A missense mutation in SLC33A1, which encodes the acetyl-CoA transporter, causes autosomal-dominant spastic paraplegia (SPG42)

Abstract

Hereditary spastic paraplegias (HSPs), characterized by progressive and bilateral spasticity of the legs, are usually caused by developmental failure or degeneration of motor axons in the corticospinal tract. There are considerable interfamilial and intrafamilial variations in age at onset and severity of spasticity. Genetic studies also showed that there are dozens of genetic loci, on multiple chromosomes, that are responsible for HSPs. Through linkage study of a pedigree of HSP with autosomal-dominant inheritance, we mapped the causative gene to 3q24-q26. Screening of candidate genes revealed that the HSP is caused by a missense mutation in the gene for acetyl-CoA transporter (SLC33A1). It is predicted that the missense mutation, causing the change of the highly conserved serine to arginine at the codon 113 (p. S113R), disrupts the second transmembrane domain in the transporter and reverses the orientation of all of the descending domains. Knockdown of Slc33a1 in zebrafish caused a curve-shaped tail and defective axon outgrowth from the spinal cord. Although the wild-type human SLC33A1 was able to rescue the phenotype caused by Slc33a1 knockdown in zebrafish, the mutant SLC33A1 (p.S113R) was not, suggesting that S113R mutation renders SLC33A1 nonfunctional and one that wild-type allele is not sufficient for sustaining the outgrowth and maintenance of long motor axons in human heterozygotes. Thus, our study illustrated a critical role of acetyl-CoA transporter in motor-neuron development and function.

Figure 1

The Pedigree of a Large Family with Hereditary Spastic Paraplegia and the Haplotypes of Markers Spanning the Linked Region on Chromosome 3q24-q26 Filled squares and circles indicate definitely affected, hatched squares indicate mildly affected. The haplotype segregating with HSP is boxed. SPG42 is flanked by D3S2326 and D3S3053.

Figure 2

Multipoint Linkage Analysis for Markers on Chromosome 3q24-q26

Figure 3

Mutation in SLC33A1 Causes HSP (A) The candidate region of 27.54 Mb between D3S2326 and D3S3053. The candidate genes screened are shown at the bottom. The location of SLC33A1 is highlighted. (B) Partial sequence chromatograms of exon 1 of SLC33A1 in a patient and a healthy control. The arrows mark the position of the SLC33A1 mutation. Sequence analysis was done on an ABI 3100 automated sequencer (Applied Biosystems). (C) Confirmation of the c.339T→G mutation by allele-specific tetra-primer PCR assay. All definitely affected and mildly affected individuals exhibited the G allele. Primers sequences are as follows: forward outer primer 5′-GTGCCCTTATCGCTCTGA-3′, reverse outer primer 5′-GTGTTATTTGATGGGTTGC-3′, forward inner primer (G allele) 5′-TTAGCTATACAGACCAAGCTTTCTTCGGG-3′, and reverse inner primer (T allele) 5′-TTGAGACTGAAGGGCCAAAAGACAGAA-3′. (D) Topological prediction of the mutant SLC33A1 protein, with use of the SOSUI program. The S113R mutation resulted in the loss of the original second TM motif and reversed the orientation of all domains, starting from 113. Upper panel, normal SLC33A1; bottom panel, mutant SLC33A1 (S113R). The mutated amino acid is indicated by the red arrow. (E) Amino acid sequence alignment of human SLC33A1 and orthologs from other species, showing phylogenetic conservation of S113. The sequences were retrieved from the Entrez protein database and aligned to each other with the use of Clustal W.

Figure 4

Rescue of slc33a1 Knockdown-Induced Abnormal-Tail Phenotype in Zebrafish by Wild-Type, But Not Mutant, Human SLC33A1 mRNA (A) Morphological features of zebrafish at 36 hpf from knockdown and rescue experiments. a, Untreated wild-type zebrafish; b, Zebrafish injected with mismatch control MO; c and d, Slc33a1 MO-injected zebrafish with severely curved tail (c) and slightly curved tail (d); e and f, zebrafish coinjected with slc33a1 MO and wild-type human SLC33A1 mRNA—the curly-tail phenotype was rescued completely (e) and partially (f); g and h, zebrafish coinjected with slc33a1 MO and mutated human SLC33A1 mRNA, with severely curved tail (g) or slightly curved tail (h). Scale bar: represents 1 mm. (B) Phenotype profile from zebrafish knockdown and rescue experiments. The embryos were classified as normal, slightly curly tail, severely curly tail, and lethality, and the percentage for each group is shown. (C) Defective motor-neuron outgrowth in the reduction of slc33a1. Spinal motor axons were stained with an anti-acetylated tubulin antibody at 36 hr after fertilization. a, motor neurons in spinal cord of zebrafish injected with mismatch control MO; b, injection of slc33a1 MO dramatically impaired outgrowth of motor axons from the spinal cord; c, coinjection of human wild-type SLC33A1 mRNA with slc33a1 MO rescued motor-axon defects; d, Coinjection of mutated human SLC33A1 mRNA did not rescue motor axon defects. Scale bar represents 50 μm.