Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.
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All data and material generated and analyzed within the main figures and supplementary material of this study are available upon request from the authors. All requests for raw and analyzed data and materials related to this article will be reviewed by the respective institution to verify whether the request is subject to any intellectual property or confidentiality obligations. Some patient-related data, including genetic sequencing data, not included in the paper or its supplements were generated as part of clinical care and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. The following databases were searched for the presence of variants reported in this paper: the Genome aggregation database (https://gnomad.broadinstitute.org), the Decipher database (https://www.deciphergenomics.org), the Leiden database (https://www.lovd.nl) and the TopMed database (https://bravo.sph.umich.edu/freeze8/hg38/).
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We thank the patients and their families for participating in our research study, C. Mendoza (NINDS/NNDCS) and G. Averion (NINDS/NNDCS) for their help in supporting the clinic, F. Eichler (Massachusetts General Hospital) for helpful discussions, and B. Smith (NINDS) and A. Nath (NINDS) for providing control serum samples. We also thank the NIH Intramural Sequencing Center for performing exome sequencing; K. Linask and J. Zhou from the NHLBI iPSC core for generating the isogenic mutant iPSC lines; and S. Lara and S. J. H. Tao Cheng in the NINDS electron microscopy facility. Work in C.G.B.’s laboratory is supported by intramural funds of NINDS/NIH. Work in T.M.D.’s laboratory was supported by CDMRP grant W81XWH-20-1-0219. C.V.L is supported by a career development grant from NIH/NINDS (K08 NS10762). T.H. is supported by Swiss National Science Foundation grant 31003A_179371. M.A.L is supported by Swiss Foundation for Research on Muscle Diseases (FSRMM). R.H.B. is funded by NIH/NINDS (R01 NS072446) and the Deater foundation. Sequencing and analysis provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) was funded by the National Human Genome Research Institute, the National Eye Institute and the National Heart, Lung and Blood Institute (grant UM1 HG008900) and, in part, by the National Human Genome Research Institute (grant R01 HG009141).
The authors declare no competing interests.
Peer review information Nature Medicine thanks Merit Cudkowicz, Ammar Al-Chalabi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Jerome Staal was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended Data Fig. 1 Sphingolipid biosynthesis pathway.
Serine palmitoyltransferase (SPT) catalyses the first and rate-limiting step in sphingolipid biosynthesis. The most abundant acyl-CoA used by SPT is palmitoyl-CoA, which after condensation with L-serine results in an 18-carbon long-chain base, 3-dehydrosphinganine. 3-dehydrosphinganine is rapidly reduced by 3-dehydrosphinganine reductase (KDSR) to form dihydrosphingosine (sphinganine). Sphinganine is acylated by a variety of ceramide synthases (CerS) to form dihydroceramides, which can be converted to ceramides by sphingolipid desaturase (DEGS). Many complex sphingolipids can be synthesized from a ceramide backbone. However, the only true exit from the pathway depends on activity of sphingosine kinase (SK) and sphingosine-1-phosphate aldolase (SGPL) to form phosphoethanolamine. SGPP = sphingosine-1-phosphate phosphatase.
Extended Data Fig. 2 SPTLC1 variant associated with amyotrophic lateral sclerosis and their effects on splicing.
a, Gel electrophoresis of PCR amplification of cDNA obtained from patient fibroblast cultures or whole blood. Fibroblasts were not available for c.58G>T patient. PCR primers were in exon 1 and 6 of SPTLC1 (NM_006415.4). SPTLC1 c.58G>T variant resulted in two bands with an apparent difference of ~100 bp. All other variants and controls resulted in a single band. b, Sequencing of the gel purified PCR product showed that the c.58G>T variant resulted in complete skipping of exon 2 (lower band). Neither the full-length product (upper band) nor the internally deleted transcript (lower band) contained the c.58G>T variant. However, it is possible that the missense variant exists in very small amounts and escaped amplification by PCR and sequencing. All other variants were confirmed and do not affect splicing.
Extended Data Fig. 3 Amyotrophic lateral sclerosis associated variant SPTLC1 cellular localization.
a, Immunostaining for SPTLC1 in patient derived fibroblast cultures shows normal subcellular localization in these cells, with an apparent distribution in the endoplasmic reticulum. Immunostaining for TDP43 also appears normal. Scale bar=40 μm b, Immunostaining for SPTLC1 in iPSC-derived human motor neurons with p.F40_S41 variant do not show any changes in its subcellular localization. Scale bar=20 μm c, Western blot of SPTLC1 in iPSC-derived human motor neurons confirms comparable expression levels in p.F40_S41 line and its isogenic control. Two independent differentiation experiments followed by western blotting were performed with similar results.
Extended Data Fig. 4 Induced pluripotent stem cell-derived human motor neurons (iPSC-hMN) with SPTLC1 ALS variants.
a, After differentiation by addition of doxycycline, SPTLC1 mutant iPSC-hMNs, one with heterozygous SPTLC1 p.F40_S41del variant and the other with compound heterozygous variants SPTLC1 p.F40_S41del; E2del (complete deletion of exon 2) and their isogenic control assume neuronal morphology. They express choline acetyltransferase (ChAT), Hb9 (a motor neuron specific transcription factor) as well as Tuj1 (neuronal microtubule marker). Scale bar = 20 μm b, Electron microscopy of iPSC-hMNs shows normal neuronal cell body morphology, nuclear heterochromatin, and organelles. In the processes (right), microtubules and occasional mitochondria and vesicles are noted. Scale bar left = 1 μm, middle and right = 200 nm. c, Sphingolipidomic analysis of de novo sphingolipid synthesis using isotope labelling (with 3,3-D2 L-serine) depicted in the form a heatmap with each row representing a sphingolipid species. Row Z-scores were calculated and depicted in colour. Canonical sphingolipids ceramides and glucosyl ceramides (glucCer) are increased in p.F40_S41del iPSC-hMNs compared to their isogenic control and increase further in the double mutant iPSC-hMNs. The bar graphs show the increase in total 2+ long-chain bases (LCB), ceramides (Cer), and glucosyl ceramides (GlucCer).
Extended Data Fig. 5 Allele specific knockdown of variant SPTLC1 mRNA.
a, The two allele-specific siRNAs targeting the L39del and F40_S41del alleles do not reduce SPTLC1 mRNA levels in control cells at concentrations as high as 100nM. Error bars are SEM. Four replicates were performed. b, Sphingolipidomic analysis of patient derived fibroblasts with SPTLC1 ALS variants (F40_S41del and two patient lines with L39del) after treatment with 10 nM allele-specific siRNA targeting each corresponding variant were analysed and compared to control fibroblasts. Following the knockdown, the cells were labeled with D3,15N-L-serine and D4-L-alanine. The de novo synthesized +3 ceramide and sphingomyelin levels show a reduction in patient cells when treated with variant-specific siRNAs. Error bars are SEM. Three replicates were performed.
Supplementary Tables 1–6.
Supplementary Video 1
Clinical findings on bedside neurologic evaluation of patients with SPTLC1-associated amyotrophic lateral sclerosis. Patient 1 (p.Y23F) and patient 3 (p.F40_S41del) are depicted, highlighting tongue fasciculations, severe upper extremity weakness and ankle clonus.
Supplementary Video 2
Skeletal muscle ultrasound in a patient with SPTLC1-associated amyotrophic lateral sclerosis. Ultrasound of patient 1 (p.Y23F) shows diffuse fasciculations. In addition, skeletal muscles appear atrophied and show increased echogenicity with a mixed granular and streaking pattern.
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Mohassel, P., Donkervoort, S., Lone, M.A. et al. Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis. Nat Med 27, 1197–1204 (2021). https://doi.org/10.1038/s41591-021-01346-1
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