PCR-based molecular diagnosis of Prader-Willi and Angelman syndromes using restriction analysis after bisulfite treatment. Potential for quantitative estimation

Abstract

Assays to estimate the DNA methylaion in the Prader-Willi (PWS)/Angelman syndrome (AS) region on chromosome 15q are widely used and are recommended for the initial screening of individuals with suspected PWS and AS. (ASHG/ACMG Test and Technology Transfer Committee. A.J.H.G., 58:1085, 1996). The Southern blotting-based methylation tests currently used can identify 100% of the patients with PWS and about 75% of the patients with AS. Recently, a methylation specific PCR (mPCR) technique for PWS/AS diagnosis was developed in order to increase the rapidity of the test and to minimize the amount of the genomic DNA necessary for testing. (Kubota T. et al. Nature Genetics, 16:16, 1997).

We have developed a PCR-based method to detect the DNA mehylation at the proximal 15q region, using restriction analysis after bisulfite treatment of total genomic DNA. Unlike the mPCR test, our method uses only one set of primers to amplify the two subpopulations of fragments from the methylated and non-methylated portion of the genomic DNA. We thus avoid the need to adjust the relative primer amounts required for the mPCR method and we have been able to overcome the potential problems that may occur while using the mPCR technique related to the non-specific inhibition of the PCR process. In addition our protocol, unlike the mPCR method, can be further developed to quantitatively assess the methylated versus non-methylated DNA ratios (Xiong Z. et al. Nucl.Ac.Res., 25:2532-2534, 1997). Such quantitative estimation may be used to detect mosaic PWS/AS patients, as well as patients with duplications of the proximal region of chromosome 15q.

We are currently using this protocol to test control and proven PWS and AS specimens in order to establish the reliability of the method for clinical application.