Development of a TaqMan-based dosage analysis PCR assay for the molecular diagnosis of 22q11.2 deletion syndrome

A 1.5 to 3 Mb microdeletion of chromosome 22q11.2 with loss of multiple genes including histone cell cycle regulator (HIRA) causes 22q11.2 deletion syndrome (22q11.2 DS), a common disorder with variable manifestations including congenital malformations affecting the heart, palate and kidneys in association with neurodevelopmental, psychiatric, endocrine and autoimmune abnormalities. The aim of this study was to develop a TaqMan based dosage analysis PCR (TaqMan qPCR) for use as a rapid, cost-effective test for clinically suspected patients fulfilling previously described criteria for molecular diagnosis of 22q11.2 DS in a lower middle-income country where the cost of testing limits its use in routine clinical practice. Nineteen patients were recruited with informed consent following ethical approval from the Ethics Review Committee (ERC), Lady Ridgway hospital, Colombo. Dosage analysis of extracted DNA was performed using a TaqMan qPCR assay by amplifying regions within the target (HIRA) and control [Testin LIM domain protein (TES)] genes of a suspected patient (P) and unaffected person (N). For detection of a deletion, the normalized values (HIRA / TES dosage) of a patient were compared with normalized values of an unaffected person. A ratio of P:N of 0.5 confirmed presence of a deletion while a ratio of 1.0 refuted this. Seven of 19 (37%) cases were confirmed to have a HIRA deletion, confirming the diagnosis of 22q11.2 DS, with these results being in complete agreement with those of fluorescence in-situ hybridization (FISH), (performed in 9/19 (47.3%) of recruited cases) and whole exome sequencing (WES) (all 19 samples tested). This TaqMan qPCR assay was able to reliably distinguish HIRA deleted cases from the non-deleted ones. The assay was both less expensive and faster compared to commercially available alternatives in our setting, including FISH and multiple ligation-dependent probe amplification (MLPA).