Recurrent de novo SPTLC2 variant causes childhood-onset amyotrophic lateral sclerosis (ALS) by excess sphingolipid synthesis

Abstract

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the upper and lower motor neurons with varying ages of onset, progression and pathomechanisms. Monogenic childhood-onset ALS, although rare, forms an important subgroup of ALS. We recently reported specific SPTLC1 variants resulting in sphingolipid overproduction as a cause for juvenile ALS. Here, we report six patients from six independent families with a recurrent, de novo, heterozygous variant in SPTLC2 c.778G>A [p.Glu260Lys] manifesting with juvenile ALS.

Methods: Clinical examination of the patients along with ancillary and genetic testing, followed by biochemical investigation of patients' blood and fibroblasts, was performed.

Results: All patients presented with early-childhood-onset progressive weakness, with signs and symptoms of upper and lower motor neuron degeneration in multiple myotomes, without sensory neuropathy. These findings were supported on ancillary testing including nerve conduction studies and electromyography, muscle biopsies and muscle ultrasound studies. Biochemical investigations in plasma and fibroblasts showed elevated levels of ceramides and unrestrained de novo sphingolipid synthesis. Our studies indicate that SPTLC2 variant [c.778G>A, p.Glu260Lys] acts distinctly from hereditary sensory and autonomic neuropathy (HSAN)-causing SPTLC2 variants by causing excess canonical sphingolipid biosynthesis, similar to the recently reported SPTLC1 ALS associated pathogenic variants. Our studies also indicate that serine supplementation, which is a therapeutic in SPTLC1 and SPTCL2-associated HSAN, is expected to exacerbate the excess sphingolipid synthesis in serine palmitoyltransferase (SPT)-associated ALS.

Conclusions: SPTLC2 is the second SPT-associated gene that underlies monogenic, juvenile ALS and further establishes alterations of sphingolipid metabolism in motor neuron disease pathogenesis. Our findings also have important therapeutic implications: serine supplementation must be avoided in SPT-associated ALS, as it is expected to drive pathogenesis further.

Keywords: ALS; BIOCHEMISTRY; MOTOR NEURON DISEASE; NEUROGENETICS; NEUROMUSCULAR.

Figure 1

Clinical phenotypic characteristics of SPTLC2 patients. (A) Patient P1 showing spasticity and toe walking. (B) P5 with significant axial (neck extension) weakness. (C) Patient P4 showing positive Babinski sign. (D) Patient P4 and P6 showing scalloped tongue (with active fasciculations on clinical examination). (E) Ultrasound of vastus lateralis (VL) muscle in an unaffected control (normal) compared with patient P1, which shows increased echogenicity with a characteristic ‘streaky’ pattern suggestive of neurogenic atrophy. Similar changes are noted in tibialis anterior muscle (TA) in patient P2. (F=Femur, T=Tibia). (F) Muscle biopsy of patient P1 stained with H&E and showing a group of atrophic muscle fibres (arrow). Scale bar=50 um. (G) ATPase 4.3 stain in P1 muscle biopsy shows severe chronic neurogenic changes with fibre type grouping and holofascicular atrophy (arrow). Note the adjacent fascicle with hypertrophic fibres (arrowhead). Scale bar=1—um. (H) Nicotinamide adenine dinucleotide stain in patient P5 showing targetoid fibres (arrow) indicative of neurogenic changes in muscle fibres.

Figure 2

Pedigrees, schematic mapping of SPTLC2 E260K variant and in silico cryo-EM structure of SPT enzyme complex. (A) Pedigree of the families of six patients with de novo SPTLC2 E260K variants. (B) Schematic of SPTLC2 and mapping of HSAN (black) and E260K (red) variants. (C) E260 is highly evolutionarily conserved across different species. (D) Cryo-EM structure of SPT enzyme complex showing its subunits, SPTLC1, SPTLC2 and ssSPTa in complex with ORMDL3. Note the direct contact of E260 residue of SPTLC2 with H7/S8 of ORMDL3 subunit. HSAN, hereditary sensory and autonomic neuropathy; SPT, serine palmitoyltransferase.

Figure 3

Sphingolipidomic profiling of E260K patient plasma. (A) Heat map cluster analysis of SL species from plasma of E260K patients compared with the controls and patients with SPTLC1-HSAN1 (C133W) and ALS (F40_S41del) variants. Absolute levels of each SL species were measured relative to internal lipid standard. Shown is the plot of the Log transformed (base 10) data with Pearson distance measure. Comparison of total plasma dihydroceramide (dhCeramide) and ceramides (B), and 1-deoxydihydroceramides (1-deoxydhceramide) and 1-deoxyceramides (C) from SPTLC2 E260K patients, related controls, SPTLC1 HSAN1 (C133W) and SPTLC1-ALS (F40_S41del) patients. For bar graphs, data are represented as mean ± SD, n=4 independent replicates, one-way ANOVA with Dunnett adjustment was used for comparisons, * p<0.05, * p<0.05, *** p<0.001**** p<0.0001, n.s, not significant. ALS, amyotrophic lateral sclerosis.

Figure 4

Spingolipidomic analysis showing increased synthesis of canonical sphingolipids in E260K patient fibroblast lines. (A) Serine palmitoyltransferase (SPT) catalyses the formation of sphingolipids by condensing serine and fattyacyl CoAs (typically palmitoyl CoA). SPTLC2 E260K variants, similar to SPTLC1-ALS variants, result in unrestrained production of canonical sphingolipids (sphinganine and ceramides) and decreased ORMDL/ceramide mediated inhibition. HSAN1-causing variants increase alanine (or glycine) usage by SPT leading to increased synthesis of 1-deoxysphingolipids. (B) Heatmap representation of individual sphingolipid species in SPTLC2 ALS patient fibroblast lines (E260K) after stable radioisotope labelling with equimolar D2-serine and D4-alanine show increased production of canonical sphingolipids such as long chain bases (LCBs), ceramides, and to a lesser extent sphingomyelins and glucosyl ceramides. In contrast, there is no overproduction of 1-deoxysphingolipids (1-deoxySLs). (C) Total levels of de novo synthesised dihydrosphingosine (DHS), dihydroceramide (dhCer), Ceramide (Cer), 1-deoxyDHS (1-dDHS), 1-deoxydihydroceramide (1-ddhCer) and 1-deoxyceramide (1-dCer) in patient fibroblasts suggest a nearly twofold overproduction of canonical sphingolipids without notable overproduction of deoxySLs. Each data point represents a technical replicate of the control or patient fibroblasts from P1 and P5. (D) Addition of serine to the media exacerbates the overproduction of canonical sphingolipids, suggesting lack of homoeostatic negative feedback regulation of variant SPTLC2-containing SPT. Serine supplementation suppresses the 1-deoxySL production in E260K fibroblast lines, suggesting the overactivity is specific to canonical SL species. (E) SPT activity in control and patient derived fibroblasts after treatment with increasing doses of cell permeable C₆-Ceramide (C₆-Cer). The activities were recorded as total labelled SL produced relative to vehicle treated cells. SL de novo synthesis and activity measurements were performed in presence of D₃,¹⁵N-L-Ser and D₄-L-Ala. HSAN, hereditary sensory and autonomic neuropathy type 1.