Rapid detection of subtelomeric deletion/duplication by novel real-time quantitative PCR using SYBR-green dye

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, miscarriages, and hematological malignancies. Automated detection of subtle deletions and duplications involving telomeres is essential for high-throughput screening procedures, but impractical when conventional cytogenetic methods are used. Novel real-time PCR quantitative genotyping of subtelomeric amplicons using SYBR green dye allows high-resolution screening of single copy number gains and losses by their relative quantification against a diploid genome. To assess the applicability of the technique in the screening and diagnosis of subtelomeric imbalances, we describe here a blinded study in which DNA from 20 negative controls and 20 patients with known unbalanced cytogenetic abnormalities involving at least one or more telomeres were analyzed using a novel human subtelomere-specific primer set, producing altogether 86 amplicons, in the SYBR-green I-based real, time quantitative PCR screening approach. Screening of the DNA samples from 20 unrelated controls for copy number polymorphism do not detect any polymorphism in the set of amplicons, but single-copy-number gains and losses were accurately detected by quantitative PCR in all patients, except the copy number alterations of the subtelomeric p-arms of the acrocentric chromosomes in two cases. Furthermore, a detailed mapping of the deletion/translocation breakpoint was demonstrated in two cases by novel real,time PCR primer-jumping. Because of the simplicity and flexibility of the SYBR-green I-based real,time detection, the primer-set can easily be extended, either to perform further detailed molecular characterization of breakpoints or to include amplicons for the detection and/or analysis of syndromes that are associated with genomic copy number alterations, e.g., detetion/duplication-syndromes and malignant cancers. (C) 2004 Wiley-Liss, Inc.