Combining newborn metabolic and DNA analysis for second-tier testing of methylmalonic acidemia

Purpose: Improved second-tier tools are needed to reduce false-positive outcomes in newborn screening (NBS) for inborn metabolic disorders on the Recommended Universal Screening Panel (RUSP). Methods: We designed an assay for multiplex sequencing of 72 metabolic genes (RUSPseq) from newborn dried blood spots. Analytical and clinical performance was evaluated in 60 screen-positive newborns for methylmalonic acidemia (MMA) reported by the California Department of Public Health NBS program. Additionally, we trained a Random Forest machine learning classifier on NBS data to improve prediction of true and false-positive MMA cases. Results: Of 28 MMA patients sequenced, we found two pathogenic or likely pathogenic (P/LP) variants in a MMA-related gene in 24 patients, and one pathogenic variant and a variant of unknown significance (VUS) in 1 patient. No such variant combinations were detected in MMA false positives and healthy controls. Random Forest–based analysis of the entire NBS metabolic profile correctly identified the MMA patients and reduced MMA false-positive cases by 51%. MMA screen-positive newborns were more likely of Hispanic ethnicity. Conclusion: Our two-pronged approach reduced false positives by half and provided a reportable molecular finding for 89% of MMA patients. Challenges remain in newborn metabolic screening and DNA variant interpretation in diverse multiethnic populations.