Purpose: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. Methods: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres.

Background: In classical homocystinuria (HCU, MIM# 236200) due to the deficiency of cystathionine bsynthase (EC 4.2.1.22) there is a clear evidence for the success of early treatment. The aim of this study was to develop and evaluate a two-tier strategy for HCU newborn screening.Methods: We reevaluated data from our newborn screening programme for Qatar in a total number of 125,047 neonates including 30 confirmed HCU patients. Our hitherto existing screening strategy includes homocysteine (Hcy) measurements in every child, resulting in a unique dataset for evaluation of two-tier strategies. Reevaluation included methionine (Met) levels, Met to phenylalanine (Phe) ratio, and Hcy. Four HCU cases identified after database closure were also included in the evaluation. In addition, dried blood spot samples selected by Met values >P97 in the newborn screening programs in Austria, Australia, the Netherlands, and Taiwan were analyzed for Hcy.Results: Met to Phe ratio was found to be more effective for first sieve than Met, sorting out nearly 90% of normal samples. Only 10% of the samples would have to be processed by second-tier measurement of Hcy in dried blood spots. As no patient with HCU was found neither in the samples investigated for HCU, nor by clinical diagnosis in the other countries, the generalization of our two-tier strategy could only be tested indirectly.Conclusion: The finally derived two-tier algorithm using Met to Phe ratio as first-and Hcy as second-tier requires 10% first-tier positives to be transferred to Hcy measurement, resulting in 100% sensitivity and specificity in HCU newborn screening. Abbreviations CBSCystathionine b-synthase DBS Dried blood spots ESI-MS/ MS Electrospray ionization-tandem mass spectrometry HCU Classical homocystinuria Hcy Homocysteine

Due to the favourable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, e.g. birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.