RESUMO
Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.
Assuntos
5-Aminolevulinato Sintetase/genética , Hidroximetilbilano Sintase/biossíntese , Fígado/metabolismo , Porfiria Aguda Intermitente/genética , 5-Aminolevulinato Sintetase/biossíntese , Adulto , Ácido Aminolevulínico/sangue , Ácido Aminolevulínico/urina , Feminino , Heme/metabolismo , Humanos , Hidroximetilbilano Sintase/antagonistas & inibidores , Fígado/patologia , Transplante de Fígado , Porfobilinogênio/sangue , Porfobilinogênio/urina , Porfiria Aguda Intermitente/enzimologia , Porfiria Aguda Intermitente/patologia , RNA Mensageiro/biossíntese , Uroporfirinas/metabolismoRESUMO
Erythrodontia is the hallmark of human congenital erythropoietic porphyria (CEP), but is also a major phenotypic feature of acute intermittent porphyria (AIP) in cats. In this study, detailed biochemical and molecular analyses were performed on two unrelated cats with autosomal dominant AIP that presented with erythrodontia, yellow-brown urine and mild changes in erythrocytes. The cats had elevated concentrations of urinary 5-aminolevulinic acid and porphobilinogen, and half normal erythrocytic hydroxymethylbilane synthase (HMBS) activity. Two novel HMBS mutations were detected; one cat had a deletion (c.107_110delACAG) and one cat had a splicing alteration (c.826-1G>A), both leading to premature stop codons and truncated proteins (p.D36Vfs 6 and p.L276Efs 6, respectively). These studies highlight the importance of appropriate biochemical and molecular genetic analyses for the accurate diagnoses of porphyrias in cats and extend the molecular genetic heterogeneity of feline AIP. Thus, although erythrodontia is a classic sign of congenital erythropoietic porphyria in human beings, cats with erythrodontia may have acute intermittent porphyria, a hepatic porphyria.
Assuntos
Doenças do Gato/diagnóstico , Hidroximetilbilano Sintase/genética , Porfiria Aguda Intermitente/veterinária , Descoloração de Dente/veterinária , Animais , Gatos , Feminino , Florida , Hidroximetilbilano Sintase/metabolismo , Dados de Sequência Molecular , Mutação , Porfiria Aguda Intermitente/diagnóstico , Análise de Sequência de DNA/veterinária , Tennessee , Descoloração de Dente/diagnósticoRESUMO
Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder due to the deficient activity of uroporphyrinogen III synthase (UROS). Knock-in mouse models were generated for the common, hematologically severe human genotype, C73R/C73R, and milder genotypes (C73R/V99L and V99L/V99L). The specific activities of the UROS enzyme in the livers and erythrocytes of these mice averaged approximately 1.2%, 11% and 19% of normal, respectively. C73R/C73R mice that survived fetal life to weaning age (~12%) had a severe microcytic hypochromic anemia (hemoglobin 7.9 g/dL, mean cellular volume 26.6 fL, mean cellular hemoglobin content 27.4 g/dL, red cell distribution width 37.7%, reticulocytes 19%) and massively accumulated isomer I porphyrins (95, 183 and 44 µmol/L in erythrocytes, spleen and liver, respectively), but a nearly normal lifespan. In adult C73R/C73R mice, spleen and liver weights were 8.2- and 1.5-fold increased, respectively. C73R/V99L mice were mildly anemic (hemoglobin was 14.0 g/dL and mean cellular hemoglobin was 13.3), with minimally accumulated porphyrins (0.10, 5.54 and 0.58 µmol/L in erythrocytes, spleen and liver, respectively), whereas adult V99L/V99L mice were normal. Of note, even the mildest genotype, V99L/V99L, exhibited porphyria in utero, which disappeared by 2 months of age. These severe and mild mouse models inform therapeutic interventions and permit further investigation of the porphyrin-induced hematopathology, which leads to photo-induced cutaneous lesions. Of significance for therapeutic intervention, these mouse models suggest that only 11% of wild-type activity might be needed to reverse the pathology in CEP patients.
Assuntos
Modelos Animais de Doenças , Porfiria Eritropoética/genética , Uroporfirinogênio III Sintetase/genética , Animais , Animais Recém-Nascidos , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Genótipo , Humanos , Rim/enzimologia , Rim/metabolismo , Rim/patologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Fenótipo , Porfiria Eritropoética/enzimologia , Porfirinas/metabolismo , Baço/enzimologia , Baço/metabolismo , Baço/patologia , Fatores de Tempo , Uroporfirinogênio III Sintetase/metabolismoRESUMO
Hereditary coproporphyria (HCP) is an autosomal dominant acute hepatic porphyria due to the half-normal activity of the heme biosynthetic enzyme, coproporphyrinogen oxidase (CPOX). The enzyme catalyzes the step-wise oxidative decarboxylation of the heme precursor, coproporphyrinogen III, to protoporphyrinogen IX via a tricarboxylic intermediate, harderoporphyrinogen. In autosomal dominant HCP, the deficient enzymatic activity results primarily in the accumulation of coproporphyrin III. To date, only a few homozygous HCP patients have been described, most having Harderoporphyria, a rare variant due to specific CPOX mutations that alter enzyme residues D400-K404, most patients described to date having at least one K404E allele. Here, we describe a Turkish male infant, the product of a consanguineous union, who presented with the Harderoporphyria phenotype including neonatal hyperbilirubinemia, hemolytic anemia, hepatosplenomegaly, and skin lesions when exposed to UV light. He was homoallelic for the CPOX missense mutation, c.980A>G (p.H327R), and had massively increased urinary uroporphyrins I and III (9,250 and 2,910 µM, respectively) and coproporphyrins I and III (895 and 19,400 µM, respectively). The patient expired at 5 months of age from an apparent acute neurologic porphyric attack. Structural studies predicted that p.H327R interacts with residue W399 in the CPOX active site, thereby accounting for the Harderoporphyria phenotype.
Assuntos
Coproporfiria Hereditária/diagnóstico , Coproporfiria Hereditária/genética , Coproporfirinogênio Oxidase/genética , Porfirinogênios/metabolismo , Substituição de Aminoácidos/genética , Consanguinidade , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto , Porfirinogênios/genéticaRESUMO
The first feline model of human congenital erythropoietic porphyria (CEP) due to deficient uroporphyrinogen III synthase (URO-synthase) activity was identified by its characteristic clinical phenotype, and confirmed by biochemical and molecular genetic studies. The proband, an adult domestic shorthair cat, had dark-red urine and brownish discolored teeth with red fluorescence under ultraviolet light. Biochemical studies demonstrated markedly increased uroporphyrinogen I in urine and plasma (2,650- and 10,700-fold greater than wild type, respectively), whereas urinary 5-aminolevulinic acid and porphobilinogen were lower than normal. Erythrocytic URO-synthase activity was <1% of mean wild-type activity, confirming the diagnosis and distinguishing it from feline phenocopies having acute intermittent porphyria. Sequencing of the affected cat's UROS gene revealed two missense mutations, c.140C>T (p.S47F) in exon 3 and c.331G>A (p.G111S) in exon 6, both of which were homozygous, presumably owing to parental consanguinity. Neither was present in 100 normal cat alleles. Prokaryotic expression and thermostability studies of the purified monomeric wild-type, p.S47F, p.G111S, and p.S47F/G111S enzymes showed that the p.S47F enzyme had 100% of wild-type specific activity but ~50% decreased thermostability, whereas the p.G111S and p.S47F/G111S enzymes had about 60% and 20% of wild-type specific activity, respectively, and both were markedly thermolabile. Molecular modeling results indicated that the less active/less stable p.G111S enzyme was further functionally impaired by a structural interaction induced by the presence of the S47F substitution. Thus, the synergistic interaction of two rare amino acid substitutions in the URO-synthase polypeptide caused the feline model of human CEP.
Assuntos
Doenças do Gato/enzimologia , Doenças do Gato/genética , Homozigoto , Mutação de Sentido Incorreto/genética , Porfiria Eritropoética/veterinária , Porfirinas/metabolismo , Uroporfirinogênio III Sintetase/genética , Animais , Doenças do Gato/sangue , Doenças do Gato/urina , Gatos , Eritrócitos/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Porfiria Eritropoética/sangue , Porfiria Eritropoética/enzimologia , Porfiria Eritropoética/urina , Porfirinas/sangue , Porfirinas/urina , Uroporfirinogênio III Sintetase/química , Uroporfirinogênio III Sintetase/metabolismoRESUMO
Human acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn error of heme biosynthesis due to the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Here, we describe the first naturally occurring animal model of AIP in four unrelated cat lines who presented phenotypically as congenital erythropoietic porphyria (CEP). Affected cats had erythrodontia, brownish urine, fluorescent bones, and markedly elevated urinary uroporphyrin (URO) and coproporphyrin (COPRO) consistent with CEP. However, their uroporphyrinogen-III-synthase (URO-synthase) activities (deficient in CEP) were normal. Notably, affected cats had half-normal HMB-synthase activities and elevated urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), the deficient enzyme and accumulated metabolites in human AIP. Sequencing the feline HMB-synthase gene revealed different mutations in each line: a duplication (c.189dupT), an in-frame 3 bp deletion (c.842_844delGAG) identical to that causing human AIP and two missense mutations, c.250G>A (p.A84T) and c.445C>T (p.R149W). Prokaryotic expression of mutations c.842_844delGAG and c.445C>T resulted in mutant enzymes with <1% wild-type activity, whereas c.250G>A expressed a stable enzyme with approximately 35% of wild-type activity. The discolored teeth from the affected cats contained markedly elevated URO I and III, accounting for the CEP-like phenocopy. In three lines, the phenotype was an autosomal dominant trait, while affected cats with the c.250G>A (p.A84T) mutation were homozygous, a unique recessive form of AIP. These animal models may permit further investigation of the pathogenesis of the acute, life-threatening neurological attacks in human AIP and the evaluation of therapeutic strategies. GenBank Accession Numbers: GQ850461-GQ850464.
Assuntos
Doenças do Gato/enzimologia , Gatos/genética , Modelos Animais de Doenças , Hidroximetilbilano Sintase/genética , Mutação , Porfiria Aguda Intermitente/enzimologia , Porfiria Eritropoética/enzimologia , Animais , Osso e Ossos/metabolismo , Doenças do Gato/genética , Doenças do Gato/metabolismo , Gatos/metabolismo , Coproporfirinas/urina , Feminino , Humanos , Hidroximetilbilano Sintase/química , Hidroximetilbilano Sintase/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Porfiria Aguda Intermitente/genética , Porfiria Aguda Intermitente/metabolismo , Porfiria Eritropoética/genética , Porfiria Eritropoética/metabolismo , Porfirinas/metabolismo , Dente/metabolismo , Uroporfirinas/urinaRESUMO
Splicing mutations account for approximately 10% of lesions causing genetic diseases, but few branchpoint sequence (BPS) lesions have been reported. In 3 families with autosomal recessive congenital erythropoietic porphyria (CEP) resulting from uroporphyrinogen III synthase (URO-synthase) deficiency, sequencing the promoter, all 10 exons and the intron/exon boundaries did not detect a mutation. Northern analyses of lymphoblast mRNAs from 2 patients and reverse-transcribed polymerase chain reaction (RT-PCR) of lymphoblast mRNAs from all 3 patients revealed multiple longer transcripts involving intron 9 and low levels of wild-type message. Sequencing intron 9 RT-PCR products and genomic DNA in each case revealed homozygosity for a novel BPS mutation (c.661-31T-->G) and alternatively spliced transcripts containing 81, 246, 358, and 523 nucleotides from intron 9. RT-PCR revealed aberrant transcripts in both wild-type and CEP lymphoblasts, whereas BPS mutation reduced the wild-type transcript and enzyme activity in CEP lymphoblasts to approximately 10% and 15% of normal, respectively. Although the +81-nucleotide alternative transcript was in-frame, it only contributed approximately 0.2% of the lymphoblast URO-synthase activity. Thus, the BPS mutation markedly reduced the wild-type transcript and enzyme activity, thereby causing the disease. This is the first BPS mutation in the last intron, presumably accounting for the observed 100% intron retention without exon skipping.
Assuntos
Processamento Alternativo/genética , Mutação , Porfiria Eritropoética/genética , Uroporfirinogênio III Sintetase/genética , Adolescente , Adulto , Northern Blotting , Análise Mutacional de DNA , Éxons/genética , Saúde da Família , Feminino , Humanos , Íntrons/genética , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Porfiria Eritropoética/enzimologia , Porfiria Eritropoética/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Adulto JovemRESUMO
Propionic acidemia results from mutations in either of the two genes, PCCA or PCCB, that encode the two subunits of the propionyl-CoA carboxylase (PCC) enzyme. In this study, we report the identification and analysis of seven novel splicing mutations involving consensus donor and acceptor splice sites. Most of them were identified in patients with a Central Asian origin, and some present in several alleles, probably reflecting founder effects. The functional consequences of the splicing mutations were analyzed in patients' fibroblasts, as well as transcript quantification using real-time PCR methods. In the PCCA gene, two mutations were demonstrated to affect 5' splice sites (c.231+1G>C and c.1209+3A>G) and two 3' acceptor splice sites (c.1210delG and c.1430G>T), all causing skipping of the exons involved, with no detectable levels of normally spliced transcript. In the PCCB gene, all three mutations involved 5' donor splice sites-two affected exon 1 splicing (c.154_183+17del46 and c.183+2T>C), the latter activating a cryptic splice site in intron 1, and the remaining mutation (c.1498+2T>C) resulted in exon 14 skipping. The results highlight the necessity to perform transcript analysis in addition to genomic DNA sequencing to characterize the effect of splicing mutations and add relevant information on the genetic epidemiology of the disease.
Assuntos
Processamento Alternativo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Carbono-Carbono Ligases/genética , Mutação , Erros Inatos do Metabolismo dos Aminoácidos/epidemiologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Sequência de Bases , Éxons , Fibroblastos/metabolismo , Efeito Fundador , Genótipo , Heterozigoto , Humanos , Íntrons , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
In this work we analyze splicing mutations identified in propionic acidemia patients to clarify their functional effects and their involvement in the disease phenotype. Two mutations in the PCCA gene detected in homozygous patients and involving consensus splice sequences (IVS21+3del4 and IVS22-2A>G) were shown to produce some normal splicing in patients' cells, at very low levels, which were quantitated by real-time PCR methods, and which presumably are sufficient to moderate the phenotype. We have also analysed the effect of mutations c.653A>G and IVS10-11del6 in the PCCB gene present in heterozygous patients with mild phenotype. The c.653A>G mutation is located in the last codon of exon 6 and interferes with the correct spliceosomal assembly activating a cryptic splice site within exon 6, which leads to an in-frame six-nucleotide deletion (delV217-K218). Minigene analysis and sequence-specific hybridization probes using real-time PCR methods showed that no normally spliced transcript is detectable in the patients' fibroblasts. The IVS10-11del6 mutation shortens the polypyrimidine tract of the 3'-splice site of exon 11, resulting in exon skipping. Some normal transcript is detectable by allele-specific hybridization probes. These analyses suggest that, in some cases, the regulation of gene splicing can potentially play an important role in human disease influencing phenotypic parameters.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Cromonas , Splicing de RNA/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Análise Mutacional de DNA , Fibroblastos , Humanos , Fenótipo , Propionatos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de DoençaRESUMO
Propionic acidemia (PA, MIM 232000 and 232050) is caused by a deficiency of mitochondrial biotin-dependent propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric enzyme composed of alpha and beta subunits, which are encoded by the PCCA and PCCB genes, respectively. The PCCA protein (alpha subunit) is responsible for the formation of carboxybiotin upon hydrolysis of ATP and contains a C-terminal biotin-binding domain and a biotin carboxylase domain, defined by homology with other biotin-dependent carboxylases, some of them characterized structurally. More than 24 mutations have been found in the PCCA gene in patients with PA, among them 14 missense mutations and one in-frame deletion, for which the precise molecular effect is unknown. In this study, we have established the pathogenicity of 11 PCCA mutations (10 missense and an in-frame deletion) by expression studies in deficient fibroblasts and in a cell-free in vitro system, and analyzed the effect of each mutation on PCC activity, protein stability and domain structure. The results show that most mutant proteins show an increased turnover and are functionally deficient, suggesting that the structural alterations they cause are incompatible with normal assembly to produce a stable, functional PCC oligomer. These results are discussed in the context of the genotype-phenotype correlations in PCCA-deficient PA patients.