Subject(s)
Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/genetics , Porphyrias, Hepatic/genetics , Chromatography, High Pressure Liquid , DNA Mutational Analysis/methods , Electrophoresis , Exons/genetics , Flavoproteins , Humans , Mitochondrial Proteins , Nucleic Acid Denaturation , Polymerase Chain Reaction , Porphyrias, Hepatic/enzymology , Protoporphyrinogen OxidaseABSTRACT
The knowledge of at least 21 different mutations and several polymorphisms in the coproporphyrinogen oxidase (CPO) gene demonstrates that the molecular basis of hereditary coproporphyria is heterogeneous. We developed a DGGE-based assay for the analysis of exons 2 to 7, including 14-96 nucleotides of the flanking intronic sequences of the CPO gene. To render it suitable for the clinical diagnostic laboratory, we designed the assay to allow use of identical PCR conditions and the same DGGE gel for analyses of all the regions. Using this assay, and subsequent sequencing of gene regions containing interallelic variations, two novel mutations in the CPO gene were identified: a missense mutation (607G-->A), leading to the substitution of an alanine with a threonine, and a nonsense mutation (1281G-->A), giving rise to a stop codon 28 codons upstream to the wild-type stop codon.
