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.

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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.

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