Newborn Screening for Metachromatic Leukodystrophy: Care Pathway and Early Clinical Outcomes from German and Austrian Pilot Programs

Newborn Screening for Metachromatic Leukodystrophy: Care Pathway and Early Clinical Outcomes from German and Austrian Pilot Programs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Newborn Screening for Metachromatic Leukodystrophy: Care Pathway and Early Clinical Outcomes from German and Austrian Pilot Programs Lucia Laugwitz, Pascal Martin, Nils Janzen, Sebastian Hegert, and 25 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9496450/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Metachromatic leukodystrophy (MLD) is a rapidly progressive leukodystrophy that leads to severe disability and early death if untreated. Autologous hematopoietic stem and progenitor cell gene therapy (HSPC-GT, atidarsagene autotemcel, arsa-cel) for early onset subtypes and allogenic hematopoietic stem cell transplantation (HSCT) for late onset disease substantially alters disease progression for early onset disease when administered before symptom onset, creating a strong rationale for newborn screening (NBS). At the same time, NBS technique for MLD in dried blood spots has recently been demonstrated to be robust and highly accurate. The aim was to give real-world results from the world’s first NBS pilots for clinical management and treatment of identified children. Methods Between September 2021 and July 2025, 359,282 newborns underwent NBS for MLD in two different laboratories in Germany and Austria using a three-tier algorithm integrating sulfatide quantification, arylsulfatase A (ARSA) activity measurement, and ARSA sequencing. Screen-positive infants underwent a predefined care pathway including standardized confirmatory diagnostics, genotype-based and biochemical prediction of disease onset, clinical assessment and management guiding early treatment and surveillance at the qualified treatment center (QTC) in Tübingen. Results Nine newborns screened positive and all were confirmed to have MLD (detection rate approximately 1 per 40,000). Based on genotype and leukocyte ARSA enzyme activity, disease onset prediction was possible in all of them. Seven infants were classified as having pre-symptomatic early-onset MLD and were referred for HSPC-GT. All treated infants showed preserved neurological function at follow-up 30 months after treatment. Two infants predicted to develop late-onset MLD entered structured surveillance for treatment with HSCT and have remained clinically stable. No false-positive or known false-negative results were observed. Conclusion These results from our pilot programs demonstrate that NBS enables reliable early identification of MLD and support streamlined care pathways leading to timely intervention. Importantly, this study provides real-world evidence illustrating that NBS for MLD can enable timely, pre-symptomatic treatment and structured surveillance within standard national healthcare systems. These findings further substantiate the value of NBS for MLD at a critical moment as several countries consider national implementation of MLD screening. metachromatic leukodystrophy newborn screening autologous hematopoietic stem and progenitor cell gene therapy lysosomal storage diseas Figures Figure 1 Figure 2 1. Introduction Metachromatic leukodystrophy (MLD) (MIM#250100) is a rare lysosomal storage disorder caused by biallelic clinically relevant variants in ARSA (MIM*607574) encoding the lysosomal enzyme arylsulfatase A (ARSA). 1–3 ARSA deficiency leads to a progressive accumulation of sulfatides, in particular in the central and peripheral nervous system, resulting in demyelination and neurodegeneration. 1 The overall birth prevalence of MLD in Europe is approximately 1 in 40,000 to 1 in 100,000. 2 Although disease onset spans infancy to adulthood, early-onset forms (i.e. late infantile and early juvenile) are most common and are associated with more rapid neurodevelopmental regression, severe disability, and early death if untreated. 4 For these children, irreversible neurological injury begins early in life, making timely intervention critical. Late onset MLD (i.e. late juvenile and adult) may present later in childhood, adolescence, or adulthood, with a more gradual progression of severe neurological or psychiatric symptoms. 4,5 Until recently, therapeutic options for early-onset MLD were limited to supportive care and, in late onset cases diagnosed in an early symptomatic or pre-symptomatic stage, allogeneic hematopoietic stem cell transplantation (HSCT). 6–8 The development of autologous hematopoietic stem and progenitor cell gene therapy (HSPC-GT, atidarsagene autotemcel, arsa-cel) has fundamentally altered the treatment landscape for early onset MLD. 9,10 When administered before the onset of clinical signs, HSPC-GT in early onset MLD and HSCT in late onset MLD are associated with preservation of early neurodevelopment and substantial modification of disease course. 6–10 However, treatment efficacy is highly time dependent: once neurological symptoms emerge, therapeutic benefit is markedly reduced. 9 This narrow therapeutic window means that for most children diagnosed with MLD, the diagnosis comes too late for currently available interventions. 11 Long-term outcomes are highly encouraging, with infants maintaining near-normal development when treated pre-symptomatically. 9,10 Newborn screening (NBS) provides a population-based strategy to identify infants with MLD pre-symptomatically and before the onset of irreversible neurological injury. 12 Over the past decade, biochemical screening approaches for MLD have been systematically developed and validated in retrospective studies. 13–15 Prospective screening pilot programs have demonstrated that MLD screening can be integrated into established NBS infrastructures with high analytical performance. 16,17 These advances have driven growing international momentum, with several countries initiating or expanding pilot programs and some incorporating MLD into national screening panels. 18 However, the availability of treatment options and screening alone does not ensure improved clinical outcomes. As a rapidly emerging field of science, gaps in evidence remain. For NBS for MLD this primarily relates to the downstream consequences of population-based detection: data are scarce on the real-world clinical management of infants identified through NBS for MLD, including confirmatory diagnostic workflows, prediction of disease onset, decision-making around presymptomatic treatment, and the feasibility of delivering time-sensitive interventions within routine healthcare systems. Equally important is our understanding how infants predicted to develop late-onset disease can be monitored safely over time without exposing families to unnecessary intervention or harm before they are eligible for treatment. 19,20 In this work, we report the first larger clinical experiences from prospective NBS pilot programs for MLD in Germany and Austria. We evaluate previously established care pathways from screening to confirmatory diagnosis, consensus-based prediction of disease onset, and subsequent clinical management. We further report the clinical care pathway of pre-symptomatic infants with MLD who underwent HSPC-GT and those managed through structured surveillance before HSCT. Taken together, these findings will offer preliminary evidence for the feasibility and potential clinical impact of NBS-enabled care for MLD in routine pediatric healthcare practice. This study advances the field by providing the first prospective real-world data on early clinical trajectories after NBS for MLD, including confirmatory diagnostic pathways, treatment decisions and outcomes. 2. Methods 2.1 Pilot Program Design Prospective NBS pilot programs for MLD were conducted in North-West Germany and nationwide in Austria between September 2021 and July 2025 through coordinated collaborations involving the Screening Laboratory Hannover, the Medical University of Vienna, and ARCHIMEDlife. In Germany, the pilot operated from September 2021 until December 2024 as a joint initiative between the Screening Laboratory Hannover and ARCHIMEDlife; thereafter, the program was fully transferred to the Screening Laboratory Hannover and continues independently. In Austria, the pilot is ongoing, and since September 2023 has been conducted as a partnership between the Medical University of Vienna and ARCHIMEDlife. German newborns born in the capture area of the Screening Laboratory Hannover were enrolled under an Institutional Review Board-approved protocol (Ärztekammer Niedersachsen; BO/39/2021). Austrian screening was conducted under approval from the Ethics Committee of the Medical University of Vienna (1961/2022). Further biochemical and clinical investigations of infants identified through screening were approved by the local ethics committee of the Medical Faculty of the University of Tübingen, Germany (948/2018BO2). All participants, or their respective legal guardians, provided written informed consent for participation and publication in accordance with the Declaration of Helsinki. Clinical trial number: not applicable. The Hannover cohort includes three screen positive infants previously reported in Laugwitz et al. 2024 (index 1 to 3) 16 , with extended follow-up and additional cases included here. The Austrian cohort has not been reported before. All analyses reported in the present study were prospectively defined and conducted within the approved protocols. The primary outcome of the pilot programs was the detection rate of confirmed MLD cases, with an expected incidence of approximately 1 per 40,000 to 100,000 newborns based on European prevalence estimates. 3 The clinical data collection for this study was completed in December 2025. 2.2 Screening Algorithm NBS for MLD was implemented using a three-tier screening strategy, as previously described. 15,16,21 The screening workflow evolved during the pilot phase to optimize analytical performance and minimize false-positive results. Since early 2024, the fully standardized three-tier algorithm has been applied to dried blood spots (DBS) obtained for routine NBS in both Germany and Austria, and consists of: (1) quantification of sulfatides (C16:0 and C16:1-OH) in dried blood spots (DBS), (2) measurement of ARSA enzymatic activity in DBS, and (3) next generation sequencing of ARSA , SUMF1 , and PSAP in DBS. Detailed analytical methods, assay cut-offs, and quality control procedures have been reported previously. 15,16,21 2.3 Clinical Management Pathway A central aim of the pilot programs was not only case detection but also the prospective evaluation of a standardized, consensus-based care pathway following a positive NBS result. Management of infants identified through NBS followed a predefined, standardized care pathway aligned with European expert consensus recommendations (Fig. 1). 20 After confirmation of the diagnosis of MLD, families received multidisciplinary counselling regarding disease characteristics, prognosis, and available management options. Care was coordinated by the QTC in Tübingen in collaboration with metabolic pediatricians, pediatric neurologists, clinical geneticists, transplant physicians, and allied health professionals. Psychosocial support and access to patient advocacy resources were offered throughout the diagnostic and management process in accordance with consensus guidance. Cases were discussed within the treatment eligibility panel of the MLDInitiative (MLDi). 22 2.4 Confirmatory Diagnostics and Molecular Assessment For all screen-positive infants, families were informed by MLD-specialized treatment centers in line with European recommendations (i.e. the QTC in Tübingen, Germany, and medical centers in Graz and Innsbruck, Austria), and infants were referred promptly for confirmatory diagnostic evaluation. Confirmatory testing was coordinated through specialized centers in collaboration with the QTC at the University Hospital Tübingen, designated as a QTC for Germany and Austria. Confirmatory diagnostics included measurement of ARSA activity in leukocytes 23,24 and repeated quantification of urinary sulfatide levels using liquid chromatography-tandem mass spectrometry 25 . Genetic sequencing was repeated using freshly obtained EDTA blood samples from the infant and both parents to confirm biallelic ARSA variant localization. Following biochemical confirmation, ARSA variants were classified according to American College of Medical Genetics and Genomics (ACMG). 26 2.5 Clinical Evaluation Baseline clinical evaluation at the QTC included a comprehensive neuropediatric examination and assessment of developmental milestones. Instrumental investigations comprised brain magnetic resonance imaging, nerve conduction velocity studies, peripheral nerve ultrasound, acoustic evoked potentials, and gallbladder ultrasound. The scope and timing of assessments were adapted to age and clinical context while adhering to consensus recommendations. 20 2.6 Prediction of Disease Onset Each infant was then classified into an MLD subtype risk category - late infantile/early juvenile, and late juvenile/adult onset based on their genotype. Additional parameters were residual ARSA activity in leukocytes 24 . Published consensus criteria for phenotype prediction in pre-symptomatic infants were applied: 20 In brief, infants carrying two predicted protein truncating variants (pPTVs) in ARSA , known variants associated with late infantile onset or who exhibited a residual ARSA activity in leukocytes of less than 1% on repeated measurements were predicted to develop the late infantile form of MLD. 24 In line with European consensus recommendation in newborns with pPTVs in trans with another clinically relevant missense variant were predicted as early onset MLD (i.e. late infantile or early juvenile). Newborns with genotypes consistently associated with late onset disease such c.542T > G (p.Ile181Ser) in trans with any other clinically relevant ARSA variant or c.1283C > T (p.Pro428Leu) in homozygous state were predicted as late onset, recognizing that distinguishing late juvenile versus adult onset is not possible with genotype alone. 20 Newborns with other well-documented ARSA genotypes reported in the literature were classified accordingly. 2.7 Treatment Decision All cases were reviewed in multidisciplinary European expert rounds of MLDi. Eligibility for treatment with HSPC-GT (arsa-cel) was assessed according to European Medicines Agency criteria. 27 Consensus decisions regarding treatment, timing, and monitoring were reached by a multidisciplinary expert panel. Infants predicted to develop early-onset MLD were referred for gene therapy within the first 12 months of life. Infants predicted to develop late-onset disease or for whom disease onset was uncertain, were enrolled in a structured long-term surveillance program, which includes regular neurological examinations, developmental assessments, and periodic neuroimaging and neurophysiological studies. Escalation to therapeutic intervention during surveillance followed consensus recommendations and was based on emerging clinical, neurophysiological, imaging, or biomarker evidence of disease activity. Outcome data were collected prospectively. 2.8 Statistical Methods and Graphics Descriptive statistics were applied to summarize the screened population, biochemical and genetic findings, and diagnostic outcomes. Illustrations were generated using Biorender and the Python package matplotlib (version 3.10.3). Descriptive statistics were calculated using Python (version 3.13.0) primarily utilizing the packages pandas and numpy . 3. Results 3.1 Screening Algorithm Performance Between September 2021 and July 2025, a total of 359,282 dried blood spot samples were analyzed across NBS pilots in Germany and Austria. Nine newborns screened positive and were subsequently confirmed to have MLD, corresponding to a prevalence of 2.51 per 100,000 live births and an incidence of approximately one in 39,920 screened newborns. No false-positive recalls occurred during the study period. During the study period, no additional infants diagnosed with MLD within the screening catchment areas were reported to the QTC in Tübingen. . 3.2 Management Timeline Screen-positive results for the nine index cases identified in the NBS stuedies were communicated to the designated QTC in Tübingen at a median age of 3 months (range 1.0–5.8 months) following NBS sample collection at birth (Fig. 2). In accordance with established European NBS recommendations, families were contacted within one week of NBS result receipt by an designated MLD expert center. In eight cases, initial disclosure was conducted by the MLD expert center. In one case, families were contacted directly by the screening laboratory owing to a miscommunication with the referring laboratory; this represented a protocol deviation. All families were offered rapid access to the QTC through video or telephone consultation or in-person outpatient visits within one week after notification of positive NBS results. Confirmatory diagnostic testing was completed within a median of 0.3 months after recall (range 0.2–1.9 months). Comprehensive clinical evaluations at the expert center were conducted within a median of 1.0 month following diagnosis (range 0–2.3 months), with timing adjusted according to predicted disease subtype. All cases were subsequently reviewed by a multidisciplinary treatment eligibility panel convened by the MLDi within 1 to 4 months after recall. For infants predicted to develop late-infantile disease, panel review was prioritized and scheduled within 1 to 2 weeks after completion of confirmatory diagnostics. Treatment recommendations were reached by consensus in eight of nine cases at the initial panel meeting. One case (index 7) required additional diagnostic clarification and repeat review before final consensus was achieved (Supplement, Case reports). Subsequent clinical assessments at the QTC were completed within 0 to 3 months for all infants and within 4 to 6 weeks for those with predicted early-onset disease. Stem cell apheresis was performed a median of 2.2 months after treatment decision (range 1.2–6.6 months), corresponding to a median of 3.6 months after NBS results (range 2.7–7.9 months). In one case (index 8), apheresis was repeated because the initial drug product did not meet release criteria, consistent with standard manufacturing protocols. HSPC-GT infusion was administered a median of 2.5 months after apheresis (range 1.8–2.5 months). Following treatment, infants were discharged a median of 1.0 month after infusion (range 0.8–1.2 months; Fig. 2). 3.3 Confirmatory Diagnostics and Molecular Findings All nine infants classified as screen positive were confirmed to have a diagnosis of MLD through biochemical and molecular testing (Table 1 ). Each infant harbored biallelic clinically relevant variants in ARSA , accompanied by markedly reduced ARSA activity in leukocytes and elevated sulfatide excretion in urine (Table 1 ; Figure S1 ). To account for biological variability, leukocyte ARSA activity and urinary sulfatides were assessed repeatedly at multiple time points (Table 1 ; Figure S1 ). Although some interday variability was observed, all repeated measurements remained consistently within the pathological range. Genotypes spanned the known phenotypic spectrum of MLD and included both previously reported pathogenic and novel variants. Three previously unreported ARSA variants were identified, including a splice donor variant, a frameshift and a missense variant. Based on ACMG criteria, these were classified as likely pathogenic, supported by functional biochemical confirmation (Table 1 ). Inclusion of the third-tier genetic testing in the NBS algorithm was critical for screening specificity. No infants with pseudodeficiency alleles or isolated heterozygous variants in ARSA , or variants in SUMF1 or PSAP fulfilled criteria for a positive screen, and no biochemical mimics of MLD were identified. Together, the combined biochemical-genetic approach in the screening and confirmatory testing algorithm enabled unambiguous diagnosis in all cases and avoided variants of uncertain clinical significance as standalone findings. Table 1 Confirmatory Diagnostics of Newborns identified Index Genotype (HGVS, GRCh38) ACMG Class ClinVar ARSA activity in leukocytes 24 Normal range: 1.5 to 6.1 E 514 nm/10 6 cells Urinary Sulfatides Normal range: T];[1283C > T],p.[(Pro138Ser)];[(Pro428Leu)] 5;5 P 1 ;P 0.007 ± 0.008 (n = 4) a Pathologically elevated b Early juvenile 2 ARSA :c.[465 + 1G > A];[1283C > T],p.[(?)];[(Pro428Leu)] 5;5 P 1 ;P 0.048 ± 0.013 (n = 6) 549.823 ± 265.427 (n = 4) Early juvenile 3 ARSA :c.[465 + 1G > A];[542T > G],p.[(?)];[(Ile181Ser)] 5;5 P;P 0.034 ± 0.029 (n = 8) 3216.248 ± 2723.202 (n = 4) Late juvenile/ adult 4 ARSA :c.[465 + 1G > A];[465 + 1G > A],p.[(?)];[(?)] 5;5 P.;P 0.014 ± 0.017 (n = 4) a 1245.992 ± 892.734 (n = 4) Late infantile 5 ARSA :c.[465 + 1G > A];[899del],p.[(?)];[(Leu300Cysfs*29)] 5; 5 P;n.r. 0.035 ± 0.034 (n = 5) a 1960.298 ± 1045.793 (n = 6) Late infantile 6 ARSA :c.[931G > A];[931G > A],p.[(Gly311Ser)];[(Gly311Ser)] 4;4 LP;LP 0.022 ± 0.018 (n = 4) a 2324.697 ± 753.985 (n = 3) Late infantile 7 ARSA :c.[465 + 1G > A];[494C > T];p.[(?)];[(Pro165Leu)] 5;3 -> 4 P;n.r. 0.087 ± 0.018 (n = 6) 1933.71 ± 1679.955 (n = 8) Early juvenile 8 ARSA :c.[684 + 1G > A];[1223_1231del],p.[(?)];[(Val408Serfs*21)] 5;5 n.r.;P 0.015 ± 0.022 (n = 6) a 1149.3 ± 1068.438 (n = 2) Late infantile 9 ARSA :c.[1283C > T];[1283C > T],p.[(Pro428Leu)];[(Pro428Leu)] 5;5 P;P 0.047 ± 0.042 (n = 4) 1680.425 ± 1254.938 (n = 2) Late juvenile/ adult n.r., not reported; P, pathogenic; LP, likely pathogenic; a below the predictive threshold of < 1% of mean activity in healthy controls according to prior reports 1921 , suggestive for early onset. b no quantification possible due to lack of stored biomaterial, sulfatides were measured qualitatively instead. 3.4 Clinical Baseline Evaluation Baseline clinical assessments were completed in all infants at the QTC in Tübingen prior to treatment or enrolment into surveillance (Supplement Table S2, Case Reports). At initial evaluation (between 3 and 6 months of age), neurological examination and developmental assessments were unremarkable in all cases, with achievement of age-appropriate milestones reported by caregivers and primary care physicians. MRI of the brain was conducted in eight infants and showed no imaging evidence of leukodystrophy-related white matter changes, for one child with late onset-MLD the first MRI evaluation is planned but not yet performed. MRI severity scores were 0 in all cases (Supplement Table S1 ). Gallbladder ultrasound revealed no gallstones or premalignant lesions. Mild gallbladder wall thickening or sludge was observed in two infants without associated clinical signs (Supplement Table S1 ). Neurophysiological studies demonstrated greater variability. Nerve conduction studies were within age-adjusted reference ranges in three infants, while six infants showed evidence of demyelinating peripheral neuropathy, including infants predicted to develop both early- and late-onset disease. Peripheral nerve ultrasound was normal in all infants examined. Auditory evoked potentials were abnormal in two cases, showing prolonged latencies without corresponding clinical hearing impairment (Supplement Table S1 ). Overall, baseline evaluations confirmed the absence of overt neurological disease at the time of diagnosis while identifying subclinical abnormalities in selected instrumental assessments. 3.5 Prediction of Disease Onset Based on the consensus criteria, the patients were stratified into predicted phenotypic subtypes: Late infantile MLD Out of the nine cases, four infants were predicted to have late infantile onset MLD. These infants harbored truncating ARSA variants known to abolish enzyme function (Table 1 ). Besides the common splice site variant c.465 + 1G > T and known frameshift variant (c.1223_1231del), a novel splice donor variant (c.684 + 1G > A) and a novel frameshift variant were identified (c.899del). Aligning with ACMG criteria these novel pPTVs were classified as likely pathogenic (class 4). Biallelic pPTVs strongly indicated symptom onset before 30 months of age and the most aggressive disease course without intervention (Table 1 ). All four had very low mean ARSA activity in leukocytes (< 1% of healthy controls) and pathologically elevated urinary sulfatide excretion (Table 1 , Figure S1 ). Early juvenile MLD Three infants were classified as early juvenile onset forms based on genotype data. Each of these cases carried one missense variant in trans with a pPTV in ARSA (Table 1 ). Two of these cases (index 1 and 2) carried ARSA genotypes previously reported multiple times in the literature, with mostly early juvenile onset (i.e. >80% of cases with comparable genotypes), and only few late juvenile cases in literature. 24 Their leukocyte ARSA activities, while clearly deficient, were somewhat higher than those of the late infantile cohort (Supplement Figure S1 ). Based on the European consensus criteria these cases were classified as early juvenile despite recognizing the variable onset due to certain genotypes even amongst siblings. 28 The third case (index 7) harbored one missense variant of uncertain significance (VUS, class 3) in trans with a well-known pathogenic pPTV. The prediction of early juvenile onset was based on data from individuals with comparable ARSA genotypes as well as subclinical findings such as demyelinating neuropathy (Supplement, Case reports). Based on the reduced ARSA activity in leukocytes and elevated urinary sulfatides in urine the variant was subsequently re-classified as likely pathogenic (class 4). Late Onset MLD two infants were classified as late-onset MLD based on the well reported genotype applying to European consensus recommendations. One infant harbored the c.542C > T ARSA variant exclusively present in late onset cases in trans with a pathogenic pPTV (index 3). 5,24 One infant carried the c.1283C > T variant in the homozygous state, also associated with a disease onset later that 6 years of age (index 9). 24 3.6 Treatment Allocation and Course The multidisciplinary treatment eligibility panel achieved a unanimous consensus regarding the respective therapeutic intervention, timing of intervention and monitoring schedule before and after treatment aligned with European consensus recommendations. Seven infants predicted to develop early-onset disease were referred for HSPC-GT. At the time of reporting, five infants had received treatment, one infant was awaiting infusion following apheresis, and one infant was undergoing treatment preparation (Fig. 2). Treatment was initiated at a median age of 11.0 months (range 7.8–13.4 months), corresponding to a median of 6.5 months after receipt of screening results. Conditioning and HSPC-GT infusion were generally well tolerated. Catheter-associated adverse events occurred in three infants and resolved without long-term sequelae (Supplement Table S1 , Case Reports). No serious adverse events related to the gene therapy product were observed. Two infants predicted to develop late-onset disease were enrolled in structured longitudinal surveillance planning to undergo HSCT according to the care pathway. 20 3.7 Early Outcomes Following HSPC-GT Early clinical outcomes following presymptomatic identification and treatment were encouraging and in line with results from the previous treatment trial. 29 Follow-up after HSPC-GT ranged from 6.3 to 30.2 months (median 18.5 months). Neurological examinations during follow-up did not reveal clinical signs of disease progression in treated infants. Four of five treated infants demonstrated age-appropriate cognitive, motor, and language development at last assessment (Supplement Table S1 ). One infant with predicted late-infantile disease showed mild motor delay at 12 months post-treatment in the context of otherwise continuous developmental progress without signs of stagnation (Supplement, Case Reports, index 4). Brain MRI performed up to 24 months post-treatment showed no leukodystrophy-related white matter changes in any treated infant. Nerve conduction studies showed variable trajectories, including progression in two infants and stability or partial improvement in five children. Of note, the treated child with mild developmental delay revealed clearly progressing demyelinating neuropathy (Supplement, Case Reports, index 4). Leukocyte ARSA enzyme activity remained within normal or supranormal ranges following treatment (Figure S1 ). 3.8 Outcomes Under Structured Surveillance The two infants predicted to develop late-onset MLD remained clinically asymptomatic at last follow-up (age range 7.85 to 37.49 months). Serial neurological examinations and developmental assessments were within age-appropriate ranges. Neurophysiological findings showed mild abnormalities without progression, and no infant met criteria for therapeutic intervention during the observation period (Supplement Table S1 ). HSCT is scheduled for both children in the pre-school period. 4. Discussion These findings have direct implications not only for the clinical management of infants identified through NBS for MLD, but also for the ongoing evaluation and implementation of MLD screening within national programs. In particular, they provide real-world evidence that population-based screening can be effectively translated into timely, coordinated, and clinically actionable care pathways within established healthcare systems. Beyond demonstrating feasibility, our results address key questions that are central to current policy and public health discussions, including analytical performance, diagnostic certainty, clinical decision-making, and long-term management strategies. To facilitate interpretation of these issues in the context of the existing evidence base, we have summarized the most relevant data and considerations in a structured Frequently Asked Questions (FAQ) section (textbox) and discuss our results further in the following sections. 4.1 Screening Feasibility and Analytical Performance The German and Austrian pilot programs show that population-based NBS for MLD is feasible and can achieve high analytical specificity in routine settings. Among 359,282 screened newborns, nine infants were identified and subsequently confirmed to have MLD, corresponding to an incidence of 1 in 39,920. No false-positive referrals were recorded during the study period. Although the small number of affected infants limits precision estimates and long-term sensitivity will require continued surveillance, the observed incidence aligns with the upper range of reported European prevalence. 3 The three-tier screening algorithm performed reliably across participating laboratories. Crucially, these performance metrics were achieved in routine screening laboratories and linked to real clinical decision-making. The findings extend earlier pre-pilot and pilot data and indicate that robust analytical performance can be maintained under real-world screening conditions. 13,14 4.2 Diagnostic Validity and Avoidance of Ambiguous Findings This three-tiered NBS algorithm not only minimized ambiguity in variant interpretation but also prevented incidental findings that could complicate counselling and clinical decision-making at a population level. The integration of biochemical and genetic data is a key strength of this screening approach. All screen-positive infants showed concordant elevations in sulfatides in urine and reductions in leukocyte ARSA activity, allowing confident classification of ARSA variants including those previously unreported alleles within ACMG frameworks. All novel variants could be classified as likely pathogenic through biochemical confirmation. Importantly, no individual harboring solely pseudodeficiency alleles screened positive in line with prior reports, despite their high population frequency. 14 Heterozygous carriers of variants in ARSA , SUMF1 , or PSAP exhibited isolated sulfatide species elevations (C16:0 or C16:1-OH) in the first tier and variably reduced ARSA activity in DBS, but lacked MLD-related genotypes, and were thus appropriately filtered by the three-tier workflow as reported before. 16 In contrast, the Italian prospective screening pilot used a two-tiered approach and repeat DBS sampling to mitigate false positive referrals. This protocol resulted in additional contact with 0.02% of neonates screened, which may have been mitigated by using the three tier algorithm. 17 Conditions with overlapping biochemical signatures, such as saposin B deficiency or multiple sulfatase deficiency, were not detected as screen-positives. Due to the current lack of disease-modifying therapeutic options these biochemically related disorders need to be excluded from recall. Therefore, the inclusion of ARSA sequencing as a third tier was essential for excluding these disorders and for minimizing unnecessary recalls, supporting the superiority of a three-tier approach over purely biochemical strategies. Overall, this design limited incidental findings and avoided unnecessary interpretive challenges. 4.3 Prediction of Disease Onset and Biomarker Limitations Early identification of MLD through NBS necessitates reliable strategies for predicting disease onset and guiding clinical management. In this cohort, genotype-based prediction, supported by residual enzyme activity and consensus criteria, enabled stratification into early-onset and late-onset phenotypes shortly after diagnosis. 19 All infants predicted to develop late-infantile or early juvenile MLD were referred promptly for evaluation for HSPC-GT, while those predicted to develop late-onset disease entered structured surveillance programs. This stratified approach allowed timely initiation of therapy for infants at highest risk of rapid disease progression while avoiding premature intervention in those likely to remain clinically presymptomatic for years. Although genotype-based prediction cannot fully capture individual variability, its application within a standardized consensus framework proved clinically actionable and feasible in routine care settings. Additional biochemical markers used for confirmation and monitoring have important limitations. Urinary sulfatide concentrations show substantial biological and pre-analytical variability and limited utility for longitudinal assessment as they do not decrease after treatment when measured in urine. Measurement of ARSA activity in leukocytes likewise exhibit biological variability, underscoring the need for iterative measurements. However, an activity below 1% in a standardized assay is predictive for early onset. 24 Of note, we detected slightly elevated neurofilament light chain and glial fibrillary acidic protein levels in blood of predicted early-onset cases, while the levels in a child with predicted late-onset remained within normal range during follow up (unpublished data). However, data on the predictive value of these biomarkers in MLD remain limited. As demyelinating neuropathy and subtle gallbladder abnormalities could be detected in infants of all subtypes, these are considered unlikely predictors for central disease onset (Supplement, Case reports). These findings highlight the need for complementary markers and longitudinal datasets to refine risk prediction in presymptomatic infants. 4.4 Early Clinical Management and Treatment Feasibility Early clinical outcomes following presymptomatic identification were encouraging but must be interpreted with caution given the short follow-up duration. Infants who underwent gene therapy during the presymptomatic period demonstrated preserved neurological examinations and age-appropriate developmental trajectories in all but one child during early follow-up, consistent with findings from controlled clinical trials. 29 Treatment delivery within the first year of life was feasible with manageable peri-transplant complications and no unexpected safety signals. However, follow-up remains limited in duration, and subtle abnormalities - including peripheral neuropathy and mild motor delay in one case - underscore that presymptomatic treatment does not necessarily prevent all disease manifestations. 30 Infants managed through surveillance for predicted late-onset disease have remained clinically asymptomatic, supporting the short-term safety of a monitoring-based approach when guided by consensus recommendations. 4.5 Monitoring Challenges and Unmet Needs Monitoring presymptomatic infants identified through NBS remains a major challenge. While urinary sulfatides are indispensable for diagnosis, their longitudinal utility for monitoring is limited by biological and pre-analytical variability. Plasma biomarkers such as neurofilament light chain and glial fibrillary acidic protein show promise as indicators of early neuroaxonal injury, but age-specific reference ranges and validated thresholds for clinical decision-making are lacking. 31 These biomarkers do not reliably distinguish between central and peripheral demyelination. This distinction is clinically important for treatment timing and post-treatment monitoring, as current therapies primarily affect central nervous system disease, whereas peripheral demyelination often continues. 30 Conventional neuroimaging was largely unremarkable during early follow-up and showed in this presymptomatic stage no correlation with predicted disease subtype. Although it was particularly pronounced in late infantile cases, peripheral demyelinating neuropathy could be detected in all subtypes within the first year of life and was not halted by HSPC-GT. These findings highlight the current absence of sensitive and reliable tools to detect early subclinical progression, particularly in infants predicted to develop late-onset disease. Longitudinal studies with larger cohorts will be essential to define biomarker trajectories, refine monitoring strategies, and optimize the timing of therapeutic intervention. 4.6 Public health implications Beyond individual clinical outcomes, these findings have important public health implications. The biochemical-first screening approach integrates into existing NBS laboratories and provides equitable detection across diverse populations, independent of genetic ancestry. The demonstrated feasibility of downstream care pathways from confirmatory diagnostics to specialist referral and treatment addresses a key requirement for population screening programs: that a positive NBS result leads to timely and meaningful clinical action. While broader implementation will require health-economic and policy considerations specific to each system, this pilot provides evidence that cross-border NBS-enabled care for MLD, as was done in Germany and Austria, is achievable. The timing of these findings is particularly relevant in light of recent policy developments. MLD has now been accepted for inclusion in the Recommended Uniform Screening Panel (RUSP) in the United States as well as into national screening programs in Norway and Sweden, reflecting growing international consensus on the benefit of early detection through NBS. A central concern during such policy deliberations is whether screening can be translated into timely, effective care without undue harm. The present study directly addresses this by demonstrating that NBS for MLD can be operationalized within nationwide healthcare systems and linked to meaningful early clinical outcomes. A recent perspective article highlighted the challenges of early detection in late-onset disease. 19 In MLD, it is important to emphasize that there is no “late mild” phenotype; rather, current evidence shows that even late-onset MLD, with symptom onset ranging from school age to adulthood, is associated with severe and clinically meaningful disease burden. Nevertheless, health surveillance and decisions regarding the timing of intervention in late-onset MLD should consider the particular challenges faced by identified individuals and their families and should incorporate as well as further develop psychosocial support strategies for this unique context. 4.8 Strengths and Limitations This work reports prospective real-world outcomes following population-based NBS for MLD, encompassing the entire pathway from population screening and confirmatory diagnostics to phenotype-based treatment stratification and early clinical follow-up. Key strengths include the large, screened population, the prospective design, the integration of standardized, consensus-based care pathways, and the linkage of screening results to real-world clinical decision-making and early outcomes across two national healthcare systems. This study also has important limitations. First, the workflow during these pilot programs did not reflect that expected once MLD NBS is fully implemented in the public health environment. For example, the time to obtain definitive positive screen results and communicate them to the QTC Tübingen was longer and more variable than desired, based on the stage of development of the algorithm. Second, the number of affected infants is small, reflecting the rarity of MLD, which limits statistical precision and precludes formal comparisons between management strategies. Third, follow-up duration remains limited, particularly for those under surveillance for late-onset disease; consequently, long-term neurological, cognitive, and quality-of-life outcomes cannot yet be assessed. Fourth, phenotype prediction relied primarily on genotype-based criteria and residual enzyme activity, which, although supported by expert consensus, do not fully capture intrafamilial and individual variability. Finally, these findings originate from well-resourced healthcare systems with established specialist networks, which may limit generalizability to settings with fewer resources or differing care structures. 5. Conclusion These German and Austrian pilot programs demonstrate that NBS for MLD can be implemented within existing healthcare infrastructures and linked to standardized diagnostic and clinical care pathways. A three-tier screening algorithm enabled early identification of affected infants and timely clinical stratification, supporting presymptomatic intervention for early-onset disease and structured surveillance for late-onset phenotypes. During early follow-up, infants managed through these pathways showed preserved neurological and developmental status. Together, these findings provide real-world evidence that NBS-enabled care for MLD is feasible and clinically actionable. Ongoing follow-up and broader implementation studies will be essential to define long-term outcomes and inform future screening policy decisions. Abbreviations The following abbreviations are used in this manuscript: ACMG American College of Medical Genetics and Genomics ARSA Arylsulfatase A CNS Central nervous system DBS Dried blood spot EJ Early juvenile EMA European Medicines Agency HSPC-GT Haematopoietic stem and progenitor cell gene therapy HSCT Haematopoietic stem cell transplantation LI Late infantile LJ Late juvenile LS-MS/MS Liquid chromatography–tandem mass spectrometry MLD Metachromatic leukodystrophy MLDi Metachromatic Leukodystrophy Initiative MRI Magnetic resonance imaging MSD Multiple sulfatase deficiency NBS Newborn screening NCV Nerve conduction velocity NGS Next-generation sequencing pPTV Predicted protein-truncating variant PSAP Prosaposin gene QTC Qualified treatment center RUSP Recommended Uniform Screening Panel SUMF1 Sulfatase-modifying factor 1 gene VUS Variant of uncertain significance Declarations Funding Information: MLD NBS studies led by D.K. and N.J. were carried out with support to Archimed life and the Hannover NBS laboratory from Orchard Therapeutics, a Kyowa Kirin company. Declaration of Interests: D.K., S.H. ands P.O. are employees of Archimed life. L.L. and S.G. received institutional research support by Orchard Therapeutics, unrelated to this study. Conflicts of Interest: All other co-authors have no conflicts of interest to declare. Author Contribution Conceptualization, L.L.,S.G., H.R.; data curation, L.L., A.C.J., P.J. M.W., T.L.; investigation, L.L., M.W., S.H., S.H., N.H., P.O., A.C.J., T.L.,V.K., M.Z.,J.K., N.K.; methodology, L.L., M.W., S.H., S.H., N.J. , D.K., P.J.; supervision, S.G.,H.R.; validation, L.L.,S.G.; visualization, P.J.; L.L.; writing – original draft, S.G., L.L.; writing – review and editing, L.L, S.G., S.H., S.H. H.R., B.P.,L.N., C.K. D.K. All authors have read and agreed to the final version of the manuscript. Acknowledgement Acknowledgments: We extend our deepest gratitude to the members of the MLD NBS Alliance and the MLDi for their pivotal contributions to the generation of crucial evidence and consensus guidelines. 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N Engl J Med 391 , 1256-1258 (2024). Trinidad, M. , et al. Predicting disease severity in metachromatic leukodystrophy using protein activity and a patient phenotype matrix. Genome Biol 24 , 172 (2023). Groeschel, S. , et al. Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Patients With Juvenile Metachromatic Leukodystrophy Compared With Nontransplanted Control Patients. JAMA Neurol 73 , 1133-1140 (2016). Wilson, J.M.G., Jungner, G. & Organization, W.H. Principles and practice of screening for disease. (1968). Mohajer, A. , et al. Characterizing Diagnostic Delays in Metachromatic Leukodystrophy: A Real-World Data Approach. J Inherit Metab Dis 48 , e70049 (2025). Bean, K., Preston, B., Adang, L.A. & Pang, F. Exploring the net monetary benefit of implementing newborn screening for metachromatic leukodystrophy in California. Mol Genet Metab 144 , 108651 (2025). Bean, K. , et al. Exploring the Cost-Effectiveness of Newborn Screening for Metachromatic Leukodystrophy (MLD) in the UK. Int J Neonatal Screen 10 (2024). Bean, K., Gelb, M.H., Adang, L.A., Chanson, C. & Pang, F. Cost-effectiveness framework by tandem mass spectrometry (TMS) for newborn screening of metachromatic leukodystrophy (MLD) in the United States (US). Mol Genet Metab 141 , 107769 (2024). Kehrer, C. , et al. Healthcare utilization and disease burden in children with metachromatic leukodystrophy in Germany. Orphanet J Rare Dis 20 , 242 (2025). Additional Declarations No competing interests reported. Supplementary Files SupplementV24SG.docx FrequentlyAskedQuestionsonNBSforMLD.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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The diagram illustrates the stepwise clinical workflow following NBS for MLD. After detection via three-tiered screening of dried blood spots, confirmatory diagnostics and clinical evaluation are initiated to predict disease onset. Confirmatory diagnostics includes ARSA activity in leukocytes, urinary sulfatide levels and genetic testing of the index and its parents. Based on the predicted subtype – early, late, or uncertain onset - a tailored treatment decision is made. Presymptomatic early-onset cases are eligible for HSPC-GT within the first year of life, while late-onset forms are scheduled for HSCT at pre-school ages. In cases with late or uncertain onset, longitudinal surveillance is mandatory. All affected individuals continue under structured follow-up to assess long-term outcomes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9496450/v1/856c6b3925a36b52974e24d8.png"},{"id":108013032,"identity":"a205e7f7-b4e2-4f48-9aab-93eebb3190fe","added_by":"auto","created_at":"2026-04-28 13:17:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTimeline of Patient Management. \u003c/strong\u003eTimeline of key clinical milestones for nine patients identified by NBS for MLD, showing the course from birth through current age. Each horizontal line represents an individual patient, with markers indicating specific events such as screening results, diagnosis confirmation, treatment decisions, medical procedures (apheresis, conditioning, Arsa-cel infusion), discharge, and neurological follow-ups. The x-axis measures months after birth, while the legend identifies each event type by symbol and color. Event metrics are listed in Supplement Table S2.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9496450/v1/783ef6b99d20680be8ad2203.png"},{"id":108490884,"identity":"d8ab92f6-76e5-4799-a963-9ce8719fb760","added_by":"auto","created_at":"2026-05-05 09:49:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":648763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9496450/v1/43fd77a8-14c8-46c6-bf52-61ee758be400.pdf"},{"id":108012938,"identity":"cfc16768-7740-47b8-a69e-488787ef0209","added_by":"auto","created_at":"2026-04-28 13:16:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":108571,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementV24SG.docx","url":"https://assets-eu.researchsquare.com/files/rs-9496450/v1/997e67004100b80d6fc468f9.docx"},{"id":108012925,"identity":"de5872c0-9dc8-4817-a52e-c5ca67744a81","added_by":"auto","created_at":"2026-04-28 13:16:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":63087,"visible":true,"origin":"","legend":"","description":"","filename":"FrequentlyAskedQuestionsonNBSforMLD.docx","url":"https://assets-eu.researchsquare.com/files/rs-9496450/v1/12e116991ff81a7c643b29c4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Newborn Screening for Metachromatic Leukodystrophy: Care Pathway and Early Clinical Outcomes from German and Austrian Pilot Programs","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMetachromatic leukodystrophy (MLD) (MIM#250100) is a rare lysosomal storage disorder caused by biallelic clinically relevant variants in \u003cem\u003eARSA\u003c/em\u003e (MIM*607574) encoding the lysosomal enzyme arylsulfatase A (ARSA).\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e ARSA deficiency leads to a progressive accumulation of sulfatides, in particular in the central and peripheral nervous system, resulting in demyelination and neurodegeneration.\u003csup\u003e1\u003c/sup\u003e The overall birth prevalence of MLD in Europe is approximately 1 in 40,000 to 1 in 100,000.\u003csup\u003e2\u003c/sup\u003e Although disease onset spans infancy to adulthood, early-onset forms (i.e. late infantile and early juvenile) are most common and are associated with more rapid neurodevelopmental regression, severe disability, and early death if untreated.\u003csup\u003e4\u003c/sup\u003e For these children, irreversible neurological injury begins early in life, making timely intervention critical. Late onset MLD (i.e. late juvenile and adult) may present later in childhood, adolescence, or adulthood, with a more gradual progression of severe neurological or psychiatric symptoms.\u003csup\u003e4,5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eUntil recently, therapeutic options for early-onset MLD were limited to supportive care and, in late onset cases diagnosed in an early symptomatic or pre-symptomatic stage, allogeneic hematopoietic stem cell transplantation (HSCT).\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e The development of autologous hematopoietic stem and progenitor cell gene therapy (HSPC-GT, atidarsagene autotemcel, arsa-cel) has fundamentally altered the treatment landscape for early onset MLD.\u003csup\u003e9,10\u003c/sup\u003e When administered before the onset of clinical signs, HSPC-GT in early onset MLD and HSCT in late onset MLD are associated with preservation of early neurodevelopment and substantial modification of disease course.\u003csup\u003e6\u0026ndash;10\u003c/sup\u003e However, treatment efficacy is highly time dependent: once neurological symptoms emerge, therapeutic benefit is markedly reduced.\u003csup\u003e9\u003c/sup\u003e This narrow therapeutic window means that for most children diagnosed with MLD, the diagnosis comes too late for currently available interventions.\u003csup\u003e11\u003c/sup\u003e Long-term outcomes are highly encouraging, with infants maintaining near-normal development when treated pre-symptomatically.\u003csup\u003e9,10\u003c/sup\u003e Newborn screening (NBS) provides a population-based strategy to identify infants with MLD pre-symptomatically and before the onset of irreversible neurological injury.\u003csup\u003e12\u003c/sup\u003e Over the past decade, biochemical screening approaches for MLD have been systematically developed and validated in retrospective studies.\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e Prospective screening pilot programs have demonstrated that MLD screening can be integrated into established NBS infrastructures with high analytical performance.\u003csup\u003e16,17\u003c/sup\u003e These advances have driven growing international momentum, with several countries initiating or expanding pilot programs and some incorporating MLD into national screening panels.\u003csup\u003e18\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, the availability of treatment options and screening alone does not ensure improved clinical outcomes. As a rapidly emerging field of science, gaps in evidence remain. For NBS for MLD this primarily relates to the downstream consequences of population-based detection: data are scarce on the real-world clinical management of infants identified through NBS for MLD, including confirmatory diagnostic workflows, prediction of disease onset, decision-making around presymptomatic treatment, and the feasibility of delivering time-sensitive interventions within routine healthcare systems. Equally important is our understanding how infants predicted to develop late-onset disease can be monitored safely over time without exposing families to unnecessary intervention or harm before they are eligible for treatment.\u003csup\u003e19,20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this work, we report the first larger clinical experiences from prospective NBS pilot programs for MLD in Germany and Austria. We evaluate previously established care pathways from screening to confirmatory diagnosis, consensus-based prediction of disease onset, and subsequent clinical management. We further report the clinical care pathway of pre-symptomatic infants with MLD who underwent HSPC-GT and those managed through structured surveillance before HSCT. Taken together, these findings will offer preliminary evidence for the feasibility and potential clinical impact of NBS-enabled care for MLD in routine pediatric healthcare practice. This study advances the field by providing the first prospective real-world data on early clinical trajectories after NBS for MLD, including confirmatory diagnostic pathways, treatment decisions and outcomes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Pilot Program Design\u003c/h2\u003e \u003cp\u003eProspective NBS pilot programs for MLD were conducted in North-West Germany and nationwide in Austria between September 2021 and July 2025 through coordinated collaborations involving the Screening Laboratory Hannover, the Medical University of Vienna, and ARCHIMEDlife. In Germany, the pilot operated from September 2021 until December 2024 as a joint initiative between the Screening Laboratory Hannover and ARCHIMEDlife; thereafter, the program was fully transferred to the Screening Laboratory Hannover and continues independently. In Austria, the pilot is ongoing, and since September 2023 has been conducted as a partnership between the Medical University of Vienna and ARCHIMEDlife.\u003c/p\u003e \u003cp\u003eGerman newborns born in the capture area of the Screening Laboratory Hannover were enrolled under an Institutional Review Board-approved protocol (\u0026Auml;rztekammer Niedersachsen; BO/39/2021). Austrian screening was conducted under approval from the Ethics Committee of the Medical University of Vienna (1961/2022). Further biochemical and clinical investigations of infants identified through screening were approved by the local ethics committee of the Medical Faculty of the University of T\u0026uuml;bingen, Germany (948/2018BO2). All participants, or their respective legal guardians, provided written informed consent for participation and publication in accordance with the Declaration of Helsinki. Clinical trial number: not applicable.\u003c/p\u003e \u003cp\u003eThe Hannover cohort includes three screen positive infants previously reported in Laugwitz et al. 2024 (index 1 to 3)\u003csup\u003e16\u003c/sup\u003e, with extended follow-up and additional cases included here. The Austrian cohort has not been reported before. All analyses reported in the present study were prospectively defined and conducted within the approved protocols.\u003c/p\u003e \u003cp\u003eThe primary outcome of the pilot programs was the detection rate of confirmed MLD cases, with an expected incidence of approximately 1 per 40,000 to 100,000 newborns based on European prevalence estimates.\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe clinical data collection for this study was completed in December 2025.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Screening Algorithm\u003c/h2\u003e \u003cp\u003eNBS for MLD was implemented using a three-tier screening strategy, as previously described.\u003csup\u003e15,16,21\u003c/sup\u003e The screening workflow evolved during the pilot phase to optimize analytical performance and minimize false-positive results. Since early 2024, the fully standardized three-tier algorithm has been applied to dried blood spots (DBS) obtained for routine NBS in both Germany and Austria, and consists of:\u003c/p\u003e \u003cp\u003e(1) quantification of sulfatides (C16:0 and C16:1-OH) in dried blood spots (DBS),\u003c/p\u003e \u003cp\u003e(2) measurement of ARSA enzymatic activity in DBS, and\u003c/p\u003e \u003cp\u003e(3) next generation sequencing of \u003cem\u003eARSA\u003c/em\u003e, \u003cem\u003eSUMF1\u003c/em\u003e, and \u003cem\u003ePSAP\u003c/em\u003e in DBS.\u003c/p\u003e \u003cp\u003eDetailed analytical methods, assay cut-offs, and quality control procedures have been reported previously.\u003csup\u003e15,16,21\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Clinical Management Pathway\u003c/h2\u003e \u003cp\u003eA central aim of the pilot programs was not only case detection but also the prospective evaluation of a standardized, consensus-based care pathway following a positive NBS result. Management of infants identified through NBS followed a predefined, standardized care pathway aligned with European expert consensus recommendations (Fig.\u0026nbsp;1).\u003csup\u003e20\u003c/sup\u003e After confirmation of the diagnosis of MLD, families received multidisciplinary counselling regarding disease characteristics, prognosis, and available management options. Care was coordinated by the QTC in T\u0026uuml;bingen in collaboration with metabolic pediatricians, pediatric neurologists, clinical geneticists, transplant physicians, and allied health professionals. Psychosocial support and access to patient advocacy resources were offered throughout the diagnostic and management process in accordance with consensus guidance. Cases were discussed within the treatment eligibility panel of the MLDInitiative (MLDi).\u003csup\u003e22\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Confirmatory Diagnostics and Molecular Assessment\u003c/h2\u003e \u003cp\u003eFor all screen-positive infants, families were informed by MLD-specialized treatment centers in line with European recommendations (i.e. the QTC in T\u0026uuml;bingen, Germany, and medical centers in Graz and Innsbruck, Austria), and infants were referred promptly for confirmatory diagnostic evaluation. Confirmatory testing was coordinated through specialized centers in collaboration with the QTC at the University Hospital T\u0026uuml;bingen, designated as a QTC for Germany and Austria. Confirmatory diagnostics included measurement of ARSA activity in leukocytes\u003csup\u003e23,24\u003c/sup\u003e and repeated quantification of urinary sulfatide levels using liquid chromatography-tandem mass spectrometry\u003csup\u003e25\u003c/sup\u003e. Genetic sequencing was repeated using freshly obtained EDTA blood samples from the infant and both parents to confirm biallelic \u003cem\u003eARSA\u003c/em\u003e variant localization. Following biochemical confirmation, \u003cem\u003eARSA\u003c/em\u003e variants were classified according to American College of Medical Genetics and Genomics (ACMG).\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Clinical Evaluation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBaseline clinical evaluation at the QTC included a comprehensive neuropediatric examination and assessment of developmental milestones. Instrumental investigations comprised brain magnetic resonance imaging, nerve conduction velocity studies, peripheral nerve ultrasound, acoustic evoked potentials, and gallbladder ultrasound. The scope and timing of assessments were adapted to age and clinical context while adhering to consensus recommendations.\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Prediction of Disease Onset\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach infant was then classified into an MLD subtype risk category - late infantile/early juvenile, and late juvenile/adult onset based on their genotype. Additional parameters were residual ARSA activity in leukocytes\u003csup\u003e24\u003c/sup\u003e. Published consensus criteria for phenotype prediction in pre-symptomatic infants were applied:\u003csup\u003e20\u003c/sup\u003e In brief, infants carrying two predicted protein truncating variants (pPTVs) in \u003cem\u003eARSA\u003c/em\u003e, known variants associated with late infantile onset or who exhibited a residual ARSA activity in leukocytes of less than 1% on repeated measurements were predicted to develop the late infantile form of MLD.\u003csup\u003e24\u003c/sup\u003e In line with European consensus recommendation in newborns with pPTVs \u003cem\u003ein trans\u003c/em\u003e with another clinically relevant missense variant were predicted as early onset MLD (i.e. late infantile or early juvenile). Newborns with genotypes consistently associated with late onset disease such c.542T\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Ile181Ser) \u003cem\u003ein trans\u003c/em\u003e with any other clinically relevant \u003cem\u003eARSA\u003c/em\u003e variant or c.1283C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Pro428Leu) in homozygous state were predicted as late onset, recognizing that distinguishing late juvenile versus adult onset is not possible with genotype alone.\u003csup\u003e20\u003c/sup\u003e Newborns with other well-documented \u003cem\u003eARSA\u003c/em\u003e genotypes reported in the literature were classified accordingly.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Treatment Decision\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll cases were reviewed in multidisciplinary European expert rounds of MLDi. Eligibility for treatment with HSPC-GT (arsa-cel) was assessed according to European Medicines Agency criteria.\u003csup\u003e27\u003c/sup\u003e Consensus decisions regarding treatment, timing, and monitoring were reached by a multidisciplinary expert panel. Infants predicted to develop early-onset MLD were referred for gene therapy within the first 12 months of life. Infants predicted to develop late-onset disease or for whom disease onset was uncertain, were enrolled in a structured long-term surveillance program, which includes regular neurological examinations, developmental assessments, and periodic neuroimaging and neurophysiological studies. Escalation to therapeutic intervention during surveillance followed consensus recommendations and was based on emerging clinical, neurophysiological, imaging, or biomarker evidence of disease activity. Outcome data were collected prospectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Methods and Graphics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDescriptive statistics were applied to summarize the screened population, biochemical and genetic findings, and diagnostic outcomes. Illustrations were generated using Biorender and the Python package \u003cem\u003ematplotlib\u003c/em\u003e (version 3.10.3). Descriptive statistics were calculated using Python (version 3.13.0) primarily utilizing the packages \u003cem\u003epandas\u003c/em\u003e and \u003cem\u003enumpy\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Screening Algorithm Performance\u003c/h2\u003e \u003cp\u003eBetween September 2021 and July 2025, a total of 359,282 dried blood spot samples were analyzed across NBS pilots in Germany and Austria. Nine newborns screened positive and were subsequently confirmed to have MLD, corresponding to a prevalence of 2.51 per 100,000 live births and an incidence of approximately one in 39,920 screened newborns. No false-positive recalls occurred during the study period. During the study period, no additional infants diagnosed with MLD within the screening catchment areas were reported to the QTC in T\u0026uuml;bingen. .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Management Timeline\u003c/h2\u003e \u003cp\u003eScreen-positive results for the nine index cases identified in the NBS stuedies were communicated to the designated QTC in T\u0026uuml;bingen at a median age of 3 months (range 1.0\u0026ndash;5.8 months) following NBS sample collection at birth (Fig.\u0026nbsp;2). In accordance with established European NBS recommendations, families were contacted within one week of NBS result receipt by an designated MLD expert center. In eight cases, initial disclosure was conducted by the MLD expert center. In one case, families were contacted directly by the screening laboratory owing to a miscommunication with the referring laboratory; this represented a protocol deviation. All families were offered rapid access to the QTC through video or telephone consultation or in-person outpatient visits within one week after notification of positive NBS results. Confirmatory diagnostic testing was completed within a median of 0.3 months after recall (range 0.2\u0026ndash;1.9 months). Comprehensive clinical evaluations at the expert center were conducted within a median of 1.0 month following diagnosis (range 0\u0026ndash;2.3 months), with timing adjusted according to predicted disease subtype. All cases were subsequently reviewed by a multidisciplinary treatment eligibility panel convened by the MLDi within 1 to 4 months after recall. For infants predicted to develop late-infantile disease, panel review was prioritized and scheduled within 1 to 2 weeks after completion of confirmatory diagnostics. Treatment recommendations were reached by consensus in eight of nine cases at the initial panel meeting. One case (index 7) required additional diagnostic clarification and repeat review before final consensus was achieved (Supplement, Case reports). Subsequent clinical assessments at the QTC were completed within 0 to 3 months for all infants and within 4 to 6 weeks for those with predicted early-onset disease. Stem cell apheresis was performed a median of 2.2 months after treatment decision (range 1.2\u0026ndash;6.6 months), corresponding to a median of 3.6 months after NBS results (range 2.7\u0026ndash;7.9 months). In one case (index 8), apheresis was repeated because the initial drug product did not meet release criteria, consistent with standard manufacturing protocols. HSPC-GT infusion was administered a median of 2.5 months after apheresis (range 1.8\u0026ndash;2.5 months). Following treatment, infants were discharged a median of 1.0 month after infusion (range 0.8\u0026ndash;1.2 months; Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Confirmatory Diagnostics and Molecular Findings\u003c/h2\u003e \u003cp\u003eAll nine infants classified as screen positive were confirmed to have a diagnosis of MLD through biochemical and molecular testing (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Each infant harbored biallelic clinically relevant variants in \u003cem\u003eARSA\u003c/em\u003e, accompanied by markedly reduced ARSA activity in leukocytes and elevated sulfatide excretion in urine (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To account for biological variability, leukocyte ARSA activity and urinary sulfatides were assessed repeatedly at multiple time points (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Although some interday variability was observed, all repeated measurements remained consistently within the pathological range. Genotypes spanned the known phenotypic spectrum of MLD and included both previously reported pathogenic and novel variants. Three previously unreported \u003cem\u003eARSA\u003c/em\u003e variants were identified, including a splice donor variant, a frameshift and a missense variant. Based on ACMG criteria, these were classified as likely pathogenic, supported by functional biochemical confirmation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Inclusion of the third-tier genetic testing in the NBS algorithm was critical for screening specificity. No infants with pseudodeficiency alleles or isolated heterozygous variants in \u003cem\u003eARSA\u003c/em\u003e, or variants in \u003cem\u003eSUMF1\u003c/em\u003e or \u003cem\u003ePSAP\u003c/em\u003e fulfilled criteria for a positive screen, and no biochemical mimics of MLD were identified. Together, the combined biochemical-genetic approach in the screening and confirmatory testing algorithm enabled unambiguous diagnosis in all cases and avoided variants of uncertain clinical significance as standalone findings.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConfirmatory Diagnostics of Newborns identified\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003cp\u003e(HGVS, GRCh38)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACMG Class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClinVar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eARSA activity in leukocytes\u003csup\u003e\u003cem\u003e24\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eNormal range:\u003c/p\u003e \u003cp\u003e1.5 to 6.1 E\u003csub\u003e514\u003c/sub\u003e nm/10\u003csup\u003e6\u003c/sup\u003e cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUrinary Sulfatides\u003c/p\u003e \u003cp\u003eNormal range: \u0026lt;300nmol/l\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePredicted Onset\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[412C\u0026thinsp;\u0026gt;\u0026thinsp;T];[1283C\u0026thinsp;\u0026gt;\u0026thinsp;T],p.[(Pro138Ser)];[(Pro428Leu)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003csup\u003e1\u003c/sup\u003e;P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 (n\u0026thinsp;=\u0026thinsp;4)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePathologically elevated\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEarly juvenile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A];[1283C\u0026thinsp;\u0026gt;\u0026thinsp;T],p.[(?)];[(Pro428Leu)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003csup\u003e1\u003c/sup\u003e;P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013 (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e549.823\u0026thinsp;\u0026plusmn;\u0026thinsp;265.427 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEarly juvenile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A];[542T\u0026thinsp;\u0026gt;\u0026thinsp;G],p.[(?)];[(Ile181Ser)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP;P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029 (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3216.248\u0026thinsp;\u0026plusmn;\u0026thinsp;2723.202 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLate juvenile/ adult\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A];[465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A],p.[(?)];[(?)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP.;P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 (n\u0026thinsp;=\u0026thinsp;4)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1245.992\u0026thinsp;\u0026plusmn;\u0026thinsp;892.734 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLate infantile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A];[899del],p.[(?)];[(Leu300Cysfs*29)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5; 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP;n.r.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.035\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034 (n\u0026thinsp;=\u0026thinsp;5)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1960.298\u0026thinsp;\u0026plusmn;\u0026thinsp;1045.793 (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLate infantile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[931G\u0026thinsp;\u0026gt;\u0026thinsp;A];[931G\u0026thinsp;\u0026gt;\u0026thinsp;A],p.[(Gly311Ser)];[(Gly311Ser)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLP;LP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 (n\u0026thinsp;=\u0026thinsp;4)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2324.697\u0026thinsp;\u0026plusmn;\u0026thinsp;753.985 (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLate infantile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A];[494C\u0026thinsp;\u0026gt;\u0026thinsp;T];p.[(?)];[(Pro165Leu)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;3 -\u0026gt; 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP;n.r.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.087\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1933.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1679.955 (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEarly juvenile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[684\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A];[1223_1231del],p.[(?)];[(Val408Serfs*21)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.r.;P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022 (n\u0026thinsp;=\u0026thinsp;6)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1149.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1068.438 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLate infantile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eARSA\u003c/em\u003e:c.[1283C\u0026thinsp;\u0026gt;\u0026thinsp;T];[1283C\u0026thinsp;\u0026gt;\u0026thinsp;T],p.[(Pro428Leu)];[(Pro428Leu)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP;P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1680.425\u0026thinsp;\u0026plusmn;\u0026thinsp;1254.938 (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLate juvenile/ adult\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003en.r., not reported; P, pathogenic; LP, likely pathogenic; \u003csup\u003ea\u003c/sup\u003ebelow the predictive threshold of \u0026lt;\u0026thinsp;1% of mean activity in healthy controls according to prior reports\u003csup\u003e1921\u003c/sup\u003e, suggestive for early onset.\u003csup\u003eb\u003c/sup\u003e no quantification possible due to lack of stored biomaterial, sulfatides were measured qualitatively instead.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Clinical Baseline Evaluation\u003c/h2\u003e \u003cp\u003eBaseline clinical assessments were completed in all infants at the QTC in T\u0026uuml;bingen prior to treatment or enrolment into surveillance (Supplement Table S2, Case Reports). At initial evaluation (between 3 and 6 months of age), neurological examination and developmental assessments were unremarkable in all cases, with achievement of age-appropriate milestones reported by caregivers and primary care physicians.\u003c/p\u003e \u003cp\u003eMRI of the brain was conducted in eight infants and showed no imaging evidence of leukodystrophy-related white matter changes, for one child with late onset-MLD the first MRI evaluation is planned but not yet performed. MRI severity scores were 0 in all cases (Supplement Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Gallbladder ultrasound revealed no gallstones or premalignant lesions. Mild gallbladder wall thickening or sludge was observed in two infants without associated clinical signs (Supplement Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeurophysiological studies demonstrated greater variability. Nerve conduction studies were within age-adjusted reference ranges in three infants, while six infants showed evidence of demyelinating peripheral neuropathy, including infants predicted to develop both early- and late-onset disease. Peripheral nerve ultrasound was normal in all infants examined. Auditory evoked potentials were abnormal in two cases, showing prolonged latencies without corresponding clinical hearing impairment (Supplement Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Overall, baseline evaluations confirmed the absence of overt neurological disease at the time of diagnosis while identifying subclinical abnormalities in selected instrumental assessments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Prediction of Disease Onset\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on the consensus criteria, the patients were stratified into predicted phenotypic subtypes:\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLate infantile MLD\u003c/strong\u003e \u003cp\u003eOut of the nine cases, four infants were predicted to have late infantile onset MLD. These infants harbored truncating \u003cem\u003eARSA\u003c/em\u003e variants known to abolish enzyme function (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Besides the common splice site variant c.465\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;T and known frameshift variant (c.1223_1231del), a novel splice donor variant (c.684\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A) and a novel frameshift variant were identified (c.899del). Aligning with ACMG criteria these novel pPTVs were classified as likely pathogenic (class 4). Biallelic pPTVs strongly indicated symptom onset before 30 months of age and the most aggressive disease course without intervention (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All four had very low mean ARSA activity in leukocytes (\u0026lt;\u0026thinsp;1% of healthy controls) and pathologically elevated urinary sulfatide excretion (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEarly juvenile MLD\u003c/strong\u003e \u003cp\u003eThree infants were classified as early juvenile onset forms based on genotype data. Each of these cases carried one missense variant \u003cem\u003ein trans\u003c/em\u003e with a pPTV in \u003cem\u003eARSA\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two of these cases (index 1 and 2) carried \u003cem\u003eARSA\u003c/em\u003e genotypes previously reported multiple times in the literature, with mostly early juvenile onset (i.e. \u0026gt;80% of cases with comparable genotypes), and only few late juvenile cases in literature.\u003csup\u003e24\u003c/sup\u003e Their leukocyte ARSA activities, while clearly deficient, were somewhat higher than those of the late infantile cohort (Supplement Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Based on the European consensus criteria these cases were classified as early juvenile despite recognizing the variable onset due to certain genotypes even amongst siblings.\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe third case (index 7) harbored one missense variant of uncertain significance (VUS, class 3) \u003cem\u003ein trans\u003c/em\u003e with a well-known pathogenic pPTV. The prediction of early juvenile onset was based on data from individuals with comparable \u003cem\u003eARSA\u003c/em\u003e genotypes as well as subclinical findings such as demyelinating neuropathy (Supplement, Case reports). Based on the reduced ARSA activity in leukocytes and elevated urinary sulfatides in urine the variant was subsequently re-classified as likely pathogenic (class 4).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLate Onset MLD\u003c/strong\u003e \u003cp\u003etwo infants were classified as late-onset MLD based on the well reported genotype applying to European consensus recommendations. One infant harbored the c.542C\u0026thinsp;\u0026gt;\u0026thinsp;T \u003cem\u003eARSA\u003c/em\u003e variant exclusively present in late onset cases \u003cem\u003ein trans\u003c/em\u003e with a pathogenic pPTV (index 3). \u003csup\u003e5,24\u003c/sup\u003e One infant carried the c.1283C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the homozygous state, also associated with a disease onset later that 6 years of age (index 9).\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Treatment Allocation and Course\u003c/h2\u003e \u003cp\u003eThe multidisciplinary treatment eligibility panel achieved a unanimous consensus regarding the respective therapeutic intervention, timing of intervention and monitoring schedule before and after treatment aligned with European consensus recommendations. Seven infants predicted to develop early-onset disease were referred for HSPC-GT. At the time of reporting, five infants had received treatment, one infant was awaiting infusion following apheresis, and one infant was undergoing treatment preparation (Fig.\u0026nbsp;2). Treatment was initiated at a median age of 11.0 months (range 7.8\u0026ndash;13.4 months), corresponding to a median of 6.5 months after receipt of screening results. Conditioning and HSPC-GT infusion were generally well tolerated. Catheter-associated adverse events occurred in three infants and resolved without long-term sequelae (Supplement Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Case Reports). No serious adverse events related to the gene therapy product were observed. Two infants predicted to develop late-onset disease were enrolled in structured longitudinal surveillance planning to undergo HSCT according to the care pathway.\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Early Outcomes Following HSPC-GT\u003c/h2\u003e \u003cp\u003eEarly clinical outcomes following presymptomatic identification and treatment were encouraging and in line with results from the previous treatment trial.\u003csup\u003e29\u003c/sup\u003e Follow-up after HSPC-GT ranged from 6.3 to 30.2 months (median 18.5 months). Neurological examinations during follow-up did not reveal clinical signs of disease progression in treated infants. Four of five treated infants demonstrated age-appropriate cognitive, motor, and language development at last assessment (Supplement Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). One infant with predicted late-infantile disease showed mild motor delay at 12 months post-treatment in the context of otherwise continuous developmental progress without signs of stagnation (Supplement, Case Reports, index 4). Brain MRI performed up to 24 months post-treatment showed no leukodystrophy-related white matter changes in any treated infant. Nerve conduction studies showed variable trajectories, including progression in two infants and stability or partial improvement in five children. Of note, the treated child with mild developmental delay revealed clearly progressing demyelinating neuropathy (Supplement, Case Reports, index 4). Leukocyte ARSA enzyme activity remained within normal or supranormal ranges following treatment (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Outcomes Under Structured Surveillance\u003c/h2\u003e \u003cp\u003eThe two infants predicted to develop late-onset MLD remained clinically asymptomatic at last follow-up (age range 7.85 to 37.49 months). Serial neurological examinations and developmental assessments were within age-appropriate ranges. Neurophysiological findings showed mild abnormalities without progression, and no infant met criteria for therapeutic intervention during the observation period (Supplement Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). HSCT is scheduled for both children in the pre-school period.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThese findings have direct implications not only for the clinical management of infants identified through NBS for MLD, but also for the ongoing evaluation and implementation of MLD screening within national programs. In particular, they provide real-world evidence that population-based screening can be effectively translated into timely, coordinated, and clinically actionable care pathways within established healthcare systems.\u003c/p\u003e \u003cp\u003eBeyond demonstrating feasibility, our results address key questions that are central to current policy and public health discussions, including analytical performance, diagnostic certainty, clinical decision-making, and long-term management strategies. To facilitate interpretation of these issues in the context of the existing evidence base, we have summarized the most relevant data and considerations in a structured Frequently Asked Questions (FAQ) section (textbox) and discuss our results further in the following sections.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Screening Feasibility and Analytical Performance\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe German and Austrian pilot programs show that population-based NBS for MLD is feasible and can achieve high analytical specificity in routine settings. Among 359,282 screened newborns, nine infants were identified and subsequently confirmed to have MLD, corresponding to an incidence of 1 in 39,920. No false-positive referrals were recorded during the study period. Although the small number of affected infants limits precision estimates and long-term sensitivity will require continued surveillance, the observed incidence aligns with the upper range of reported European prevalence.\u003csup\u003e3\u003c/sup\u003e The three-tier screening algorithm performed reliably across participating laboratories. Crucially, these performance metrics were achieved in routine screening laboratories and linked to real clinical decision-making. The findings extend earlier pre-pilot and pilot data and indicate that robust analytical performance can be maintained under real-world screening conditions.\u003csup\u003e13,14\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Diagnostic Validity and Avoidance of Ambiguous Findings\u003c/h2\u003e \u003cp\u003eThis three-tiered NBS algorithm not only minimized ambiguity in variant interpretation but also prevented incidental findings that could complicate counselling and clinical decision-making at a population level. The integration of biochemical and genetic data is a key strength of this screening approach.\u003c/p\u003e \u003cp\u003eAll screen-positive infants showed concordant elevations in sulfatides in urine and reductions in leukocyte ARSA activity, allowing confident classification of \u003cem\u003eARSA\u003c/em\u003e variants including those previously unreported alleles within ACMG frameworks. All novel variants could be classified as likely pathogenic through biochemical confirmation.\u003c/p\u003e \u003cp\u003eImportantly, no individual harboring solely pseudodeficiency alleles screened positive in line with prior reports, despite their high population frequency.\u003csup\u003e14\u003c/sup\u003e Heterozygous carriers of variants in \u003cem\u003eARSA\u003c/em\u003e, \u003cem\u003eSUMF1\u003c/em\u003e, or \u003cem\u003ePSAP\u003c/em\u003e exhibited isolated sulfatide species elevations (C16:0 or C16:1-OH) in the first tier and variably reduced ARSA activity in DBS, but lacked MLD-related genotypes, and were thus appropriately filtered by the three-tier workflow as reported before.\u003csup\u003e16\u003c/sup\u003e In contrast, the Italian prospective screening pilot used a two-tiered approach and repeat DBS sampling to mitigate false positive referrals. This protocol resulted in additional contact with 0.02% of neonates screened, which may have been mitigated by using the three tier algorithm.\u003csup\u003e17\u003c/sup\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eConditions with overlapping biochemical signatures, such as saposin B deficiency or multiple sulfatase deficiency, were not detected as screen-positives. Due to the current lack of disease-modifying therapeutic options these biochemically related disorders need to be excluded from recall. Therefore, the inclusion of \u003cem\u003eARSA\u003c/em\u003e sequencing as a third tier was essential for excluding these disorders and for minimizing unnecessary recalls, supporting the superiority of a three-tier approach over purely biochemical strategies. Overall, this design limited incidental findings and avoided unnecessary interpretive challenges.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Prediction of Disease Onset and Biomarker Limitations\u003c/h2\u003e \u003cp\u003eEarly identification of MLD through NBS necessitates reliable strategies for predicting disease onset and guiding clinical management. In this cohort, genotype-based prediction, supported by residual enzyme activity and consensus criteria, enabled stratification into early-onset and late-onset phenotypes shortly after diagnosis.\u003csup\u003e19\u003c/sup\u003e All infants predicted to develop late-infantile or early juvenile MLD were referred promptly for evaluation for HSPC-GT, while those predicted to develop late-onset disease entered structured surveillance programs. This stratified approach allowed timely initiation of therapy for infants at highest risk of rapid disease progression while avoiding premature intervention in those likely to remain clinically presymptomatic for years. Although genotype-based prediction cannot fully capture individual variability, its application within a standardized consensus framework proved clinically actionable and feasible in routine care settings. Additional biochemical markers used for confirmation and monitoring have important limitations. Urinary sulfatide concentrations show substantial biological and pre-analytical variability and limited utility for longitudinal assessment as they do not decrease after treatment when measured in urine. Measurement of ARSA activity in leukocytes likewise exhibit biological variability, underscoring the need for iterative measurements. However, an activity below 1% in a standardized assay is predictive for early onset.\u003csup\u003e24\u003c/sup\u003e Of note, we detected slightly elevated neurofilament light chain and glial fibrillary acidic protein levels in blood of predicted early-onset cases, while the levels in a child with predicted late-onset remained within normal range during follow up (unpublished data). However, data on the predictive value of these biomarkers in MLD remain limited. As demyelinating neuropathy and subtle gallbladder abnormalities could be detected in infants of all subtypes, these are considered unlikely predictors for central disease onset (Supplement, Case reports).\u003c/p\u003e \u003cp\u003eThese findings highlight the need for complementary markers and longitudinal datasets to refine risk prediction in presymptomatic infants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Early Clinical Management and Treatment Feasibility\u003c/h2\u003e \u003cp\u003eEarly clinical outcomes following presymptomatic identification were encouraging but must be interpreted with caution given the short follow-up duration. Infants who underwent gene therapy during the presymptomatic period demonstrated preserved neurological examinations and age-appropriate developmental trajectories in all but one child during early follow-up, consistent with findings from controlled clinical trials.\u003csup\u003e29\u003c/sup\u003e Treatment delivery within the first year of life was feasible with manageable peri-transplant complications and no unexpected safety signals. However, follow-up remains limited in duration, and subtle abnormalities - including peripheral neuropathy and mild motor delay in one case - underscore that presymptomatic treatment does not necessarily prevent all disease manifestations.\u003csup\u003e30\u003c/sup\u003e Infants managed through surveillance for predicted late-onset disease have remained clinically asymptomatic, supporting the short-term safety of a monitoring-based approach when guided by consensus recommendations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Monitoring Challenges and Unmet Needs\u003c/h2\u003e \u003cp\u003eMonitoring presymptomatic infants identified through NBS remains a major challenge. While urinary sulfatides are indispensable for diagnosis, their longitudinal utility for monitoring is limited by biological and pre-analytical variability. Plasma biomarkers such as neurofilament light chain and glial fibrillary acidic protein show promise as indicators of early neuroaxonal injury, but age-specific reference ranges and validated thresholds for clinical decision-making are lacking.\u003csup\u003e31\u003c/sup\u003e These biomarkers do not reliably distinguish between central and peripheral demyelination. This distinction is clinically important for treatment timing and post-treatment monitoring, as current therapies primarily affect central nervous system disease, whereas peripheral demyelination often continues.\u003csup\u003e30\u003c/sup\u003e Conventional neuroimaging was largely unremarkable during early follow-up and showed in this presymptomatic stage no correlation with predicted disease subtype. Although it was particularly pronounced in late infantile cases, peripheral demyelinating neuropathy could be detected in all subtypes within the first year of life and was not halted by HSPC-GT. These findings highlight the current absence of sensitive and reliable tools to detect early subclinical progression, particularly in infants predicted to develop late-onset disease. Longitudinal studies with larger cohorts will be essential to define biomarker trajectories, refine monitoring strategies, and optimize the timing of therapeutic intervention.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e4.6\u003c/em\u003e Public health implications\u003c/h2\u003e \u003cp\u003eBeyond individual clinical outcomes, these findings have important public health implications. The biochemical-first screening approach integrates into existing NBS laboratories and provides equitable detection across diverse populations, independent of genetic ancestry. The demonstrated feasibility of downstream care pathways from confirmatory diagnostics to specialist referral and treatment addresses a key requirement for population screening programs: that a positive NBS result leads to timely and meaningful clinical action. While broader implementation will require health-economic and policy considerations specific to each system, this pilot provides evidence that cross-border NBS-enabled care for MLD, as was done in Germany and Austria, is achievable. The timing of these findings is particularly relevant in light of recent policy developments. MLD has now been accepted for inclusion in the Recommended Uniform Screening Panel (RUSP) in the United States as well as into national screening programs in Norway and Sweden, reflecting growing international consensus on the benefit of early detection through NBS. A central concern during such policy deliberations is whether screening can be translated into timely, effective care without undue harm. The present study directly addresses this by demonstrating that NBS for MLD can be operationalized within nationwide healthcare systems and linked to meaningful early clinical outcomes.\u003c/p\u003e \u003cp\u003eA recent perspective article highlighted the challenges of early detection in late-onset disease.\u003csup\u003e19\u003c/sup\u003e In MLD, it is important to emphasize that there is no \u0026ldquo;late mild\u0026rdquo; phenotype; rather, current evidence shows that even late-onset MLD, with symptom onset ranging from school age to adulthood, is associated with severe and clinically meaningful disease burden. Nevertheless, health surveillance and decisions regarding the timing of intervention in late-onset MLD should consider the particular challenges faced by identified individuals and their families and should incorporate as well as further develop psychosocial support strategies for this unique context.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Strengths and Limitations\u003c/h2\u003e \u003cp\u003eThis work reports prospective real-world outcomes following population-based NBS for MLD, encompassing the entire pathway from population screening and confirmatory diagnostics to phenotype-based treatment stratification and early clinical follow-up. Key strengths include the large, screened population, the prospective design, the integration of standardized, consensus-based care pathways, and the linkage of screening results to real-world clinical decision-making and early outcomes across two national healthcare systems.\u003c/p\u003e \u003cp\u003eThis study also has important limitations. First, the workflow during these pilot programs did not reflect that expected once MLD NBS is fully implemented in the public health environment. For example, the time to obtain definitive positive screen results and communicate them to the QTC T\u0026uuml;bingen was longer and more variable than desired, based on the stage of development of the algorithm. Second, the number of affected infants is small, reflecting the rarity of MLD, which limits statistical precision and precludes formal comparisons between management strategies. Third, follow-up duration remains limited, particularly for those under surveillance for late-onset disease; consequently, long-term neurological, cognitive, and quality-of-life outcomes cannot yet be assessed. Fourth, phenotype prediction relied primarily on genotype-based criteria and residual enzyme activity, which, although supported by expert consensus, do not fully capture intrafamilial and individual variability. Finally, these findings originate from well-resourced healthcare systems with established specialist networks, which may limit generalizability to settings with fewer resources or differing care structures.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThese German and Austrian pilot programs demonstrate that NBS for MLD can be implemented within existing healthcare infrastructures and linked to standardized diagnostic and clinical care pathways. A three-tier screening algorithm enabled early identification of affected infants and timely clinical stratification, supporting presymptomatic intervention for early-onset disease and structured surveillance for late-onset phenotypes. During early follow-up, infants managed through these pathways showed preserved neurological and developmental status. Together, these findings provide real-world evidence that NBS-enabled care for MLD is feasible and clinically actionable. Ongoing follow-up and broader implementation studies will be essential to define long-term outcomes and inform future screening policy decisions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eACMG\u003c/b\u003e American College of Medical Genetics and Genomics\u003c/p\u003e\u003cp\u003e\u003cb\u003eARSA\u003c/b\u003e Arylsulfatase A\u003c/p\u003e\u003cp\u003e\u003cb\u003eCNS\u003c/b\u003e Central nervous system\u003c/p\u003e\u003cp\u003e\u003cb\u003eDBS\u003c/b\u003e Dried blood spot\u003c/p\u003e\u003cp\u003e\u003cb\u003eEJ\u003c/b\u003e Early juvenile\u003c/p\u003e\u003cp\u003e\u003cb\u003eEMA\u003c/b\u003e European Medicines Agency\u003c/p\u003e\u003cp\u003e\u003cb\u003eHSPC-GT\u003c/b\u003e Haematopoietic stem and progenitor cell gene therapy\u003c/p\u003e\u003cp\u003e\u003cb\u003eHSCT\u003c/b\u003e Haematopoietic stem cell transplantation\u003c/p\u003e\u003cp\u003e\u003cb\u003eLI\u003c/b\u003e Late infantile\u003c/p\u003e\u003cp\u003e\u003cb\u003eLJ\u003c/b\u003e Late juvenile\u003c/p\u003e\u003cp\u003e\u003cb\u003eLS-MS/MS\u003c/b\u003e Liquid chromatography\u0026ndash;tandem mass spectrometry\u003c/p\u003e\u003cp\u003e\u003cb\u003eMLD\u003c/b\u003e Metachromatic leukodystrophy\u003c/p\u003e\u003cp\u003e\u003cb\u003eMLDi\u003c/b\u003e Metachromatic Leukodystrophy Initiative\u003c/p\u003e\u003cp\u003e\u003cb\u003eMRI\u003c/b\u003e Magnetic resonance imaging\u003c/p\u003e\u003cp\u003e\u003cb\u003eMSD\u003c/b\u003e Multiple sulfatase deficiency\u003c/p\u003e\u003cp\u003e\u003cb\u003eNBS\u003c/b\u003e Newborn screening\u003c/p\u003e\u003cp\u003e\u003cb\u003eNCV\u003c/b\u003e Nerve conduction velocity\u003c/p\u003e\u003cp\u003e\u003cb\u003eNGS\u003c/b\u003e Next-generation sequencing\u003c/p\u003e\u003cp\u003e\u003cb\u003epPTV\u003c/b\u003e Predicted protein-truncating variant\u003c/p\u003e\u003cp\u003e\u003cb\u003ePSAP\u003c/b\u003e Prosaposin gene\u003c/p\u003e\u003cp\u003e\u003cb\u003eQTC\u003c/b\u003e Qualified treatment center\u003c/p\u003e\u003cp\u003e\u003cb\u003eRUSP\u003c/b\u003e Recommended Uniform Screening Panel\u003c/p\u003e\u003cp\u003e\u003cb\u003eSUMF1\u003c/b\u003e Sulfatase-modifying factor 1 gene\u003c/p\u003e\u003cp\u003e\u003cb\u003eVUS\u003c/b\u003e Variant of uncertain significance\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Information:\u003c/h2\u003e\n\u003cp\u003eMLD NBS studies led by D.K. and N.J. were carried out with support to Archimed\u003cem\u003elife\u003c/em\u003e and the Hannover NBS laboratory from Orchard Therapeutics, a Kyowa Kirin company.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Interests:\u003c/h2\u003e\n\u003cp\u003eD.K., S.H. ands P.O. are employees of Archimed\u003cem\u003elife.\u003c/em\u003e L.L. and S.G. received institutional research support by Orchard Therapeutics, unrelated to this study. Conflicts of Interest: All other co-authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, L.L.,S.G., H.R.; data curation, L.L., A.C.J., P.J. M.W., T.L.; investigation, L.L., M.W., S.H., S.H., N.H., P.O., A.C.J., T.L.,V.K., M.Z.,J.K., N.K.; methodology, L.L., M.W., S.H., S.H., N.J. , D.K., P.J.; supervision, S.G.,H.R.; validation, L.L.,S.G.; visualization, P.J.; L.L.; writing \u0026ndash; original draft, S.G., L.L.; writing \u0026ndash; review and editing, L.L, S.G., S.H., S.H. H.R., B.P.,L.N., C.K. D.K. All authors have read and agreed to the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eAcknowledgments: We extend our deepest gratitude to the members of the MLD NBS Alliance and the MLDi for their pivotal contributions to the generation of crucial evidence and consensus guidelines. We thank Michael Gelb for reviewing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGieselmann, V. \u0026amp; Kr\u0026auml;geloh-Mann, I. Metachromatic Leukodystrophy. in \u003cem\u003eThe Online Metabolic and Molecular Bases of Inherited Disease\u003c/em\u003e (eds. Valle, D.L., Antonarakis, S., Ballabio, A., Beaudet, A.L. \u0026amp; Mitchell, G.A.) (McGraw-Hill Education, New York, NY, 2019).\u003c/li\u003e\n\u003cli\u003eAsbreuk, M.\u003cem\u003e, et al.\u003c/em\u003e Metachromatic Leukodystrophy: New Therapy Advancements and Emerging Research Directions. \u003cem\u003eNeurology\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, e213817 (2025).\u003c/li\u003e\n\u003cli\u003eChang, S.C., Bergamasco, A., Bonnin, M., Bison\u0026oacute;, T.A. \u0026amp; Moride, Y. 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Vol. 2025.\u003c/li\u003e\n\u003cli\u003eElg\u0026uuml;n, S.\u003cem\u003e, et al.\u003c/em\u003e Phenotypic variation between siblings with Metachromatic Leukodystrophy. \u003cem\u003eOrphanet journal of rare diseases\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 136-136 (2019).\u003c/li\u003e\n\u003cli\u003eFumagalli, F.\u003cem\u003e, et al.\u003c/em\u003e Long-term effects of atidarsagene autotemcel for metachromatic leukodystrophy. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e392\u003c/strong\u003e, 1609\u0026ndash;1620 (2025).\u003c/li\u003e\n\u003cli\u003eZambon, A.A.\u003cem\u003e, et al.\u003c/em\u003e Effects of atidarsagene autotemcel gene therapy on peripheral nerves in late-infantile metachromatic leukodystrophy. \u003cem\u003eBrain\u003c/em\u003e (2025).\u003c/li\u003e\n\u003cli\u003eBeerepoot, S.\u003cem\u003e, et al.\u003c/em\u003e Neurofilament light chain and glial fibrillary acidic protein levels in metachromatic leukodystrophy. \u003cem\u003eBrain\u003c/em\u003e (2021).\u003c/li\u003e\n\u003cli\u003eLaugwitz, L.\u003cem\u003e, et al.\u003c/em\u003e Newborn Screening and Presymptomatic Treatment of Metachromatic Leukodystrophy. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e391\u003c/strong\u003e, 1256-1258 (2024).\u003c/li\u003e\n\u003cli\u003eTrinidad, M.\u003cem\u003e, et al.\u003c/em\u003e Predicting disease severity in metachromatic leukodystrophy using protein activity and a patient phenotype matrix. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 172 (2023).\u003c/li\u003e\n\u003cli\u003eGroeschel, S.\u003cem\u003e, et al.\u003c/em\u003e Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Patients With Juvenile Metachromatic Leukodystrophy Compared With Nontransplanted Control Patients. \u003cem\u003eJAMA Neurol\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 1133-1140 (2016).\u003c/li\u003e\n\u003cli\u003eWilson, J.M.G., Jungner, G. \u0026amp; Organization, W.H. 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Exploring the net monetary benefit of implementing newborn screening for metachromatic leukodystrophy in California. \u003cem\u003eMol Genet Metab\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 108651 (2025).\u003c/li\u003e\n\u003cli\u003eBean, K.\u003cem\u003e, et al.\u003c/em\u003e Exploring the Cost-Effectiveness of Newborn Screening for Metachromatic Leukodystrophy (MLD) in the UK. \u003cem\u003eInt J Neonatal Screen\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e(2024).\u003c/li\u003e\n\u003cli\u003eBean, K., Gelb, M.H., Adang, L.A., Chanson, C. \u0026amp; Pang, F. Cost-effectiveness framework by tandem mass spectrometry (TMS) for newborn screening of metachromatic leukodystrophy (MLD) in the United States (US). \u003cem\u003eMol Genet Metab\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 107769 (2024).\u003c/li\u003e\n\u003cli\u003eKehrer, C.\u003cem\u003e, et al.\u003c/em\u003e Healthcare utilization and disease burden in children with metachromatic leukodystrophy in Germany. \u003cem\u003eOrphanet J Rare Dis\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 242 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"metachromatic leukodystrophy, newborn screening, autologous hematopoietic stem and progenitor cell gene therapy, lysosomal storage diseas","lastPublishedDoi":"10.21203/rs.3.rs-9496450/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9496450/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMetachromatic leukodystrophy (MLD) is a rapidly progressive leukodystrophy that leads to severe disability and early death if untreated. Autologous hematopoietic stem and progenitor cell gene therapy (HSPC-GT, atidarsagene autotemcel, arsa-cel) for early onset subtypes and allogenic hematopoietic stem cell transplantation (HSCT) for late onset disease substantially alters disease progression for early onset disease when administered before symptom onset, creating a strong rationale for newborn screening (NBS). At the same time, NBS technique for MLD in dried blood spots has recently been demonstrated to be robust and highly accurate. The aim was to give real-world results from the world\u0026rsquo;s first NBS pilots for clinical management and treatment of identified children.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBetween September 2021 and July 2025, 359,282 newborns underwent NBS for MLD in two different laboratories in Germany and Austria using a three-tier algorithm integrating sulfatide quantification, arylsulfatase A (ARSA) activity measurement, and \u003cem\u003eARSA\u003c/em\u003e sequencing. Screen-positive infants underwent a predefined care pathway including standardized confirmatory diagnostics, genotype-based and biochemical prediction of disease onset, clinical assessment and management guiding early treatment and surveillance at the qualified treatment center (QTC) in T\u0026uuml;bingen.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNine newborns screened positive and all were confirmed to have MLD (detection rate approximately 1 per 40,000). Based on genotype and leukocyte ARSA enzyme activity, disease onset prediction was possible in all of them. Seven infants were classified as having pre-symptomatic early-onset MLD and were referred for HSPC-GT. All treated infants showed preserved neurological function at follow-up 30 months after treatment. Two infants predicted to develop late-onset MLD entered structured surveillance for treatment with HSCT and have remained clinically stable. No false-positive or known false-negative results were observed.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese results from our pilot programs demonstrate that NBS enables reliable early identification of MLD and support streamlined care pathways leading to timely intervention. Importantly, this study provides real-world evidence illustrating that NBS for MLD can enable timely, pre-symptomatic treatment and structured surveillance within standard national healthcare systems. These findings further substantiate the value of NBS for MLD at a critical moment as several countries consider national implementation of MLD screening.\u003c/p\u003e","manuscriptTitle":"Newborn Screening for Metachromatic Leukodystrophy: Care Pathway and Early Clinical Outcomes from German and Austrian Pilot Programs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 13:14:15","doi":"10.21203/rs.3.rs-9496450/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d44d4102-651d-4e47-9bd1-4cbff772b7b1","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T13:14:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 13:14:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9496450","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9496450","identity":"rs-9496450","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}