Infantile Krabbe disease (IKD) is a rapidly progressive leukodystrophy for which hematopoietic stem cell transplantation (HCT) offers the only disease-modifying treatment. HCT must occur presymptomatically, ideally by 30 days of life, to optimize survival and neurodevelopmental outcomes. With the expansion of newborn screening for Krabbe disease (KD), pediatric neurologists and geneticists will be responsible for obtaining urgent diagnostic testing and connecting families to experts for information and treatment. Given the variability in state-specific screening protocols, clinicians must be familiar with their local algorithms to act quickly on receiving abnormal results and refer to nearby, specialty treatment centers. Informed consent discussions must clearly outline the natural history of untreated IKD, the requirement for rapid, presymptomatic treatment, and provide a realistic view of the benefits and limitations of HCT. Supportive and palliative care should be prioritized regardless of treatment choice. This publication provides expert guidance on interpreting newborn screening results, conducting rapid confirmatory testing, and referring to and/or establishing treatment centers ready to care for IKD. With anticipatory planning and interdisciplinary collaboration, pediatric specialists can provide accurate diagnosis, compassionate counseling, and expedited evaluation to meet the narrow window for effective HCT.

Newborn screening (NBS) for X-linked adrenoleukodystrophy (ALD) enables early identification of boys at risk for adrenal insufficiency (AI) and cerebral ALD (CALD). However, NBS frequently identifies ABCD1 variants of uncertain significance (VUS), which are associated with only borderline-elevated C26:0-lysophosphatidylcholine (LPC(26:0)) levels. Traditional American College of Medical Genetics and Genomics (ACMG) pathogenicity classification does not account for age-dependent penetrance or the broader phenotypic spectrum, complicating risk assessment and clinical management. Through the Grey Zone Project, we developed a risk-stratification framework using a receiver operating characteristic (ROC)-based approach prioritizing 95% sensitivity. This framework incorporates biochemical and longitudinal clinical data from 1627 control subjects and 196 confirmed ALD patients. Three pediatric risk categories were defined: "no ALD" (<110 nmol/L LPC(26:0)), "lower-risk AI/CALD" (110-177 nmol/L), and "at-risk AI/CALD" (>177 nmol/L). When applied to 108 samples carrying 51 unique ABCD1 VUSs, 26 variants were reclassified as "no ALD," 15 as "lower-risk AI/CALD," and 10 as "at-risk AI/CALD." The framework reclassifies ABCD1 variants based on biochemical risk profiles, reducing false-positive referrals, avoiding unnecessary MRI surveillance, and alleviating parental anxiety by identifying children who are unlikely to develop childhood-onset disease. Integrating biochemical thresholds with genetic and longitudinal clinical data improves the specificity of NBS without compromising its sensitivity. Providing systematic feedback on false-positive cases to screening laboratories will further refine cut-offs. This framework provides a scalable, evidence-based model for interpreting variants and enabling personalized follow-up in ALD and other disorders with a variable age of onset.

BACKGROUND AND OBJECTIVES:Cerebral adrenoleukodystrophy (CALD) is a common manifestation of adrenoleukodystrophy (ALD) in men. Early detection of CALD lesions through MRI screening is critical to allow for therapeutic action preventing severe disability and death. While the frequency of brain MRI monitoring has been addressed by international recommendations, no consensus currently exists regarding which MRI sequences should be used in a real-world setting for screening and follow-up of CALD lesions. The aim of this study was to establish guidelines for the MRI protocol in clinical practice and to identify priority sequences for research use, thereby promoting intercenter harmonization. METHODS:A modified Delphi procedure was used to achieve consensus on MRI protocols for ALD screening, lesion monitoring, and research applications among experts with experience in brain imaging in ALD. Questionnaires allowed experts to indicate whether they considered sequences as core, optional, or research, or to express agreement (5-point scale ranging from completely disagree to completely agree) with specific statements. Topics where no agreement was reached were discussed during online consensus meetings. RESULTS:Thirty experts from 9 countries participated and agreed that the core screening protocol for ALD in adults and children should include at least 3D T1-weighted, spin-echo T2-weighted, 3D fluid-attenuated inversion recovery, and diffusion-weighted imaging (DWI). Postcontrast T1-weighted imaging should be performed systematically in specific clinical scenarios. Experts supported using DWI alongside the Loes score and postcontrast imaging to assess lesion progression. A research protocol was defined, prioritizing diffusion tensor imaging, MR perfusion, and quantitative volumetric analyses. DISCUSSION:This international project harmonizes the ALD MRI protocol, thus offering a practical framework to screen and monitor lesions, which will improve clinical decision making. It also identifies MRI sequences that should be prioritized in future research. Future research on MRI in ALD should focus on topics where no consensus has yet been reached in this project.

Background and ObjectivesCerebral adrenoleukodystrophy (CALD) is a common manifestation of adrenoleukodystrophy (ALD) in men. Early detection of CALD lesions through MRI screening is critical to allow for therapeutic action preventing severe disability and death. While the frequency of brain MRI monitoring has been addressed by international recommendations, no consensus currently exists regarding which MRI sequences should be used in a real-world setting for screening and follow-up of CALD lesions. The aim of this study was to establish guidelines for the MRI protocol in clinical practice and to identify priority sequences for research use, thereby promoting intercenter harmonization.MethodsA modified Delphi procedure was used to achieve consensus on MRI protocols for ALD screening, lesion monitoring, and research applications among experts with experience in brain imaging in ALD. Questionnaires allowed experts to indicate whether they considered sequences as core, optional, or research, or to express agreement (5-point scale ranging from completely disagree to completely agree) with specific statements. Topics where no agreement was reached were discussed during online consensus meetings.ResultsThirty experts from 9 countries participated and agreed that the core screening protocol for ALD in adults and children should include at least 3D T1-weighted, spin-echo T2-weighted, 3D fluid-attenuated inversion recovery, and diffusion-weighted imaging (DWI). Postcontrast T1-weighted imaging should be performed systematically in specific clinical scenarios. Experts supported using DWI alongside the Loes score and postcontrast imaging to assess lesion progression. A research protocol was defined, prioritizing diffusion tensor imaging, MR perfusion, and quantitative volumetric analyses.DiscussionThis international project harmonizes the ALD MRI protocol, thus offering a practical framework to screen and monitor lesions, which will improve clinical decision making. It also identifies MRI sequences that should be prioritized in future research. Future research on MRI in ALD should focus on topics where no consensus has yet been reached in this project.

BACKGROUND:Seizures and epilepsy in leukodystrophies are poorly understood. This multisite study aimed to capture the prevalence of seizures in a nationwide cohort of patients, identify factors associated with increased seizure and epilepsy risk, and quantify prevalence of refractory seizures. METHODS:Retrospective data was extracted from leukodystrophy clinics across eight pediatric health centers from January 1, 2013, to March 31, 2020. Patients 18 years old and younger and diagnosed with leukodystrophy, including those identified by newborn screening, were eligible. Epilepsy was defined as two or more unprovoked seizures. Clinical variables including patient age, diagnosis, imaging features (binary: normal vs abnormal), electroencephalogram (EEG) findings, the presence/absence, and diagnoses of seizures/epilepsy were obtained. RESULTS:Data from 704 patients were analyzed. At least one seizure occurred in 29% of all patients. Epilepsy developed in 80% of patients with at least one seizure. EEG abnormalities were present in 84% of patients with seizures versus 54% without (P < 0.001). In a multivariate analysis of seizure risk, higher increased likelihoods were associated with every one-year increase in age (1.13 times higher; 95% confidence interval [CI]: [1.07, 1.19], P < 0.001), generalized epileptiform discharges (13.4 times higher; 95% CI: [2.22, 259], P = 0.019), focal and multifocal epileptiform discharges (3.56 times higher; 95% CI: [2.04, 6.36], P < 0.001), and focal slowing (3.57 times higher; 95% CI: [1.58, 8.58], P = 0.003). Diffuse slowing was not significantly associated with seizure in the multivariate analysis (odds ratio 1.45, 95% CI: [0.85, 2.45], P = 0.170). In a comparison of different pathological classifications, seizures, but not epilepsy, were more common in astrocytopathies compared to myelinopathies (seizures 41% vs 28%, P = 0.046; epilepsy 89% vs 79%, P = 0.376). Two or more antiseizure medications were required to control seizures in multiple leukodystrophies. CONCLUSIONS:Age, focal slowing, and epileptiform discharges, but not generalized slowing or abnormal brain magnetic resonance imaging, were significant predictors for seizure in this population. Patients with generalized discharges have a higher risk of seizures compared to those with focal discharges. Seizure burden, age at first seizure, number of antiseizure medications needed, and presence of EEG abnormalities vary based on underlying leukodystrophy. Our findings suggest the need for targeted strategies for monitoring seizures and epilepsy in the leukodystrophies.

Background and ObjectivesX-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy with an incidence of 1:15,000 births. Newborn screening has enabled widespread use of serial brain MRI scans for early detection and treatment of cerebral ALD (cALD). Differentiating early cALD lesions from incidental findings remains challenging. We sought to characterize incidental MRI findings during cALD surveillance and describe features that may help distinguish them from early cALD lesions.MethodsMRI reports from boys with ALD followed at the Lucile Packard Children's Hospital and Weill Cornell Medicine from 2012 to 2023 were reviewed. Inclusion criteria were boys with ALD who had at least 1 presymptomatic MRI before the age of 12 and no neurologic symptoms. We analyzed narrative content of available MRI reports, manually verified definite cALD lesions, and classified all other findings as incidental.ResultsOur study included 69 boys with ALD (median age of 6.9 years, interquartile range 4.0-9.5),, with a total of 390 MRI reports reviewed. Participants had either pathogenic ABCD1 variants (45%, n = 31), likely pathogenic variants (9%, n = 6), variants of uncertain significance (39%, n = 27), or a positive family history with confirmatory biochemical testing (7%, n = 5). During surveillance, 11% (n = 8) developed cALD lesions with classical cALD neuroimaging features. Incidental findings were common (74%, n = 51). The most frequent incidental findings included punctate T2/fluid-attenuated inversion recovery (FLAIR) hyperintensities in the frontal lobe (29%, n = 20), abnormal T2/FLAIR signal in the periatrial or periventricular white matter (19%, n = 13), prominent perivascular spaces (17%, n = 12), T2/FLAIR hyperintensities in non-frontal lobe regions (14%, n = 10), delayed myelination (14%, n = 10), and stable curvilinear T2/FLAIR hyperintensity in the splenium or genu (13%, n = 9), with the latter requiring follow-up imaging to distinguish from cALD. Only 23% (n = 16) had neither cALD nor incidental findings reported across all time points. Incidental findings were neither statistically associated with genotype classification nor predictive of cALD.DiscussionIncidental findings were commonly reported during cALD surveillance, possibly reflecting radiologists' intent to differentiate them from cALD. Incidental findings were distinguishable from true cALD lesions based on anatomic location, stability over time, and persistent absence of contrast enhancement. Our findings provide a practical framework for differentiating incidental findings from cALD lesions during surveillance.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients’ SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3–/–) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

A 7-month-old boy presented to our clinic with developmental delay, Magnetic Resonance Imaging (MRI) features of delayed myelination and diffusion restriction, and a homozygous variant of uncertain significance (c.4T>G, p.Phe2Val) in HIKESHI, a gene associated with autosomal-recessive hypomyelinating leukodystrophy 13. We hypothesized that the variant is disease-causing and aimed to rescue the cellular phenotype with vector-mediated gene replacement. HIKESHI mediates heat-induced nuclear accumulation of heat-shock proteins, including HSP70, to protect cells from stress. We generated skin fibroblasts from the proband and proband's mother (heterozygous) to compare protein expression and subcellular localization of HSP70 under heat stress conditions, and the effect of vector-mediated overexpression of HIKESHI in the proband's cells under the same heat stress conditions. Western blot analysis revealed absent HIKESHI protein from proband fibroblasts, contrasted with ample expression in parental cells. Under heat stress conditions, while the mother's cells displayed appropriate nuclear localization of HSP70, the proband's cells displayed impaired nuclear translocalization. When patient fibroblasts were provided exogenous HIKESHI, the transfected proband's cells showed restored heat-induced nuclear translocalization of HSP70 under conditions of heat stress. These functional data establish that the patient's variant is a pathogenic loss-of-function mutation, thus confirming a diagnosis of hypomyelinating leukodystrophy 13 and that vector-mediated gene replacement may be an effective treatment approach for patients with this disorder.

Identify seizure risk factors and quantify the efficacy of anti-seizure medications in patients with leukodystrophy

Metachromatic leukodystrophy (MLD) is a fatal, progressive neurodegenerative disorder caused by biallelic pathogenic mutations in the ARSA (Arylsulfatase A) gene. With the advent of presymptomatic diagnosis and the availability of therapies with a narrow window for intervention, it is critical to define a standardized approach to diagnosis, presymptomatic monitoring, and clinical care. To meet the needs of the MLD community, a panel of MLD experts was established to develop disease-specific guidelines based on healthcare resources in the United States. This group developed a consensus opinion for best-practice recommendations, as follows: (i) Diagnosis should include both genetic and biochemical testing; (ii) Early diagnosis and treatment for MLD is associated with improved clinical outcomes; (iii) The panel supported the development of newborn screening to accelerate the time to diagnosis and treatment; (iv) Clinical management of MLD should include specialists familiar with the disease who are able to follow patients longitudinally; (v) In early onset MLD, including late infantile and early juvenile subtypes, ex vivo gene therapy should be considered for presymptomatic patients where available; (vi) In late-onset MLD, including late juvenile and adult subtypes, hematopoietic cell transplant (HCT) should be considered for patients with no or minimal disease involvement. This document summarizes current guidance on the presymptomatic monitoring of children affected by MLD as well as the clinical management of symptomatic patients. Future data-driven evidence and evolution of these recommendations will be important to stratify clinical treatment options and improve clinical care.

Background X-linked adrenoleukodystrophy (X-ALD) is highly variable, ranging from slowly progressive adreno-myeloneuropathy to severe brain demyelination and inflammation (cerebral ALD, CALD) affecting males with childhood peak onset. Risk models integrating blood-based biomarkers to indicate CALD onset, enabling timely interventions, are lacking. Therefore, we evaluated the prognostic value of blood biomarkers in addition to current neuroimaging predictors for early detection of CALD.Methods We measured blood biomarkers in a retrospective, male CALD risk-assessment cohort consisting of 134 X-ALD patients and 66 controls and in a phenotype-blinded validation set (25 X-ALD boys, 4-13 years) using Simoa (R) and Luminex (R) technologies.Findings Among 25 biomarkers indicating axonal damage, astrocye/microglia activation, or immune-cell recruitment, neurofilament light chain (NfL) had the highest prognostic value for early indication of childhood/adolescent CALD. A plasma NfL cut-off level of 8.33 pg/mL, determined in the assessment cohort, correctly discriminated CALD with an accuracy of 96% [95% CI: 80-100] in the validation group. Multivariable logistic regression models revealed that combining NfL with GFAP or cytokines/chemokines (IL-15, IL-12p40, CXCL8, CCL11, CCL22, and IL-4) that were significantly elevated in CALD vs healthy controls had no additional benefit for detecting neuroinflammation. Some cytokines/chemokines were elevated only in childhood/adolescent CALD and already upregulated in eBioMedicineasymptomatic X-ALD children (IL-15, IL-12p40, and CCL7). In adults, NfL levels distinguished CALD but were lower than in childhood/adolescent CALD patients with similar (MRI) lesion severity. Blood GFAP did not differentiate CALD from non-inflammatory X-ALD.Interpretation Biomarker-based risk prediction with a plasma NfL cut-off value of 8.33 pg/mL, determined by ROC analysis, indicates CALD onset with high sensitivity and specificity in childhood X-ALD patients. A specific pro -inflammatory cytokine/chemokine profile in asymptomatic X-ALD boys may indicate a primed, immanent inflammatory state aligning with peak onset of CALD. Age-related differences in biomarker levels in adult vs childhood CALD patients warrants caution in predicting onset and progression of CALD in adults. Further evaluations are needed to assess clinical utility of the NfL cut-off for risk prognosis of CALD onset.

Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls.ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes.We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10–30 kDa ACE effector which is deficient in Gaucher spleen.The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease.

Brief summary: This paper describes consensus expert recommendations for the diagnosis and management of patients with Adrenoleukodystrophy.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease 1 – 7 , evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA 8 , 9 and binds to guanosine 10 – 12 . We identified a de novo, previously undescribed missense TLR7 Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7 Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP 10 – 12 , and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c + age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7 Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

BACKGROUND AND OBJECTIVES:We sought to characterize the natural history and standard-of-care practices between the radiologic appearance of brain lesions, the appearance of lesional enhancement, and treatment with hematopoietic stem-cell transplant or gene therapy among boys diagnosed with presymptomatic childhood-onset cerebral adrenoleukodystrophy (CCALD). METHODS:We analyzed a multicenter, mixed retrospective/prospective cohort of patients diagnosed with presymptomatic CCALD (Neurologic Function Score = 0, Loes Score [LS] = 0.5-9.0, and age <13 years). Two time-to-event survival analyses were conducted: (1) time from CCALD lesion onset-to-lesional enhancement and (2) time from enhancement-to-treatment. The analysis was repeated in the subset of patients with (1) the earliest evidence of CCALD, defined as an MRI LS ≤ 1, and (2) patients diagnosed between 2016 and 2021. RESULTS:Seventy-one boys were diagnosed with presymptomatic cerebral lesions at a median age of 6.4 years [2.4-12.1] with a LS of 1.5 [0.5-9.0]. Fifty percent of patients had lesional enhancement at diagnosis. In the remaining 50%, the median Kaplan-Meier (KM)-estimate of time from diagnosis-to-lesional enhancement was 6.0 months (95% CI 3.6-17.8). The median KM-estimate of time from enhancement-to-treatment is 3.8 months (95% CI 2.8-5.9); 2 patients (4.2%) developed symptoms before treatment. Patients with a diagnostic LS ≤ 1 were younger (5.8 years [2.4-11.5]), had a time-to-enhancement of 4.7 months (95% CI 2.7-9.30), and were treated in 3.8 months (95% CI 3.1-7.1); no patients developed symptoms before treatment. Time from CCALD diagnosis-to-treatment decreased over the course of the study (ρ = -0.401, p = 0.003). DISCUSSION:Our findings offer a more refined understanding of the timing of lesion formation, enhancement, and treatment among boys with presymptomatic CCALD. These data offer benchmarks for standardizing clinical care and designing future clinical trials.