Four common mutations of the cystathionine beta-synthase gene detected by multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Background. A deficiency of cystathionine beta-synthase (CBS) is the most f requent cause of homocystinuria. The effect of therapy is related to the un derlying CBS genotype, which makes early diagnosis of this genetic defect i mportant. Our aim was to develop a fast and reliable method based on matrix -assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spect rometry for the determination of common mutations of the CBS gene. Methods: We used MALDI-TOF mass spectrometry to detect four common CBS muta tions (G307S, T272M, I278T, and V320A). The method is based on multiplex PC R of exons 7, 8, and 9, followed by single nucleotide extension in the pres ence of dideoxy NTPs of four primers targeted at the separate mutation site s. The extension products, as well as the 3-hydroxypicolinic acid matrix, w ere incubated with cation-exchange beads to remove disturbing salt contamin ants. Results: The above-mentioned mutations were determined in samples from 12 h omocystinuria patients. The MALDI-TOF spectra allowed unambiguous discrimin ation between primers and extension products (>9 Da) in the mass range betw een 4500 and 7500 Da. No labeled primers or ddNTPs were required. The genot yping was verified by reference technique. Conclusion: Our results demonstrate fast, simple, and unambiguous multiplex genotyping of four common CBS mutations by MALDI-TOF mass spectrometry. (C ) 1999 American Association for Clinical Chemistry.