Insertion of Alu element responsible for acute intermittent porphyria

In this study, we report a large Finnish family in which an Alu element int erferes with the coding region of the porphobilinogen deaminase (PBGD) gene resulting in acute intermittent porphyria (AIP), Polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) analysis of exon 5 among patients showed an abnormal band around 350 bp apart from the norma l bands, Subcloning and sequencing of the fragment revealed a 333-bp Alu se quence that was directly inserted into exon 5 in antisense orientation, The junction sequences included a 13-bp target site duplication. This Alu cass ette belongs to a Ya5 subfamily, one of the youngest and currently most act ive Alu subfamilies in evolution, The Alu insertion resulted in a dramatica lly decreased steady-state level of the allelic transcript, as this Alu seq uence could not be demonstrated by direct sequencing of the amplified cDNA synthesized from total RNA extracted from the patients' lymphoblast cell li nes. A stop codon present in the reading frame causes premature termination of PBGD synthesis. The predicted polypeptide contains 64 of the 361 amino acids of PBGD, followed by 13 amino acids that are not identical to the PBG D polypeptide, To further characterize the consequences of the insertion, t he Alu sequence was inserted into exon 5 of the PBGD cDNA and expressed in the eukaryotic COS-1 cell line. The mutated construct expressed no enzyme a ctivity comparable to that of the wild type PBGD; furthermore, fro mutant p rotein could be detected by Western blot analysis. Hum Mutat 13:431-438, 19 99, (C) 1999 Wiley-Liss, Inc.