Mutation Screening of the Entire Coding Region of the Protoporphyrinogen Oxidase Gene Using Denaturing Gradient Gel Electrophoresis and Denaturing HPLC

Abstract

Variegate porphyria (VP) is characterized by decreased activity of protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway. VP belongs to the group of mixed porphyrias and presents with both cutaneous manifestations and acute neurovisceral attacks. Genetically, VP is associated with mutations in the gene encoding PPOX and is usually inherited as an autosomal dominant trait with low clinical penetrance (1)(2). Although a few founder mutations predominate in certain populations, VP generally is a heteroallelic disease, with a variety of mutations in the PPOX gene being responsible for the decreased activity of PPOX (3)(4). The 5.5-kb PPOX gene is located on chromosome 1q22-23 and contains 1 noncoding and 12 coding exons (5). To date, >80 different mutations and polymorphisms have been identified in the PPOX gene (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Characterization by mutational analysis of suspected cases of VP should be considered an important clinical diagnostic tool because of the similarities in clinical presentation of VP, porphyria cutanea tarda (PCT), and hereditary coproporphyria. Furthermore, the use of DNA diagnostic methods enables detection of latent or asymptomatic mutation carriers at risk of developing VP. Here we describe assays for easy and reliable screening of the entire coding region of the PPOX gene as well as 74 bp of the noncoding exon 1 and 8–41 bp of the flanking intron sequences, using denaturing gradient gel electrophoresis (DGGE) and denaturing HPLC (dHPLC). Because VP frequently is confused with the more common PCT, we screened for PPOX mutations in DNA from 40 sporadic PCT patients, using established assays to detect possible misclassified VP cases. The study protocol was approved by the relevant Regional Ethical Committees. All participants received written information and gave …