Characterization of Three Variegate Porphyria

BÁRBARA X GRANATA; MARCO BARALLE; FRANCISCO BARALLE; MARÍA VICTORIA ROSSETTI

Lucerna

Congreso; Annual Assembly of the Swiss Society of Clinical Chemistry and International Congress of Porphyrins and Porphyrias and International meeting of Porphyria Patients; 2013

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008 three unrelated patients were diagnosed as Variegate Porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the portoporphirinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338 + 3insT, c.807 G > A, c.808-1 G > C. These mutations would not affect the protein sequence leading us to think that they might be splicing mutations. RT-PCRs performed with patient ’ s mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patient ’ s disease by causing aberrant splicing, we set out a minigene approach. We found an exon skipping of 90 ± 10 % for the mutation c.808-1 G > C, consistent with the interruption of the 100 % conserved AG dinucleotide present at the intron-exon junction. The mutation c.338 + 3insT was assayed in two different minigenes because the WT profile was not clear, finding in both cases an exon skipping of 100 ± 0 % and 98.5 ± 1.5 % , respectively in the presence of  the mutation. Finally, the mutation c.807 G > A showed a 100 ± 0 % of exclusion and the study was complemented with a U1 snRNA splicing  rescue assay finding that the mutation might be disrupting U1 snRNA binding site. In sum, all the mutations lead to exon skipping and as a consequence a premature stop codon appears in the following exon. Therefore the abnormal mRNAs are most likely degraded by a mechanism such as nonsense mediated decay, which recognizes premature stop codons. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the manifestation of the disease and highlighting the use of minigene functional assays to complement transcript analysis.  . .