INVESTIGADORES
DE SIERVI Adriana
artículos
Título:
Two new mutations (H106P and L178V) in the protoporphyrinogen oxidase gene in Argentineanpatients with variegate porphyria
Autor/es:
DE SIERVI A; PARERA V; BATLLE AMC; ROSSETTI MV
Revista:
HUMAN MUTATION
Editorial:
WILEY-LISS, DIV JOHN WILEY & SONS INC
Referencias:
Año: 2000 vol. 181 p. 1 - 3
ISSN:
1059-7794
Resumen:
Protoporphyrinogen oxidase (PPOX) (EC1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway and catalyses the six-electron oxidation conversion of protoporphyrinogen IX to protoporphyrin IX. A defect in this enzyme results in variegate porphyria (VP) of which typical clinical signs are skin photosensitivity and/or acute neurovisceral attacks. Human PPOX has been characterized (Dailey and Dailey, 1996). The enzyme defect is inherited as an autosomal dominant trait with incomplete penetrance. The 8-Kb PPOX gene is situated on chromosome 1q22-23 (Roberts et al, 1995) and contains one noncoding and twelve coding exons. Human cDNA encoding PPOX has been cloned and sequenced (Nishimura et al, 1995; MIM#176200). Sequence analysis revealed that PPOX consists of 477 amino acids with a calculated molecular mass of 50.8kDa. Northern blot analysis revealed the synthesis of a 1.8 Kb pair mRNA for PPOX (Nishimura et al, 1995). So far, 77 different mutations were identified in the PPOX gene causing VP. We report here the biochemical and genetic studies performed in Argentinean VP patients. In the patient of family #1 direct DNA sequencing revealed a heterozygous A to C mutation at the nucleotide 317 if we start counting the first base of the initiating methionine as number 1. The base change results in a histidine to proline substitution at position 106 (H106P) in the deduced aminoacid sequence in exon 4. To confirm this point mutation, enzyme restriction analysis was performed in the proband and 50 unrelated normal subjects. The restriction studies were assayed on a DNA fragment of 758 bp length obtained after amplification, with Sph I restriction enzyme. Digestion produces two fragments of 687 and 71 bp in length, respectively, in a normal individual. H106P mutation abolishes the restriction site for Sph I obtaining three fragments of 758, 687 and 71 bp in length in the heterozygous individual. Sph I digestion revealed that H106P was absent in the 50 unrelated normal controls. The 8-Kb PPOX gene is situated on chromosome 1q22-23 (Roberts et al, 1995) and contains one noncoding and twelve coding exons. Human cDNA encoding PPOX has been cloned and sequenced (Nishimura et al, 1995; MIM#176200). Sequence analysis revealed that PPOX consists of 477 amino acids with a calculated molecular mass of 50.8kDa. Northern blot analysis revealed the synthesis of a 1.8 Kb pair mRNA for PPOX (Nishimura et al, 1995). So far, 77 different mutations were identified in the PPOX gene causing VP. We report here the biochemical and genetic studies performed in Argentinean VP patients. In the patient of family #1 direct DNA sequencing revealed a heterozygous A to C mutation at the nucleotide 317 if we start counting the first base of the initiating methionine as number 1. The base change results in a histidine to proline substitution at position 106 (H106P) in the deduced aminoacid sequence in exon 4. To confirm this point mutation, enzyme restriction analysis was performed in the proband and 50 unrelated normal subjects. The restriction studies were assayed on a DNA fragment of 758 bp length obtained after amplification, with Sph I restriction enzyme. Digestion produces two fragments of 687 and 71 bp in length, respectively, in a normal individual. H106P mutation abolishes the restriction site for Sph I obtaining three fragments of 758, 687 and 71 bp in length in the heterozygous individual. Sph I digestion revealed that H106P was absent in the 50 unrelated normal controls. In the patient of family #2 we found another new missense mutation: L178V. Direct sequencing of purified PCR products from patient revealed a heterozygous C to G mutation at the nucleotide 532 if we start counting the first base of the initiating methionine as number 1. The base change results in a leucine to valine substitution at position 178 (L178V) in the deduced aminoacid sequence in exon 6. To confirm this point mutation, enzyme restriction analysis was performed in the proband and 50 unrelated normal subjects. The restriction studies were assayed on a DNA fragment of 680 bp length obtained after amplification, with Blp I  restriction enzyme. Digestion produces three fragments of 543, 100 and 37 bp in length, respectively, in a normal individual. L178V mutation abolishes one restriction site for Blp I obtaining four fragments of 580, 543, 100 and 37 bp in length in the heterozygous individual. Blp I digestion revealed that L178V was absent in the 50 unrelated normal controls.  PCR products from patient revealed a heterozygous C to G mutation at the nucleotide 532 if we start counting the first base of the initiating methionine as number 1. The base change results in a leucine to valine substitution at position 178 (L178V) in the deduced aminoacid sequence in exon 6. To confirm this point mutation, enzyme restriction analysis was performed in the proband and 50 unrelated normal subjects. The restriction studies were assayed on a DNA fragment of 680 bp length obtained after amplification, with Blp I  restriction enzyme. Digestion produces three fragments of 543, 100 and 37 bp in length, respectively, in a normal individual. L178V mutation abolishes one restriction site for Blp I obtaining four fragments of 580, 543, 100 and 37 bp in length in the heterozygous individual. Blp I digestion revealed that L178V was absent in the 50 unrelated normal controls.  PCR products from patient revealed a heterozygous C to G mutation at the nucleotide 532 if we start counting the first base of the initiating methionine as number 1. The base change results in a leucine to valine substitution at position 178 (L178V) in the deduced aminoacid sequence in exon 6. To confirm this point mutation, enzyme restriction analysis was performed in the proband and 50 unrelated normal subjects. The restriction studies were assayed on a DNA fragment of 680 bp length obtained after amplification, with Blp I  restriction enzyme. Digestion produces three fragments of 543, 100 and 37 bp in length, respectively, in a normal individual. L178V mutation abolishes one restriction site for Blp I obtaining four fragments of 580, 543, 100 and 37 bp in length in the heterozygous individual. Blp I digestion revealed that L178V was absent in the 50 unrelated normal controls.  PCR products from patient revealed a heterozygous C to G mutation at the nucleotide 532 if we start counting the first base of the initiating methionine as number 1. The base change results in a leucine to valine substitution at position 178 (L178V) in the deduced aminoacid sequence in exon 6. To confirm this point mutation, enzyme restriction analysis was performed in the proband and 50 unrelated normal subjects. The restriction studies were assayed on a DNA fragment of 680 bp length obtained after amplification, with Blp I  restriction enzyme. Digestion produces three fragments of 543, 100 and 37 bp in length, respectively, in a normal individual. L178V mutation abolishes one restriction site for Blp I obtaining four fragments of 580, 543, 100 and 37 bp in length in the heterozygous individual. Blp I digestion revealed that L178V was absent in the 50 unrelated normal controls.