Characterization of Variegate Porphyria mutations using a minigene approach

BÁRBARA X GRANATA; MARCO BARALLE; LAURA DE CONTI; PARERA, VICTORIA ESTELA; ROSSETTI MARÍA VICTORIA

JOURNAL OF INHERITED METABOLIC DISEASE

SPRINGER

Lugar: Berlin; Año: 2015 vol. 20 p. 30 - 44

0141-8955

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 protoporphirinogen 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. As these mutations do not affect the protein sequence we hypothesized that they might be splicing mutations. RT-PCRs performed on the 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 utilized a minigene approach. We found that the three mutations lead to exon skipping, therefore the abnormal mRNAs are most likely degraded by a mechanism such as nonsense mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.