Functional characterization of 42 GAA missense variants using a validated in vitro cellular functional assay

The advent of newborn screening for Pompe disease has led to a massive increase in the number of missense variants of uncertain significance (VUS) reported in GAA. Molecular diagnostic testing may be inconclusive in the presence of one or more VUS, posing a significant challenge since most positive NBS occur in healthy infants with no apparent disease-related symptoms. It then becomes challenging to distinguish between pre-symptomatic infants with late-onset Pompe disease who require close monitoring and follow-up, and those who may just be carriers or have a partially functional pseudodeficiency variant. Missense variants are notoriously difficult to interpret in a clinical setting. Thus, there is a high demand for functional evidence to predict their impact in the absence of documented clinical evidence. To address this rapidly growing need, we have developed and validated a cell-based in vitro assay to test individual missense variants and their impacts on GAA enzyme activity and protein processing. Using this assay, we characterized 42 missense GAA VUS. We found that residual enzyme activity spanned the full GAA activity range, from complete loss of product to near wild type, with 66% of these variants falling below established cutoffs for a potentially deleterious effect. In addition, we provide case examples from individuals evaluated at the Duke Pediatric Medical Genetics clinic with GAA VUS and describe the utility of this functional assay in solving clinical conundrums. While variant classification cannot rely on a single line of evidence, our assay adds functional evidence to support classifications and clinical decision making, especially in the setting of novel variants not previously reported in patients with Pompe disease.