Characterization of new mutations in the coding sequence and 5'-untranslated region of the human prostacyclin synthase gene (CYP8A1)

Inheritable interindividual differences in prostacyclin production may be implicated in the pathogenesis of several human vascular diseases. Using a polymerase chain reaction/single-strand conformation polymorphism strategy, we screened for mutations in the gene encoding cytochrome P450 prostacyclin synthase (CYP8A1). DNA samples from healthy French volunteers (n=130) of Caucasian origin were examined. Five mutations, comprising two previously reported silent mutations and three novel rare missense mutations (P38L, S118R, and R379S), were identified in the coding sequence of the gene. In the 5'-proximal region, we also found a variable number of tandem repeats (VNTR) polymorphism that consisted of four different alleles with 4-6 tandem repeats of a 9-bp unit containing a putative Spl transcriptional factor binding site. One of these (R6), a frequent allele (23.6% of alleles tested) harboring six repeats, is novel, whereas the other three are known. In vitro analysis of the effect of each VNTR allele on promoter activity of a reporter gene was performed by a transient transfection assay. Data confirmed the modulator effect of the VNTR polymorphism on reporter gene transcription. Furthermore, the data demonstrate that allele R6 has the most potent inducing effects in the A549 cell line and, after IL-6 stimulation, in human pulmonary artery endothelial cells. Overall, the data demonstrate that CYP8A1 is polymorphic in Caucasians, and that a polymorphism affecting the 5'-proximal region may result in interindividual differences in CYP8A1 transcriptional regulation in vivo. Additional factors, such as the presence of inflammatory mediators, may be required to modulate transcription of the CYP8A1 gene.