Acute intermittent porphyria (AIP) represents the most frequent type of acute porphyria. The underlying cause is a defect in the hydroxymethylbilane synthase (HMBS) gene. Diagnosis of AIP is crucial for preventing life-threatening, acute attacks among both symptomatic and asymptomatic carriers. We established the diagnostic tool, high-resolution melting (HRM), for diagnosing AIP. Of 13 amplicons amplified by PCR in the presence of the LCGreen Plus dye, 4 showed polymorphic backgrounds. The ability of the HRM method to detect DNA variations in the HMBS gene was tested on a DNA sample with 10 known mutations by a curve shape scan using the LightScanner instrument. Furthermore, genomic DNA (gDNA) samples from 97 individuals with suspected hepatic porphyria were tested. All possible genotypes from each of four polymorphic amplicons were detected. Each of the 10 mutations tested had an altered melting profile compared with the melting profile of the controls. Screening the group of subjects with suspected hepatic porphyria revealed nine different DNA variations, four of which were novel. In conclusion, HRM is a fast, cost-effective prescreening method for detecting DNA variations in the HMBS gene. Therefore, the screening can be easily applied to a porphyria family if misdiagnosis or rare dual porphyria is suspected.
- MeSH
- akutní intermitentní porfyrie diagnóza genetika MeSH
- algoritmy MeSH
- analýza polymorfismu délky amplifikovaných restrikčních fragmentů MeSH
- denaturace nukleových kyselin MeSH
- detekce genetických nosičů metody MeSH
- DNA sondy diagnostické užití krev MeSH
- fluorescenční barviva diagnostické užití MeSH
- fluorescenční spektrometrie přístrojové vybavení MeSH
- genetické testování metody MeSH
- hydroxymethylbilansynthasa genetika MeSH
- interkalátory diagnostické užití MeSH
- jaterní porfyrie diagnóza genetika MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus genetický MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Mutations in the hydroxymethylbilane synthase (HMBS) gene are responsible for the inherited disorder of acute intermittent porphyria (AIP). AIP is diagnosed on the basis of characteristic clinical symptoms, elevated levels of urinary porphyrin precursors aminolevulinic acid (ALA) and porphobilinogen (PBG) and a decreased erythrocytic HMBS activity, although an identifiable HMBS mutation provides the ultimate proof for AIP. Six Israeli AIP families underwent biochemical and mutation analysis in order to establish an AIP diagnosis. Variability with respect to the ALA/PBG levels and HBMS activity was found among the index patients. Indeed, each family carried a unique mutation in the HMBS gene. A novel missense c.95G>C (p.R32P) was shown to be a de novo mutation in one family, along with five known mutations p.T59I, p.D178N, p.V215M, c.730_731delCT and c.982_983delCA identified in the rest of the families. Both R32P and D178N were expressed in a prokaryotic system. Recombinant p.R32P was enzymatically inactive as demonstrated by a <1% residual activity, whereas p.D178N possessed 81% of the activity of the wild type enzyme. However, the p.D178N mutant did display a shift in optimal pH and was thermo labile compared to the wild type. Among the four missense mutations, p.R32P and p.V215M had not only harmful effects on the enzyme in vitro but also were associated with high levels of ALA/PBG in patients. On the other hand, the in vitro effect of both p.T59I and p.D178N, and the impact of these mutations on the enzyme structure and function as interpreted by the 3-D structure of the Escherichia coli enzyme, were weaker than that of p.R32P and p.V215M. Concomitantly, patients carrying the p.T59I or p.D178N had normal or borderline increases in ALA/PBG concentrations although they presented characteristic clinical symptoms. These findings provided further insights into the causal relationship between HMBS mutations and AIP.
- MeSH
- akutní intermitentní porfyrie etnologie genetika MeSH
- bodová mutace MeSH
- dospělí MeSH
- exony genetika MeSH
- financování organizované MeSH
- hydroxymethylbilansynthasa genetika chemie MeSH
- konformace proteinů MeSH
- kyselina aminolevulová moč MeSH
- lidé středního věku MeSH
- lidé MeSH
- missense mutace MeSH
- mladiství MeSH
- mladý dospělý MeSH
- molekulární modely MeSH
- mutační analýza DNA MeSH
- porfobilinogen metabolismus MeSH
- proteiny z Escherichia coli chemie MeSH
- sekvenční delece MeSH
- senioři nad 80 let MeSH
- stabilita proteinů MeSH
- substituce aminokyselin MeSH
- vztahy mezi strukturou a aktivitou MeSH
- židé genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Evropa MeSH
- Indie MeSH
- Izrael MeSH
Acute intermittent porphyria is an autosomal dominantly inherited disorder, classified as acute hepatic porphyria, caused by a deficiency of hydroxymethylbilane synthase (EC 2.5.1.61, EC 4.3.1.8, also known as porphobilinogen deaminase, uroporphyrinogen I synthase), the third enzyme in heme biosynthesis. Clinical features include autonomous, central, motor or sensory symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. A diagnosis of acute intermittent porphyria is crucial to prevent life-threatening acute attacks. Detection of DNA variations by molecular techniques allows a diagnosis of acute intermittent porphyria in situations where the measurement of porphyrins and precursors in urine and faeces and erythrocyte hydroxymethylbilane synthase activity is inconclusive. In the present study, we identified gene defects in six Czech patients with acute intermittent porphyria, as diagnosed based on biochemical findings, and members of their families to confirm the diagnosis at the molecular level and/or to provide genetic counselling. Molecular analyses of the hydroxymethylbilane synthase gene revealed seven mutations. Four were previously reported: c.76C>T, c.77G>A, c.518G>A, c.771 + 1G>T (p.Arg26Cys, p.Arg26His, p.Arg173Gln). Three were novel mutations: c.610C>A, c.675delA, c.750A>T (p.Gln204Lys, p.Ala226ProfsX28, p.Glu250Asp). Of particular interest, one patient had two mutations (c.518G>A; c.610C>A), both located in exon 10 of the same allele. To establish the effects of the mutations on enzyme function, biochemical characterization of the expressed normal recombinant and mutated proteins was performed. Prokaryotic expression of the mutant alleles of the hydroxymethylbilane synthase gene revealed that, with the exception of the p.Gln204Lys mutation, all mutations resulted in little, if any, enzymatic activity. Moreover, the 3D structure of the Escherichia coli and human protein was used to interpret structure-function relationships for the mutations in the human isoform.
- MeSH
- akutní intermitentní porfyrie diagnóza enzymologie genetika MeSH
- Escherichia coli enzymologie genetika metabolismus MeSH
- financování organizované MeSH
- hydroxymethylbilansynthasa genetika metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- mutační analýza DNA MeSH
- sekvence nukleotidů MeSH
- substituce aminokyselin MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
The porphyrias are group of mostly inherited disorders in which a specific spectrum of accumulated and excreted porphyrins and heme precursors are associated with characteristic clinical features. There are eight enzymes involved in the heme synthesis and defects in seven of them cause porphyria. Four of them are described as acute hepatic porphyrias, which share possible precipitation of acute attacks with symptoms engaging the nervous system. Acute intermittent porphyria (AIP), caused by partial deficiency of the porphobilinogen deaminase (PBGD), is the most frequent among hepatic porphyrias. Clinical expression is highly variable and ~ 90 % of AIP heterozygotes remain asymptomatic throughout life. During systematic genetic analysis of the AIP patients diagnosed in the Czech and Slovak Republics, we found a special case of AIP. In a 15-year-old boy with abdominal and subsequent neurological symptomatology, we identified de novo mutation 966insA within the PBGD gene leading to a stop codon after 36 completely different amino acids compared to the wt-sequence. To establish the effects of this mutation on the protein structure, we expressed mutant constructs with described mutation in E. coli and analyzed their biochemical and enzymatic properties. Moreover, computer-assisted protein structure prediction was performed.
- MeSH
- akutní intermitentní porfyrie diagnóza enzymologie genetika MeSH
- běloši MeSH
- Escherichia coli metabolismus MeSH
- hydroxymethylbilansynthasa chemie genetika izolace a purifikace MeSH
- lidé MeSH
- mladiství MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutace * MeSH
- mutační analýza DNA MeSH
- rodokmen MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
The porphyrias arise from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. All genes encoding these enzymes have been cloned and several mutations underlying the different types of porphyrias have been reported. Traditionally, the diagnosis of porphyria is made on the basis of clinical symptoms, characteristic biochemical findings, and specific enzyme assays. In some cases however, these diagnostic tools reveal overlapping findings, indicating the existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultaneously. Recently, it was reported that the so-called Chester porphyria shows features of both variegate porphyria and acute intermittent porphyria. Linkage analysis revealed a novel chromosomal locus on chromosome 11 for the underlying genetic defect in this disease, suggesting that a gene that does not encode one of the enzymes of heme biosynthesis might be involved in the pathogenesis of the porphyrias. After excluding candidate genes within the linkage interval, we identified a nonsense mutation in the porphobilinogen deaminase gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria. However, we could not detect a mutation in the coding or the promotor region of the protoporphyrinogen oxidase gene that is mutated in variegate porphyria. Our results indicate that Chester porphyria is neither a dual porphyria, nor a separate type of porphyria, but rather a variant of acute intermittent porphyria. Further, our findings largely exclude the possibility that a hitherto unknown gene is involved in the pathogenesis of the porphyrias.
- MeSH
- akutní intermitentní porfyrie klasifikace genetika MeSH
- ferredoxiny genetika MeSH
- flavoproteiny genetika MeSH
- hydroxymethylbilansynthasa genetika MeSH
- lidé MeSH
- mitochondriální proteiny genetika MeSH
- molekulární sekvence - údaje MeSH
- mutační analýza DNA MeSH
- nesmyslný kodon * MeSH
- protoporfyrinogenoxidasa genetika MeSH
- sekvence nukleotidů MeSH
- sukcinátdehydrogenasa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Acute intermittent porphyria (AIP) is an autosomal dominant disorder of heme biosynthesis caused by molecular defects in the porphobilinogen deaminase (PBGD) gene. This paper reviews published mutations, their types, and polymorphisms within the PBGD gene. To date, 301 different mutations and 21 polymorphisms have been identified in the PBGD gene in AIP patients and individuals from various countries and ethnic groups. During the search for mutations identified among Slavic AIP patients we found 65 such mutations and concluded that there is not a distinct predominance of certain mutations in Slavs.
- MeSH
- akutní intermitentní porfyrie epidemiologie genetika MeSH
- běloši genetika MeSH
- hydroxymethylbilansynthasa genetika MeSH
- lidé MeSH
- mutace * MeSH
- polymorfismus genetický * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- srovnávací studie MeSH
- MeSH
- akutní intermitentní porfyrie genetika krev moč MeSH
- exony MeSH
- genetické testování MeSH
- hydroxymethylbilansynthasa genetika krev MeSH
- introny MeSH
- lidé MeSH
- mutace MeSH
- porfobilinogen moč MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
