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X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder resulting from pathogenic variants in the ABCD1 gene that primarily affects the nervous system and is characterized by progressive axonal degeneration in the spinal cord and peripheral nerves and leukodystrophy. Dysfunction of peroxisomal very long-chain fatty acid (VLCFA) degradation has been implicated in ALD pathology, but the impact on astrocytes, which critically support neuronal function, remains poorly understood. Fibroblasts from four ALD patients were reprogrammed to generate human-induced pluripotent stem cells (hiPSC). hiPSC-derived astrocytes were generated to study the impact of ALD on astrocytic fatty acid homeostasis. Our study reveals significant changes in the lipidome of ALD hiPSC-derived astrocytes, characterized by an enrichment of VLCFAs across multiple lipid classes, including triacylglycerols, cholesteryl esters, and phosphatidylcholines. Importantly, ALD hiPSC-derived astrocytes not only exhibit intrinsic lipid dysregulation but also affect the dendritic tree complexity of neurons in co-culture systems. These findings highlight the cell-autonomous effects of pathogenic variants in the ABCD1 protein on astrocytes and their microenvironment, shed light on potential mechanisms underlying ALD neuropathology, and underscore the critical role of astrocytes in neuronal health., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Lipids play pivotal roles in an extensive range of metabolic and physiological processes. In recent years, the convergence of trapped ion mobility spectrometry and MS has enabled 4D-lipidomics, a highly promising technology for comprehensive lipid analysis. 4D-lipidomics assesses lipid annotations across four distinct dimensions-retention time, collisional cross section, m/z (mass-to-charge ratio), and MS/MS spectra-providing a heightened level of confidence in lipid annotation. These advantages prove particularly valuable when investigating complex disorders involving lipid metabolism, such as adrenoleukodystrophy (ALD). ALD is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) due to pathogenic variants in the ABCD1 gene. A comprehensive 4D-lipidomics strategy of ALD fibroblasts demonstrated significant elevations of various lipids from multiple classes. This indicates that the changes observed in ALD are not confined to a single lipid class and likely impacts a broad spectrum of lipid-mediated physiological processes. Our findings highlight the incorporation of mainly saturated and monounsaturated VLCFA variants into a range of lipid classes, encompassing phosphatidylcholines, triacylglycerols, and cholesterol esters. These include ultra-long-chain fatty acids with a length of up to thirty carbon atoms. Lipid species containing C26:0 and C26:1 were the most frequently detected VLCFA lipids in our study. Furthermore, we report a panel of 121 new candidate biomarkers in fibroblasts, exhibiting significant differentiation between controls and individuals with ALD. In summary, this study demonstrates the capabilities of a 4D-lipid profiling workflow in unraveling novel insights into the intricate lipid modifications associated with metabolic disorders like ALD., Competing Interests: Conflict of interest Sven W. Meyer is employed at Bruker Daltonics GmbH. All the other authors declarethat they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity., (© 2022. The Author(s).)

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1 . Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1 , were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D 3 -C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D 3 -C16:0/D 3 -C22:0 ratio in the D 3 -C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.

X-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy with a birth incidence of 1:14,700 live births. The disease is caused by mutations in ABCD1 and characterized by very long-chain fatty acids (VLCFA) accumulation. In childhood, male patients are at high-risk to develop adrenal insufficiency and/or cerebral demyelination. Timely diagnosis is essential. Untreated adrenal insufficiency can be life-threatening and hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. For this reason, ALD is being added to an increasing number of newborn screening programs. ALD newborn screening involves the quantification of C26:0-lysoPC in dried blood spots which requires a dedicated method. C26:0-carnitine, that was recently identified as a potential new biomarker for ALD, has the advantage that it can be added as one more analyte to the routine analysis of amino acids and acylcarnitines already in use. The first objective of this study was a comparison of the sensitivity of C26:0-carnitine and C26:0-lysoPC in dried blood spots from control and ALD newborns both in a case-control study and in newborns included in the New York State screening program. While C26:0-lysoPC was elevated in all ALD newborns, C26:0-carnitine was elevated only in 83%. Therefore, C26:0-carnitine is not a suitable biomarker to use in ALD newborn screen. In women with ALD, plasma VLCFA analysis results in a false negative result in approximately 15-20% of cases. The second objective of this study was to compare plasma VLCFA analysis with C26:0-carnitine and C26:0-lysoPC in dried blood spots of women with ALD. Our results show that C26:0-lysoPC was elevated in dried blood spots from all women with ALD, including from those with normal plasma C26:0 levels. This shows that C26:0-lysoPC is a better and more accurate biomarker for ALD than plasma VLCFA levels. We recommend that C26:0-lysoPC be added to the routine biochemical array of diagnostic tests for peroxisomal disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)