Investigation of a mutation in CYP26B1

Abstract

Retinoic acid is known to have several important roles in embryonic development, including limb and hindbrain development and has been shown to induce neural tube defects when it is present at teratogenic levels. The CYP26 enzyme family are known to metabolise retinoic acid and also play an important role in embryonic development as has been illustrated through several animal knockout studies.

A distinct phenotype was observed in two fetuses of a consanguineous Somalian family. The phenotype displayed severe congenital abnormalities of the skeleton and the brain. A sequence variant was identified in CYP26B1 and was investigated as the cause of the observed phenotype. The 1088G>T sequence variant predicts an amino acid substitution, R363L, within the highly conserved EXXR motif that is present in almost all of the enzymes belonging to the cytochrome super family.

The aim of this study is to determine whether the CYP26B1 R363L variant identified lacks normal enzymatic activity. Stable expression constructs of the wildtype, mutant and empty control vector were transfected into HEK293 cells. The stable cell lines were treated with retinoic acid, RNA extracted and used to create cDNA. qRT-PCR was used to measure the up regulation of the RA responsive genes, ICER and CYP26A1. This showed that the mutant does indeed lack activity when using CYP26A1 as a test gene and that ICER was not a good gene for measuring the catabolism of RA by CYP26B1. This evidence is consistent with the hypothesis that CYP26B1 R363L is the cause of the phenotype observed.