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. 2025 Apr 24;11(2):30.
doi: 10.3390/ijns11020030.

Newborn Screening for Metachromatic Leukodystrophy in Tuscany: The Paradigm of a Successful Preventive Medicine Program

Sabrina Malvagia  1 , Alessandra Bettiol  2 , Margherita Porcaro  1 , Massimo Mura  1 , Silvia Funghini  1 , Daniela Ombrone  1 , Giulia Forni  1 , Emanuela Scolamiero  1 , Filippo Coppi  1 , Roberta Damiano  1 , Cristina Cereda  3 , Simonetta Simonetti  4 , Annalisa Lonetti  5 , Marta Daniotti  6 , Anna Caciotti  7 , Amelia Morrone  7   8 , Valeria Calbi  9   10 , Francesca Fumagalli  9   10   11 , Alessandro Aiuti  9   10   12 , Elena Procopio  6 , Renzo Guerrini  8   13 , Giancarlo la Marca  1   2
Affiliations

Newborn Screening for Metachromatic Leukodystrophy in Tuscany: The Paradigm of a Successful Preventive Medicine Program

Sabrina Malvagia et al. Int J Neonatal Screen. .

Abstract

Metachromatic leukodystrophy (MLD) is a rare inherited disorder of lysosomal storage, caused by a deficiency in the arylsulfatase A (ARSA) enzyme, leading to toxic accumulation of sulfatides, which progressively impair motor and cognitive function. MLD is a candidate for inclusion in newborn screening (NBS) programs, due to the narrow pre-symptomatic window for effective therapeutic intervention. We set up a prospective pilot NBS program for MLD in Tuscany, based on a two-step approach. The first-tier test quantified four sulfatides; if levels exceeded the cut-off, we performed the second-tier test by measuring ARSA activity on the same neonatal dried blood spot (DBS). We performed the first-tier test on 42,262 newborns over two years and the second-tier test on residual neonatal DBS from 90 of them (0.21%). We recalled 10 newborns (0.02%) for an additional DBS, due to insufficient residual material for a second-tier test (n = 4) or to low ARSA activity (n = 6). We found normal ARSA activity in all new DBS and identified no new cases of MLD. Retrospective analysis of eight neonatal and fifteen non-neonatal DBS from patients with genetically confirmed MLD showed that the algorithm accurately identified MLD patients. This diagnostic algorithm proved feasible and accurate for early detection of MLD in prospective NBS.

Keywords: arylsulfatase A; lysosomal storage disorder; metachromatic leukodystrophy; newborn screening; tandem mass spectrometry.

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Conflict of interest statement

F.F., V.C. and A.A. are investigators of clinical trials sponsored by Orchard Therapeutics. The other authors declare no conflicts of interest.

Figures

Figure A1
Figure A1
Stability of the four sulfatides at four temperatures over 33 days.
Figure A2
Figure A2
Extracted ion chromatogram comparing a negative control newborn and an MLD case.
Figure 1
Figure 1
Proposed algorithm for the MLD newborn screening.
Figure 2
Figure 2
Schematic representation of the routine laboratory procedures for the first-tier test used in MLD newborn screening.
Figure 3
Figure 3
Overall data from the MLD newborn screening program in Tuscany over two years. We performed the second-tier test (2TT) on 90 newborns (0.21% of the screened neonatal population). The recall rate was approximately 0.02% (n = 10). * In one case, since all the quantified sulfatides also remained increased on the second sample (analyzed in duplicate), we proceeded with the genetic study, in agreement with the family. No genetic alterations were highlighted, and the subject was classified as a false positive. Since then, the diagnostic flow-chart has been changed, and genetic testing is now performed only in the case of ARSA enzymatic deficiency in both samples (neonatal DBS and second DBS after recall).
Figure 4
Figure 4
ARSA enzyme activity in a negative control newborn and a MLD case. The red peak represents the internal standard (IS), and the blue peak corresponds to the product of the ARSA activity (ARSA-P) in a negative control newborn (on the left) and in a MLD patient (on the right).
Figure 5
Figure 5
Summarizes the distribution of the four sulfatides (A–D) and their sum (E) in the neonatal DBS from 90 false positive cases, eight confirmed MLD patients and controls (n = 3154). (F) compares ARSA activity measured in fresh DBS samples from false positive cases (n = 90), non-neonatal MLD patients (n = 15) and controls (n = 120). Red dashed lines represent the current cut-off values for each of the four sulfatides and the sum.
See this image and copyright information in PMC

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