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Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria
Constanza Solis, … , David F. Bishop, Robert J. Desnick
Constanza Solis, … , David F. Bishop, Robert J. Desnick
Published March 15, 2001
Citation Information: J Clin Invest. 2001;107(6):753-762. https://doi.org/10.1172/JCI10642.
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Article

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

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Abstract

Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined 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 unrelated patients, and (d) a –90C to A transversion in a putative CP2 binding motif. Also, a –224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase 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. Electrophoretic mobility shift assays indicated that the –70C mutation altered GATA1 binding, whereas the adjacent –76A mutation did not. Similarly, the –90C mutation altered CP2 binding, whereas the –86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.

Authors

Constanza Solis, Gerardo I. Aizencang, Kenneth H. Astrin, David F. Bishop, Robert J. Desnick

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Figure 1

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EMSAs of GATA1 and CP2 binding by the URO-synthase erythroid promoter an...
EMSAs of GATA1 and CP2 binding by the URO-synthase erythroid promoter and the α-globin promoter, respectively. EMSAs were performed as described in Methods. (a) Partial sequence of the human and murine (36) URO-synthase erythroid-specific promoters. The location and orientation (<, >) of the GATA1, E-box, and CP2 erythroid binding elements are indicated, as are the four novel promoter mutations. Dots are placed every tenth nucleotide. The heterologous CP2 site from the murine α-globin gene promoter and the GATA1 site from the human SCL gene promoter are shown. (b) The radiolabeled probe is GATA1-1 (see a). K562 nuclear extract was present in lanes 2–8. A 100-fold mass excess of unlabeled competitor is included in the following lanes: lane 3, GATA1-1; lane 4, GATA1-SCL; lane 6, GATA1-1, –70 mutation; lane 7, GATA1-1, –76 mutation. (c) The radiolabeled probe is CP2-α-globin (see a). K562 nuclear extract was present in lanes 2–6. Unlabeled competitor is included in the following lanes: lane 3, CP2-α-globin; lane 4, CP2-1; lane 5, CP2-1(–90); lane 6, CP2-1(–86). (d) The radiolabeled probe was CP2-α-globin (see a). K562 nuclear extract was present in lanes 1 to 3. Lane 2: incubation with preimmune serum and Protein A. Lane 3: incubation with anti-CP2 antibody and Protein A.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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