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.

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.

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.

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.

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.

Retinal Morphology in Sjögren-Larsson Syndrome on OCT: From Metabolic Crystalline Maculopathy to Early-Onset Macular Degeneration.

PURPOSE: To study long-term macular changes by spectral-domain (SD) OCT in patients with Sjögren-Larsson syndrome (SLS). DESIGN: Retrospective cohort study. PARTICIPANTS: Twenty-two patients with genetically proven SLS (12 female, 10 male; median age, 21 years; range, 3-47 years) were included in the study. One or more SD-OCT scans were available from the period 2008 to 2017. METHODS: Seventeen patients underwent SD-OCT imaging of the macula in 2017. Earlier scans were available of the other 5 patients. The latest available SD-OCT scans were qualitatively assessed for morphologic changes in 19 patients. In addition, retinal layer thickness could be measured in 15 patients. The latest scans were compared with previous scans to assess the course of the disease qualitatively (n = 15 patients) and quantitatively (n = 10 patients). MAIN OUTCOME MEASURES: Macular morphology and retinal layer thickness on SD-OCT scans during the study period. RESULTS: In all patients, abnormal macular morphology was observed in both eyes, already from the youngest age. Intraretinal crystals, visible as hyperreflective dots, were visible in all eyes and located in the retinal nerve fiber layer, inner plexiform layer, and outer plexiform layer. Furthermore, the photoreceptor (PR) layer lacked the physiologic thickness amplification beneath the fovea. Pseudocysts with limited interruption of the underlying PR layer were observed in half of the patients, all younger than 30 years of age. Frank atrophy of the retinal pigment epithelium (RPE) and a neovascular complex were seen in 3 older patients and 1 older patient, respectively. The fovea was significantly thinner compared with healthy controls and decreased even further during the study period. CONCLUSIONS: Macular dystrophy in SLS may initially comprise an arrest in foveal development. Because of the absence of macular pigment, phototoxic damage may then cause central retinal degeneration of the vulnerable macula, marked by the development of pseudocysts. Eventually, the young adult patients may proceed to an early-onset end-stage macular degeneration with frank atrophy of the RPE or neovascularization.

Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.

Insulation by myelin lipids is essential to fast action potential conductivity: changes in their quality or amount can cause several neurologic disorders. Sjögren-Larsson syndrome (SLS) is one such disorder, which is caused by mutations in the fatty aldehyde dehydrogenase ALDH3A2. To date, the molecular mechanism underlying SLS pathology has remained unknown. In this study, we found that Aldh3a2 is expressed in oligodendrocytes and neurons in the mouse brain, and neurons of Aldh3a2 knockout (KO) mice exhibited impaired metabolism of the long-chain base, a component of sphingolipids. Aldh3a2 KO mice showed several abnormalities corresponding to SLS symptoms in behavioral tests, including increased paw slips on a balance beam and light-induced anxiety. In their brain tissue, 2-hydroxygalactosylceramide, an important lipid for myelin function and maintenance, was reduced by the inactivation of fatty acid 2-hydroxylase. Our findings provide important new insights into the molecular mechanisms responsible for neural pathogenesis caused by lipid metabolism abnormalities.-Kanetake, T., Sassa, T., Nojiri, K., Sawai, M., Hattori, S., Miyakawa, T., Kitamura, T., Kihara, A. Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.

Genotype and phenotype variability in Sjögren-Larsson syndrome.

The Sjögren-Larsson syndrome (SLS) is a rare autosomal recessive disorder caused by pathogenic variants in the ALDH3A2 gene, which codes for fatty aldehyde dehydrogenase (FALDH). FALDH prevents the accumulation of toxic fatty aldehydes by converting them into fatty acids. Pathogenic ALDH3A2 variants cause symptoms such as ichthyosis, spasticity, intellectual disability, and a wide range of less common clinical features. Interpreting patient-to-patient variability is often complicated by inconsistent reporting and negatively impacts on establishing robust criteria to measure the success of SLS treatments. Thus, with this study, patient-centered literature data was merged into a concise genotype-based, open-access database (www.LOVD.nl/ALDH3A2). One hundred and seventy eight individuals with 90 unique SLS-causing variants were included with phenotypic data being available for more than 90%. While the three lead symptoms did occur in almost all cases, more heterogeneity was observed for other frequent clinical manifestations of SLS. However, a stringent genotype-phenotype correlation analysis was hampered by the considerable variability in reporting phenotypic features. Consequently, we compiled a set of recommendations of how to generate comprehensive SLS patient descriptions in the future. This will be of benefit on multiple levels, for example, in clinical diagnosis, basic research, and the development of novel treatment options for SLS.

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide.

Lipids are the primary components of the skin permeability barrier, which is the body's most powerful defensive mechanism against pathogens. Acylceramide (ω-O-acylceramide) is a specialized lipid essential for skin barrier formation. Here, we identify PNPLA1 as the long-sought gene involved in the final step of acylceramide synthesis, esterification of ω-hydroxyceramide with linoleic acid, by cell-based assays. We show that increasing triglyceride levels by overproduction of the diacylglycerol acyltransferase DGAT2 stimulates acylceramide production, suggesting that triglyceride may act as a linoleic acid donor. Indeed, the in vitro analyses confirm that PNPLA1 catalyses acylceramide synthesis using triglyceride as a substrate. Mutant forms of PNPLA1 found in patients with ichthyosis exhibit reduced or no enzyme activity in either cell-based or in vitro assays. Altogether, our results indicate that PNPLA1 is directly involved in acylceramide synthesis as a transacylase, and provide important insights into the molecular mechanisms of skin barrier formation and of ichthyosis pathogenesis.

Changes in the Submandibular Salivary Gland Epithelial Cell Subpopulations During Progression of Sjögren's Syndrome-Like Disease in the NOD/ShiLtJ Mouse Model.

Sjögren's syndrome (SS), an autoimmune exocrinopathy, is associated with dysfunction of the secretory salivary gland epithelium, leading to xerostomia. The etiology of SS disease progression is poorly understood as it is typically not diagnosed until late stage. Since mouse models allow the study of disease progression, we investigated the NOD/ShiLtJ mouse to explore temporal changes to the salivary epithelium. In the NOD/ShiLtJ model, SS presents secondary to autoimmune diabetes, and SS disease is reportedly fully established by 20 weeks. We compared epithelial morphology in the submandibular salivary glands (SMG) of NOD/ShiLtJ mice with SMGs from the parental strain at 12, 18, and 22 weeks of age and used immunofluorescence to detect epithelial proteins, including the acinar marker, aquaporin 5, ductal cell marker, cytokeratin 7, myoepithelial cell marker, smooth muscle α-actin, and the basal cell marker, cytokeratin 5, while confirming immune infiltrates with CD45R. We also compared these proteins in the labial salivary glands of human SS patients with control tissues. In the NOD/ShiLtJ SMG, regions of lymphocytic infiltrates were not associated with widespread epithelial tissue degradation; however, there was a decrease in the area of the gland occupied by secretory epithelial cells in favor of ductal epithelial cells. We observed an expansion of cells expressing cytokeratin 5 within the ducts and within the smooth muscle α-actin(+) basal myoepithelial population. The altered acinar/ductal ratio within the NOD/ShiLtJ SMG likely contributes to salivary hypofunction, while the expansion of cytokeratin 5 positive-basal cells may reflect loss of function or indicate a regenerative response.

Plasmalogens and fatty alcohols in rhizomelic chondrodysplasia punctata and Sjögren-Larsson syndrome.

Plasmalogens are a special class of ether-phospholipids, best recognized by their vinyl-ether bond at the sn-1 position of the glycerobackbone and by the observation that their deficiency causes rhizomelic chondrodysplasia punctata (RCDP). The complex plasmalogen biosynthetic pathway involves multiple enzymatic steps carried-out in peroxisomes and in the endoplasmic reticulum. The rate limiting step in the biosynthesis of plasmalogens resides in the formation of the fatty alcohol responsible for the formation of an intermediate with an alkyl-linked moiety. The regulation in the biosynthesis of plasmalogens also takes place at this step using a feedback mechanism to stimulate or inhibit the biosynthesis. As such, fatty alcohols play a relevant role in the formation of ether-phospholipids. These advances in our understanding of complex lipid biosynthesis brought two seemingly distinct disorders into the spotlight. Sjögren-Larsson syndrome (SLS) is caused by defects in the microsomal fatty aldehyde dehydrogenase (FALDH) leading to the accumulation of fatty alcohols and fatty aldehydes. In RCDP cells, the defect in plasmalogens is thought to generate a feedback signal to increase their biosynthesis, through the activity of fatty acid reductases to produce fatty alcohols. However, the enzymatic defects in either glyceronephosphate O-acyltransferase (GNPAT) or alkylglycerone phosphate synthase (AGPS) disrupt the biosynthesis and result in the accumulation of the fatty alcohols. A detailed characterization on the processes and enzymes that govern these intricate biosynthetic pathways, as well as, the metabolic characterization of defects along the pathway should increase our understanding of the causes and mechanisms behind these disorders.

Method to simultaneously determine the sphingosine 1-phosphate breakdown product (2E)-hexadecenal and its fatty acid derivatives using isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight mass spectrometry.

Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjögren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatography-electrospray ionization-quadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjögren-Larsson syndrome, in more detail.

Multimodal imaging of the macula in hereditary and acquired lack of macular pigment.

PURPOSE: Macular pigment (MP) deficit has been described in macular teleangiectasia type 2 (MTA; acquired MP loss) and in Sjögren-Larsson syndrome (SLS; hereditary MP deficiency). Central blue light-induced fundus autofluorescence (FAF) and blue light fundus reflectance (BLR) are thought to reflect MP distribution. This study was performed to describe the macular morphology in SLS and MTA by multimodal imaging to further investigate the causes of FAF and BLR changes in these disorders. METHODS: This was a single-centre, cross-sectional, retrospective, observational study on SLS and MTA patients treated at our institution. In a multimodal retinal imaging dataset, patterns of BLR and FAF changes were compared with the optical coherence tomography (OCT) and clinical appearance of the patients' retinas. RESULTS: Multimodal image sets of seven eyes of four patients with SLS and of 25 eyes of 15 patients with MTA were included in this study. In MTA, areas of focal FAF increase were mainly associated with retinal pseudocysts and photoreceptor loss and were co-located with regions of increased BLR. In SLS, areas of focally decreased FAF correlated with the typical intraretinal glistening dots. Frequently, a spot of focally increased FAF was visible at the fovea of SLS patients, often independent of the presence of pseudocysts or photoreceptor loss on OCT. CONCLUSION: In MTA and SLS different patterns of FAF alterations could be observed. The areas of increased BLR, which are thought to correlate with MP loss, appeared to have only restricted correlation with FAF appearance.

Fatty aldehyde and fatty alcohol metabolism: review and importance for epidermal structure and function.

Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren-Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Novel mutation in Sjogren-Larsson syndrome is associated with divergent neurologic phenotypes.

Sjögren-Larsson syndrome is an inherited disorder of lipid metabolism caused by mutations in the ALDH3A2 gene that codes for fatty aldehyde dehydrogenase, which results in accumulation of fatty aldehydes and alcohols and is characterized by ichthyosis, intellectual disability, and spastic diplegia/quadriplegia. The authors describe 2 unrelated Honduran patients who carried the same novel homozygous nonsense mutation (c.1309A>T, p.K437X) and ALDH3A2 DNA haplotype, but widely differed in disease severity. One patient exhibited spastic quadriplegia with unusual neuroregression, whereas the other patient had the usual static form of spastic diplegia with neurodevelopmental disabilities. Biochemical analyses showed a similar profound deficiency of fatty aldehyde dehydrogenase activity and impaired fatty alcohol metabolism in both patients' cultured fibroblasts. These results indicate that variation in the neurologic phenotype of Sjögren-Larsson syndrome is not strictly determined by the ALDH3A2 mutation or the biochemical defect as expressed in cultured fibroblasts, but by unidentified epigenetic/environmental factors, gene modifiers, or other mechanisms.

The Sjögren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the sphingosine 1-phosphate degradation pathway.

Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS.

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds.

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.