The sensitivity of single-strand conformation polymorphism (SSCP) analysis for the detection of mutations in the porphobilinogen deaminase (PBGD) gene among Finnish patients with acute intermittent porphyria (AIP) was studied. 13 novel mutations including one de novo event, and six previously characterized mutations were identified among AIP patients. The 19 mutations reported here for 28 families cover 72% of all the AIP families in the Finnish population of five million. When compared to direct sequencing, SSCP-analysis detected 17 (89%) of the 19 mutations when a combination of various electrophoretic conditions were used. The most informative electrophoretic condition was a gel run without glycerol in the coldroom (11/18 mutations). 86% of mutations were identified from amplified fragments greater than 300 bp and detection was dependent on both the amount of glycerol in the gel and the running temperature, but seemed to be independent of the size of the analyzed fragment or the type of mutation. The diagnostic efficiency of biochemical assays versus mutation screening in the PBGD gene was studied in three large AIP families, each representing different CRIM subtypes of AIP. The results demonstrated that using assays of erythrocyte PBGD activity, the majority (82%) of family members (n = 51) were diagnosed correctly. Of a total of 81 family members, 30 of whom had deficiency of PBGD confined to non-erythroid tissues, diagnosis at the asymptomatic stage of disease in 11 individuals (14%) required the application of mutation screening.

Three splicing defects (IVS1+3G-->T, 86A-->T, IVS13-2A-->G), an insertion (416insCA), and two missense mutations (664G-->A and 833T-->G) in the porphobilinogen deaminase (PBGD) gene were identified in six unrelated Finnish patients with acute intermittent porphyria (AIP). The IVS1+3G-->T substitution resulted in activation of a cryptic splice site in intron 1 and retention of a 67-bp fragment in the mutant transcript. The 86A-->T mutation at the end of exon 3 was predicted to cause an amino acid substitution (E29L). However, sequencing of the cDNA sample of the proband revealed exon 3 skipping from the mutant transcript. The IVS13-2A-->G substitution caused retention of intron 13 in the mutant transcript. In exon 8, 416insCA resulted in a frameshift. All three splicing defects and the CA insertion resulted in a truncated protein and thus, probably the loss of PBGD activity. The two novel missense mutations, 664G-->A in exon 12 and 833T-->C in exon 14 caused a single amino acid substitution (V222M and L278P). So far 25 different mutations have been characterized from 37 (93%) of a total of 40 unrelated Finnish AIP families, confirming the genetic heterogeneity of the disease even in a previously isolated area of Finland.

We investigated the effects of heme on metabolism of coumarin, debrisoquin, caffeine, and lidocaine in seven female patients with variegate porphyria and in 10 healthy men. During baseline conditions metabolism of the drugs was identical in the two groups. Compared with the results without heme, a single infusion of heme arginate (3 mg/kg heme) significantly decreased the debrisoquin/4-hydroxy-debrisoquin metabolic ratio in subjects with porphyria (p = 0.016) and in the control subjects (p = 0.016) and increased formation of monoethylglycinexylidide from lidocaine (p = 0.016 and p = 0.004, respectively). Metabolism of coumarin and caffeine was not affected by heme. Our results show that, in patients with porphyria and in healthy subjects, exogenous heme is able to accelerate the reactions mediated by the cytochrome isozymes CYP2D6 (debrisoquin) and CYP3A4 (lidocaine) but not reactions mediated by CYP1A2 (caffeine) and CYP2A6 (coumarin). This suggests that influence of heme on drug metabolism is P450 isozyme-specific.

Background: Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of porphobilinogen deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symptoms. Although molecular biological studies on the porphobilinogen deaminase (PBGD) gene have revealed several mutations responsible for AIP, the properties of mutant PBGD in eukaryotic expression systems have not been studied previously. Materials and Methods: Seven mutations were analyzed using transient expression of the mutated polypeptides in COS-1 cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot analysis, pulse-chase experiments, and immunofluorescence staining. Results: Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resulted in a decreased enzyme activity (0-5%), but R225G and 1073delA (elongated protein) displayed a significant residual activity of 16% and 50%, respectively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a truncated protein. In the pulse-chase experiment, the mutant polypeptides were as stable as the wild-type enzyme. In the immunofluorescence staining both wild-type and mutant polypeptides were diffusely dispersed in the cytoplasm and, thus, no accumulation of mutated proteins in the cellular compartments could be observed. Conclusions: The results confirm the causality of mutations for the half normal enzyme activity measured in the patients' erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a detectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBGD and independent of the cross-reacting immunological material (CRIM) class.