Identification and characterization of novel uroporphyrinogen decarboxylase gene mutations in a large series of porphyria cutanea tarda patients and relatives

Abstract

Porphyria cutanea tarda (PCT) arises from decreased hepatic activity of uroporphyrinogen decarboxylase (UROD). Both genetic and environmental factors interplay in the precipitation of clinically overt PCT, but these factors may vary between different geographic areas. Decreased activity of UROD in erythrocytes was used to identify patients with UROD mutations among a group of 130 Spanish PCT patients. Nineteen patients (14.6%) were found to harbor a mutation in the UROD gene. Eight mutations were novel: M1I, 5del10, A22V, D79N, F84I, Q116X, T141I and Y182C. Five others were previously described: F46L, V134Q, R142Q, P150L and E218G. The new missense mutations and P150L were expressed in Escherichia coli. D79N and P150L resulted in proteins that were localized to inclusion bodies. The other mutations produced recombinant proteins that were purified and showed reduced activity (range: 2.3-73.2% of wild type). These single amino acid changes were predicted to produce complex structural alterations and/or reduced stability of the enzyme. Screening of relatives of the probands showed that 37.5% of mutation carriers demonstrated increased urinary porphyrins. This study emphasizes the role of UROD mutations as a strong risk factor for PCT even in areas where environmental factors (hepatitis C virus) have been shown to be highly associated with the disease.

Fig. 1

Receiver operating characteristic curve for UROD activity in erythrocytes. Area under the curve (95% CI): 0.9625 (0.9314–0.9935).

Fig. 2

Expression and purification of UROD proteins in Escherichia coli. (a) Recombinant proteins were purified using an Ni-NTA resin and separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Recombinant human UROD runs at an apparent molecular weight of 42 kDa. The UROD without a mutation (wild type, WT) is run for comparison. (b) The insoluble pellet (I) and the soluble supernatant (S) were separated on a 10% SDS-PAGE gel; D79N, P150L and WT were compared with the purified recombinant human UROD (rhUROD). The UROD protein from D79N and P150L is present in the insoluble fraction, while WT is primarily present in the soluble fraction. EV, empty vector control.

Fig. 3

Location of UROD mutants at the base of the TIM barrel. Mutants D79N and P150L destabilize the core of the barrel and lead to misfolding of the proteins. The hydrogen bonding distances of D79 are shown as dashed yellow lines.

Fig. 4

Location of UROD missense mutants identified in this study. A monomer of UROD with β-sheets in red, α-helices in blue and stands in purple is shown in the cartoon. The side chains of amino acids mutated are labeled and shown in green.