Genetic and biochemical studies in Argentinean patients with variegate porphyria

Abstract

BackgroundA partial deficiency in Protoporphyrinogen oxidase (PPOX) produces the mixed disorder Variegate Porphyria (VP), the second acute porphyria more frequent in Argentina. Identification of patients with an overt VP is absolutely important because treatment depends on an accurate diagnosis but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death.MethodsWe have studied at molecular level 18 new Argentinean patients biochemically diagnosed as VP. PPOX gene was amplified in one or in twelve PCR reactions. All coding exons, flanking intronic and promoter regions were manual or automatically sequenced. For RT-PCR studies RNA was retrotranscripted, amplified and sequenced. PPOX activity in those families carrying a new and uncharacterized mutation was performed.ResultsAll affected individuals harboured mutations in heterozygous state. Nine novel mutations and 3 already reported mutations were identified. Six of the novel mutations were single nucleotide substitutions, 2 were small deletions and one a small insertion. Three single nucleotide substitutions and the insertion were at exon-intron boundaries. Two of the single nucleotide substitutions, c.471G>A and c.807G>A and the insertion (c.388+3insT) were close to the splice donor sites in exons 5, 7 and intron 4 respectively. The other single nucleotide substitution was a transversion in the last base of intron 7, g.3912G>C (c.808-1G>C) so altering the consensus acceptor splice site. However, only in the first case the abnormal band showing the skipping of exon 5 was detected. The other single nucleotide substitutions were transversions: c.101A>T, c.995G>C and c.670 T>G that result in p.E34V, p.G332A and W224G aminoacid substitutions in exons 3, 10 and 7 respectively. Activity measurements indicate that these mutations reduced about 50% PPOX activity and also that they co-segregate with this reduced activity value. Two frameshift mutations, c.133delT and c.925delA, were detected in exons 3 and 9 respectively. The first leads to an early termination signal 22 codons downstream (p.S45fsX67) and the second leads to a stop codon 5 codons downstream (p.I309fsX314). One reported mutation was a missense mutation (p.G232R) and 2 were frameshift mutations: c.1082insC and 1043insT. The last mutation was detected in six new apparently unrelated Argentinean families.ConclusionMolecular analysis in available family members revealed 14 individuals who were silent carriers of VP. Molecular techniques represent the most accurate approach to identify unaffected carriers and to provide accurate genetic counselling for asymptomatic individuals. The initial screening includes the insertion search.

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

“…She has four daughters; two of them also carry the mutation with a reduction of PPOX activity of about 50%, and only one has biochemically VP features (Table 1). The already reported mutations characterized in this study were three missense mutations c.101A>T (p.E34V), c.670T>G (p.W224G), and c.995G>C (p.G332A) indentified in Argentinean families (Rossetti et al 2008): mutation c.101A>T (p.E34V) was identified in two members of a family who presented cutaneous symptoms alone, and in an unrelated patient who developed only acute symptoms. Mutation c.670T>G (p.W224G) was identified in two members of a family who experienced both cutaneous and acute symptoms, and in an asymptomatic relative.…”

“…Two of these mutations are novel and three were previously identified in our laboratory (Rossetti et al 2008). …”

“…Moreover, the molecular defect was characterized in these and four families previously studied in our laboratory (Rossetti et al 2008). All patients gave informed consent prior to their inclusion in the study.…”

“…Plasma porphyrin index (PPI) was determined as described by Rossetti et al, 2008. Lymphocyte PPOX activity was measured as described by Brenner andBloomer, 1980 andDeybach et al, 1981.…”

“…The clinical and biochemical profiles of these patients are present at the age of diagnosis and the genotype of the novel patients studied is shown in Table 1. Data from the families carrying the reported mutations were previously published (Rossetti et al 2008). …”