Feasibility and validation of an integrated newborn screening algorithm with the targeted Next Generation Sequencing (tNGS) technology as part of a 2nd tier test for Pompe and MPS I

ABSTRACT Due to the lack of widespreadnd tier screening testsnewborn screening providers and parents struggle to understand the ramification of genetic conditions detected during the neonatal periodThis is especially true in newborn screening for Lysosomal Storage DiseasesLSDssuch as Pompe and Mucopolysaccharidosis Type IMPS Iwhere pseudodeficient variants result in a lack of enzyme activity in the newborn dried blood spotDBSscreening testbut present with normal endogenous enzyme activity and with no overt disease manifestationsCurrent screening algorithms do not routinely identify or provide molecular genetic etiology for LSDs because the genotype is not routinely monitoredHoweverfrequency of pseudodeficiency mutations can be as high asin certain populations and can be a significant source of false positiveresultsEnhanced sensitivity and specificity in newborn screening for Pompe and MPS I is essentialbut extremely challengingCorrect diagnosis of a diseaseand its underlying biochemical and molecular basisis not only critical for successful differential diagnosis and treatment early in life but it is also important in reducing anxiety in parents and unnecessary burdens on follow up programsCurrent gene sequencing methods including genome scale sequencingWhole Exome and Whole GenomeWES and WGSrespectivelyused for confirmatory diagnosis are impractical in newborns and do not scaleA targeted next generation sequencingtNGSpanel method to address deficiencies in current testing can be used as a routinend tier newborn screen to significantly improve sensitivity and specificityThe panel designs of single genes are impractical for scaling in screening conditions due to the rare nature of LSDs and maintenance of multiple assaysGenome scale sequencing approaches are not comprehensive across non coding regionsintronspromotersetcwith gaps in coverage or high costs that are prohibitive for screening paradigmsWe will create a comprehensive newborn specific gene panel for LSDs by establishing sample collection and processing workflows more appropriate for newbornsWe will demonstrate the power of tNGS for reducing falseresults by identifying clinical variants and pseudodeficiency mutations in Pompe and MPS IOur goal is to develop an assay with high sensitivity and specificityWe will demonstrate the value of the assay when used as and tier screen followingst tier enzyme analysis via the BaebiesandaposFDA cleared SEEKER device for high throughput newborn screening of LSDsWe will use the results to develop an algorithm that can be used by newborn screening programs for accurate interpretation of screening resultsThese studies will eventually allow CLIA CAP validation of our tNGS methodology and permit us to offer our screening panel for LSDs on a commercial basisOur approach has the potential to rapidly and simultaneously screen for hundreds of other LSD or newborn conditions with future developmentEarly identification of many of these additional disorders is critical for rapid and appropriate management PROJECT NARRATIVE Newborn screening programs are currently expanding their test offerings to include enzyme measurements for the identification of lysosomal storage disordersLSDswith available treatments that can improve clinical outcome when started early in lifeWe are developing and tier targeted Next Generation Sequencing DNA test that will improve the primary screening algorithm and help in precise diagnosis and treatment of newborns