Pompe disease (PD) is screened by a two tier newborn screening (NBS) algorithm, the first tier of which is an enzymatic assay performed on newborn dried blood spots (DBS). As first tier enzymatic screening tests have false positive results, an immediate second tier test on the same sample is critical in resolving newborn health status. Two methodologies have been proposed for second tier testing: (a) measurement of enzymatic activities such as of Creatine/Creatinine over alpha-glucosidase ratio, and (b) DNA sequencing (a molecular genetics approach), such as targeted next generation sequencing. (tNGS). In this review, we discuss the tNGS approach, as well as the challenges in providing second tier screening and follow-up care. While tNGS can predict genotype-phenotype effects when known, these advantages may be diminished when the variants are novel, of unknown significance or not discoverable by current test methodologies. Due to the fact that criticisms of screening algorithms that utilize tNGS are based on perceived complexities, including variant detection and interpretation, we clarify the actual limitations and present the rationale that supports optimizing a molecular genetic testing approach with tNGS. Second tier tNGS can benefit clinical decision-making through the use of the initial NBS DBS punch and rapid turn-around time methodology for tNGS, that includes copy number variant analysis, variant effect prediction, and variant ‘cut-off’ tools for the reduction of false positive results. The availability of DNA sequence data will contribute to the improved understanding of genotype-phenotype associations and application of treatment. The ultimate goal of second tier testing should enable the earliest possible diagnosis for the earliest initiation of the most effective clinical interventions in infants with PD.
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