Expression and characterization of six mutations in the protoporphyrinogenoxidase gene among Finnish variegate porphyria patients

Background: Variegate porphyria (VP) is an inherited disorder of heme biosy nthesis that results from a partial deficiency of protoporphyrinogen oxidas e (PPOX). Patients with VP may experience acute neurovisceral attacks and c utaneous photosensitivity. To date we have characterized 109 VP patients re presenting 19 VP families in the Finnish population of 5 million, both bioc hemically and clinically. Materials and Methods: Mutations were identified by direct sequencing of th e patients' genomic DNA. The effect of the mutations was determined by sequ encing the reverse transcriptase polymerase chain reaction (RT-PCR) product amplified from total RNA extracted from the patients' lymphoblast cell lin es and expressing the mutations in E. coli and COS-1 cells. Results: Of the six mutations identified in the PPOX gene, three mutations (IVS2-2a -->c, 338G -->C, and 470A -->C) caused splicing defects, one produ ced a frameshift (78insC) and two mutations (R152C and L401F) caused amino acid substitutions. In RT-PCR, the IVS2-2a -->c mutation caused a retention of a 36-bp fragment in the 3 ' end of intron 2, the 338G -->C mutation cau sed an exon 4 deletion, and the 470A -->C mutation caused an exon 5 deletio n with retention of a 19-bp fragment of the 3 ' end or intron 5. In both pr okaryotic and eukaryotic expression systems, the PPOX activities of five mu tants were decreased to 0-5% of the normal activity. Conclusions: This study describes five novel mutations and one earlier desc ribed major mutation among Finnish VP patients. All mutations produced dete ctable transcripts, but resulted in decreased PPOX activity confirming the causality of the mutations and the biochemical defects in these patients.