Loss-of-function ferrochelatase and gain-of-function erythroid-specific 5-aminolevulinate synthase mutations causing erythropoietic protoporphyria and x-linked protoporphyria in North American patients reveal novel mutations and a high prevalence of X-linked protoporphyria

Abstract

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inborn errors of heme biosynthesis with the same phenotype but resulting from autosomal recessive loss-of-function mutations in the ferrochelatase (FECH) gene and gain-of-function mutations in the X-linked erythroid-specific 5-aminolevulinate synthase (ALAS2) gene, respectively. The EPP phenotype is characterized by acute, painful, cutaneous photosensitivity and elevated erythrocyte protoporphyrin levels. We report the FECH and ALAS2 mutations in 155 unrelated North American patients with the EPP phenotype. FECH sequencing and dosage analyses identified 140 patients with EPP: 134 with one loss-of-function allele and the common IVS3-48T>C low expression allele, three with two loss-of-function mutations and three with one loss-of-function mutation and two low expression alleles. There were 48 previously reported and 23 novel FECH mutations. The remaining 15 probands had ALAS2 gain-of-function mutations causing XLP: 13 with the previously reported deletion, c.1706_1709delAGTG, and two with novel mutations, c.1734delG and c.1642C>T(p.Q548X). Notably, XLP represented ~10% of EPP phenotype patients in North America, two to five times more than in Western Europe. XLP males had twofold higher erythrocyte protoporphyrin levels than EPP patients, predisposing to more severe photosensitivity and liver disease. Identification of XLP patients permits accurate diagnosis and counseling of at-risk relatives and asymptomatic heterozygotes.

Figure 1

FECH loss-of-function mutations causing autosomal-recessive EPP. Mutations in black and blue type were previously reported and listed in the Human Gene Mutation Database, release date 29 June 2012. Previously published mutations in blue were those found in the patients of this study. Novel mutations are indicated in red. Asterisks indicate splicing mutations caused by exonic mutations. The gene depicted in the top line shows exonic mutations, and mutations in intronic sequences are shown on the bottom line.

Figure 2

Large FECH deletions and complex deletion/insertion rearrangements causing EPP. The Human Gene Mutation Database (HGMD) accession numbers are provided, and the extent of the deletion is listed to the right of each deleted region. In parentheses, the number of patients previously reported with the deletion is followed by the number of patients with the deletion in the present study. Novel mutations are those with only one incidence in this study. Question marks indicate the unknown positions of the breakpoints. Alu sequences and their orientations are indicated with open arrows. The published breakpoints have been mapped to the FECH sequence provided in GenBank accession number NG_008175 from the February 2009 HG-19 human genome assembly. HGMD accession numbers for mutations with identical breakpoints have been listed together with the respective deletion region.

Figure 3

FECH three-dimensional structure and the location of novel deleterious FECH mutations. The crystal structure of one of the homodimeric FECH monomers is shown by using secondary structure cartoons in green and the surface in light gray. The view is from the side of the protein that is membrane-bound, looking into the active site region containing protoporphyrin IX and the iron-sulfur cluster. The residues changed by the novel mutations are shown as space-filling spheres and are the indicated native residues, not the mutations.

Figure 4

XLP mutations and gene structure. (A) ALAS2 loss-of-function and gain-of-function mutations causing X-linked sideroblastic anemia and EPP. Mutations in black and blue are those reported in the Human Gene Mutation Database, release date 29 June 2012. The mutations in bold are published mutations also found in patients of this study. The mutations in bold italics are the novel mutations. (B) Variations in the C-terminal sequences of the XLP gain-of-function mutations. The partial wild-type exon 11 ALAS2 sequence is boxed. The hybrid sequences of wild-type ALAS2 and the sequence following the mutation sites are aligned below the wild-type sequence. The termination codons are denoted by asterisks. Note that for the pE569GfsX24 and pQ581SfsX13 mutations, the last 12 mutated residues are identical to each other and different from the wild-type.