Molecular analysis of acute intermittent porphyria: mutation screening in 20 patients in Germany reveals 11 novel mutations
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].
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Patients
For our study, blood samples of 20 symptomatic unrelated patients were provided after informed consent. Seventeen were females and three were males (Table 1). Clinical data were obtained for the majority of the patients. Seventeen individuals were of German origin, one patient was from Afghanistan, one from former Yugoslavia and one of Turkish origin. Carrier screening was performed in the family of patient nos. 1, 6, 10, 14 and 19 .
First approach: DNA preparation and strategy
DNA was prepared from lymphocytes or lymphoblastoid cells
Results and discussion
The gene sequence for hydroxymethylbilane synthase was examined in 17 patients of German origin, one of Afghan descent (patient no. 8), one patient from former Yugoslavia (patient no. 18) and one from Turkey (patient no. 4). Of the total of 20 mutations characterized, 11 are novel mutations, 9 have been reported previously (Table 4).
The mutations identified represent nine missense mutations (MS), eight splicing defects (SD), two frameshifts (FS) and one nonsense mutation (NS). Similar to the
Acknowledgements
We thank the patients who donated their blood samples for molecular analysis and our colleagues who provided clinical data. Special thanks to Laboratory Medicine Dortmund, Dr. A. Eberhard and Partners for their collaboration.
Financial support was obtained through a grant from Charité, Humboldt-University.
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