Multiplex, quantitative, real-time PCR is a sensitive, inexpensive and rapid assay to detect DiGeorge Syndrome (DGS1) and can be adapted to newborn screening

  • Joshua Larson, Medical College of Wisconsin, United States
  • Aoy Tomita-Mitchell, Medical College of Wisconsin, United States
  • Sujana Ghanta, University of Louisville, United States
  • John Routes, Medical College of Wisconsin, United States
  • Ulrich Broeckel, Medical College of Wisconsin, United States
  • William Grossman, Medical College of Wisconsin, United States
  • James Tweddell, Medical College of Wisconsin, United States
  • Michael Mitchell, Medical College of Wisconsin, United States

    • Objective: DiGeorge Syndrome (DGS1), also known as 22q11.2 deletion syndrome, affects 1:4000 live births. The gold standard for detection of DGS1 is fluorescence in situ hybridization (FISH), with a probe at the TUPLE1 gene. However, FISH has a high false negative rate (15%), is costly, and time-consuming. The goal of this study was to determine if multiplexed quantitative real-time PCR (MQPCR), a rapid and inexpensive assay, could be used to determine haploinsufficiency in DGS1.

    Methods: A blinded study was performed on 384 patients following congenital heart surgery. 14 were FISH-positive for DGS1, and seven additional patients were diagnosed clinically. In the remaining 363, 68 were FISH-negative. Using real-time PCR, the cycle threshold (Ct) value was used to calculate the relative gene copy number (rGCN) in the region of the TBX1 gene.

    Results: 363 patients were identified as normal, mean rGCN value 1.00 (95%CI 0.71-1.3), indicating two copies, and 21 patients were identified as DGS1, mean rGCN value 0.51 (95%CI 0.33- 0.69), indicating a deletion (p<0.0001). All 14 (FISH-positive) DGS1 patients test were successfully identified. Six of seven clinically diagnosed DGS1 patients were found to carry a deletion (one did not), all confirmed via microarray (Affymetrix 6.0). One patient not previously identified as DGS1 was detected and confirmed via microarray. The sensitivity (21/21) and specificity (363/363) of the assay was 100%.

    Conclusion: In summary, the MQPCR assay is rapid (<4hrs), inexpensive (<$1), sensitive, and specific for DGS1. The assay is readily adaptable to high-throughput and ideal for population-based newborn screening.