Validation of a targeted metabolomics panel for improved second‐tier newborn screening. Issue 2 (2nd February 2023)

• Record Type: Journal Article
• Authors: Mak, Justin; Peng, Gang; Le, Anthony; Gandotra, Neeru; Enns, Gregory M.; Scharfe, Curt; Cowan, Tina M.
• Is Part Of: Journal of inherited metabolic disease. Volume 46:Issue 2(2023)
• Page Start: 194
• Page End: 205
• Publication Date: 2023-02-02
• Subjects: inborn metabolic disorders -- machine learning -- metabolomics -- newborn screening -- second‐tier testing -- supervised machine learning -- tandem mass spectrometry; Metabolism, Inborn errors of -- Periodicals; Metabolism -- Disorders -- Periodicals; 616.39042
• DOI: 10.1002/jimd.12591
• Languages: English
• ISSNs: 0141-8955

Abstract

Abstract: Improved second‐tier assays are needed to reduce the number of false positives in newborn screening (NBS) for inherited metabolic disorders including those on the Recommended Uniform Screening Panel (RUSP). We developed an expanded metabolite panel for second‐tier testing of dried blood spot (DBS) samples from screen‐positive cases reported by the California NBS program, consisting of true‐ and false‐positives from four disorders: glutaric acidemia type I (GA1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long‐chain acyl‐CoA dehydrogenase deficiency (VLCADD). This panel was assembled from known disease markers and new features discovered by untargeted metabolomics and applied to second‐tier analysis of single DBS punches using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in a 3‐min run. Additionally, we trained a Random Forest (RF) machine learning classifier to improve separation of true‐ and false positive cases. Targeted metabolomic analysis of 121 analytes from DBS extracts in combination with RF classification at a sensitivity of 100% reduced false positives for GA1 by 83%, MMA by 84%, OTCD by 100%, and VLCADD by 51%. This performance was driven by a combination of known disease markers (3‐hydroxyglutaric acid, methylmalonic acid, citrulline, and C14:1), other amino acids and acylcarnitines, and novel metabolites identified to be isobaric to several long‐chain acylcarnitine and hydroxy‐acylcarnitine species. These findings establish the effectiveness of this second‐tier test to improve screening for these four conditions and demonstrate the utility of supervised machine learning in reducing false‐positives for conditions lacking clearly discriminating markers, with future studies aimed at optimizing and expanding the panel to additional disease targets. Abstract : We developed a 121‐analyte LC‐MS/MS metabolomics panel in combination with supervised machine learning to separate false‐positive from true‐positive results for four metabolic disorders. This approach is expandable for inclusion of additional disease markers with the goal of reducing diagnostic delays and streamlining second‐tier newborn screening for metabolic disorders.