Genetic and functional analyses of SPTLC1 in juvenile amyotrophic lateral sclerosis

Abstract

Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of the motor system. Pathogenic variants in SPTLC1, encoding a subunit of serine palmitoyltransferase, cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), and have recently been associated with juvenile ALS. SPTLC1 variants associated with ALS cause elevated levels of sphinganines and ceramides. Reports on ALS associated with SPTLC1 remain limited. This study aimed to investigate the frequency of SPTLC1 variants in ALS and relevant clinical characteristics.

Methods: We analyzed whole-exome and whole-genome sequence data from 40 probands with familial ALS and 413 patients with sporadic ALS without previously identified causative variants. Reverse transcription polymerase chain reaction (RT-PCR) analysis and droplet digital PCR (ddPCR) were used to assess splicing and mosaicism, respectively. Plasma sphingolipid levels were quantified to analyze biochemical consequences.

Results: The heterozygous c.58G>A, p.Ala20Thr variant was identified in a 21-year-old Japanese female patient presenting with symmetric weakness which slowly progressed over 15 years. RT-PCR analysis showed no splice defects. Plasma sphingolipid levels in the patient were significantly increased compared to her asymptomatic parents. ddPCR revealed that the asymptomatic father harbored a mosaic variant with 17% relative mutant allele abundance in peripheral blood leukocytes.

Conclusions: We identified a pathogenic c.58G>A, p.Ala20Thr SPTLC1 variant in a patient with juvenile ALS, likely inherited from an asymptomatic parent with mosaicism. Lipid analysis results are consistent with previous findings on SPTLC1-associated ALS. Further studies are necessary to determine the clinical effect of mosaic variants of SPTLC1.

Keywords: SPTLC1; Juvenile amyotrophic lateral sclerosis; Mosaicism; Sphingolipids.

Fig. 1

a Pedigree chart of a patient with early-onset amyotrophic lateral sclerosis (ALS) carrying the serine palmitoyltransferase long chain base subunit 1 (SPTLC1) variant. Arrows indicate the proband. Filled and open symbols represent ALS-affected and ALS-unaffected individuals, respectively, and slashed symbols indicate deceased individuals. Squares represent males, and circles denote females. b Physical map of SPTLC1 and primers used for polymerase chain reaction analysis. Variants associated with juvenile ALS and HSAN1 are shown in red and black, respectively. Variants in the S331 residue, indicated in blue, demonstrate an overlapping phenotype of HSAN and motor neuron disease. c Magnetic resonance imaging (MRI) studies of the brain. Sagittal view of T1-weighted imaging (left), and axial view of susceptibility weighted imaging (middle, right) are shown

Fig. 2

Genetic analysis of pedigree 1. a Direct nucleotide sequence analysis of the proband, father, and mother, in pedigree 1. A heterozygous variant (c.58G>A, p.Ala20Thr) in serine palmitoyltransferase long chain base subunit 1 (SPTLC1) was identified in the proband. The electropherogram showed a low-amplitude signal of c.58G>A in the father, suggesting a mosaic variant. b Reverse transcription-polymerase chain reaction (PCR) analysis of the c.58G>A variant. Electrophoresis of complementary DNA (cDNA) products. cDNA derived from the c.58G>A variant showed one band corresponding to that of the wildtype allele (374 bp). A band corresponding to exon 2 skipping (266 bp) was not observed. c Subsequent direct nucleotide sequence analysis of the cDNA product. The whole length of exon 2 was preserved in the c.58G>A variant. d Results of droplet digital PCR analysis. A duplicate assay was conducted for each sample, corresponding to the two dots. Mutant allele frequencies were calculated as 0.17, 0.50, and 0.00 in the father, proband, and mother, respectively

Fig. 3

Results of the sphingolipid analysis. a Sphinganine (SA), b sphingosine (SO), c d18:1 sphingosine-1-phosphate (S1P), d total ceramides, including all lengths of fatty acid chains (Cer). SA, SO, d18:1 S1P, and Cer levels were significantly higher in the proband compared with the asymptomatic parents. d20:0 SA, d20:1 SO, deoxymethyl SO, and deoxy SO were not detected in any of the samples. ***p < 0.005, ****p < 0.0005

Fig. 4

Metabolic pathway of sphingolipids. Normal serine palmitoyltransferase (SPT) uses l-serine and palmitoyl-CoA as substrates to form 3-keto-dihydrosphingosine. Abnormal SPT associated with HSAN1 uses l-alanine instead of l-serine as substrates, resulting in deoxysphinganine formation. In variants associated with amyotrophic lateral sclerosis (ALS), the abnormal SPT results in excessive production of sphynganines and ceramides, instead of deoxyphinganine formation. ASA arylsulfatase A, CERK ceramide kinase, CERS ceramide synthase, CGT ceramide galactosyltransferase, CST cerebroside sulfotransferase, DEGS dehydroceramide desaturase, GCS glucosylceramide synthase, 3-KSR 3-ketosphinganine reductase, SGPP sphingosine 1-phosphate phosphatase, SPT serine palmitoyltransferase