Newborn Screening for Mucopolysaccharidoses: Results of a Pilot Study with 100 000 Dried Blood Spots

Abstract

Objective-To test, in a newborn screening (NBS) laboratory, the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay 5 enzymatic activities in dried blood spots (DBS) for NBS of 5 lysosomal storage diseases (mucopolysaccharidosis [MPS]-II, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII).Study design-Three mm punches from de-identified DBS were obtained from the Washington NBS laboratory and submitted to the 5-plex LC-MS/MS assay. Screen cut-offs were established by analyzing the enzymatic activity in patients confirmed to have the MPS disorder. DNA sequencing of the relevant gene was performed on a second DBS punch for all samples with enzyme activity below 10% of the mean daily activity. Results-(1)For MPS-II, 18 below cut-off samples, 1 pathogenic genotype, and 2 "high risk" genotypes; (2) For MPS-IIIB, no below cut-off samples; (3) For MPS-IVA, (4) For MPS-VI, 4 below cut-off samples and no high-risk genotypes; (5) MPS-VII, 1 below cut-off sample confirmed by genotype and clinical report to be affected.

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses

“…The mean enzyme activity for each MPS type in this study tended to be lower than those reported previously [18,29,31] (Table 2). Because the DBSs used in this study were frozen for more than 9 months after collection, prolonged storage may have led to some loss of enzyme activity.…”

“…This fluctuation could be due to subtle changes in the methods used for the enzyme reaction, such as fluctuations in reaction temperature or reaction time from day to day. In the actual operation of NBS, quality control standards can be run each day along with newborn samples, and enzyme activities can be expressed as a percentage of the population mean or percentage of the daily population mean [31].…”

“…It has been reported that measuring enzyme activity by LC-MS/MS significantly reduces the number of PD cases compared to that using 4-methylumbelliferone [33]. However, it remains difficult to completely distinguish PD cases from controls [31]. It is also possible that the enzyme activity, which was originally in the lower limit of the normal range, decreased to the patient range because of long-term storage.…”

“…A recent NBS study of 100,000 newborns using LC-MS/MS to assay the enzymes involved in MPS II, MPS IIIB, MPS IVA, MPS VI, and MPS VII revealed the following number of newborns with enzyme activities below the cutoffs: MPS II (n = 18), MPS IIIB (n = 0), MPS IVA (n = 8), MPS VI (n = 4), and MPS VII (n = 1) [31]. This is a manageable number of first-tier screen-positive samples.…”

“…For MPS II and MPS VII, 2 of 18 screen-positives and 1 of 1 screen-positives, respectively, were likely to be disease-affected based on subsequent genotype analysis [31]. Zero of four MPS VI screen-positive samples were predicted to be true positives based on genotype, and no genotype data could be obtained for the MPS IVA screen-positives [31]. It is anticipated that second-tier analysis by measuring GAGs in DBSs will significantly reduce the false-positive rate.…”