Characterization of missense mutations in Argentinian patients with variegate porphyria

GRANATA, BARBARA XOANA; MENDEZ, MANUEL; PARERA, VICTORIA; BATLLE, ALCIRA; ENRÍQUEZ DE SALAMANCA, RAFAEL; ROSSETTI, MARIA VICTORIA

Cardiff

Congreso; International Porphyrins and Porphyrias Meeting; 2011

British Association of Dermatologists

Porphyrias are a group of metabolic disorders that result from a partial deficiency of one of the enzymes involved in the haem biosynthetic pathway. Variegate porphyria (VP) develops when the defective enzyme is protoporphyrinogen oxidase (PPOX). This disease is an autosomal dominant disorder with incomplete penetrance, associated to a 50% decrease of the enzyme activity. Clinically it is considered to be a mixed porphyria and patients can present acute and/or cutaneous symptoms. Recently, in 18 apparently nonrelated VP patients we found four new splicing defects, three new missense mutations and two novel small deletions. The three new missense mutations (p.E34V, W224G and G332A) were absent in 50 normal individuals and the PPOX activity in the probands and in their asymptomatic relatives was reduced to approximately 50%. The aim of the work was to corroborate that the missense mutations p.E34V, W224G and G332A were responsible for the manifestation of VP. In order to achieve this we expressed them in a prokaryotic system, using as template the pTRC?PPOX-wt vector, which contains the wild-type sequence of PPOX. To generate each mutant construct a fragment of PPOX cDNA, containing the desired mutation and restriction sites for cloning, was generated by polymerase chain reactionbased site-directed mutagenesis in one or two amplification steps. Once each construct was obtained, they were used to transform competent Escherichia coli strain JM109. Bacterial clones were then grown to log phase and induced with 5 mmol L)1 isopropyl-b-D-thiogalactoside for 3 h. Cells were harvested, resuspended in buffer and disrupted by sonication. They were centrifuged once more and the supernatant was used as source of enzyme to determine the PPOX activity in strict conditions of darkness and anaerobiosis. According to the results (see Table 1) all the mutations studied generate a considerable decrease in the enzyme activity; therefore, they are certainly the cause of VP in the patients who carry them.