Multiplexed quantitative real-time PCR to detect 22q11.2 deletion in patients with congenital heart disease. Physiol Genomics 2010 Sep;42A(1):52-60
Date
06/17/2010Pubmed ID
20551144Pubmed Central ID
PMC2957771DOI
10.1152/physiolgenomics.00073.2010Scopus ID
2-s2.0-77957122165 (requires institutional sign-in at Scopus site) 39 CitationsAbstract
22q11.2 Deletion syndrome (22q11.2 DS) [DiGeorge syndrome type 1 (DGS1)] occurs in ∼1:3,000 live births; 75% of children with DGS1 have severe congenital heart disease requiring early intervention. The gold standard for detection of DGS1 is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH is costly and is typically ordered in conjunction with a karyotype analysis that takes several days. Therefore, FISH is underutilized and the diagnosis of 22q11.2 DS is frequently delayed, often resulting in profound clinical consequences. Our goal was to determine whether multiplexed, quantitative real-time PCR (MQPCR) could be used to detect the haploinsufficiency characteristic of 22q11.2 DS. A retrospective blinded study was performed on 382 subjects who had undergone congenital heart surgery. MQPCR was performed with a probe localized to the TBX1 gene on human chromosome 22, a gene typically deleted in 22q11.2 DS. Cycle threshold (C(t)) was used to calculate the relative gene copy number (rGCN). Confirmation analysis was performed with the Affymetrix 6.0 Genome-Wide SNP Array. With MQPCR, 361 subjects were identified as nondeleted with an rGCN near 1.0 and 21 subjects were identified as deleted with an rGCN near 0.5, indicative of a hemizygous deletion. The sensitivity (21/21) and specificity (361/361) of MQPCR to detect 22q11.2 deletions was 100% at an rGCN value drawn at 0.7. One of 21 subjects with a prior clinical (not genetically confirmed) DGS1 diagnosis was found not to carry the deletion, while another subject, not previously identified as DGS1, was detected as deleted and subsequently confirmed via microarray. The MQPCR assay is a rapid, inexpensive, sensitive, and specific assay that can be used to screen for 22q11.2 deletion syndrome. The assay is readily adaptable to high throughput.
Author List
Tomita-Mitchell A, Mahnke DK, Larson JM, Ghanta S, Feng Y, Simpson PM, Broeckel U, Duffy K, Tweddell JS, Grossman WJ, Routes JM, Mitchell MEAuthors
Ulrich Broeckel MD Chief, Center Associate Director, Professor in the Pediatrics department at Medical College of WisconsinDonna K. Mahnke Research Scientist I in the Pediatrics department at Medical College of Wisconsin
Aoy Tomita Mitchell PhD Professor in the Surgery department at Medical College of Wisconsin
John Routes MD Professor in the Pediatrics department at Medical College of Wisconsin
Pippa M. Simpson PhD Adjunct Professor in the Pediatrics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Chromosomes, Human, Pair 22DNA Copy Number Variations
DiGeorge Syndrome
Heart Defects, Congenital
Humans
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Retrospective Studies
