Evaluating the feasibility and diagnostic performance of a multi-tier newborn screening algorithm for X-linked adrenoleukodystrophy in Denmark

Newborn screening (NBS) for X-linked adrenoleukodystrophy (ALD) has been implemented in several countries enabling early monitoring and treatment before irreversible disease develops. Important questions remain regarding the optimal analytical strategy to identify which newborns should be identified and reported through screening. We conducted a retrospective study using dried blood spots (DBS) from newborns undergoing routine NBS and archived DBS from patients with ALD and other peroxisomal diseases. A three-tier screening algorithm was evaluated: first-tier flow-injection analysis-tandem mass spectrometry (FIA-MS/MS) measurement of C26:0-lysophosphatidylcholine (C26:0-LPC), second-tier liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of C26:0-LPC, and third-tier next generation sequencing (NGS) with targeted analysis of ABCD1 variants and X-chromosome copy number. We included 61,431 newborns, 22 ALD patients, and 20 patients with other peroxisomal diseases in the retrospective cohort. First- and second-tier cut-offs of 0.33 μmol/L and 0.15 μmol/L identified 21 of 22 ALD patients in the retrospective cohort, with one missed case being a female whose second-tier C26:0-LPC concentration was below the cut-off, and one previously undiagnosed male with ALD from the newborn cohort. Demographic and perinatal characteristics were associated with first-tier C26:0-LPC concentrations, but each explained less than 3% of the variation in first-tier C26:0-LPC concentrations. In males, both first- and second-tier screening achieved a sensitivity of 100%, while the positive predictive value was 4.8% in first-tier and 82.4% after second-tier analysis. Third-tier NGS identified the new ALD case and all known ALD cases. The study found that a three-tier strategy combining FIA-MS/MS, LC-MS/MS, and targeted NGS is a feasible approach for NBS of ALD in Denmark. Second-tier LC-MS/MS maintained 100% sensitivity in males while positive predictive value was 82.4%, and NGS-based X-chromosome copy number analysis enabled identification of males with ALD.