title: Rapid detection of subtelomeric deletion/duplication by novel real-time quantitative PCR using SYBR-green dye.
abstract: 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., deletion/duplication-syndromes and malignant cancers.
authors.affiliation: Institute of Human Genetics, University of Goettingen, Goettingen, Germany. dboehm@gwdg.de
authors.forename: Detlef
authors.initials: D
authors.lastname: Boehm
authors.forename: Sabine
authors.initials: S
authors.lastname: Herold
authors.forename: Alma
authors.initials: A
authors.lastname: Kuechler
authors.forename: Thomas
authors.initials: T
authors.lastname: Liehr
authors.forename: Franco
authors.initials: F
authors.lastname: Laccone
mesh_terms: D052160:Benzothiazoles; D002869:Chromosome Aberrations; D019457:Chromosome Breakage; D025063:Chromosome Disorders / Q000175:diagnosis* / Q000235:genetics; D002877:Chromosomes, Human; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D017384:Sequence Deletion; D016615:Telomere / Q000235:genetics*; D013997:Time Factors; D014178:Translocation, Genetic
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I