Victoria Estela Parera

Genetic and biochemical studies in Argentinean patients with variegate porphyria

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.

Hepatitis C Virus and GBV-C/Hepatitis G Virus in Argentine Patients with Porphyria Cutanea Tarda

To investigate hepatitis C virus (HCV) and GBV-C/hepatitis G virus (HGV) genotype prevalence among HCV-infected porphyria cutanea tarda (PCT) patients, 19 HCV-infected patients with associated PCT were studied. A control group of 53 age-matched HCV-infected patients without associated PCT was selected. Eighteen of the 19 serologically positive HCV-PCT patients showed HCV RNA in serum. Genotype 1b was the most prevalent among both HCV-PCT patients (72.2%; 13/18) and age-matched HCV controls (50.9%; 27/53). Such different genotypic prevalence failed to reach statistical significance (χ2 with Yates’ correction, p = 0.19). The single HCV-PCT patient without detectable HCV RNA was also infected with genogroup 3 GBV-C/HGV. This GBV-C/HGV RNA prevalence (5.3%) among HCV-PCT patients is not statistically different from that observed among Argentine blood donors (5.5%; 11/200). To our knowledge, these results show for the first time the molecular epidemiology of both HCV and GBV-C/HGV associated to PCT in America.

Mutations in familial porphyria cutanea tarda: Two novel and two previously described for hepatoerythropoietic porphyria Communicated by: Mark H. Paalman Online Citation: Human Mutation, Mutation in Brief #354 (2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/354.pdf

Uroporphyrinogen decarboxylase (URO-D) deficiency is responsible for two forms of genetic cutaneous porphyria: familial porphyria cutanea tarda (f-PCT) and hepatoerythropoietic porphyria (HEP). The f-PCT transmitted as an autosomal dominant trait, is characterized by photosensitive cutaneous lesions frequently associated to hepatic dysfunction and is precipitated by various ecogenic factors. The HEP, transmitted as a recessive trait, is more severe than f-PCT and would be considered as the homozygous form of f-PCT. For the mutational analysis of f-PCT patients, the entire URO-D gene was amplified and each exon, intron-exon boundaries and the promoter region were cycle sequenced. Five mutations were found in 6 unrelated families studied, of these, two were new: a nonsense mutation in exon 6 (W159X) and a splice defect in intron 9 (IVS9(-1)G-->C). The other two missense mutations, P62L and A80G, had been previously reported in the homozygous state in HEP families. The g10insA, reported in our laboratory, was again identified in other two unrelated families. In addition 3 novel URO-D polymorphisms in non-coding regions were found. The reverse transcription-PCR and sequencing of the splice mutation carriers RNA did not reveal the presence of an abnormal mRNA, suggesting that no stable transcript from the mutated allele is synthesized. These results increase to 39 the number of mutations identified in the URO-D gene; 4 of them causing both HEP and f-PCT.

Identification of CYP3A5 and CYP2B6 Polymorphisms in Porphyria Cutanea Tarda Associated to Human Immunodeficiency Virus

To date, few or no data concerning the prevalence of polymorphisms in drug metabolism genes of antiretroviral drugs have been reported in the Argentinean population or in porphyric individuals worldwide. The purpose of the current investigation was to determine whether interindividual differences in cytochrome P450 3A5 (CYP3A) and 2B6 (CYP2B6) genes could influence the triggering of Porphyria Cutanea Tarda (PCT) in subjects with human immunodeficiency virus (HIV) after antiretroviral exposure. A total of 141 subjects, 60 control volunteers and 81 unrelated individuals with PCT were included in the study. In the porphyric group, 21 individuals were HIV positive. To evaluate the presence of the alleles CYP3A5*3, CYP3A5*6 and CYP2B6*6 a polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis was performed. The frequencies of CYP3A5*3 were 0.91 in control group, 0.89 in PCT patients and 0.89 in PCT-HIV. CYP2B6*6 frequencies were 0.31 in control group, 0.34 in PCT group and 0.30 in PCT-HIV group. We have shown that the allelic frequencies of CYP3A5*3 or CYP2B6*6 in our population were similar to those reported for other Caucasian populations. Although, we have not found significant differences in polymorphisms of CYP3A5 and CYP2B6 between the different groups analyzed, there are an enormous number of biological variables that may influence antiretroviral treatment, like other genetic polymorphisms of phase I or phase II enzymes, or transporters like multidrug resistance transporter gene (MDR1), which can contribute to antiretroviral drug toxicities and response or even it is possible that the PCT-HIV association has more than one factor responsible for the onset of PCT symptoms.

Diagnosis of latent acute intermittent porphyria by genetic analysis

Porphyrias, a group of inherited enzyme disorders of the haem biosynthetic pathway, can be clinically classi®ed into acute and non-acute types. Acute intermittent porphyria (AIP), the most common acute type, is an autosomal dominant disorder caused by a partial de®ciency of porphobilinogen deaminase (PBG-D). Acute attacks of this disease are often precipitated by factors such as drugs, alcohol and caloric deprivation, and early detection is important for the prevention of acute attacks. In asymptomatic carriers the excretion of haem precursors is often normal in between attacks, and diagnosis is based on the determination of erythrocyte PBG-D activity. There is, however, some overlap in erythrocyte PBG-D activity between normal individuals and AIP patients. These limitations prompted us to undertake the study of the mutations responsible for AIP at the DNA level. To date, about 159 mutations in the PBG-D gene have been reported, including missense, non-sense, frameshift and splice site mutations. These may lead to a decrease in PBG-D levels, the complete absence of detectable protein, the production of an altered but stable protein with abnormal catalytic properties, or an absolutely unstable protein. In this study we compared the biochemical results with the genetic data of a group of patients with AIP.

Functional associations of genetic variants involved in the clinical manifestation of erythropoietic protoporphyria in the Argentinean population

Background  Combined inheritance of genetic variants in ferrochelatase gene (FECH) are implicated in clinical manifestation of Erythropoietic Protoporphyria (EPP).

Acute Intermittent Porphyria in Argentina: An Update

Porphyrias are a group of metabolic diseases that arise from deficiencies in the heme biosynthetic pathway. A partial deficiency in hydroxymethylbilane synthase (HMBS) produces a hepatic disorder named Acute Intermittent Porphyria (AIP); the acute porphyria is more frequent in Argentina. In this paper we review the results obtained for 101 Argentinean AIP families and 6 AIP families from foreign neighbour countries studied at molecular level at Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP). Thirty-five different mutations were found, of which 14 were described for the first time in our population. The most prevalent type of mutations was the missense mutations (43%) followed by splice defects (26%) and small deletions (20%). An odd case of a double heterozygous presentation of AIP in a foreign family from Paraguay is discussed. Moreover, it can be noted that 38 new families were found carrying the most frequent mutation in Argentina (p.G111R), increasing to 55.66% the prevalence of this genetic change in our population and adding further support to our previous hypothesis of a founder effect for this mutation in Argentina. Identification of patients with an overt AIP is 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.

Characterization of Variegate Porphyria Mutations Using a Minigene Approach

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008, three unrelated patients were diagnosed with variegate porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the protoporphyrinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338+3insT, c.807G>A, and c.808-1G>C. As these mutations do not affect the protein sequence, we hypothesized that they might be splicing mutations. RT-PCRs performed on the patients mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patients disease by causing aberrant splicing, we utilized a minigene approach. We found that the three mutations lead to exon skipping; therefore, the abnormal mRNAs are most likely degraded by a mechanism such as nonsense-mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.

Haplotype Study in Argentinean Variegate Porphyria Patients.

Background/Aims: The porphyrias are genetically heterogeneous diseases, and each mutation is exclusive to one or two families. Among the mutations responsible for variegate porphyria in our country, c.1042_1043insT stands out, since it was described only in Argentina and is present in about 40% of genetically diagnosed families. Thus, we hypothesized the possible existence of a common ancestor for the mutation in our population. Methods: We conducted a study based on microsatellite (short tandem repeats) haplotypes. Results: We found a common haplotype in all of the patients carrying the common mutation. The age of the mutation was estimated to be about 375 years. Conclusion: There is a recent founder effect in our population for this particular genetic alteration in variegate porphyria.

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

A partial deficiency in protoporphyrinogen oxidase (PPOX) produces the acute/cutaneous (or mixed) variegate porphyria (VP), the third most frequent porphyria in Argentina. This autosomal dominant disorder is clinically characterized by skin lesions and/or acute neurovisceral attacks. The precise diagnosis of patients with a symptomatic VP is essential to provide accurate treatment. It is also critical to identify asymptomatic relatives to avoid precipitating factors and prevent acute attacks.Functional consequences of five PPOX missense mutations were evaluated in a prokaryotic expression system. Three mutations were found in families previously reported c.101A>T (p.E34V), c.670T>G (W224G), c.995G>C (G332A) and two were novel findings c.227C>T (p.S76F), c.1265A>G (p.Y422C). All mutations were identified in heterozygotes with reduced PPOX activity and variable clinical expression of the disease, including asymptomatic cases. Prokaryotic expression showed that all five missense mutations decreased the PPOX activity, demonstrating their detrimental effect on enzyme function, and thus, providing evidence for their causative role in VP. These results reinforce the importance of molecular genetic analysis for VP diagnosis and especially the usefulness of prokaryotic expression of missense mutations to assess their deleterious effect on PPOX activity.MM and BXG contributed equally to the publication. RES and MVR share senior authorship.