Targeted resequencing of FECH locus reveals that a novel deep intronic pathogenic variant and eQTLs may cause erythropoietic protoporphyria (EPP) through a methylation-dependent mechanism



Existing data do not explain the reason why some individuals homozygous for the hypomorphic FECH allele develop erythropoietic protoporphyria (EPP) while the majority are completely asymptomatic. This study aims to identify novel possible genetic variants contributing to this variable phenotype.


High-throughput resequencing of the FECH gene, qualitative analysis of RNA, and quantitative DNA methylation examination were performed on a cohort of 72 subjects.


A novel deep intronic variant was found in four homozygous carriers developing a clinically overt disease. We demonstrate that this genetic variant leads to the insertion of a pseudo-exon containing a stop codon in the mature FECH transcript by the abolition of an exonic splicing silencer site and the concurrent institution of a new methylated CpG dinucleotide. Moreover, we show that the hypomorphic FECH allele is linked to a single haplotype of about 20 kb in size that encompasses three noncoding variants that were previously associated with expression quantitative trait loci (eQTLs).


This study confirms that intronic variants could explain the variability in the clinical manifestations of EPP. Moreover, it supports the hypothesis that the control of the FECH gene expression can be mediated through a methylation-dependent modulation of the precursor messenger RNA (pre-mRNA) splicing pattern.

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The authors are grateful to all patients and their families who donated samples for this study. We thank Pasquale Missineo for biochemical analysis, Valeria Fiorentino for support in the panel design, Luca Turchet for graphic assistance, and David Horner for English editing and comments that greatly improved the manuscript. We also thank M.D. Cappellini for her constant and continuous support in the research activity. This research was supported by grants from the Italian Ministry of Health (GR–2011–02347129 to E.D.P.) and in part from Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (RC2019).

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Correspondence to Elena Di Pierro PhD.

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  • erythropoietic protoporphyria
  • deep intronic pathogenic variant
  • eQTLs
  • CpG sites
  • pre-mRNA splicing pattern