Erythropoietic protoporphyria is an autosomal dominant disease with incomplete penetrance, due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the final step of the heme biosynthetic pathway. The clinical phenotype of EPP results from coinheritance of a mutated allele and a wild-type low expressed allele of the FECH gene. To date, more than 100 different mutations have been identified in the FECH gene of EPP patients. Different evidence suggests that an entire haplotype (−251G, IVS1-23T, and IVS3-48C) reduces allele expression. In two Italian EPP patients carring the −250G>C mutation in the promoter region we recently demonstrated the loss of an SP1 binding site causing a strong impairment of promoter activity. In both patients we searched for the GTC haplotype on the other allele. Family segregation studies established that the GTC haplotype was in trans to the −250C mutation. Indeed, asymptomatic parents carried either the three polymorphisms or the point mutation. In both patients only the IVS3-48C polymorphism was in apparent homozygosity since family studies established the absence of Mendelian segregation. To reduce the possibility of interfering polymorphisms in the primer sites, a second set of sequencing primers was designed. Apparent homozygosity was confirmed. We assumed the presence of an intronic deletion on the same allele carrying the −250G>C mutation. In order to establish the size of the deletion we performed a semiquantitative RT-PCR analysis on fresh RNA and XL-PCR analysis on DNA. In both cases the primers were situated in regions outside the heterozygotic polymorphisms. As expected RNA analysis revealed the presence of a normal fragment of 612bp and a shorter fragment of 343bp that was less abundant compared to controls. Sequence analysis on the isolated abnormal fragment showed loss of exons 3 and 4. DNA analysis revealed the presence of two fragments of 21440bp and 15864bp respectively. We performed an internal long PCR to obtain two shorter fragments 9717bp and 4141bp respectively. Sequence analysis on the isolated abnormal fragment showed a 5576bp deletion defined by two short direct repeats of about 40bp. The deleted region includes both a first repeat and interposed sequence. These results showed the presence of two functional mutations on the same allele. Deletion contributed to create a null allele preventing formation of the correct mRNA that is already strongly impaired by the −250G>C mutation. The deletion is caused by slipped strand mispairing or more probably by unequal intragenic recombination. These two mutations in the promoter region and intron 3 respectively, are located in the same regions as the −251G and ivs3-48C polymorphisms, strongly suggesting a functional relationship between these two regions.

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