Detection of DNA variations in the polymorphic hydroxymethylbilane synthase gene by high-resolution melting analysis

Acute intermittent porphyria (AIP) represents the most frequent type of acute porphyria. The underlying cause is a defect in the hydroxymethylbilane synthase (HMBS) gene. Diagnosis of AIP is crucial for preventing life-threatening, acute attacks among both symptomatic and asymptomatic carriers. We established the diagnostic tool, high-resolution melting (HRM), for diagnosing AIP. Of 13 amplicons amplified by PCR in the presence of the LCGreen Plus dye, 4 showed polymorphic backgrounds. The ability of the HRM method to detect DNA variations in the HMBS gene was tested on a DNA sample with 10 known mutations by a curve shape scan using the LightScanner instrument. Furthermore, genomic DNA (gDNA) samples from 97 individuals with suspected hepatic porphyria were tested. All possible genotypes from each of four polymorphic amplicons were detected. Each of the 10 mutations tested had an altered melting profile compared with the melting profile of the controls. Screening the group of subjects with suspected hepatic porphyria revealed nine different DNA variations, four of which were novel. In conclusion, HRM is a fast, cost-effective prescreening method for detecting DNA variations in the HMBS gene. Therefore, the screening can be easily applied to a porphyria family if misdiagnosis or rare dual porphyria is suspected.