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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.

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&#95;731delCT and c.982&#95;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.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder of heme biosynthesis caused by molecular defects in the hydroxymethylbilane synthase (HMBS) gene. In this study, we report two novel missense sequence variations in the HMBS gene, T59I (C176T) and V215M (G643A), in two patients with clinical symptoms compatible with acute attacks of porphyria. However, only the patient who carried V215M presented with full AIP-affirming biochemical evidence. Both variant proteins were expressed in a prokaryotic system and characterized in vitro. Recombinant T59I and V215M had residual activity of 80.6% and 19.4%, respectively, of that of the wild type enzyme. Moreover, changes in K(m), V(max) and thermostability observed in the recombinant V215M suggest a causal relationship between V215M and AIP. The association between the T59I substitution and AIP is less obvious. Based on our investigation, substitution T59I is more likely to be a mutation with a weak effect than a rare form of polymorphism. This study demonstrates that in vitro characterization of missense variations in the HMBS gene can provide valuable information for the interpretation of clinical, biochemical and genetic data, for establishing a diagnosis of AIP. It also highlights the fact that there are still many aspects to be investigated concerning AIP and corroborates the need to report new data that can help to clarify the genotype-phenotype relationship.

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.