Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP)

• Published in: Human molecular genetics 2013-04, Vol.22 (7), p.1280-1288
• Main Authors: Ducamp, Sarah, Schneider-Yin, Xiaoye, de Rooij, Felix, Clayton, Jerome, Fratz, Erica J, Rudd, Alice, Ostapowicz, George, Varigos, George, Lefebvre, Thibaud, Deybach, Jean-Charles, Gouya, Laurent, Wilson, Paul, Ferreira, Gloria C, Minder, Elisabeth I, Puy, Hervé
• Format: Article
• Language: English
• Subjects: 5-Aminolevulinate synthase; 5-Aminolevulinate Synthetase - chemistry; 5-Aminolevulinate Synthetase - deficiency; 5-Aminolevulinate Synthetase - genetics; 5-Aminolevulinate Synthetase - metabolism; Amino Acid Sequence; Amino acids; Base Sequence; C-Terminus; Child, Preschool; Codon, Nonsense; DNA Mutational Analysis; Enzymatic activity; Exons; Female; Frameshift Mutation; Genetic Association Studies; Genetic Diseases, X-Linked - enzymology; Genetic Diseases, X-Linked - genetics; Humans; Infant; Kinetics; Molecular Sequence Data; Mosaicism; Mutagenesis, Site-Directed; Nonsense mutant; Pedigree; Point mutation; Protein Structure, Tertiary; Protoporphyria, Erythropoietic - enzymology; Protoporphyria, Erythropoietic - genetics; Protoporphyrin; Sequence Analysis, DNA; X chromosome; Young Adult
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/dds531

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.