Allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), a powerful method for diagnosing loss of imprinting of the 11p15 region in Russell Silver and Beckwith Wiedemann syndromes

• Published in: Human mutation Vol. 32; no. 2; pp. 249 - 258
• Main Authors: Azzi, Salah, Steunou, Virginie, Rousseau, Alexandra, Rossignol, Sylvie, Thibaud, Nathalie, Danton, Fabienne, Le Jule, Marilyne, Gicquel, Christine, Le Bouc, Yves, Netchine, Irène
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.02.2011; Hindawi Limited; Wiley
• Subjects: Beckwith-Wiedemann; Beckwith-Wiedemann Syndrome - diagnosis; Beckwith-Wiedemann Syndrome - genetics; CDKN1C; Chromosomes, Human, Pair 11; DNA Methylation; Genomic Imprinting; H19; Humans; ICR1; ICR2; IGF2; imprinting disorders; KCNQ1; Molecular Diagnostic Techniques - methods; Polymerase Chain Reaction - methods; Reproducibility of Results; Russell-Silver; Sensitivity and Specificity; Silver-Russell Syndrome - diagnosis; Silver-Russell Syndrome - genetics; Life Sciences
• ISSN: 1059-7794; 1098-1004; 1098-1004
• DOI: 10.1002/humu.21403

Many human syndromes involve a loss of imprinting (LOI) due to a loss (LOM) or a gain of DNA methylation (GOM). Most LOI occur as mosaics and can therefore be difficult to detect with conventional methods. The human imprinted 11p15 region is crucial for the control of fetal growth, and LOI at this locus is associated with two clinical disorders with opposite phenotypes: Beckwith-Wiedemann syndrome (BWS), characterized by fetal overgrowth and a high risk of tumors, and Russell-Silver syndrome (RSS), characterized by intrauterine and postnatal growth restriction. Until recently, we have been using Southern blotting for the diagnosis of RSS and BWS. We describe here a powerful quantitative technique, allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), for the diagnosis of these two complex disorders. We first checked the specificity of the probes and primers used for ASMM RTQ-PCR. We then carried out statistical validation for this method, on both retrospective and prospective populations of patients. This analysis demonstrated that ASMM RTQ-PCR is more sensitive than Southern blotting for detecting low degree of LOI. Moreover, ASMM RTQ-PCR is a very rapid, reliable, simple, safe, and cost effective method. Hum Mutat 32:249-258, 2011.