Abstract

Bietti's crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germ-line mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations; two splice site alterations and one missense mutation, viz, c.992C>A translating to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes to better understand the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1-4 genes showed CYP4V2 to be a major P450 in ARPE-19 cells, a human cell line spontaneously generated from normal human retinal pigment epithelium, and the only detectable CYP4 transcript. Immunohistochemical analysis demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea, and that the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid, the latter an important constituent of the retina, to their respective ω-hydroxylated products at rates similar to purified CYP4F2, an established hepatic PUFA hydroxylase. The disease-associated p.H331P variant was undetectable by Western blot in HepG2 cells stably transduced with lentiviral expression vectors. Finally, over expression of functional CYP4V2 in HepG2 cells alters lipid homeostasis. Therefore, we have demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active towards PUFAs and that the functional deficit in BCD patients carrying the H331P variant is most likely a consequence of the instability of the mutant protein.