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Case Reports
. 2017 Feb 7;88(6):533-542.
doi: 10.1212/WNL.0000000000003595. Epub 2017 Jan 11.

Sphingosine 1-phosphate lyase deficiency causes Charcot-Marie-Tooth neuropathy

Derek Atkinson  1 , Jelena Nikodinovic Glumac  1 , Bob Asselbergh  1 , Biljana Ermanoska  1 , David Blocquel  1 , Regula Steiner  1 , Alejandro Estrada-Cuzcano  1 , Kristien Peeters  1 , Tinne Ooms  1 , Els De Vriendt  1 , Xiang-Lei Yang  1 , Thorsten Hornemann  1 , Vedrana Milic Rasic  2 , Albena Jordanova  2
Affiliations
Case Reports

Sphingosine 1-phosphate lyase deficiency causes Charcot-Marie-Tooth neuropathy

Derek Atkinson et al. Neurology. .

Abstract

Objective: To identify the unknown genetic cause in a nuclear family with an axonal form of peripheral neuropathy and atypical disease course.

Methods: Detailed neurologic, electrophysiologic, and neuropathologic examinations of the patients were performed. Whole exome sequencing of both affected individuals was done. The effect of the identified sequence variations was investigated at cDNA and protein level in patient-derived lymphoblasts. The plasma sphingoid base profile was analyzed. Functional consequences of neuron-specific downregulation of the gene were studied in Drosophila.

Results: Both patients present an atypical form of axonal peripheral neuropathy, characterized by acute or subacute onset and episodes of recurrent mononeuropathy. We identified compound heterozygous mutations cosegregating with disease and absent in controls in the SGPL1 gene, encoding sphingosine 1-phosphate lyase (SPL). The p.Ser361* mutation triggers nonsense-mediated mRNA decay. The missense p.Ile184Thr mutation causes partial protein degradation. The plasma levels of sphingosine 1-phosphate and sphingosine/sphinganine ratio were increased in the patients. Neuron-specific downregulation of the Drosophila orthologue impaired the morphology of the neuromuscular junction and caused progressive degeneration of the chemosensory neurons innervating the wing margin bristles.

Conclusions: We suggest SPL deficiency as a cause of a distinct form of Charcot-Marie-Tooth disease in humans, thus extending the currently recognized clinical and genetic spectrum of inherited peripheral neuropathies. Our data emphasize the importance of sphingolipid metabolism for neuronal function.

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Figures

Figure 1
Figure 1. Pedigree and clinical features of the studied autosomal recessive Charcot-Marie-Tooth 2 family
(A) Family tree. Square symbols represent males and circles females. Black symbols indicate affected individuals. (B) Serial images of the patients. Patient II-1 at age 32 presents with unilateral mild hypotrophy and weakness of the muscles in the first interosseal space, bilateral atrophy, and severe weakness (Medical Research Council scale 0–2) of all distal leg muscles. Patient II-2 at age 28 has normal appearance of the distal muscles of the hands, unilateral severe weakness of hallux extensor, and normal standing on tiptoe.
Figure 2
Figure 2. Sphingosine 1-phosphate (S1P) lyase (SPL): Transcript, protein, and metabolite analysis
(A) Electropherograms around the 2 mutations in SGPL1 (c.551T>C and c.1082C>G) upon analyzing genomic DNA (gDNA) or complementary DNA (cDNA) isolated from peripheral mononuclear blood cells, before and after cycloheximide (CHX) treatment. (B) Crystal structure of the dimeric human sphingosine 1-phosphate lyase, adapted from pdb 4Q6R. The 2 subunits (A, B) of the dimeric protein are displayed in tan and blue ribbons, respectively. Top right: zoom in view shows the Ile184 residue that is surrounded by 7 other hydrophobic residues (i.e., Ala163, Phe167, Leu181, Ala402, Trp405 from subunit A; Leu130, Tyr133 from subunit B) to constitute a hydrophobic core. Bottom right: the p.Ile184Thr mutation substitutes the long aliphatic side chain of isoleucine with a small and polar hydroxyl group of threonine in the middle of the hydrophobic core. (C) Immunoblotting analysis of SPL in 20 μg of protein lysates of lymphoblast cultures from all family members and 7 healthy control individuals. β-actin is used as loading control. Chemiluminescence exposure time of 60 seconds. (D) Immunoblotting analysis of SPL in 30 μg of protein lysates of lymphoblasts of patient II-2 incubated with vehicle alone (DMSO), with the MG-132 proteasome inhibitor, or nontreated (NT). SPL accumulates in a time-dependent manner in cells from the patient, compared to the control. β-actin demonstrates equal loading. Chemiluminescence exposure time of 600 seconds. (E) S1P levels in plasma of patents, patients, and unrelated controls (n = 24). (F) Total sphingosine/sphinganine (SO/SA) ratio after hydrolysis in plasma of patients, parents, and unrelated controls (n = 3). (E, F) Error bars represent the SD. ***p < 0.001; ns = nonsignificant (one-way analysis of variance with Bonferroni multiple comparison test against the control group).
Figure 3
Figure 3. Analysis of neuron-specific Sply deficiency in Drosophila
(A) Immunolabeling of presynaptic (anti-HRP antibody, green) and postsynaptic (anti-DLG antibody, magenta) compartments of the neuromuscular junctions (NMJs) in third instar larvae, pan-neuronally (nsyb-Gal4) expressing RNAi constructs against Sply (Sply knockdown; RNAiSply[1], RNAiSply[2]), neuronally relevant genes (RNAiSmn, RNAiMyc), genes not expressed in neurons (RNAi controls; RNAiCup, RNAiS-Lap5[1], RNAiS-Lap[2]), or the driver alone (control genetic background; nsyb-Gal4/+). Scale bar, 20 μm. (B–D) Quantitative analysis of the NMJ phenotypes. Maximum intensity projections of Z-stacks comprising the full NMJ were used. The number of boutons per NMJ (B) was counted using the Cell Counter plugin. ImageJ was used to count the number of individual branch segments (C) and calculate the total NMJ length (D). Error bars represent the SD of at least 13 NMJs per genotype. *p < 0.05; ***p < 0.001; ns = nonsignificant (one-way analysis of variance with Bonferroni multiple comparison test against the control genetic background). (E) Nerve tract along the wing L1 vein visualized by mCherry, where RNAi constructs are expressed using the dpr-Gal4 driver. Representative images of wings are shown for day 1 and day 20+ old flies expressing the driver alone (Dpr-Gal4/+), neuronally relevant gene (RNAiSmn), or Sply (RNAiSply[1]). Scale bar, 20 μm. (F) Quantification of the number of wings showing axonal fragmentation per genotype (n = 30–60/genotype).
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