Gene Dosage Analysis Identifies Large Deletions of the FECH Gene in 10% of Families with Erythropoietic Protoporphyria

Abstract

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.

Diagnostic Strategies for Autosomal Dominant Acute Porphyrias: Retrospective Analysis of 467 Unrelated Patients Referred for Mutational Analysis of the HMBS, CPOX, or PPOX Gene

“…NM_000309), or CPOX (GenBank accession no. NM_000097) by direct genomic sequencing, as has previously been described (28 ). The following regions were sequenced: HMBS exons 1 and 3-15, 30 -250 bp of their flanking regions, and 1000 bp of the 5Ј untranslated region (UTR); CPOX exons 1-7, 30 -200 bp of their flanking regions, and 700 bp of the 5Ј UTR; PPOX exons 1-13 and 30 -300 bp of their flanking regions.…”

“…The following regions were sequenced: HMBS exons 1 and 3-15, 30 -250 bp of their flanking regions, and 1000 bp of the 5Ј untranslated region (UTR); CPOX exons 1-7, 30 -200 bp of their flanking regions, and 700 bp of the 5Ј UTR; PPOX exons 1-13 and 30 -300 bp of their flanking regions. When bidirectional sequencing identified no mutation, quantitative PCR was used for gene-dosage analysis to identify large deletions (28 ).…”

Multiplex ligation-dependent probe amplification: a novel approach for genetic diagnosis of Porphyria

“…In the recent years, deletions of single genes or parts of them have been reported as responsible for different forms of Porphyria. 11,13,14 Chromosome deletions of megabases have also been reported in late-onset porphyria caused by clonal expansion in the myelodysplastic process, 20 suggesting that heme genes are prone to this type of molecular abnormality. Considering the high molecular heterogeneity and the technical difficulties in determining insertions, deletions or complex rearrangements of a few kilobase pairs, it is quite possible that patients with clinical symptoms of porphyria remain undiagnosed even in specialized centers, because of undetected mutations.…”

“…Moreover, the feasibility of these techniques depends on the availability of DNA from biological parents, good quality DNA, and polymorphic sites with high frequency and efficiency of multiplex PCR. 14,15 For all these reasons, it is possible that long gene deletions have gone undetected in patients with clinical symptoms and biochemical signs of porphyria. Recently, a rapid and relatively easy technique for DNA quantitative analysis, named multiplex ligation-dependent probe amplification (MLPA), has been described.…”

Eryhtropoietic Protoporphyria

“…18 Berroeta reported a UK case with late onset of EPP and identified a mutation (1001C→T; P334L). 19 In Canada, Pierro identified a 10,376 bp deletion (c.1-7887_67+2422del) including a portion of the upstream intergenic region, the promoter, the exon 1 and a portion of intron 1 in a Canadian EPP patient of Italian origin.…”