Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria

Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporp hyrinogen III synthase. Extensive mutation analyses of 40 unrelated patient s only identified approximately 90% of mutant alleles. Sequencing the recen tly discovered erythroid-specific promoter in six patients with a single un defined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelate d patients, and (d) a -90C to A transversion in a putative CP2 binding moti f. Also, a -224T to C polymorphism was present in approximately 4% of 200 u nrelated Caucasian alleles. We inserted these mutant sequences into lucifer ase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophore tic mobility shift assays indicated that the -70C mutation altered GATA1 bi nding, whereas the adjacent -76A mutation did not. Similarly, the -90C muta tion altered CP2 binding, whereas the -86A mutation did not. Thus, these fo ur pathogenic erythroid promoter mutations impaired erythroid-specific tran scription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional-binding elements for erythroid-specific heme biosynthesis.