Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterizationof six novel mutations associated with familial PCT

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoeryth ropoietic porphyria (HEP), are associated with mutations in the gene encodi ng the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, mos t of which are private, have been identified in HEP and fPCT patients, conf irming the heterogeneity of the underlying genetic defects of these disease s. We have established a denaturing gradient gel electrophoresis (DGGE) ass ay for mutation detection in the UROD gene, enabling the simultaneous scree ning for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M 01T, F229L, and M324T), two splice mutations (IVS3-2A-->T and IVS5-2A-->G) leading to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous ex pression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein hav e no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a cons iderable increase in thermolability, The six new mutations bring to a total of 29 the number of disease-related mutations in the UROD gene. The DGGE a ssay presented greatly improves the genetic diagnosis of fPCT and HEP, ther eby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases. Hum Mutat 14 :222-232, 1999. (C) 1999 Wiley-Liss, Inc.