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.

Department of Pediatrics and Center for Applied Genomics 1st School of Medicine Charles University Prague Czech Republic