title: A pyrosequencing-based assay for the rapid detection of the 22q11.2 deletion in DNA from buccal and dried blood spot samples.
abstract: The 22q11.2 deletion syndrome is one of the most common deletion syndromes in newborns. Some affected newborns may be diagnosed shortly after birth because of the presence of heart defects, palatal defects, or severe immune deficiencies. However, diagnosis is often delayed in patients presenting with other associated conditions that would benefit from early recognition and treatment, such as speech delays, learning difficulties, and schizophrenia. Fluorescence in situ hybridization (FISH) is the gold standard for deletion detection, but it is costly and time consuming and requires a whole blood specimen. Our goal was to develop a suitable assay for population-based screening of easily collectible specimens, such as buccal swabs and dried blood spots (DBS). We designed a pyrosequencing assay and validated it using DNA from FISH-confirmed 22q11 deletion syndrome patients and normal controls. We tested DBS from nine patients and paired buccal cell and venous blood specimens from 20 patients. Results were 100% concordant with FISH assay results. DNA samples from normal controls (n = 180 cell lines, n = 15 DBS, and n = 88 buccal specimens) were negative for the deletion. Limiting dilution experiments demonstrated that accurate results could be obtained from as little as 1 ng of DNA. This method represents a reliable and low-cost alternative for detection of the common 22q11.2 microdeletions and can be adapted to high-throughput population screening.
authors: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia. Electronic address: duk5@cdc.gov.
authors: Deborah
authors: D
authors: Koontz
authors: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia.
authors: Kirsten
authors: K
authors: Baecher
authors: Allergy and Immunology Section, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia.
authors: Lisa
authors: L
authors: Kobrynski
authors: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia.
authors: Stanimila
authors: S
authors: Nikolova
authors: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia.
authors: Margaret
authors: M
authors: Gallagher
mesh_terms: D015342:DNA Probes; D004062:DiGeorge Syndrome / Q000175:diagnosis* / Q000235:genetics*; D059788:Dried Blood Spot Testing; D023061:Gene Order; D056426:Genetic Loci; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009061:Mouth Mucosa / Q000166:cytology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D015342:DNA Probes