Newborn Screening for Vitamin B6 Non-responsive Classical Homocystinuria: Systematical Evaluation of a Two-Tier Strategy

Abstract

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

Newborn screening for homocystinurias: Recent recommendations versus current practice

“…In the past several decades, different NBS strategies for the homocystinurias have been developed. Several recent publications have proposed to adopt two‐tier screening strategies . These approaches recommended to assess in a first step the primary markers: (a) elevated methionine (Met) and/or methionine‐to‐phenylalanine (Met/Phe) ratio for CBSD; (b) low Met and/or Met/Phe levels for RMD; and (c) elevated propionylcarnitine (C3), propionyl/acetylcarnitine (C3/C2) ratio and possibly C3/Met or heptadecanoylcarnitine for cRMD from dried blood spots (DBS).…”

A Rapid Screening Method for the Measurement of Neonatal Total Homocysteine in Dried Blood Spots by Liquid Chromatography-Tandem Mass Spectrometry

“…Methods with shorter retention times may produce less specific and inaccurate results that are caused by merging of neighboring interfering peaks with tHCY. Rapid LC-MS/MS methods using silica based and reversed phase columns for separation and quantitation of tHCY, as the free acid have been previously reported [5,7,28,29]. Our experience with chromatography of tHCY as both the free acid and as the butyl ester showed a significant improvement in peak symmetry and reproducibility of retention time and peak area for the latter: the butylation reduces the polarity and increases the interaction of the HCY with the column matrix.…”

“…Peterschmitt et al, observed double the detection rate by lowering the methionine cut-off from 138 to 67 µmol/L, and cut-off values as low as 40 µmol/L have been proposed to improve sensitivity [26]. However, changing cut-offs to increase the diagnostic sensitivity will reduce specificity, resulting in an increased need of unnecessary follow-up [7,27]. Okun et al, recently proposed using methionine and methionine/phenylalanine ratio as a primary screens with tHCY as a secondary screen [7].…”

“…However, changing cut-offs to increase the diagnostic sensitivity will reduce specificity, resulting in an increased need of unnecessary follow-up [7,27]. Okun et al, recently proposed using methionine and methionine/phenylalanine ratio as a primary screens with tHCY as a secondary screen [7]. In the confirmed positive cases that are presented in this paper, two of the nine cases would have been missed if a 90th percentile of the normal population was applied as a cutoff for the methionine or the methionine/phenylalanine ratio.…”

“…For example, the incidence ranges between 1:6400 to 1:20,500 in European populations and between 1:1800 to 1:3000 in the Qatari population [2][3][4][5][6][7]. The true worldwide prevalence is likely underestimated because of undiagnosed cases [8].…”