Clinical implications of discordant massarray and sanger sequencing results in cystic fibrosis newborn screening

Background: Agena’s MassArray platform is used for high throughput genotyping in our service. This method combines primer extension termination polymerase chain reaction (PCR) with mass spectrometry (MADI-TOF) and proprietary genotype calling algorithms.

Method: The Agena iPLEX CFTR 74 v3 off-the-shelf assay is used as a ‘common variant screen’ for cystic fibrosis (CFTR) genotyping, including infants with an immunoreactive trypsin in the top 1% on newborn blood spot screening. Variants detected by this assay are not routinely confirmed by Sanger sequencing.

Case report: Cascade testing identified a variant called by the MassArray platform as c.2175dupA to be discordant with capillary electrophoresis sequencing, which identified this as c.2173G>A. Many of the variants on this screening panel are rare in our experience. The pathogenic c.2175dupA variant had not been previously detected in clinical testing in our service. The c.2173G>A variant is classified as a variant of unknown significance and would not usually be reported in a newborn screening patient.

Discussion: A primer extension product with a specific mass to charge ratio can detect different PCR products. The presence of rare SNPs is listed as a method limitation, but in this Case, resulted in variant re-classification.