Familial Porphyria Cutanea Tarda: Characterization of Seven Novel Uroporphyrinogen Decarboxylase Mutations and Frequency of Common Hemochromatosis Alleles

Abstract

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Functional consequences of naturally occurring mutations in human uroporphyrinogen decarboxylase

“…on April 4, 2019. by guest www.bloodjournal.org From previously described by Mendez et al, 13 who could detect no activity attributable to the mutant protein in an E coli expression system. Although Met165 was remote from the active site, it was packed in a hydrophobic pocket that appeared unable to accommodate a charged arginine side chain ( Figure 3C).…”

“…[3][4][5][6][7][8][9][10][11][12][13][14] Ten of these mutant alleles are predicted to generate shorter proteins as a result of a premature stop codon or a variation in a splice site. [3][4][5][6][7][8][9][10][11][12][13] The remaining 20 previously reported mutations are missense mutations listed in Table 4 and mapped onto the URO-D monomer in Figure 3C. The structural location of these mutations and the predicted structural effects are described in Table 4.…”

“…More than 30 different URO-D mutations have been identified in unrelated patients. [3][4][5][6][7][8][9][10][11][12][13][14] Most carriers of mutant URO-D alleles do not express clinical phenotypes unless additional factors are present that further reduce the activity of URO-D in the liver. 15,16 Factors shown to be important include alcohol abuse, exposure to the hepatitis C virus, use of medicinal estrogens, and development of hepatic siderosis (often caused by mutation of the hemochromatosis gene).…”

A mouse model of familial porphyria cutanea tarda

“…Environmental factors such as alcohol abuse, hepatitis C virus, and estrogen use also play a role in clinical expression (1,5). Numerous mutations of the URO-D gene have been identified in patients with F-PCT (6)(7)(8)(9)(10)(11)(12)(13)(14)(15) but the most common is a splice-site mutation resulting in the deletion of exon 6 (16).…”

Functional Characterization of Five Protoporphyrinogen oxidase Missense Mutations Found in Argentinean Variegate Porphyria Patients

“…To generate each mutant constructs, a fragment of the PPOX cDNA, containing the desired mutation and restriction sites for cloning, was generated by PCR-based sitedirected mutagenesis in one or two amplification steps (Cormack 1991;Méndez et al 1998). PCR reactions were performed using primers indicated in Table 2 and the pTrc-PPOX-wt as template.…”