Volume 129 Number 3 March 2019 conjugation of L-serine and palmitoyl-CoA, the rate-limiting step catalyzed by serine palmitoyltransferase (SPT). The immediate product 3-keto-sphinganine is reduced to sphinganine (SA), which is then N-acylated to dihydroceramide (dhCer) by 1 of 6 ceramide synthase isoforms (CerS1-6) (4). In the final step, dhCer is converted to ceramide by the insertion of a Δ4,5 trans (Δ4E) double bond into the SA backbone. This final conversion is catalyzed by the Δ4-dihydroceramide desaturase DEGS1 (5). On the catabolic side, ceramides are deacylated by ceramidases to form sphingosine (SO), which can be either recycled back to ceramides (sal-BACKGROUND. Sphingolipids are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling, and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids leads to various neurological pathologies; however, the entire spectrum of sphingolipid metabolism disorders remains elusive. METHODS.A combined approach of genomics and lipidomics was applied to identify and characterize a human sphingolipid metabolism disorder. RESULTS.By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous systems, we identified a homozygous p.Ala280Val variant in DEGS1, which catalyzes the last step in the ceramide synthesis pathway. The blood sphingolipid profile in the patient showed a significant increase in dihydro sphingolipid species that was further recapitulated in patient-derived fibroblasts, in CRISPR/Cas9-derived DEGS1-knockout cells, and by pharmacological inhibition of DEGS1. The enzymatic activity in patient fibroblasts was reduced by 80% compared with wild-type cells, which was in line with a reduced expression of mutant DEGS1 protein. Moreover, an atypical and potentially neurotoxic sphingosine isomer was identified in patient plasma and in cells expressing mutant DEGS1. CONCLUSION.We report DEGS1 dysfunction as the cause of a sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous systems.(OMIM #617575) (19-21), but also with axonal peripheral neuropathy without renal or adrenal deficiencies (22).Here, we identify DEGS1 dysfunction as the cause of an SL disorder with leukodystrophy and hypomyelination of the peripheral nervous system.

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile.

Hereditary neuropathies comprise a wide variety of chronic diseases associated to more than 80 genes identified to date. We herein examined 612 index patients with either a Charcot-Marie-Tooth phenotype, hereditary sensory neuropathy, familial amyloid neuropathy, or small fiber neuropathy using a customized multigene panel based on the next generation sequencing technique. In 121 cases (19.8%), we identified at least one putative pathogenic mutation. Of these, 54.4% showed an autosomal dominant, 33.9% an autosomal recessive, and 11.6% an X-linked inheritance. The most frequently affected genes were PMP22 (16.4%), GJB1 (10.7%), MPZ, and SH3TC2 (both 9.9%), and MFN2 (8.3%). We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4, AARS, BIC2, DHTKD1, FGD4, HK1, INF2, KIF5A, PDK3, REEP1, SBF1, SBF2, SCN9A, and SPTLC2 with a declining frequency. Thirty-four novel variants were considered likely pathogenic not having previously been described in association with any disorder in the literature. In one patient, two homozygous mutations in HK1 were detected in the multigene panel, but not by whole exome sequencing. A novel missense mutation in KIF5A was considered pathogenic because of the highly compatible phenotype. In one patient, the plasma sphingolipid profile could functionally prove the pathogenicity of a mutation in SPTLC2. One pathogenic mutation in MPZ was identified after being previously missed by Sanger sequencing. We conclude that panel based next generation sequencing is a useful, time- and cost-effective approach to assist clinicians in identifying the correct diagnosis and enable causative treatment considerations.

ObjectiveTo evaluate the safety and efficacy of l-serine in humans with hereditary sensory autonomic neuropathy type I (HSAN1).MethodsIn this randomized, placebo-controlled, parallel-group trial with open-label extension, patients aged 18–70 years with symptomatic HSAN1 were randomized to l-serine (400 mg/kg/day) or placebo for 1 year. All participants received l-serine during the second year. The primary outcome measure was the Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNS). Secondary outcomes included plasma sphingolipid levels, epidermal nerve fiber density, electrophysiologic measurements, patient-reported measures, and adverse events.ResultsBetween August 2013 and April 2014, we enrolled and randomized 18 participants, 16 of whom completed the study. After 1 year, the l-serine group experienced improvement in CMTNS relative to the placebo group (−1.5 units, 95% CI −2.8 to −0.1, p = 0.03), with evidence of continued improvement in the second year of treatment (−0.77, 95% CI −1.67 to 0.13, p = 0.09). Concomitantly, deoxysphinganine levels dropped in l-serine-treated but not placebo-treated participants (59% decrease vs 11% increase; p < 0.001). There were no serious adverse effects related to l-serine.ConclusionHigh-dose oral l-serine supplementation appears safe in patients with HSAN1 and is potentially effective at slowing disease progression.Clinicaltrials.gov identifierNCT01733407.Classification of evidenceThis study provides Class I evidence that high-dose oral l-serine supplementation significantly slows disease progression in patients with HSAN1.

ObjectivesHereditary sensory neuropathy type 1 (HSN1) is a rare, slowly progressive neuropathy causing profound sensory deficits and often severe motor loss. L-serine supplementation is a possible candidate therapy but the lack of responsive outcome measures is a barrier for undertaking clinical trials in HSN1. We performed a 12-month natural history study to characterise the phenotype of HSN1 and to identify responsive outcome measures.MethodsAssessments included Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), CMTNSv2-Rasch modified, nerve conduction studies, quantitative sensory testing, intraepidermal nerve fibre density (thigh), computerised myometry (lower limbs), plasma 1-deoxysphingolipid levels, calf-level intramuscular fat accumulation by MRI and patient-based questionnaires (Neuropathic Pain Symptom Inventory and 36-Short Form Health Survey version 2 [SF-36v2]).Results35 patients with HSN1 were recruited. There was marked heterogeneity in the phenotype mainly due to differences between the sexes: males generally more severely affected. The outcome measures that significantly changed over 1 year and correlated with CMTNSv2, SF-36v2-physical component and disease duration were MRI determined calf intramuscular fat accumulation (mean change in overall calf fat fraction 2.36%, 95% CI 1.16 to 3.55, p=0.0004), pressure pain threshold on the hand (mean change 40 kPa, 95% CI 0.7 to 80, p=0.046) and myometric measurements of ankle plantar flexion (median change −0.5 Nm, IQR −9.5 to 0, p=0.0007), ankle inversion (mean change −0.89 Nm, 95% CI −1.66 to −0.12, p=0.03) and eversion (mean change −1.61 Nm, 95% CI −2.72 to −0.51, p=0.006). Intramuscular calf fat fraction was the most responsive outcome measure.ConclusionMRI determined calf muscle fat fraction shows validity and high responsiveness over 12 months and will be useful in HSN1 clinical trials.

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Hereditary sensory and autonomic neuropathy 1 (HSAN1) is an autosomal dominant disorder that can be caused by variants in SPTLC1 or SPTLC2, encoding subunits of serine palmitoyl-CoA transferase. Disease variants alter the enzyme's substrate specificity and lead to accumulation of neurotoxic 1-deoxysphingolipids. We describe two families with autosomal dominant HSAN1C caused by a new variant in SPTLC2, c.547C>T, p.(Arg183Trp). The variant changed a conserved amino acid and was not found in public variant databases. All patients had a relatively mild progressive distal sensory impairment, with onset after age 50. Small fibers were affected early, leading to abnormalities on quantitative sensory testing. Sural biopsy revealed a severe chronic axonal neuropathy with subtotal loss of myelinated axons, relatively preserved number of non-myelinated fibers and no signs for regeneration. Skin biopsy with PGP9.5 labeling showed lack of intraepidermal nerve endings early in the disease. Motor manifestations developed later in the disease course, but there was no evidence of autonomic involvement. Patients had elevated serum 1-deoxysphingolipids, and the variant protein produced elevated amounts of 1-deoxysphingolipids in vitro, which proved the pathogenicity of the variant. Our results expand the genetic spectrum of HSAN1C and provide further detail about the clinical characteristics. Sequencing of SPTLC2 should be considered in all patients presenting with mild late-onset sensory-predominant small or large fiber neuropathy.

Hereditary sensory neuropathy type 1 (HSAN1) may be the first genetic neuropathy amenable to a specific mechanism-based treatment, as L-serine supplementation can be used to lower the neurotoxic levels of 1-deoxysphingolipids (1-deoxySL) that cause the neurodegeneration. The treatment is so far untested in HSAN1C caused by variants in the serine palmitoyl transferase subunit 2 (SPTLC2) gene. The aim of this study was to establish whether oral L-serine lowers 1-deoxySL in a patient with HSAN1C, to perform a dose escalation to find the minimal effective dose, and to assess the safety profile and global metabolic effects of the treatment. Our patient underwent a 52-wk treatment in which the L-serine dose was titrated up to 400 mg/kg/day. She was followed up by repeated clinical examination, nerve conduction testing, and skin biopsies to document effects on small nerve fibers. Serum was assayed for 1-deoxySL and metabolomics analysis of 111 metabolites. We found a robust lowering of 1-deoxySL, which correlated in a near-linear fashion with increased serum L-serine levels. Metabolomics analysis showed a modest elevation in glycine and a marked reduction in the level of cytosine, whereas most of the other assayed metabolites did not change. There were no direct side effects from the treatment, but the patient developed a transitory toe ulceration during the course of the study. The Charcot–Marie–Tooth neuropathy score increased by 1 point. We conclude that oral supplementation of L-serine decreases 1-deoxySL in HSAN1C without major global effects on metabolism. L-serine is therefore a potential treatment for HSAN1C.