Discovery of novel diagnostic biomarkers for Sjögren-Larsson syndrome by untargeted lipidomics.

AIM: Sjögren-Larsson syndrome (SLS) is a rare neurometabolic disorder that mainly affects brain, eye and skin and is caused by deficiency of fatty aldehyde dehydrogenase. Our recent finding of a profoundly disturbed brain tissue lipidome in SLS prompted us to search for similar biomarkers in plasma as no functional test in blood is available for SLS. METHODS AND RESULTS: We performed plasma lipidomics and used a newly developed bioinformatics tool to mine the untargeted part of the SLS plasma and brain lipidome to search for SLS biomarkers. Plasma lipidomics showed disturbed ether lipid metabolism in known lipid classes. Untargeted lipidomics of both plasma and brain (white and grey matter) uncovered two new endogenous lipid classes highly elevated in SLS. The first biomarker group were alkylphosphocholines/ethanolamines containing different lengths of alkyl-chains where some alkylphosphocholines were > 600-fold elevated in SLS plasma. The second group of biomarkers were a set of 5 features of unknown structure. Fragmentation studies suggested that they contain ubiquinol and phosphocholine and one feature was also found as a glucuronide conjugate in plasma. The plasma features were highly distinctive for SLS with levels >100-1000-fold the level in controls, if present at all. We speculate on the origin of the alkylphosphocholines/ethanolamines and the nature of the ubiquinol-containing metabolites. CONCLUSIONS: The metabolites identified in this study represent novel endogenous lipid classes thus far unknown in humans. They represent the first plasma metabolite SLS-biomarkers and may also yield more insight into SLS pathophysiology.

Sjogren-Larsson syndrome brain volumetric reductions demonstrated with an automated software.

BACKGROUND: Sjogren-Larsson syndrome (SLS) is a neurocutaneous disease with an autosomal recessive inheritance, caused by mutations in the gene that encodes fatty aldehyde dehydrogenase (ALDH3A2), clinically characterized by ichthyosis, spastic diplegia, and cognitive impairment. Brain imaging plays an essential role in the diagnosis, demonstrating a nonspecific leukoencephalopathy. Data regarding brain atrophy and grey matter involvement is scarce and discordant. OBJECTIVE: We performed a volumetric analysis of the brain of two siblings with SLS with the aim of detecting deep grey matter nuclei, cerebellar grey matter, and brainstem volume reduction in these patients. METHODS: Volume data obtained from the brain magnetic resonance imaging (MRI) of the two patients using an automated segmentation software (Freesurfer) was compared with the volumes of a healthy control group. RESULTS: Statistically significant volume reduction was found in the cerebellum cortex, the brainstem, the thalamus, and the pallidum nuclei. CONCLUSION: Volume reduction in grey matter leads to the hypothesis that SLS is not a pure leukoencephalopathy. Grey matter structures affected in the present study suggest a dysfunction more prominent in the thalamic motor pathways.

ANTECEDENTES: A Síndrome de Sjogren-Larsson (SSL) é uma doença neurocutânea de herança autossômica recessiva, causada por mutações no gene que codifica a aldeído graxo desidrogenase (ALDH3A2), caracterizada clinicamente por ictiose, diplegia espástica e comprometimento cognitivo. A imagiologia cerebral desempenha um papel essencial no diagnóstico, demonstrando uma leucoencefalopatia inespecífica. Dados sobre atrofia cerebral e envolvimento da substância cinzenta são escassos e discordantes. OBJETIVO: Realizamos uma análise volumétrica do cérebro de dois irmãos com SLS com o objetivo de detectar núcleos profundos de substância cinzenta, substância cerebral cinzenta e redução do volume do tronco encefálico nestes pacientes. MéTODOS: Os dados de volume obtidos da ressonância magnética (RM) cerebral dos dois pacientes usando um software de segmentação automática (Freesurfer) foram comparados com os volumes de um grupo controle saudável. RESULTADOS: Redução de volume estatisticamente significativa foi encontrada no córtex do cerebelo, no tronco cerebral, no tálamo e nos núcleos pálidos. CONCLUSãO: A redução do volume da substância cinzenta leva à hipótese de que a SSL não é uma leucoencefalopatia pura. As estruturas da substância cinzenta afetadas no presente estudo sugerem uma disfunção mais proeminente nas vias motoras talâmicas.

Ceramide profiling of stratum corneum in Sjögren-Larsson syndrome.

BACKGROUND: Sjögren-Larsson syndrome (SLS) is a neurocutaneous disorder whose causative gene is the fatty aldehyde dehydrogenase ALDH3A2 and of which ichthyosis is the major skin symptom. The stratum corneum contains a variety of ceramides, among which ω-O-acylceramides (acylceramides) and protein-bound ceramides are essential for skin permeability barrier formation. OBJECTIVES: To determine the ceramide classes/species responsible for SLS pathogenesis and the enzymes that are impaired in SLS. METHODS: Genomic DNA was collected from peripheral blood samples from an SLS patient and her parents, and whole-genome sequencing and Sanger sequencing were performed. Lipids were extracted from stratum corneum samples from the SLS patient and healthy volunteers and subjected to ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: A duplication (c.55_130dup) and a missense mutation (p.Lys447Glu) were found in the patient's ALDH3A2 gene. The patient had reduced levels of all acylceramide classes, with total acylceramide levels at 25 % of healthy controls. Reductions were also observed for several nonacylated ceramides: ceramides with phytosphingosine or 6-hydroxysphingosine in the long-chain base moiety were reduced to 24 % and 41 % of control levels, respectively, and ceramides with an α-hydroxy fatty acid as the fatty acid moiety were reduced to 29 %. The fatty acid moiety was shortened in many nonacylated ceramide classes. CONCLUSION: These results suggest that reduced acylceramide levels are a primary cause of the ichthyosis symptoms of SLS, but reductions in other ceramide classes may also be involved.

Small touches to big walks -the impact of rehabilitation on Sjögren-Larsson syndrome: A case report.

Sjögren-Larsson syndrome (SLS) is a rare neurocutaneous disorder characterized by the presence of congenital ichthyosis, spasticity, and mental retardation. As with other rare genetic diseases, treatment is mainly symptomatic. Due to the absence of definitive treatment, lifelong follow-up and support of patients are important to improve the quality of life. A 7-year-old female child who was diagnosed as having SLS was referred to the rehabilitation clinic. After 20 sessions of a rehabilitation program, she started walking independently with the additional contribution of ankle-foot orthoses (AFOs). The contribution of the short-term rehabilitation approach and especially the administration of AFOs to the independence level of the patient is emphasized herein.

Síndrome de Sjögren-Larsson en España; descripción de 3 nuevos casos / Sjögren-Larsson syndrome in Spain: Description of three new cases

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Impaired Skin Barrier Function Due to Reduced ω-O-Acylceramide Levels in a Mouse Model of Sjögren-Larsson Syndrome.

Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder whose causative gene encodes the fatty aldehyde dehydrogenase ALDH3A2. To date, the detailed molecular mechanism of the skin pathology of SLS has remained largely unclear. We generated double-knockout (DKO) mice for Aldh3a2 and its homolog Aldh3b2 (a pseudogene in humans). These mice showed hyperkeratosis and reduced fatty aldehyde dehydrogenase activity and skin barrier function. The levels of ω-O-acylceramides (acylceramides), which are specialized ceramides essential for skin barrier function, in the epidermis of DKO mice were about 60% of those in wild-type mice. In the DKO mice, levels of acylceramide precursors (ω-hydroxy ceramides and triglycerides) were increased, suggesting that the final step of acylceramide production was inhibited. A decrease in acylceramide levels was also observed in human immortalized keratinocytes lacking ALDH3A2. Differentiated keratinocytes prepared from the DKO mice exhibited impaired long-chain base metabolism. Based on these results, we propose that the long-chain-base-derived fatty aldehydes that accumulate in DKO mice and SLS patients attack and inhibit the enzyme involved in the final step of acylceramide production. Our findings provide insight into the pathogenesis of the skin symptoms of SLS, i.e., decreased acylceramide production, and its molecular mechanism.

A Neurodegenerative Phenotype Associated With Sjögren-Larsson Syndrome.

Sjögren-Larsson syndrome (SLS) is a rare neurologic disorder caused by pathogenic sequence variants in ALDH3A2 and characterized by ichthyosis, spasticity, intellectual disability, and a crystalline retinopathy. Neurologic symptoms develop in the first 2 years of life. Except for worsening ambulation due to spastic diplegia and contractures, the neurologic disease has been considered static and a neurodegenerative course is distinctly unusual. We describe a young child with Sjögren-Larsson syndrome who exhibited an early and severely progressive neurologic phenotype that may have been triggered by a febrile rotavirus infection. Together with 7 additional published cases of these atypical patients, we emphasize that a neurodegenerative course can be an extreme outcome for a minority of patients with Sjögren-Larsson syndrome.

Novel ALDH3A2 mutations in structural and functional domains of FALDH causing diverse clinical phenotypes in Sjögren-Larsson syndrome patients.

Mutations in ALDH3A2 cause Sjögren-Larsson syndrome (SLS), a neuro-ichthyotic condition due to the deficiency of fatty aldehyde dehydrogenase (FALDH). We screened for novel mutations causing SLS among Indian ethnicity, characterized the identified mutations in silico and in vitro, and retrospectively evaluated their role in phenotypic heterogeneity. Interestingly, asymmetric distribution of nonclassical traits was observed in our cases. Nerve conduction studies suggested intrinsic-minus-claw hands in two siblings, a novel neurological phenotype to SLS. Genetic testing revealed five novel homozygous ALDH3A2 mutations in six cases: Case-1-NM_000382.2:c.50C>A, NP_000373.1:p.(Ser17Ter); Case-2-NM_000382.2:c.199G>T, NP_000373.1:p.(Glu67Ter); Case-3-NM_000382.2:c.1208G>A, NP_000373.1:p.(Gly403Asp); Case-4-NM_000382.2:c.1325C>T, NP_000373.1:p.(Pro442Leu); Case-5 and -6 NM_000382.2:c.1349G>A, NP_000373.1:p.(Trp450Ter). The mutations identified were predicted to be pathogenic and disrupt the functional domains of the FALDH. p.(Pro442Leu) at the C-terminal α-helix, might impair the substrate gating process. Mammalian expression studies with exon-9 mutants confirmed the profound reduction in the enzyme activity. Diminished aldehyde-oxidizing activity was observed with cases-2 and 3. Cases-2 and 3 showed epidermal hyperplasia with mild intracellular edema, spongiosis, hypergranulosis, and perivascular-interstitial lymphocytic infiltrate and a leaky eosinophilic epidermis. The presence of keratin-containing milia-like lipid vacuoles implies defective lamellar secretion with p.(Gly403Asp). This study improves our understanding of the clinical and mutational diversity in SLS, which might help to fast-track diagnostic and therapeutic interventions of this debilitating disorder.

Muscle Tone Assessment under General Anesthesia for Sjögren-Larsson Syndrome and Spasticity.

INTRODUCTION: Study of muscle tone in individuals with severe spasticity (Modified Asworth Scale - MAS:3) under general anesthesia can confirm or rule out the eventual necessity of the impending spasticity relieving ablative neurosurgery by observing the hypertonia reduction and passive range of motion expansion. Therefore, what we measure under muscle relaxants is practically a fixed deformity. CASE PRESENTATION: The study was performed on a girl with Sjögren-Larsson syndrome, presenting with icthyosis and spastic diplegia. Proposed intervention was Dorsal Rhizotomy. Under general anesthesia, with and without muscle relaxants, hypertonia was significantly reduced (MAS:1), but the angle of motion did not increase much. CONCLUSION: We decided not to perform such a neurosurgical procedure. In ambiguous situations, the proposed study can help in decision-making for spasticity treatment.

Aldh1l2 knockout mouse metabolomics links the loss of the mitochondrial folate enzyme to deregulation of a lipid metabolism observed in rare human disorder.

BACKGROUND: Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation. METHODS: We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways. RESULTS: Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from ß-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired ß-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria. CONCLUSIONS: The ALDH1L2 function is important for CoA-dependent pathways including ß-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.

Sjögren-Larsson syndrome: Anesthetic considerations and practical recommendations.

BACKGROUND: Sjögren-Larsson syndrome is a rare inherited neurocutaneous disorder characterized by congenital ichthyosis, spasticity, intellectual disability, seizures, and ophthalmologic changes. Most individuals with Sjögren-Larsson syndrome live well into adulthood and often require surgical intervention to manage their symptomatology. AIMS: The aim of this work was to review the clinical aspects of Sjögren-Larsson syndrome, highlight the unique anesthetic considerations associated with this disease, and provide practical recommendations about anesthetic management. METHODS: A retrospective case review from February 2013 to October 2019 was performed based on subject participation in a Sjögren-Larsson syndrome longitudinal study at the University of Nebraska Medical Center. Anesthetic and surgical records were reviewed for the following data: age, sex, relevant comorbid conditions, anesthetic induction and maintenance agents, intravenous and oral analgesics, muscle relaxants, and anesthetic-related complications. RESULTS: Fourteen patients with Sjögren-Larsson syndrome undergoing 48 anesthetic events were identified. A variety of anesthetic techniques was utilized. No serious adverse events were encountered. The most common clinical observations were related to the ichthyosis seen in Sjögren-Larsson syndrome, which led to difficulty in adherence of electrocardiogram leads and intravenous catheter dressings. CONCLUSIONS: We found that anesthesia can be safely administered in patients with Sjögren-Larsson syndrome. Providers should be aware of anesthetic management issues in Sjögren-Larsson syndrome including challenges placing and securing lines and monitors secondary to the ichthyosis.

Sjogren-Larsson Syndrome: A case series of five members from an extended family with a novel mutation.

BACKGROUNDD: Sjogren-Larsson syndrome (SLS) is a rare autosomal recessive disorder, characterized by a triad of spastic tetraplegia or diplegia, congenital ichthyosis, and intellectual disability. METHODS: We report a seven-years-old female born to consanguineous parents who presented with erythematous dry scaly skin all over the body sparing the face, without collodion membrane which started since birth. There were associated with global developmental delay and seizure disorder. SLS was suspected and hence sequence analysis of the ALDH3A2 gene by next-generation sequencing was performed for the patient. RESULTS: A novel nucleotide exchange in homozygous state at position c.1320 in exon 9 of the ALDH3A2 gene (c.1320T>A), leading to a stop of the protein sequence (p.Tyr440) was detected in the patient. Genetic testing of the patient's extended family revealed another four affected family members with the same mutation. CONCLUSIONS: SLS should be suspected in any patient with a triad of ichthyosis, intellectual disability and spastic di/tetraplegia. Molecular genetic testing of the ALDH3A2 gene should be performed to confirm the diagnosis. Extended family screening is highly recommended.

1-O-Alkylglycerol accumulation reveals abnormal ether glycerolipid metabolism in Sjögren-Larsson syndrome.

Sjögren-Larsson syndrome (SLS) is an inherited metabolic disease characterized by ichthyosis, spasticity, intellectual disability and deficient oxidation and accumulation of of fatty aldehydes and alcohols. We investigated whether excess fatty alcohols in SLS are diverted into biosynthesis of ether glycerolipids (eGLs) by measuring the 1-O-alkylglycerol (AG) backbone of eGLs in stratum corneum, plasma and red blood cells (RBCs). In all tissues, saturated and monounsaturated AGs were detected. In stratum corneum from SLS patients, saturated AGs (C15-C20) were increased 97-fold (range: 86- to 169-fold) compared to controls. AGs were largely (67 ± 9%) derived from neutral esterified eGLs (i.e. alkyl-diacylglyerol) and free non-esterified AGs (28 ± 10%), but very little from plasmalogens (3 ± 5%). Plasma from SLS patients had 2-fold more C18:0-AG (p < 0.005) and 40% less C16:1-AG (p < 0.01) than controls but the total concentration of AGs was not increased, and the AG profile in RBCs from SLS subjects was normal. All AGs were profoundly reduced in plasma and RBCs from patients with Zellweger spectrum disorder, who have impaired eGL (i.e. plasmalogen) synthesis. The striking accumulation of AGs in stratum corneum of SLS patients constitutes a novel lipid biomarker for this disease, and may contribute to the pathogenesis of the ichthyosis. Measurement of AGs is a simple and convenient method to assess global synthesis of eGLs and potentially identify patients with defects in their metabolism.

Disturbed brain ether lipid metabolism and histology in Sjögren-Larsson syndrome.

Sjögren-Larsson syndrome (SLS) is a rare neurometabolic syndrome caused by deficient fatty aldehyde dehydrogenase. Patients exhibit intellectual disability, spastic paraplegia, and ichthyosis. The accumulation of fatty alcohols and fatty aldehydes has been demonstrated in plasma and skin but never in brain. Brain magnetic resonance imaging and spectroscopy studies, however, have shown an abundant lipid peak in the white matter of patients with SLS, suggesting lipid accumulation in the brain as well. Using histopathology, mass spectrometry imaging, and lipidomics, we studied the morphology and the lipidome of a postmortem brain of a 65-year-old female patient with genetically confirmed SLS and compared the results with a matched control brain. Histopathological analyses revealed structural white matter abnormalities with the presence of small lipid droplets, deficient myelin, and astrogliosis. Biochemically, severely disturbed lipid profiles were found in both white and gray matter of the SLS brain, with accumulation of fatty alcohols and ether lipids. Particularly, long-chain unsaturated ether lipid species accumulated, most prominently in white matter. Also, there was a striking accumulation of odd-chain fatty alcohols and odd-chain ether(phospho)lipids. Our results suggest that the central nervous system involvement in SLS is caused by the accumulation of fatty alcohols leading to a disbalance between ether lipid and glycero(phospho)lipid metabolism resulting in a profoundly disrupted brain lipidome. Our data show that SLS is not a pure leukoencephalopathy, but also a gray matter disease. Additionally, the histopathological abnormalities suggest that astrocytes and microglia might play a pivotal role in the underlying disease mechanism, possibly contributing to the impairment of myelin maintenance.

Macular crystalline inclusions in Sjögren-Larsson syndrome are dynamic structures that undergo remodeling.

BACKGROUND: Sjögren-Larsson syndrome (SLS) is a rare genetic neurocutaneous disease caused by mutations in ALDH3A2 that results in deficiency of fatty aldehyde dehydrogenase and accumulation of fatty aldehydes and alcohols. The disease is associated with ichthyosis, spasticity, and intellectual disability. Patients exhibit a characteristic retinopathy with macular crystalline inclusions that first appear in early childhood and increase with age. Once formed, the inclusions are thought to be inert and irreversible. We sought to document how the crystalline inclusions change over time. MATERIALS AND METHODS: Serial retinal photographs of 4 SLS subjects (9-23 years old) were taken over a period of 1-3 years. Images were compared by visual inspection and analyzed using ImageJ/Fiji software to observe changes. RESULTS: Visual inspection of retinal photographs of SLS subjects taken over time demonstrated distinctive changes in crystalline inclusions. New inclusions were formed and some established inclusions regressed. These changes were conveniently demonstrated with software-based photographic image analysis. CONCLUSIONS: We conclude that macular inclusions in SLS are not simply inert deposits, but are dynamic structures that form over time and are subject to remodeling. This conclusion provides new insight into the interplay between the metabolic defect and retinal pathology in SLS, and raises the potential for new therapeutic approaches to reverse some aspects of the maculopathy.

Comprehensive Molecular Profiles of Functionally Effective MSC-Derived Extracellular Vesicles in Immunomodulation.

Accumulating evidence indicates that mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) exhibit immunomodulatory effects by delivering therapeutic RNAs and proteins; however, the molecular mechanism underlying the EV-mediated immunomodulation is not fully understood. In this study, we found that EVs from early-passage MSCs had better immunomodulatory potency than did EVs from late-passage MSCs in T cell receptor (TCR)- or Toll-like receptor 4 (TLR4)-stimulated splenocytes and in mice with ocular Sjögren's syndrome. Moreover, MSC-EVs were more effective when produced from 3D culture of the cells than from the conventional 2D culture. Comparative molecular profiling using proteomics and microRNA sequencing revealed the enriched factors in MSC-EVs that were functionally effective in immunomodulation. Among them, manipulation of transforming growth factor ß1 (TGF-ß1), pentraxin 3 (PTX3), let-7b-5p, or miR-21-5p levels in MSCs significantly affected the immunosuppressive effects of their EVs. Furthermore, there was a strong correlation between the expression levels of TGF-ß1, PTX3, let-7b-5p, or miR-21-5p in MSC-EVs and their suppressive function. Therefore, our comparative strategy identified TGF-ß1, PTX3, let-7b-5p, or miR-21-5p as key molecules mediating the therapeutic effects of MSC-EVs in autoimmune disease. These findings would help understand the molecular mechanism underlying EV-mediated immunomodulation and provide functional biomarkers of EVs for the development of robust EV-based therapies.