Novel Mutation and Polymorphisms of the HMBS Gene Detected by Denaturing HPLC

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Acute intermittent porphyria (AIP) is an autosomal dominant, inborn error of the metabolism of heme biosynthesis caused by partial deficiency of hydroxymethylbilane synthase (HMBS). This enzyme catalyzes the condensation of four molecules of porphobilinogen to a tetrapyrrole, hydroxymethylbilane. AIP is characterized by acute attacks of a neurological disorder manifesting as abdominal pain, hypertension, tachycardia, peripheral neuropathy, and mental dysfunction. Biochemical diagnosis of AIP relies on increased urinary porphobilinogen and normal fecal porphyrin excretion (1). Identification of presymptomatic AIP carriers in families with affected individuals is of clinical importance because avoidance of precipitating agents (e.g., drugs and alcohol) can prevent the occurrence of the first porphyric attack, which may be life-threatening. However, there is a significant overlap between the enzyme activities of healthy individuals and patients with AIP (2). In addition, there is a variant of AIP, in which the red blood cell (RBC) HMBS activity is normal (3). DNA-based diagnosis of presymptomatic AIP has been more reliable than diagnosis by RBC HMBS activity, and, thus, direct detection of mutations in presymptomatic AIP is now the definitive approach. The human HMBS gene spans ∼10 kb of DNA on chromosome 11q24.1-24.2 and contains 15 exons (4). Extensive allelic heterogeneity has been demonstrated in this gene, and 159 mutations have been identified in the HMBS gene (5). Most of these mutations are found only in individual families, except those found in Dutch (R116W) (6) and Swedish (W198X) (7) AIP families. Although DNA sequencing can identify all the mutations, this approach is both labor-intensive and time-consuming (8). Several methods that accelerate mutational screening before sequencing have been developed, such as single-strand conformation polymorphism analysis (9), heteroduplex gel analysis (10), and denaturing gradient gel electrophoresis (11)(12). Recently, denaturing HPLC (DHPLC) appears to be more sensitive than other methods in mutation detection, as exemplified …