Spinal muscular atrophy associated with progressive myoclonic epilepsy is caused by mutations in ASAH1

Abstract

Spinal muscular atrophy (SMA) is a clinically and genetically heterogeneous disease characterized by the degeneration of lower motor neurons. The most frequent form is linked to mutations in SMN1. Childhood SMA associated with progressive myoclonic epilepsy (SMA-PME) has been reported as a rare autosomal-recessive condition unlinked to mutations in SMN1. Through linkage analysis, homozygosity mapping, and exome sequencing in three unrelated SMA-PME-affected families, we identified a homozygous missense mutation (c.125C>T [p.Thr42Met]) in exon 2 of ASAH1 in the affected children of two families and the same mutation associated with a deletion of the whole gene in the third family. Expression studies of the c.125C>T mutant cDNA in Farber fibroblasts showed that acid-ceramidase activity was only 32% of that generated by normal cDNA. This reduced activity was able to normalize the ceramide level in Farber cells, raising the question of the pathogenic mechanism underlying the CNS involvement in deficient cells. Morpholino knockdown of the ASAH1 ortholog in zebrafish led to a marked loss of motor-neuron axonal branching, a loss that is associated with increased apoptosis in the spinal cord. Our results reveal a wide phenotypic spectrum associated with ASAH1 mutations. An acid-ceramidase activity below 10% results in Farber disease, an early-onset disease starting with subcutaneous lipogranulomata, joint pain, and hoarseness of the voice, whereas a higher residual activity might be responsible for SMA-PME, a later-onset phenotype restricted to the CNS and starting with lower-motor-neuron disease.

Figure 1

Pedigrees and Linkage Analysis in SMA-PME-Affected Families (A) Pedigrees of SMA-PME-affected families. The numbers denote individuals whose DNA samples were available for genetic analysis. Linkage analysis and homozygosity mapping were performed in families D and ITA. (B) Combined results of linkage analysis of families D and ITA show a maximum LOD score Z_max = 2.7 at θ = 0.0. The x axis represents genetic distance in cM, and the y axis represents LOD scores.

Figure 2

ASAH1 Mutations Identified in SMA-PME-Affected Families (A) Sanger sequencing of ASAH1 exon 2 (genomic DNA) shows (1) a homozygous (or hemizygous) c.125C>T mutation in affected individuals of the three families (arrow); (2) a heterozygous c.125C>T mutation in both parents of families D and ITA and the mother of family ITB; and (3) a WT allele only in the father of family ITB. Reverse strands are shown. (B) Copy-number and SNP analysis of the affected child in family ITB shows a heterozygous deletion of the whole gene. Dots indicate copy number and SNP copy number (1 or 2). Vertical bars indicate copy number (dark green) or SNPs (light green). The horizontal red rectangular box indicates the physical position of the heterozygous deletion.

Figure 3

Immunoblot Analysis of ASAH1 (A) Farber fibroblasts were either not tranfected (“NT”) or cotransfected with LacZ and pcDNA5/TO expression vectors carrying the ASAH1 cDNA or not (“Empty”). The pcDNA5/TO-ASAH1 (“ASAH1”), pcDNA5/TO-ASAH1-C2 (“C2”), and pcDNA5/TO-ASAH1-C3 (“C3”) plasmids carry the WT sequence, whereas the pcDNA5/TO-ASAH1-P4 (“P4”) and pcDNA5/TO-ASAH1-P6 (“P6”) plasmids carry the mutant (c.125C>T) sequence. Forty-eight hours later, cell extracts were analyzed by immunoblotting with acid-ceramidase and β-actin antibodies. (B) The density of the bands corresponding to the precursor form (55 kDa), the β-subunit (40 kDa), and the α-subunit (13 kDa) of acid ceramidase was normalized to that of β-actin and compared to those of the WT protein (“ASAH1”). Note the reduced amount of the α-subunit in cells transfected with mutant cDNA. (C) The acid-ceramidase and bacterial β-galactosidase enzyme activities were determined on the same cell lysates as described in Table 1.

Figure 4

Morpholino Knockdown of asah1b in Zebrafish (A–C) Morphology of living embryos at 52 hpf. WT embryos, control-5-mis-MO-injected embryos, and embryos injected with 0.6 pmole of asah1b-MO (asah1b morphants) are shown. Note the curved body shape that asah1b morphants show in comparison to the body shape of both controls. Views are lateral, and the dorsal sides are at the top. The scale bar represents 200 μm. (D–F) Side views of 48 hpf living zebrafish embryos stained with acridine orange (D); some single acridine-orange-positive neurons are depicted by asterisks in WT embryos, control-5-mis-MO-injected embryos (E), or asah1b morphants (F). Asah1b morphants show substantial cell death in the spinal cord (dashed lines), whereas both controls show only a low, basal number of apoptotic cells. The scale bar represents 100 μm. The following abbreviation is used: sc, spinal cord. (G) Bar chart depicting the mean number (± SD) of acridine-orange-positive cells per surface unit for WT (2.66 ± 0.74), control 5 mis MO (3.83 ± 1.34), and asah1b morphants (16.3 ± 2.5) at 48 hpf. Data represent mean ± SD. ^∗∗∗p < 0.0001. (H–M) The asah1b morpholino decreases motor-axon collateral formation. Lateral views of znp1-antibody staining of controls (H and I) and asah1b morphants (J) are shown at 48 hpf. znp1 labels motor axons (H, arrowheads). Tracings of motor-axon tracts ventral to the spinal cord (K–M) are derived from panels directly above. Scale bars represent 50 μm. The following abbreviations are used: D, dorsal; V, ventral; A, anterior; and P, posterior. (N–P) Lateral views of acetylated-tubulin-expressing axons of the PLLn (arrow) in WT, control-5-mis-MO-injected embryos, and asah1b morphants at 48 hpf. Axonal growth is similar in all groups; the PLLn reaches the tip end of the tail (arrows, n = 10 per group). (Q) Bar chart depicting the mean number (± SD) of axon branches per fascicle for WT (18 ± 1.59), control-5-mis-MO-injected embryos (17.8 ± 1.5), and asah1b morphants (6 ± 2.2) at 48 hpf. Compared with both controls, Asah1b morphants show a significant decrease in axonal branching. ^∗∗∗p < 0.0001.