Molecular analysis of acute intermittent porphyria: mutation screening in 20 patients in Germany reveals 11 novel mutations

Abstract

Acute intermittent porphyria (AIP) is a very rare autosomal dominant disorder with low penetrance. Mutations in the gene of the porphobilinogen deaminase (PBG-D), also called hydroxymethylbilane synthase (HMBS), cause a partial deficiency of this enzyme of the heme biosynthetic pathway. Overstimulation of heme biosynthesis causes clinical symptoms. Because of the variability of the symptoms, diagnosis is often delayed. Using two approaches for genetic analysis, first in a stepwise manner, then sequencing extensive parts of the gene, the screening of the DNA of 20 unrelated individuals revealed 20 different mutations, 11 of which had not been reported previously. The novel mutations affected intron 1 (33 + 2 T→C), exon 5 (181 G→C), intron 6 (267–61 del 8 bp), intron 7 (345–1 G→C), intron 9 (498 + 15 G→T and 499–13 Δ-14 bp indel TGA), intron 13 (825 + 1 G→C and 825 + 2 T→C), exon 15 (962 G-A, 1067 del A and 1067–1068 ins 5 bp). The other nine mutations detected affected intron 14, exons 6, 7, 8, 9, 10 (3×) and 12. In the majority of AIP patients, the genotype does not predict phenotypic expression. Since the sudden manifestation of the disease maybe prevented by early diagnosis, identification of AIP gene carriers is the best preventive measure. This was performed in five families, revealing 10 additional AIP gene carriers.

Introduction

Porphobilinogen deaminase (PBG-D; E.C. 4.3.1.8) or hydroxymethylbilane synthase (HMBS; MW 176000) is the third enzyme in the heme biosynthetic pathway. It catalyzes the formation of hydroxymethylbilane (HMB) from four porphobilinogen molecules [1]. In acute intermittent porphyria (AIP), an enzymopathy with low penetrance, inherited in autosomal dominant fashion, its activity is reduced to about 50%. Most individuals remain asymptomatic throughout their lives, but 10–20% develop intermittent attacks of neurovisceral dysfunction. The prevalence of the most common of the acute hepatic porphyrias is estimated to be 6 per 10,000 in the French Caucasian population [2].

Attacks are often precipitated by heme biosynthesis stimulating agents or situations (e.g., drugs, alcohol, starvation, stress) [1]. Detection of carriers is important to prevent the occurrence of unpredictable and sometimes fatal attacks. Mutations in the 10 kb PBG-D gene in chromosomal region 11q24.1→11q24.2 [3] lead to an alteration in the conformation of the enzyme. Over 230 mutations within the 15 exons and 14 introns are identified at present [4], [5], [6], [7]. Because of different promoters, two tissue-specific isoforms of PBG-D are formed. The larger housekeeping isoform (361 amino acids, 42 kDa) encoded by exon 1 and 3–15 is produced by ubiquitous tissue cells. The promoter resides in the 5′ flanking region of the gene. Erythroblasts generate the shorter isoform (344 amino acids, 40 kDa) encoded by exons 2–15. The promoter is located 3 kb downstream in exon 1. In classical AIP, housekeeping as well as erythroid-specific enzyme show a 50% reduced activity (95–98% of all AIP cases).

Mutations are located on exons 3–15. In the variant or nonerythroid AIP, mutations are located on exon 1, the neighboring introns or on a short section of exon 3 that precedes the initiating codon of the erythroid isoform [8]. In this case, as opposed to classical AIP, the erythroid-specific PBG-D has normal activity [9], [10], [11], [12].

Detection of urinary overproduction of the porphyrin precursors porphobilinogen (PBG) and δ-aminolevulinic acid (δ-ALA) is critical for the diagnosis of AIP [1]. In the latent phase of the disease, however, excretion of these porphyrin precursors is not necessarily increased. More specific is the measurement of HMBS activity in erythrocytes. Still, definite diagnosis is difficult due to a significant overlap between findings in carriers and controls [1], [12]. Reliable results are obtained using molecular biological methods. Information is obtained rapidly by fragment amplification of the gene with PCR, followed by direct sequencing. Before sequencing of the gene, application of denaturing gel electrophoresis (DGGE) has also been used [13].