Detection of the 22q11 Deletion Using Dried Blood Spots and Digital PCR
Monday, March 7, 2016
South Exhibit Hall H (Convention Center)
Lisa J. Kobrynski, MD MPH FAAAAI
Rationale: 22q11.2 deletion syndrome (22q11DS) has an estimated prevalence of 1:3000. Infants with cardiac defects are frequently tested for 22q11DS at birth, but some are not diagnosed until adolescence.  In SCID newborn screening approximately 29% of newborns with abnormal TREC have 22q11DS. NBS for 22q11DS may aid in early diagnosis.

Methods: Primers for TUPLE and ZNF74 and FAM probes were used. RNAseP, probe labeled with HEX, was the reference gene (2N).  DNA was eluted from 2 or 3.2 mm blood spots punches into 40 µL. Normal cord blood and unaffected parents were controls. Samples from patients with 22q11DS, after consent.  Ndel enzyme was added to digest DNA. Droplets were generated using an automated droplet generator (BioRad). Plate was sealed and was heated to 95ºC for 10 minutes; 45 cycles of 95ºC for 30 seconds and 60ºC for 1 minute. Droplets were counted using Bio-Rad droplet reader. Copy number calculated using QuantaSoft (BioRad).

Results: Copy number of both TUPLE and ZNF74, separately or multiplexed was accurately measured in all control and deleted patients.  Copy number varied from 1000 to 20,000/20µL for 2 and 3 mm punches respectively. The target:RNAseP ratio was not affected.  For multiplexed PCR, nondeleted patients had 4 copies of target genes (2 for RNaseP), deleted patients had 2 copies. One patient with a deletion of TUPLE alone had 3 copies. 

Conclusions: Digital PCR provides an accurate and efficient way of detecting the 22q11 deletion using dried blood spots.  Multiplexing this assay with TREC could make this a cost effective tool for NBS.