28%, and 62% in white, Saudi Arabian, Chinese, and Tanzanian individuals, respectively. By use of human HEK293 cells, no significant differences in expression between CYP3A7.1 and CYP3A7.2 were found and fetal livers homozygous for CUpsilonP3A7*2 had similar or higher CYP3A7 protein contents than CUpsilonP3A7*1 livers. Kinetic studies showed that CYP3A7.2 was a functional enzyme with a significantly higher catalytic constant (kcat) as compared with CYP3A7.1 (P < .05). Interestingly, fetal livers that expressed CYP3A7.2 also expressed CYP3A5 protein, and we found a linkage disequilibrium between the CUpsilonP3A7*2 and CUpsilonP3A5*1 alleles that was subject to interethnic differences. Determination of the alprazolam 1-hydroxylation rate revealed that CYP3A5 plays a significant role in the metabolism of CYP3A substrates in the fetal liver. CONCLUSION: We have identified 2 different CYP3A phenotypes in the fetal liver--one that is the result of a CUpsilonP3A7*1/CUpsilonP3A5*3 haplotype causing CYP3A7.1 but no CYP3A5 expression and another with higher detoxification capacity, inherent in the UpsilonP3A7*2/CUpsilonP3A5*1 haplotype, where CYP3A5 and a more active form of CYP3A7 are expressed.