Characterization of missense mutations in Argentinean Variegate Porphyria Patients

GRANATA BX; MENDEZ, M; PARERA VE; BATLLE A; ENRIQUEZ DE SALAMANCA R; ROSSETTI MV

Cardiff

Congreso; Porphyrins and Porphyrias 2011; 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 heme biosynthetic pathway. Variegate Porphyria (VP) develops when the defective enzyme is the 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 apparentely non-related VP patients we found 4 new splicing defects, 3 new missense mutations and 2 novel small deletions. The 3 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 a 50% approximately.   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 PCR-based 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 5mM IPTG for 3 h. Cells were harvested, resuspended in buffer and disrupted by sonication. This was centrifugated once more and the supernatant was used as source of enzyme to determine the PPOX activity in strict conditions of obscurity and anaerobiosis.    According to the results (see table below) 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. Construct PPOX  specific activity (nmol PROTO/mg/h) Mean ±SD (Range) Residual Activity (%) pTRC                0.21 0 pTRC – PPOX-wt 13.27 100 pTRC – PPOX- E34V 0.51 2.29 pTRC – PPOX – W224G 0.76 1.22 pTRC – PPOX – G332A 0.20 0