Congenital erythropoietic porphyria: identification and expression of exonic mutations in the uroporphyrinogen III synthase gene.

Abstract

Congenital erythropoietic porphyria (CEP), an inborn error of heme biosynthesis, results from the deficient activity of uroporphyrinogen III synthase (URO-synthase). This autosomal recessive disorder is heterogeneous; patients with severe disease are often transfusion dependent, while milder patients primarily have cutaneous involvement. To investigate this phenotypic heterogeneity, exonic point mutations in the URO-synthase gene were identified in unrelated CEP patients. Four missense mutations were identified: (a) an A to G transition of nucleotide (nt) 184 that predicted a Thr to Ala substitution at residue 62 (designated T62A); (b) a C to T transition of nt 197 that encoded an Ala to Val replacement at residue 66 (A66V); (c) a T to C transition of nt 217 that predicted a Cys to Arg substitution at residue 73 (C73R); and (d) a C to T transition of nt 683 that resulted in a Thr to Met replacement at residue 228 (T228M). In addition, a G to A transition of nt 27 that did not change the encoded amino acid (A9A) was detected in an African patient. The T62A, C73R, and T228M alleles did not express detectable enzymatic activity, while the A66V allele expressed residual, but unstable activity. The C73R allele was present in eight of 21 unrelated CEP patients (21% of CEP alleles). In three patients, identification of both alleles permitted genotypephenotype correlations; the A66V/C73R, T228M/C73R, and C73R/C73R genotypes had mild, moderately severe, and severe disease, respectively. These findings provide the first genotype-phenotype correlations and permit molecular heterozygote detection in this inherited porphyria. (J. Clin. Invest.

Congenital erythropoietic porphyria: identification and expression of 10 mutations in the uroporphyrinogen III synthase gene.

“…Of note, only the C73R lesion was common, occurring in about one third of the CEP alleles studied, whereas the other mutations were detected only in one to three unrelated families (7,8,(18)(19)(20). Each of the missense mutations has been expressed in E. coli (8,20), and several were found to have significant residual activity which appeared to correlate with milder phenotypes (7,8,20 (25), the strands were denatured in 10 yl of 0.1 M NaOH for 30 min, and the nonbiotinylated strands were eluted with two 50-sl washes of 0.1 M NaOH. The biotinylated strands were washed with 50 MI of the binding and washing buffer (10 mM Tris-HCl, pH 7.5, containing 1.0 mM EDTA and 2.0 M NaCl) and then with 50 p1 of Tris-EDTA buffer.…”

“…Additional manifestations may include hypertrichosis, alopecia, and erythrodontia. Although milder patients have some residual activity to synthesize sufficient heme for hematopoiesis and for other essential heme-requiring proteins, the amount of residual activity appears to be correlated with clinical severity (7,8).…”

“…The availability of the URO-synthase cDNA permitted investigation of the molecular lesions causing CEP. To date, six missense mutations (7,8,(18)(19)(20), an 80-bp insertion, and a 98-bp deletion (dell48-245) (20) have been identified in unrelated CEP patients. Of note, only the C73R lesion was common, occurring in about one third of the CEP alleles studied, whereas the other mutations were detected only in one to three unrelated families (7,8,(18)(19)(20).…”

“…To date, six missense mutations (7,8,(18)(19)(20), an 80-bp insertion, and a 98-bp deletion (dell48-245) (20) have been identified in unrelated CEP patients. Of note, only the C73R lesion was common, occurring in about one third of the CEP alleles studied, whereas the other mutations were detected only in one to three unrelated families (7,8,(18)(19)(20). Each of the missense mutations has been expressed in E. coli (8,20), and several were found to have significant residual activity which appeared to correlate with milder phenotypes (7,8,20 (25), the strands were denatured in 10 yl of 0.1 M NaOH for 30 min, and the nonbiotinylated strands were eluted with two 50-sl washes of 0.1 M NaOH.…”

“…The normal and mutant URO-synthase alleles were expressed in E. coli using the pKK223-3 vector (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) as previously described (8,16). pKK-UROS-V99A, pKK-UROS-A104V, pKK-UROS-633insA, and pKK-UROS-G225S were constructed by cassette substitution of the cloned mutant cDNA into the pKK-UROS vector (8), while the pKK-UROS-L4F, pKK-UROS-Y19C, pKK-UROS-V82F, pKK-UROS-Q249X constructs were engineered by mega-primer PCR mutagenesis (27), and then were cassette-substituted into the pKK-UROS vector.…”