Abstract
Background
Hereditary spastic paraplegia type 56 (SPG56) is a rare autosomal recessive neurodegenerative disorder caused by biallelic variants in the CYP2U1 gene, which encodes a cytochrome P450 enzyme involved in fatty acid metabolism and mitochondrial function. The clinical spectrum includes progressive spasticity of the lower limbs, developmental delay or regression, cognitive impairment, and variable ophthalmological findings. Although several cases have been reported in recent years, the functional characterization of individual variants remains limited.
Case presentation
Here we describe a male patient with early-onset SPG56 carrying two CYP2U1 missense variants, NM_183075.3:c.1376 C > T p.(Pro459Leu) and NM_183075.3:c.557G > A p.(Arg186His). Combined genomic, cellular, and in silico analyses confirmed loss of enzymatic activity and protein instability, supporting the pathogenic classification of both variants. Functional validation led to reclassification of the p.(Arg186His) variant from uncertain significance to pathogenic. Further, we link specific CYP2U1 missense changes to convergent molecular defects, thereby refining genotype–phenotype correlations. From a therapeutic perspective, we highlight the relevance of experimental interventions such as folinic acid supplementation and multimodal spasticity management, while emphasizing the future promise of gene therapy for SPG56 patients.
Conclusions
Our findings highlight the value of integrating genomic, biochemical, and structural approaches in the diagnostic evaluation of rare neurogenetic disorders, and provide functional evidence that the identified CYP2U1 variants are damaging, consistent with the observed early-onset complex SPG56 phenotype.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12920-026-02394-7.
Keywords: Hereditary spastic paraplegia, SPG56, CYP2U1, Functional validation, Missense variants, Cytochrome P450, In silico modeling
Background
Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of more than 80 genetic syndromes, primarily affecting motor neurons. The hallmark symptom is progressive spasticity and weakness of the lower extremities due to neurodegeneration of the corticospinal tract. Uncomplicated (or pure) HSP manifests mainly with motor impairment, whereas complicated (or complex) HSP displays additional neurological or extra-neurological features such as intellectual disability, epilepsy, cognitive dysfunction, peripheral neuropathy, or chorioretinal dystrophy. In recent years, an increasing number of causative genes have been identified, with diverse modes of inheritance – autosomal dominant, autosomal recessive, X-linked dominant, or mitochondrial. While individual HSP subtypes are rare or ultra-rare, the overall prevalence of HSP is approximately 1 in 100,000 individuals, depending on the population studied [1–3].
Spastic paraplegia type 56 (SPG56, OMIM#615030) is an autosomal recessive HSP caused by biallelic pathogenic variants in the CYP2U1 gene, which encodes a member of the cytochrome P450 family [4, 5]. Although the exact function of CYP2U1 in humans is not fully understood, it is predominantly expressed in the central nervous system and thymus [5]. It represents a specific human enzyme that catalyzes ω- and (ω–1)-hydroxylation of long-chain fatty acids, particularly arachidonic acid and docosahexaenoic acid (DHA) [6, 7]. In experimental models, CYP2U1 has been shown to convert arachidonic acid exclusively into two regionally specific metabolites, 19-hydroxy- and 20-hydroxyarachidonic acid (19-HETE and 20-HETE). These products belong to bioactive eicosanoids, acting as local signaling molecules that influence vascular tone, neuronal excitability, Ca²⁺ channel regulation, and immune responses. 19-HETE has been demonstrated to function as an inhibitor of P/Q-type Ca²⁺ channels in Purkinje cells of the cerebellum, suggesting that CYP2U1 may indirectly modulate neurotransmitter release and cerebral microcirculation. Conversely, 20-HETE acts as a vasoconstrictor in cerebral and renal tissues, contributing to the regulation of blood flow and blood pressure. Given the high expression of CYP2U1 in the thymus, a role in immune processes has also been proposed, mediated through the metabolism of polyunsaturated fatty acids that affect lymphocyte proliferation and differentiation. Thus, CYP2U1 activity integrates lipid signaling, neurophysiology, and immune regulation, occupying a unique position among the cytochrome P450 enzymes [6].
To date, 61 pathogenic or likely pathogenic CYP2U1 variants have been reported in the ClinVar database, most of which are predicted to result in loss of function of the enzyme according to current variant annotations. Approximately 50 individuals with SPG56 have been reported in the literature [8–22]. All of them carried biallelic CYP2U1 variants, while heterozygous carriers were asymptomatic. SPG56 typically presents in infancy with progressive spastic paraplegia and variable additional features, including intellectual disability, cognitive dysfunction, upper limb involvement, psychiatric manifestations, and MRI abnormalities such as hypomyelination or brain calcifications [10–13, 16, 21]. However, a small number of cases with uncomplicated HSP have been described. In 2025, a case report documented a woman with biallelic pathogenic CYP2U1 variants who developed adult-onset symptoms at the age of 39 [22], illustrating significant phenotypic and age-related variability. Furthermore, variable maculopathies in patients with biallelic CYP2U1 variants were first described by Leonardi et al. in 2016 [9] and have since been repeatedly confirmed by other reports [18, 20]. A recent report described two siblings, both compound heterozygotes for the same pathogenic CYP2U1 variants [20]. One sibling developed neurological symptoms consistent with SPG56 from the age of two but exhibited no visual impairment, whereas the other showed reduced visual acuity at age of 15 years without neurological manifestations. Detailed ophthalmologic evaluations revealed retinal impairment in both individuals, resembling changes seen in macular telangiectasia type 2, with more severe manifestations in the visually impaired sibling. The striking phenotypic discordance, despite identical biallelic genotypes, underscores the unresolved variability of CYP2U1-related disease and the likely contribution of genetic or environmental modifiers.
All reported cases were identified through a systematic literature search using the keywords CYP2U1 AND (SPG56 OR spastic paraplegia), followed by detailed screening of the described clinical features in each report; overlapping cases were carefully excluded. A summary of the most common symptoms and their percentage occurrence, based on 52 literature-reported cases, is presented in Table 1.
Table 1.
| Lower limbs spasticity | 92% |
| Upper limbs spasticity | 27% |
| Neurodevelopmental disorders | 52% |
| Seizures | 6% |
| Dysarthria | 29% |
| Abnormal brain MRI | 29% |
| White matter hyperintensities | 19% |
| Thinning of the corpus callosum | 6% |
| Delayed myelination | 6% |
| Basal ganglia calcification | 8% |
| Unspecified visual impairment | 8% |
| Maculopathy | 32% |
| Pigmentary degenerative | 8% |
| Pseudoxanthoma elasticum | 6% |
| Macular telangiectasia | 8% |
| Unspecified maculopathy | 10% |
These observations emphasize the importance of publishing detailed case reports of individuals with CYP2U1 variants. Careful phenotypic characterization across ages and clinical presentations is crucial for identifying potential modifiers, genetic, metabolic, or environmental, that influence disease expression. Such cumulative evidence may refine genotype–phenotype correlations, improve diagnostic protocols for early detection of comorbid symptoms, and support the development of personalized therapeutic strategies.
In this study, we report a patient with early-onset complex SPG56 carrying two CYP2U1 missense variants and provide their functional characterization using biochemical and structural approaches. Our aim is to contribute additional evidence supporting variant pathogenicity and to further delineate the clinical and molecular features associated with CYP2U1-related disease.
Case presentation
We present a Czech male born to healthy non-consanguineous parents. The family does not originate from a known endogamous or genetically isolated population. Proband has an older sister who experienced a mild speech delay but is otherwise healthy, with no neurological symptoms. The mother reported use of acetylsalicylic acid during pregnancy due to a risk of placental abruption; otherwise, the perinatal history was uneventful. Early psychomotor development was within normal limits, with appropriate motor milestones. Between 3 and 4 months of age, the patient began physiotherapy for mild body asymmetry, occasional opisthotonic posturing, and central hypotonia, with limited improvement. At approximately 6 months of age, the physiotherapist noted increased spasticity in the lower limbs, prompting intensified physiotherapy. Over the next two months, motor development further deteriorated, resulting in hospitalization for complex evaluation.
Neurological examinations revealed regression of motor function to a level comparable with a newborn, axial central hypotonia combined with early-stage spasticity of the limbs (more pronounced in the lower limbs and with all typical clinical signs of central motor neuron impairment), variable dystonic posturing of the extremities, and increased regurgitation. Psychosocial development was found to be normal, or at least much less impaired than motor skills. This trend was also evident in subsequent developments. Extensive laboratory work-up, including a comprehensive selective screening for inherited metabolic disorders, revealed no major abnormalities. Only borderline vitamin B12 deficiency and mild vitamin D insufficiency were detected, both of which were corrected with appropriate supplementation. Ultrasound of the abdomen revealed no abnormalities. Additional findings included a closed anterior fontanelle, mild slowing of background activity in the EEG (Supplementary Figure S3), demyelinating changes on EMG (Supplementary Figure S4), and MRI abnormalities consistent with delayed myelination (Fig. 1A, B). Cardiac examination, including EKG and echocardiography, revealed no structural or functional abnormalities. Ophthalmologic assessments were repeatedly normal, with no abnormalities detected on fundoscopic examination.
Fig. 1.
Brain MRI at 9 months of age. (A) T1_tra: bilaterally symmetrical low signal intensity areas in the paraventricular white matter of the frontal lobes (abnormal due to age-dependent process of myelination), (B, C) T2 + FLAIR_tra: band-like hyperintensities of white matter in both sequences (predominantly in the left frontal and right occipital region), corresponding with delayed myelination, (D) T1-weighted sequence, sagittal plane
Given the unclear etiology, further metabolic work-up was performed, along with comprehensive genetic testing. No specific inborn error of metabolism was identified. Genetic testing consisted of karyotyping, array-based comparative genomic hybridization (array-CGH), whole-exome sequencing (WES), and mitochondrial genome sequencing. The patient had a normal male karyotype, and array-CGH showed no deletions or duplications. Mitochondrial sequencing revealed no variants relevant to the phenotype. WES identified two variants in the CYP2U1 gene, likely pathogenic variant NM_183075.3:c.1376 C > T p.(Pro459Leu) and variant of uncertain significance NM_183075.3:c.557G > A p.(Arg186His). Subsequent parental testing confirmed the biallelic positions, with each parent being heterozygous for one of the two variants (see Fig. 2). The sister is not a carrier of either of the described CYP2U1 variants.
Fig. 2.
Sanger sequencing electrophoretograms from segregation analysis. The likely pathogenic NM_183075.3:c.1376 C > T variant was inherited from the unaffected father, while the variant of uncertain significance, NM_183075.3:c.557G > A, was inherited from the unaffected mother
Both identified CYP2U1 variants are extremely rare in population databases. The NM_183075.3:c.1376 C > T p.(Pro459Leu) variant (rs747965749) has a reported allele frequency of approximately 0.00005–0.00006 in gnomAD, while NM_183075.3:c.557G > A p.(Arg186His) (rs746319505) is present at an allele frequency of approximately 0.00001. Given these extremely low frequencies, the probability of their co-occurrence by chance in the same individual is exceedingly low.
In addition to the protein-level effects, we considered the possibility of alternative transcript isoforms that might escape the impact of the identified variants. Interrogation of genome annotation databases (Ensembl and UCSC Genome Browser) indicates that CYP2U1 has a limited number of annotated transcript variants, with the canonical isoform containing both affected residues (Arg186 and Pro459). No well-supported protein-coding isoforms lacking these regions were identified. While the existence of low-abundance or tissue-specific transcripts cannot be entirely excluded, current evidence suggests that alternative splicing is unlikely to provide significant functional compensation for the identified variants.
This genetic finding strongly supported a diagnosis of a compound heterozygote SPG56 and was consistent with the clinical presentation, further reinforcing the genotype–phenotype correlation characteristic of CYP2U1-related disease. To further validate the diagnosis, we performed a functional study and in silico structure modelling of the NM_183075.3:c.557G > A p.(Arg186His) variant in the CYP2U1 gene, previously classified as a variant of uncertain significance, as well as confirming the likely pathogenic variant NM_183075.3:c.1376 C > T p.(Pro459Leu) by these approaches.
Functional analysis and in silico structure modelling
To determine the ability of CYP2U1 with the missense variants NM_183075.3:c.557G > A p.(Arg186His) or NM_183075.3:c.1376 C > T p.(Pro459Leu) to catalyse the hydroxylation of arachidonic acid to 19-HETE and 20-HETE isomers, we adapted a method for detecting the products of this enzymatic reaction by LC-MS as described previously [4].
Briefly, full-length human CYP2U1 cDNA was subcloned into pcDNA3.1(+)IRES_GFP plasmid (Addgene). Gene variants NM_183075.3:c.557G > A and NM_183075.3:c.1376 C > T were generated using Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and confirmed by Sanger sequencing. In three independent biological replicates, HEK293T cells (cultured in DMEM with 10% FBS and antibiotics) were seeded at 1 × 104 cells/cm2 and transfected the next day by each plasmid construct (empty plasmid, CYP2U1 c.557G > A p.(Arg186His), CYP2U1 c.1376 C > T p.(Pro459Leu), or CYP2U1 wild-type) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Two days after transfection, cells were harvested in 1×PBS with a protease inhibitor cocktail (Thermo Scientific). Similar expression of each construct in cells was verified by flow cytometry (GFP, data not shown). Cells were then lysed by sonication in lysis buffer (50 mM K-phosphate, pH 7.4, 0.5 M sucrose, 1 mM EDTA). After centrifugation at 20,000 g/4˚C/10 min, the supernatant was collected, and the protein concentration was measured by BCA Protein Assay (Thermo Scientific). 25 µg of total proteins was used for the conventional western blot analysis using anti-CYP2U1 antibody (sc-393368, Santa Cruz Biotechnology) and anti-β-actin antibody (4970 S, Cell Signaling Technology). The protein CYP2U1 was detected only in lysates from cells transfected either with wild-type or p.(Arg186His) variant, but consistently undetectable in the p.(Pro459Leu) variant across all biological replicates, suggesting that this variant results in protein instability (Fig. 3A).
Fig. 3.
Detection of protein level and enzymatic activity of CYP2U1 and its variants. (A) Western blot analysis of CYP2U1 wild-type and missense variants overexpressed in HEK293T cells; representative immunoblot (anti-CYP2U1: sc-393368, Santa Cruz Biotechnology, and anti-β-actin: 4970 S, Cell Signaling Technology) is shown. (B) Relative peak area of the metabolites 19-HETE and 20-HETE detected by UPLC-MS, determining the enzymatic activity of CYP2U1 wild-type and missense variants overexpressed in HEK293T cells; normalized to internal standard 20-HETE-d6; error bars represent SD. Empty plasmid pcDNA3.1(+)IRES_GFP was used as a control. Data were obtained from three independent biological replicates
The enzymatic activity of CYP2U1 was determined by incubating 400 µg of total proteins diluted in lysis buffer with 5 µM arachidonic acid (Sigma Aldrich). The samples were first incubated at 37 °C/10 min, then the 2×NADPH regenerating system (Promega) was added (v/v) and the incubation continued at 37 °C for the next 45 min. The reaction was terminated by acidification to pH ~ 3.5 with acetic acid, and the metabolites were extracted twice with ethyl acetate (v/v). After that, ethyl acetate was evaporated (Eppendorf Concentrator Plus), and the residues were diluted in 25 µL of absolute EtOH. Finally, the UHPLC-MS was used to clearly distinguish the 19-HETE and 20-HETE isomers (Supplementary Figure S1) and demonstrate their presence/absence in the sample, while 20-HETE-d6 (Cayman Chemicals) was used as an internal standard. The analysis was performed using an Agilent 1290 Infinity II liquid chromatograph with an Agilent QQQ6495A mass spectrometer, equipped with an Acquity BEH C18 (1.7 μm, 2.1 mm x100 mm, Waters, detailed specifications in Supplementary Methods). The 19-HETE and 20-HETE isomers were detected only in the CYP2U1 wild-type sample. Neither of the mutant proteins catalyzed the hydroxylation of arachidonic acid to these metabolites (Fig. 3B), with signals in mutant samples consistently below the limit of detection, suggesting the loss of enzymatic activity in the CYP2U1 p.(Arg186His) variant and confirming the pathogenic effect of the CYP2U1 p.(Pro459Leu) variant, probably due to the protein instability.
Next, we used in silico modelling to better understand the effect of variants on the protein structure and function. The tertiary structure of CYP2U1 was obtained from AlphaFold DB [23] (Fig. 4). The missing heme group was added using AlphaFill [24] inferred from the homologous structure PDB ID 3C6G. The active site location was determined based on the position of Pilocarpine in the analogous structure PDB ID 3T3S, identified via AlphaFill [25] with further confirmation from multiple ligands detected by AlphaFill. The protein-heme complex was also modelled using AlphaFold3 [26]. Both models were structurally aligned and compared in 3D. Overall, the models obtained from AlphaFold DB and predicted using AlphaFold3 show good structural agreement, including the location and orientation of the heme (Supplementary Figure S2). Site-directed mutations were introduced in both models using PyMOL [27], and their spatial positioning relative to the heme and catalytic pocket was critically analyzed.
Fig. 4.
The structural model of CYP2U1 and localization of mutations. The model downloaded from the AlphaFold Database (ID: AF-Q7Z449-F1-v4) is shown as a ribbon representation: the overall protein structure (left) and a close-up view (right). The location of heme (magenta stick) and the ligand Pilocarpine (yellow stick), bound in the active site, was transferred from homologous structures (PDB IDs 3C6G and 3T3S, respectively) using the AlphaFill algorithm. The localization of mutations Arg186His (red stick) and Pro459Leu (green stick) is shown in the structure of wild-type CYP2U1
The Arg186His mutation is located at the tip of the helix (Fig. 4, red stick). The substitution of a polar arginine for a polar histidine could potentially be tolerated by the structure. This is supported by the AlphaMissense prediction, which assigns a score of 0.49, suggesting an ambiguous effect. Using other prediction algorithms, the probably damaging (PolyPhen2: HDivPred score 1, HVarPred score 0.999; [28]) and deleterious (SIFT v6.2.0: score 0.00, median 4.32; [29], and MutationTaster v2021: Tree vote 70|30 del|benign [30]) effects on protein structure were predicted. The Arg186His mutation is positioned 12 Å away from the heme and is localized on the opposite side of the catalytic pocket. The helix carrying this mutation is in direct contact with the heme, and even a slight distortion due to the Arg186His substitution could impair heme binding or repositioning, leading to a detrimental effect on catalytic function [4]. Loss of heme binding renders the enzyme functionally inactive, compromising its ability to hydroxylate arachidonic acid, a key biochemical process in neuronal maintenance [6].
The Pro459Leu mutation is located on a loop, approximately 18 Å away from the heme (Fig. 4, green stick). This mutation is predicted to be poorly tolerated by the protein structure due to an apparent steric clash between the Leu side chain and adjacent residues in this region. It is expected to cause structural destabilization. This interpretation is further supported by the AlphaMissense prediction, which assigns a score of 0.67, indicating a deleterious effect. This variant is listed in the HGMD database [31] as causative for spastic paraplegia based on primary literature evidence published by Leeson et al. [32].
The NM_183075.3:c.557G > A p.(Arg186His) variant reclassification
The functional and in silico analyses complemented the genetic findings, allowing us to propose reclassification of the missense variant NM_183075.3:c.557G > A from uncertain significance to pathogenic in accordance with ACMG Standards and Guidelines criteria [33]. Firstly, WES and subsequent parental testing revealed the biallelic position of this variant with a likely pathogenic variant (NM_183075.3:c.1376 C > T) in our patient (PM3). As demonstrated by a functional study, the variant NM_183075.3:c.557G > A leads to the loss of enzymatic activity of CYP2U1 (PS3), and the deleterious effect was also predicted by in silico methods (PP3). In addition, the criteria PM2 (extremely low allele frequency; MAF < 0.01%; gnomAD) and PP2 (missense variants are a common mechanism of disease; [4, 5, 11, 22]) were fulfilled. This variant therefore meets the criteria to be classified as pathogenic (PS3, PM2, PM3, PP2, PP3), and we have accordingly submitted this variant to the ClinVar database with this classification.
Therapeutic approaches
Following the SPG56 diagnosis, we conducted a literature review to explore potential therapeutic considerations. Wong et al. reported a possible association between CYP2U1-related SPG56 and cerebral folate deficiency in two patients, who received long-term oral folinic acid supplementation [19]. The authors described clinical stabilization; however, these observations remain limited and should be interpreted with caution.
In our patient, cerebrospinal fluid (CSF) folate levels could not be assessed due to unsuccessful lumbar punctures, and no direct evidence of folate deficiency was obtained. Oral folinic acid supplementation was nevertheless initiated on an empirical basis, without objective data on treatment response to date. Symptomatic treatment with clonazepam and baclofen was introduced to manage spasticity.
Following the diagnosis, the patient’s family connected with an international network of families affected by SPG56 through social media and expressed interest in participation in an upcoming academic clinical trial of gene therapy for CYP2U1-related spastic paraplegia, which is currently being prepared in Australia. Overall, these observations highlight the need for further systematic studies to evaluate potential therapeutic strategies in SPG56.
Discussion and conclusions
Hereditary spastic paraplegia type 56 (SPG56) represents one of the most complex forms of autosomal recessive HSPs due to its variable phenotypic expression and overlapping metabolic and mitochondrial features. The present case adds to the limited number of reported patients with biallelic CYP2U1 variants and expands the clinical and molecular spectrum of the disease. The early onset and rapid progression observed in our patient are consistent with previously described infantile cases, where motor regression and severe spasticity develop within the first year of life [10, 11, 16]. The combination of NM_183075.3:c.1376 C > T p.(Pro459Leu) and NM_183075.3:c.557G > A p.(Arg186His) variants has not been reported previously, and our functional validation provides direct experimental evidence for their pathogenicity.
Previous studies have shown that CYP2U1 plays an essential role in lipid metabolism and mitochondrial homeostasis [5–7]. The enzyme’s ability to hydroxylate arachidonic acid and other polyunsaturated fatty acids generates bioactive eicosanoids (19-HETE, 20-HETE), which are key regulators of neuronal signaling and vascular tone [6]. Pathogenic variants that abolish this enzymatic activity are expected to impair neuronal membrane integrity and synaptic signaling, leading to the progressive motor neuron degeneration characteristic of SPG56. Our findings are in full agreement with earlier reports demonstrating loss of function as the main pathogenic mechanism [4, 12]. The instability of the Pro459Leu protein and the loss of catalytic activity in Arg186His further emphasize that even structurally conservative substitutions can disrupt the enzymatic function of CYP2U1.
The genotype-phenotype correlation in SPG56 remains challenging due to the small number of cases and the presence of considerable intrafamilial variability. Some patients with identical genotypes show only mild spasticity, while others develop severe neurodevelopmental regression or ocular pathology such as maculopathy or retinopathy [9, 18, 22]. In our patient, no visual abnormalities were detected, supporting the notion that ocular involvement is not a universal feature but rather a variable manifestation potentially influenced by environmental or modifier genes. This heterogeneity highlights the need for continuous phenotypic monitoring and data sharing across international registries.
From a diagnostic standpoint, this study demonstrates the importance of integrating comprehensive genomic, biochemical, and structural analyses. Whole-exome sequencing remains the most efficient approach for diagnosing SPG56 and related HSP subtypes. However, as shown here, functional validation can provide decisive evidence for reclassifying variants of uncertain significance into pathogenic categories, in accordance with ACMG guidelines. This approach not only improves diagnostic accuracy but also supports the development of potential genotype-specific therapeutic strategies.
Therapeutically, management of SPG56 remains largely symptomatic. The recently reported association between CYP2U1-related HSP and cerebral folate deficiency [19] suggests a potential metabolic target in a subset of patients. In our case, despite unsuccessful cerebrospinal folate assessment, empirical folinic acid supplementation was initiated, reflecting the current experimental approach to potentially modifiable metabolic components. The patient’s family has also been connected with an international SPG56 community and is preparing to participate in an upcoming academic gene therapy trial in Australia, expected to begin in 2026. This underlines the increasing translational momentum toward personalized treatment in ultra-rare neurogenetic disorders.
This study has several limitations. It is based on a single patient, which limits generalizability and precludes robust genotype–phenotype conclusions. Functional analyses were restricted to in vitro assays and may not fully capture in vivo disease mechanisms. In addition, CSF folate levels and objective treatment response data were not available, limiting interpretation of potential therapeutic implications.
However, our data expand the current knowledge of CYP2U1-associated hereditary spastic paraplegia type 56 (SPG56) by identifying and functionally characterizing two missense variants, NM_183075.3:c.557G > A p.(Arg186His) and NM_183075.3:c.1376 C > T p.(Pro459Leu). Our findings demonstrate an undetectable enzymatic activity for the p.(Arg186His) variant and indicate that the p.(Pro459Leu) variant leads to protein instability. Both variants disrupt CYP2U1 function through distinct molecular mechanisms. This case highlights the diagnostic and interpretative value of integrating genomic, biochemical, and in silico structural approaches in the evaluation of variant pathogenicity in rare neurogenetic disorders.
These data provide further support for the association between CYP2U1 variants and SPG56 and highlight the need for cautious consideration of experimental therapeutic approaches, such as folinic acid supplementation and symptomatic management of spasticity, while gene therapy remains a potential future direction requiring further evidence. Comprehensive functional validation should be considered an important step in assessing variants of uncertain significance, particularly in rare neurogenetic diseases.
Supplementary Information
Acknowledgements
Not applicable.
Authors’ contributions
ES and KR drafted the manuscript. OS conceptualized and designed the study and critically revised the manuscript. KS performed the patient’s diagnostic evaluation, collected clinical data, and substantially contributed to manuscript drafting and preparation of the final version. PP performed data analysis and interpretation and critically revised the manuscript. JD contributed to the study conception and design of protein analyses and critically revised the manuscript. JK, OH and RD contributed to data collection and critically revised the manuscript. EH and KK performed laboratory analysis. KR designed the functional analysis. KR, VH, MS, ZS performed the functional analysis and critically revised the manuscript. All authors read and approved the final manuscript.
Funding
Supported by the Ministry of Health of the Czech Republic – DRO (University Hospital Brno, 65269705) and by the grant of the Faculty of Medicine, Masaryk University: MUNI/A/1591/2023. This publication is also the outcome of the project CREATIC funded from the European Union’s Horizon Europe Coordination and Support Action under the Grant agreement number 101059788. Computational resources were provided by the CZECRIN and e-INFRA projects (LM2023049 and 90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic. The analytical part of the work was carried out with the support of the RECETOX Research Infrastructure (ID LM2023069, MEYS CR, 2023–2026).
Data availability
The datasets generated and/or analysed during the current study are available in the ClinVar repository, [Accession numbers: SCV006311003, SCV006586705].
Declarations
Ethics approval and consent to participate
The study was approved by the University Hospital Brno Ethics Committee (approval no. 12-071222) and conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained and signed by the patient’s parents. Patient identification and other personal information that could be used to reveal the identity of the patient were protected.
Consent for publication
Parents of the patient presented in this case report consented to the publication. The written consent form was signed.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Eva Sustrova and Kamila Rihova contributed equally to this work.
Contributor Information
Ondrej Slaby, Email: oslaby@med.muni.cz.
Katerina Slaba, Email: slaba.katerina@fnbrno.cz.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and/or analysed during the current study are available in the ClinVar repository, [Accession numbers: SCV006311003, SCV006586705].