Amplifying the spectrum of SPAST gene mutations

Abstract

Hereditary spastic paraplegias (HSPs) include a group of neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower extremities, caused by axon degeneration of corticospinal tracts. Spastic paraplegia type 4 (SPG4) is the most common autosomal dominant form of HSP and is caused by mutations in the SPAST gene. SPAST gene encodes for the protein spastin, a member of the ATPases Associated with a variety of cellular Activity (AAA) family.We describe a newly variant in SPAST gene, within an Italian family affected by pure HSP. In particular, we found a heterozygous intragenic microdeletion of 3T in exon 13 of SPG4 gene. The 3T deletion results in a mutated protein with a unique leucine residues deletion at the protein position 508, in the AAA ATPase domain. This variant is not registered in any public database either as rare normal variant nor as mutation in SPAST gene and the importance of this aminoacid is confirmed by the absolute conservation in multiple alignments with diverse species. We conclude that the novel SPAST gene variant identified is probably pathogenic and destabilizes the precise arrangement of the nucleotide binding domain, with a consequent loss-of-function of the mutated spastin protein.

Figure 1.

Family tree of the SPG4 family. Affected individuals are shown as filled symbols and unaffected individuals as unfilled symbols. The black arrow shows the proband (subject II.1)

Figure 2.

The results obtained with the Multiplex Ligation-dependent Probe Amplification, DNA sequence analysis and Single Base probe Extension tests for healthy control and affected proband are shown. A left) MLPA analysis of the SPAST gene of a healthy control showed no deletion/duplication of SPAST gene exons. A right) MLPA analysis of the SPAST gene of the proband showed heterozygous deletion of exon 13 (yellow arrow). B left and right) The coding sequence of exon 13 is shown (top left, bold letters underlined) as well as the 3T (red) deleted in the family. The partial electropherograms (forward and revers) of exon 13 of a healthy control (left) and the SPG4 affected proband (right) are shown. The rectangle shows the deleted 3T in the family. Seq. For = Forward DNA sequence; Seq. Rev = Reverse DNA sequence; W.T. allele = wild type allele; MUT. Allele = mutant allele. C left and right) The results of the SBE assay of a healthy control (left) and the affected proband (right) are shown. At the bottom of the 2C panel on the left and right respectively are represented schematically the expected single base elongated in forward and revers SBE assays for the wild type and the mutated exon 13.

Figure 3.

Figure 3: Structure of the AAA domain of the human spastin and alignment of different species. A) The Amino acid sequence of protein spastin resulting from the 3T deletion in exon 13 is represented (top of the panel). Cartoon representation of the AAA domain structure of the human spastin (pdb id.: 3vfd) (bottom of the panel). The nucleotide binding domain is highlighted in yellow. L508 = Leu 508, P383 =Pro 383 and N386 = Asn386 are shown in sticks. Details of the spatial arrangement of the three aminoacids are shown in the right panel. B) Alignment of the spastins from different species. The Swiss-Prot codes indicate the following: Human: homo sapiens; Pig: Sus scrofa; Bovin: Bos taurus; Mouse: Mus musculus; Rat: rattus norvegicus; Chick: Gallus gallus; Xentr: Xenopus Tropicalis; Xenla: Xenopus Laevis; Danre: Danio rerio; Drome: Drosophila melanogaster; Anoga: Anopheles gambiae; Ixosc: Ixodes scapularis; Nemve: Nematostella vectensis; Dicdi: Dictyostelium discoideum. The red rectangle evidences the position of the conserved Leu508 in the ATPase domain.