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Abstract
ABSTRACT
Background
We identified a novel ABCD1 variant (c.773T>G, p.Leu258Arg, NM_000033.4) in a Chinese pedigree affected by X‐linked adrenoleukodystrophy (X‐ALD). This missense variant in exon 1 is predicted to be pathogenic and likely constitutes the genetic basis of the disease phenotype in this family.
Methods
Results
Sequencing analysis identified a hemizygous missense variant in the
Conclusion
In accordance with the “Standards and Guidelines for the Interpretation of Sequence Variants” established by the American College of Medical Genetics and Genomics (ACMG), we assessed the pathogenicity of the novel
Full text
Introduction
X-linked adrenoleukodystrophy (X-ALD, OMIM: 300100) is a devastating inherited disorder predominantly affecting males, characterized by progressive demyelination and axonal degeneration in the nervous system and adrenal insufficiency. With a global prevalence of approximately 1 in 17,000 newborns (Mukherjee et al. 2024), its clinical heterogeneity poses significant diagnostic and prognostic challenges. The disease spectrum ranges from the rapidly fatal childhood cerebral form (CCALD, ~35% of cases) to the chronic adrenomyeloneuropathy (AMN, ~60%) and Addison-like presentations (Baker et al. 2022; Parasar et al. 2024; Zuo and Chen 2024). This profound variability, particularly the unpredictable progression to lethal cerebral involvement, underscores the critical need for better predictors of disease course and targets for intervention.
The core molecular defect lies in pathogenic variants of the ABCD1 gene, which encodes the peroxisomal membrane protein adrenoleukodystrophy protein (ALDP). ALDP functions as a homodimer essential for transporting VLCFA into peroxisomes for β-oxidation (Chen et al. 2022; Schleker et al. 2022). ABCD1 mutations disrupt this process, leading to toxic VLCFA accumulation primarily in the adrenal cortex, testes, and neural tissues, driving disease pathogenesis (Chen et al. 2022).
Despite the identification of over 3700 distinct ABCD1 variants cataloged in the X-ALD Mutation Database (Liu et al. 2022), a fundamental gap persists in understanding the mechanistic relationship between the spectrum of ABCD1 mutations, particularly those disrupting transmembrane and ATP-binding domains critical for transporter function (Jia et al. 2022; Schleker et al. 2022), and the extreme clinical heterogeneity observed in X-ALD. This genotype–phenotype discordance severely hinders accurate prognosis and personalized management strategies for affected individuals and families.
In this study, we identified a novel ABCD1 variant (c.773T>G). This variant meets the ACMG criteria for classification as “Likely Pathogenic”. Subsequent functional analyses conclusively demonstrated the pathogenicity of this ABCD1 variant. This finding achieves dual significance: (1) it expands the global ABCD1 pathogenic mutation database, and critically, (2) it provides essential molecular information for accurate clinical genetic diagnosis of X-ALD patients, particularly within the context of familial genetic counseling.
Materials and Methods
Ethics Approval and Consent to Participate
This study was approved by the Medical Ethics Committee of SSL, Central Hospital of Dongguan City, Affiliated Dongguan Shilong People's Hospital of Guangdong Medical University. The ethics approval reference number is V1.1 2022–074-01.
Whole-Exome Sequencing and Variant Analysis
Genomic DNA was isolated from peripheral blood specimens of clinical subjects using standardized procedures. Blood samples were drawn into EDTA-coated vacuum collection tubes and processed with the QIAamp DNA Blood Mini Kit (Qiagen) in accordance with the manufacturer's specifications. Comprehensive exome analysis was executed at AmCare Genomic Laboratory (Guangzhou, China) through next-generation sequencing technology. Target enrichment procedures employed the SureSelectXT HS Human All Exon v7 platform (Agilent Technologies, USA) followed by high-throughput sequencing on an Illumina NextSeq 550 system (USA).
Raw sequencing data underwent alignment against the GRCh38 human reference assembly through Burrows-Wheeler Aligner (BWA v0.7.13). Subsequent variant detection and interpretation utilized ANNOVAR computational pipelines integrated with population genomics resources (1000 Genomes, dbSNP, GnomAD) and clinical databases (ClinVar, HGMD, OMIM). Pathogenicity prediction algorithms including SIFT, PolyPhen-2, Provean, and MutationTaster were systematically applied to assess missense mutation effects. All genetic alterations were classified according to ACMG evidence-based guidelines for variant interpretation. Familial segregation analysis through bidirectional Sanger sequencing confirmed the inheritance patterns of candidate variants (He et al. 2025).
Vector Construction
The cDNAs encoding ABCD3, wild-type ABCD1 (ABCD1WT), and the mutant ABCD1 (c.773T>G, ABCD1Leu258Arg) were separately cloned into the pcDNA3.1-V5 and p3×FLAG-CMV-10 expression vectors. All constructs, including p3 × FLAG-CMV-10, pcDNA3.1-V5, p3×FLAG-CMV-10-ABCD1WT, p3×FLAG-CMV-10-ABCD1Leu258Arg, and pcDNA3.1-V5-ABCD3, were obtained from Paivi Biosciences Inc. (Wuhan, China). The recombinant plasmids were verified by sequencing to confirm the accuracy of the cloning.
Biochemical Testing and Neuroimaging
VLCFAs were measured using gas chromatography–mass spectrometry (Camtosun et al. 2021). Magnetic resonance imaging (MRI) was employed to identify pathological changes in the brain (Mathkour et al. 2023).
Immunofluorescence Assays
For immunofluorescence, HEK293 cells were initially washed twice with PBS, followed by fixation with 4.0% paraformaldehyde for 10 min. Permeabilization was then performed using 0.1% Triton X-100 to facilitate antibody penetration. Nonspecific binding sites were blocked by incubating the cells with 5% BSA for 30 min. The cells were subsequently incubated with primary antibodies: anti-Flag (1:1000, Cell Signaling Technology, #2368) and anti-V5 (1:1000, Cell Signaling Technology, #80076). Detection was carried out using Alexa 488-conjugated goat anti-rabbit (1:400, Abcam, ab150077) and Alexa Cy3-conjugated goat anti-mouse (1:400, Abcam, ab97035) secondary antibodies. Nuclei were stained with DAPI (Biosharp, Beijing, China). Imaging was performed using a Leica TCS SP5 confocal microscope equipped with a Plan-Apochromat 63× NA 1.4 oil immersion differential interference contrast (DIC) objective. Optical sections (0.5 μm thickness) were captured in a z-stack montage (Hu et al. 2023).
Molecular Dynamics Simulation
The ABCD1 structural topology was acquired from the AlphaFold protein structure database and rendered using the PyMOL molecular visualization system (v3.0.3). Conformational sampling of secondary structure elements was analyzed through PROCHECK's Ramachandran plot validation server (Chauhan et al. 2023). Comparative molecular dynamics (MD) simulations between wild-type and mutant ABCD1 conformers were implemented to quantify structural divergence through three principal metrics: backbone root mean square deviation (RMSD), residue-specific fluctuation (RMSF), and macromolecular compactness (radius of gyration, Rg). Simulation protocols were established using the Gromacs 2022 computational suite (Gruszczyk et al. 2022) with the following parameterization: Amber99sb-ildn force field application, SPCE explicit water solvation, and cubic periodic boundary conditions (17.695 nm3) maintaining a 1.2 nm solute-box margin (Li et al. 2022). Charge neutralization was achieved through automated ion placement. Energy minimization (50,000-step steepest descent protocol) preceded system equilibration in dual thermodynamic ensembles: NVT (300 K, V-rescale thermostat) and NPT (1 bar, Berendsen barostat) (Shino and Takada 2021). Production MD trajectories (100 ns duration) employed Particle Mesh Ewald electrostatics with a 1.0 nm cutoff for both Coulombic and Lennard-Jones interactions (Wang et al. 2021).
Statistical Analysis
Statistical computations were executed in GraphPad Prism 10 (GraphPad Software, USA) with experimental data expressed as mean ± SEM. Comparative analyses employed parametric hypothesis testing frameworks: two-tailed Student's t-test for dichotomous comparisons, whereas omnibus one-way ANOVA with Tukey's multiple comparison test addressed multigroup evaluations. Specific treatment-control contrasts were resolved using Dunnett's post hoc methodology following ANOVA. All inferential statistics maintained a predetermined alpha level of 0.05 for significance determination.
Results
Case Description
A 14-year-old male patient was admitted to the Neurology Department of Dongguan Songshan Lake Central Hospital due to progressive memory decline over a period of more than 5 months and vision impairment lasting over 4 months. The patient exhibited decreased learning ability, characterized by poor academic performance, slow reactions, recent memory issues, repeatedly asking the same questions, needing frequent reminders for daily activities, and experiencing unclear vision without expressing complaints. Over time, the memory problems worsened, and the patient began to show signs of blurred vision, difficulty reading text on the classroom blackboard, double vision, and distorted visual perception, again without complaints. He was admitted to the hospital for further investigation into the causes of his cognitive and visual impairments. The patient had no significant prior medical history. Given the suspicion of autoimmune encephalitis, he underwent a 5-day course of high-dose intravenous immunoglobulin therapy. However, his condition showed no improvement following treatment.
Clinical Diagnosis
Vital sign evaluation demonstrated normothermic status (36.5°C) with hemodynamic parameters recorded as follows: cardiac rhythm 100 bpm, respiratory cycle frequency 20 breaths/min, and normotensive blood pressure measurement (115/70 mmHg). Notable findings included partial skin hyperpigmentation (Figure 1A,B). No evidence of jaundice, rash, or petechiae was observed. Additionally, no enlargement of superficial lymph nodes was detected upon palpation. Cardiopulmonary and abdominal examinations were unremarkable. The patient exhibited clear and coherent speech. Temporal orientation was intact, while spatial orientation showed mild impairment. Arithmetic ability was tested with the calculation “86 minus 7”, which highlighted mild difficulty. Pupillary examination showed bilaterally equal and round pupils, with intact light reflexes and normal extraocular movements. No nystagmus or other cranial nerve abnormalities were noted. Limb reflexes were increased (++), while bilateral pathological reflexes were absent (−). There was no evidence of meningeal irritation, with negative results for Kernig and Brudzinski signs (−). Cranial MRI demonstrated leukodystrophy affecting the posterior horns of the bilateral lateral ventricles and the peritrigonal white matter, with involvement of the splenium of the corpus callosum, findings consistent with a diagnosis of adrenoleukodystrophy (Figure 1C–F). Electroencephalography (EEG) revealed a borderline pattern. The autoimmune encephalitis autoantibody assay yielded negative results, ruling out autoimmune encephalitis in this patient. The plasma VLCFA profile revealed markedly elevated levels, with C26:0 measured at 5.1 nmol/mL (reference range ≤ 1.3 nmol/mL) and a C26:0/C22:0 ratio of 0.087 (reference range ≤ 0.023) (Table 1). These findings, along with a characteristic peroxisomal profile, confirmed the diagnosis of X-ALD. Currently, there are no effective therapeutic interventions available for patients with XX-ALD. Upon diagnosis, hormone replacement therapy is primarily administered to alleviate certain symptoms; however, this treatment does not halt disease progression. The patient's condition typically deteriorates inexorably, with progressive development of various neurological impairments, including progressive cognitive decline, dysarthria, hearing loss, motor dysfunction, urinary incontinence, and visual loss. After a follow-up period of one and a half years, the patient ultimately succumbed to the disease.
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TABLE 1 VLCFAs in members of a family with X-ALD (nmol/mL).
| VLCFAs | Proband | Mother of the proband | Father of the proband | Re. value |
| C22:0 | 58.5 | 71.8 | 60.3 | ≤ 96.3 |
| C24:0 | 77.8 | 73.5 | 51.9 | ≤ 91.4 |
| C26:0 | 5.1↑ | 0.95 | 1.05 | ≤ 1.30 |
| C24:0/C26:0 | 1.33 | 1.19 | 1.08 | ≤ 1.39 |
| C26:0/C22:0 | 0.087↑ | 0.015 | 0.021 | ≤ 0.023 |
Genetic Analysis of X-ALD Pedigree
To investigate the molecular etiology of X-ALD in the familial cohort, comprehensive exome analysis was conducted on the index case (pedigree position II-1, Figure 2A). This genomic interrogation identified a previously unreported pathogenic variant (c.773T>G; p.Leu258Arg) in the ABCD1 gene (NM_000033), localized to exon 1. The detected substitution demonstrated complete segregation with the disease phenotype, suggesting its potential causal role in the observed X-ALD manifestation. This analysis revealed a novel missense mutation, c.773T>G (p.Leu258Arg), in exon 1 of the X-linked ABCD1 gene, which appears to be associated with the clinical presentation of X-ALD in this individual. Sanger sequencing further confirmed the presence of this mutation in both the proband and his mother (Figure 2B), suggesting that the mother is a carrier of the pathogenic ABCD1 allele. Sequence alignment across various species demonstrated that the affected site is highly conserved (Figure 2D). To assess the pathogenicity of the c.773T>G mutation, bioinformatics tools including Provean, SIFT, PolyPhen-2, and MutationTaster were employed (Figure 2E). All analyses consistently predicted that this novel mutation in ABCD1 is indeed pathogenic.
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3D Structure Prediction of ALDP
To evaluate the accuracy of the protein structure prediction, this study employed the PROCHECK tool within the SAVESv6.0 toolkit () to generate a Ramachandran plot for assessing the stereochemical quality of the predicted model (Agarwal et al. 2023). The results revealed that for the ALDP, 92.8% (1000 residues) of amino acid residues were located in most favored regions, 6.9% (74 residues) in additional allowed regions, and 0.4% (four residues) in generously allowed regions. Importantly, no residues were found in disallowed regions. Notably, LYS569 and ASP361 were positioned in generously allowed regions, suggesting that these residues may constrain the generation of models with improved stereochemical quality (Figure 3A). Computational structural analysis of ABCD1-encoded ALDP was conducted through the SWISS-MODEL homology modeling platform. This in silico approach enabled systematic evaluation of mutation-induced conformational alterations in the ALDP tertiary structure (Cheng et al. 2022). Homology modeling, using a template structure from the Protein Data Bank (PDB), was employed to predict the effects of the c.773T>G mutation on ALDP's structure (Figure 3B,C). Literature reports highlight that a high-density region of missense mutations is concentrated in exons 1 and 2 of ABCD1 (Zuo and Chen 2024). Further structural analysis revealed that the c.773T>G mutation occurs within exon 1, where the amino acid substitution from leucine 258 to arginine may induce a conformational change in the protein (Figure 2C), potentially disrupting its normal function.
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Molecular Dynamics Simulations
To investigate the structural stability and dynamic characteristics of ALDPWT and ALDPLeu258Arg in an aqueous environment, this study performed 100 ns MD simulations. The radius of gyration (Rg) for ALDPWT fluctuated between 3.6 and 4.3 nm, ultimately converging to a stable value of 3.7 nm by the end of the simulation. In contrast, ALDPLeu258Arg exhibited Rg values ranging from 3.73104 to 4.26129 nm, stabilizing at 3.82581 nm at 100 ns (Figure 3F). These results indicate that ALDPLeu258Arg has a slightly reduced compactness compared to ALDPWT. The RMSD analysis revealed a rapid increase during the initial phase of the simulation (0–10 ns), corresponding to structural equilibration. After approximately 10 ns, the RMSD values stabilized. For ALDPWT, RMSD values fluctuated between 1.2 and 1.5 nm beyond 20 ns, reflecting high structural stability (Figure 3E). The root mean square fluctuation (RMSF) profiles of both proteins exhibited comparable fluctuation patterns across most residues, suggesting similar global flexibility and stability. However, specific regions, including the N- and C-terminal regions as well as residues 53, 128, 295, 367, 442, and 567, showed significantly higher fluctuations in ALDPLeu258Arg relative to ALDPWT (Figure 3D). Increases in flexibility suggest that the p.Leu258Arg mutation induces structural destabilization in these regions. Overall, these findings demonstrate that the p.Leu258Arg mutation compromises the structural stability of ALDP, leading to reduced compactness and increased regional flexibility, which may adversely affect the protein's functional properties.
Subcellular Localization of ALDPLeu258Arg Variant In Vitro
To evaluate whether the Leu258Arg mutation affects the subcellular localization of ALDP, this study utilized immunofluorescence analysis to examine the expression patterns of ALDPLeu258Arg in HEK293 cells. As shown in Figure 4A, all experimental cells displayed fluorescent signals (red) corresponding to the peroxisomal marker ABCD3, confirming the presence of intact peroxisomes. In cells expressing ALDPWT, the fluorescent signals for ALDPWT (green) co-localized extensively with those of ABCD3 (red), resulting in a yellow-orange signal. This high degree of overlap demonstrates that ALDPWT was correctly targeted to peroxisomes, consistent with accurate expression and functional localization in HEK293 cells (Figure 4B). In contrast, cells expressing ALDPLeu258Arg exhibited a markedly different pattern. The fluorescence signal for ALDPLeu258Arg (green) was predominantly distributed in the cytoplasm, with minimal overlap with the peroxisomal marker ABCD3 (Figure 4C). This mislocalization suggests that the Leu258Arg mutation disrupts the proper peroxisomal targeting of ALDP. Such aberrant localization could compromise peroxisomal functions, including fatty acid β-oxidation, by reducing the availability of functional ALDP within the organelle.
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Discussion
X-ALD is a peroxisomal disorder characterized by neurodegeneration and adrenocortical insufficiency. The core pathogenic mechanism involves loss-of-function variants in the ABCD1 gene, leading to dysfunction of ALDP. This disrupts the peroxisomal transmembrane transport of VLCFA (C22–C26), resulting in their pathological accumulation in plasma and tissues. Global epidemiological studies indicate a male prevalence of approximately 1:20,000, a female prevalence of approximately 1:10,000, and an estimated incidence ranging between 1:20,000 and 1:30,000 (Engelen et al. 2012; Wiesinger et al. 2015).
ALDP belongs to subfamily D of the ATP-binding cassette (ABC) transporter superfamily (ABCD). The human ABCD subfamily comprises four members (ABCD1-4), with ABCD1-3 localized to the peroxisomal membrane (Tawbeh et al. 2021). These proteins contain conserved transmembrane domains (TMDs) and nucleotide-binding domains (NBDs), utilizing ATP hydrolysis to drive substrate transport (Janas et al. 2003; Kawaguchi and Imanaka 2022). To date, the X-ALD Mutation Database () has cataloged over 3700 ABCD1 gene variants, predominantly comprising missense mutations (63.8%), nonsense mutations (9.1%), frameshift mutations (15.7%), splice site mutations (4.0%), and exon deletions (2.5%). The vast majority of variants (92.9%) reside within exons, with 6.1% in introns and < 1% in untranslated regions (UTRs) (Janas et al. 2003). Studies demonstrate regional clustering of pathogenic ABCD1 variants: the TMDs (exons 1–2) harbor the highest proportion (46%), followed by the NBDs (exons 6–9, 35%), with exons 3–4 and 5 accounting for 9% and 7%, respectively (Mallack et al. 2022). Notably, Liu et al. reported that exon 1 represents a mutational hotspot (37.5%) in AMN patients worldwide, with the majority being missense mutations (84.4%) (Zhang et al. 2021). However, a study by Chen et al. on a Chinese cohort (n = 14) identified known mutations predominantly within exons 6–9 (9/12), with only a minority in exons 1–2 (3/12), suggesting potential population heterogeneity in mutation distribution and highlighting the importance of larger sample sizes (Zhang et al. 2025).
This study reports a Chinese male adolescent with the cerebral form of X-ALD (onset at age 14, presenting with progressive cognitive decline and visual/auditory loss). Genetic analysis identified a novel missense mutation in the ABCD1 gene, c.773T>G (p.Leu258Arg). This variant is located within the known mutational hotspot region of exon 1 and results in the substitution of a highly conserved, nonpolar leucine (Leu) residue at position 258 of the ALDP protein with a polar arginine (Arg). Analysis using variant pathogenicity prediction tools classified this mutation as “Likely Pathogenic”. The variant was absent in 195 healthy controls and public population genomic databases (e.g., gnomAD), excluding it as a common polymorphism. Homology modeling of the protein three-dimensional structure revealed that the p.Leu258Arg substitution induces a significant conformational change in the ABCD1 protein. Subcellular localization experiments further demonstrated that while the wild-type ALDP-GFP fusion protein co-localized efficiently with the peroxisomal marker ABCD3, co-localization of the Leu258Arg mutant with ABCD3 was markedly impaired. Integrating this evidence, in accordance with the ACMG variant interpretation guidelines, we classify c.773T>G (p.Leu258Arg) as a “Pathogenic Variant”.
X-ALD is classified into seven clinical subtypes based on age of onset and primary organ involvement: childhood cerebral, adolescent cerebral, AMN, adult cerebral, olivo-ponto-cerebellar, Addison only, and asymptomatic (Kemp et al. 2001). Cerebral ALD characterizes the pediatric population, whereas AMN primarily affects adults (Kang et al. 2024). Pathogenic variants within exon 1 of ABCD1 are associated with diverse clinical phenotypes. For instance, patients with p. Pro48Ser, p. Gly277Arg, and p.Ser290Trp variants presented with AMN; those with p.Gln266Arg and p.Ser108Leu exhibited both cerebral and AMN phenotypes; p.Asn148Asp and p.Asn214Asp were associated with the cerebral form; p. Thr254Pro manifested as AMN (Matsukawa et al. 2011). The patient with p.Leu258Arg identified in this study presented with the adolescent cerebral form. These clinical observations further underscore the complexity of genotype–phenotype correlations in ABCD1, indicating that variants within exon 1 do not strictly correlate with a single clinical subtype. Phenotypic expression is likely modulated by other genetic or environmental factors.
Overall, this study reports a novel pathogenic missense mutation, c.773T>G (p.Leu258Arg), located within the mutational hotspot exon 1 of the ABCD1 gene, thereby expanding its mutational spectrum. Through bioinformatic prediction, structural modeling, and functional assays, we demonstrate that this mutation impairs the normal conformation of ALDP and its precise localization to the peroxisomal membrane, ultimately leading to loss of its VLCFA transport function. Our findings advance the understanding of the pathogenic mechanisms underlying ABCD1 mutations and re-emphasize the high degree of heterogeneity and complexity in X-ALD genotype–phenotype relationships. These results provide important experimental evidence for elucidating the molecular pathology of X-ALD and may contribute to the future development of targeted diagnostic strategies and therapies.
Author Contributions
Designed the study: J.H., P.H., and H.F.; Performed experiments: H.F., L.H., X.L., and B.H.; Analyzed the data: J.H. and P.H.; Drafted the manuscript: J.H. and P.H. All authors read and approved the final manuscript.
Acknowledgments
We would like to convey our gratitude to the patient and his family.
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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