A novel MSP/DHPLC method for the investigation of the methylation status of imprinted genes enables the molecular detection of low cell mosaicism

We describe a new procedure for the analysis of the methlyation status of i mprinted genes based on methylation-specific PCR followed by denaturing hig h performance liquid chromatography (MSP/DHPLC). The method offers a rapid and very reliable alternative to conventional methods used for such purpose s such as Southern blots and methylation specific PCR (allele specific MSP) . The efficient resolution of the differentially methylated alleles is demo nstrated for two human imprinted genes, namely the SNRPN gene and the LIT1 gene (KCNQ1OT1). Abnormal imprinting of the two genes is associated with th e Angelman/Prader-Willi syndromes and the Beckwith-Wiedemann syndrome, resp ectively. The MSP/DHPLC method is based on PCR amplification of gene segmen ts which show parent-of-origin specific methylation. Genomic DNA is subject ed to an in vitro bisulfite treatment prior to PCR amplifications using pri mers specific for modified DNA. Both alleles are theoretically amplified wi th equal efficiency and are represented by identically sized PCR products; they differ, however, at a number of positions within the amplified DNA seg ment. The DHPLC analysis allows a very efficient resolution of the two popu lations of PCR products. The high sensitivity and quantitative properties o f the MSP/DHPLC method are illustrated based on its ability to reveal a low cell mosaicism in an infant with a maternal uniparental disomy 15 (i.e., P rader-Willi syndrome patient). The minor cell line (approximately 8% in blo od) was not detectable with conventional molecular analysis. While the dete ction of low cell mosaicisms of structurally abnormal chromosomes usually r elies on cytogenetic studies, the MSP/DHPLC method described here not only offers an alternative at the molecular level, but may also reveal mosaicism s concerning structurally intact chromosomes. Hum Mutat 17:423-430, 2001. ( C) 2001 Wiley Liss, Inc.