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.

Genetic and biochemical studies in Argentinean patients with variegate porphyria.

BACKGROUND: A 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. METHODS: We 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. RESULTS: All 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. CONCLUSION: Molecular 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.

[Identification of mutations in the protoporphyrin oxidase gene and its diagnostic implications in porphyria variegata in Chile]. / Porfiria variegata en chile: identificación de mutaciones en el gen protoporfirinógeno oxidasa y su implicancia diagnóstica.

Variegate porphyria (VP) results from a hereditary deficiency of protoporphyrinogen oxidase (PPOX) that is transmitted in an autosomal dominan fashion. The diagnosis is based on the clinical symptoms and is confirmed biochemically. Sometimes, however, these diagnostic tools reveal limitations in establishing the definitive diagnosis of the prevailing type of acute porphyria. In these patients, molecular genetic analyses can be useful. We performed molecular genetic studies in 13 Chilean families by PCR amplification of the PPOX gene, conformation sensitive gel electrophoresis, and automated DNA sequencing. In five symptomatic patients from different families, respectively, the biochemical data confirmed the diagnosis of VP. In seven other families, however, the biochemical studies were not conclusive. Furthermore, the original biochemical analysis in one clinically severely affected patient from a further family even suggested the diagnosis of erythropoietic protoporphyria (EPP). Beside the respective index patients, we studied 78 asymptomatic family members and 50 healthy, unrelated individuals for control purposes. In five families, the previous diagnosis of VP could be confirmed genetically. Further, half of the asymptomatic relatives revealed a mutation in the PPOX gene, consisting of three missense mutations and two deletion mutations. Mutation R168H that had been already described previously in German VP families was found in a Chilean family of German origin. Further, two novel missense mutations, designated L74P and G232S, could be detected. In four Chilean families, we found the deletion 1330deICT that had also been previously described in three Swedish VP families. The second deletion, 1239delTACAC, has not been described anywhere else but Chile and could be identified in seven families. One patient who was initially diagnosed with EPP turned out to be a compound heterozygote for mutations on both alleles of the PPOX gene. In conclusion, our molecular genetic analyses unequivocally confirmed the diagnosis of VP in seven families who originally had revealed inconclusive biochemical data. Further, early genetic analysis allows for the identification of asymptomatic mutation carriers, thereby offering the possibility of adequate counselling and the prevention of potentially life-threatening acute porphyric attacks.

Porfiria variegata en Chile: identificación de mutaciones en el gen protoporfirinógeno oxidasa y su implicancia diagnóstica / Identification of mutations in the protoporphyrin oxidase gene and its diagnostic implications in porphyria variegata in Chile

Variegate porphyria (VP) results from a hereditary deficiency of protoporphyrinogen oxidase (PPOX) that is transmitted in an autosomal dominan fashion. The diagnosis is based on the clinical symptoms and is confirmed biochemically. Sometimes, however, these diagnostic tools reveal limitations in establishing the definitive diagnosis of the prevailing type of acute porphyria. In these patients, molecular genetic analyses can be useful. We performed molecular genetic studies in 13 Chilean families by PCR amplification of the PPOX gene, conformation sensitive gel electrophoresis, and automated DNA sequencing. In five symptomatic patients from different families, respectively, the biochemical data confirmed the diagnosis of VP. In seven other families, however, the biochemical studies were not conclusive. Furthermore, the original biochemical analysis in one clinically severely affected patient from a further family even suggested the diagnosis of erythropoietic protoporphyria (EPP). Beside the respective index patients, we studied 78 asymptomatic family members and 50 healthy, unrelated individuals for control purposes. In five families, the previous diagnosis of VP could be confirmed genetically. Further, half of the asymptomatic relatives revealed a mutation in the PPOX gene, consisting of three missense mutations and two deletion mutations. Mutation R168H that had been already described previously in German VP families was found in a Chilean family of German origin. Further, two novel missense mutations, designated L74P and G232S, could be detected. In four Chilean families, we found the deletion 1330deICT that had also been previously described in three Swedish VP families. The second deletion, 1239delTACAC, has not been described anywhere else but Chile and could be identified in seven families. One patient who was initially diagnosed with EPP turned out to be a compound heterozygote for mutations on both alíeles of the PPOX gene. In conclusion, our molecular genetic analyses unequivocally confirmed the diagnosis of VP in seven families who originally had revealed inconclusive biochemical data. Further, early genetic analysis allows for the identification of asymptomatic mutation carriers, thereby offering the possibility of adequate counselling and the prevention of potentially life-threatening acute porphyric attacks.

La porfiria variegata (PV), enfermedad de origen genético con forma de herencia autosómica dominante, se debe a deficiencia en la actividad protoporfirinógeno oxidasa (PPOX). Su diagnóstico se basa en antecedentes clínicos y se confirma con análisis bioquímicos. Éstos, en algunos casos, pueden presentar limitaciones para establecer el diagnóstico definitivo de la variedad de porfiria aguda, situación en que el estudio genético molecular puede resultar útil. Se efectuó estudio genético en trece familias chilenas usando amplificación del gen PPOX por PCR, electroforesis conformacional y secuenciación automática de DNA. Cinco de estas familias incluían pacientes índices sintomáticos con diagnóstico bioquímico establecido de PV; otras siete familias incluían pacientes índices con estudio bioquímico no concluyente de la variedad de porfiria aguda y, finalmente, una familia con diagnóstico previo de protoporfiria eritropoyética (PPE). Además, se estudiaron 78 familiares asintomáticos y 50 personas sanas, no relacionadas, como controles. En cinco familias el estudio genético confirmó el diagnóstico bioquímico previo de PV. El 50% de los familiares asintomáticos resultaron ser portadores de una mutación en el gen PPOX. Se identificaron tres mutaciones por sustitución de bases: la R168H, descrita en familias de origen alemán y dos nuevas mutaciones, designadas L74P y G232S. También se identificaron dos mutaciones por deleción de bases designadas 1330delCT y la 1239delTACAC. La primera, que había sido descrita previamente en tres familias suecas, se encontró en cuatro familias chilenas. La segunda se encontró en siete familias y no ha sido descrita previamente. El estudio genético permitió mostrar que un paciente que originalmente fue diagnosticado con PPE correspondía a un heterocigoto compuesto para dos mutaciones en el gen PPOX. En conclusión, los estudios moleculares permitieron confirmar el diagnóstico de PV en cinco familias, efectuar diagnóstico de PV en familias en las cuales los datos bioquímicos no eran concluyentes, corregir el diagnóstico original en una familia e identificar portadores asintomáticos entre los familiares de los pacientes índices. Los estudios genéticos moleculares ayudan a realizar un adecuado consejo genético a pacientes y familiares y hace posible practicar prevención de las crisis agudas de porfiria, las que son potencialmente mortales.

A Chilean boy with severe photosensitivity and finger shortening: the first case of homozygous variegate porphyria in South America.

A 7-year-old Chilean boy presented with severe photosensitivity, blistering, erosions and scarring on sun-exposed areas of the body since the age of 6 months. Additionally, he showed a short stature and shortening of the fingers. Laboratory examination revealed greatly elevated protoporphyrin levels in the blood. Such biochemical findings can be observed in homozygous variants of usually autosomal dominantly inherited acute porphyrias such as variegate porphyria (VP) and hereditary coproporphyria, which usually do not become manifest before the second or third decade of life in heterozygotes. Using polymerase chain reaction-based techniques we identified a missense mutation in exon 7 on the paternal allele and a frameshift mutation in exon 13 on the maternal allele of the protoporphyrinogen oxidase gene that harbours the mutations underlying VP. This is the first homozygous case of VP in South America. As VP represents the most frequent type of acute porphyria not only in Chile but also in South Africa, more such cases could be expected in the future, particularly because a founder mutation for this disease has already been described in the Chilean and South African population.