case_images: PMC10771913_01
case_images: PMC10771913_02
case_images: Pedigree. Arrow = proband, black symbol = affected by SPG4, white symbols = not reported by proband to have difficulty walking, and white symbols with ? = unknown status. d = died, y = years.
case_images: CRIG2023-4553365.001.jpg
case_images: PMC10771913_02_CRIG2023-4553365.001.jpg
case_images: fig1
case_images: The pedigree is shown in Figure 1.
case_images: Sequencing data, conservation, and in silico modelling. (a) Sequencing data shown in integrative genomics viewer (Broad Institute) for the proband (upper panel) and a control (lower panel). The nucleotide substitution from adenine (A) to guanine (G) is shown in orange. (b) Evolutionary conservation of the amino acid affected by the spastin p.(Asp584Gly) variant across different species. The affected amino acid is shown in red. Blue indicates amino acids that are similar to the human sequence. Data from Alamut Visual Software (Sophia Genetics) and protein BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). (c) Spastin domain structure and paralog conservation of amino acids 571-597 among 14 related orthologues identified from the protein data bank (PDB). Alignment was created by VarSite (EMBL-EBI). The amino acids in the paralogue alignment are coloured by residue type. Shown below the alignment is the number of disease-causing variants at each position based on the p.(Asp584Gly) substitution reported in our case and reports in the human gene mutation database (HGMD) professional version 2022.2 (QiaGen). N-term = N-terminal sequence, MIT = microtubule interacting and trafficking domain, MTBD = microtubule-binding domain, and AAA = ATPase associated with various cellular activities. The asparagine (D) residue at position 584 is marked with a box. (d) Cartoon modelling of the human spastin hexamonomer. There is one colour for each monomer. The Asp584 residue is shown in red in each of the six monomers. (e) Localization of the p.Asp584 residue (dark red) and its interacting amino acids (orange). Hydrogen bonds (light blue) predicted with p.Arg578, p.Arg581, and p.Ser588. (f) Localization of the p.(Asp584Gly) substitution (dark red) and interacting amino acids (orange). Hydrogen bonds (light blue) are predicted to be formed with p.Ser588, but not with p.Arg578 and p.Arg581. The position of the new amino acid change within the structure was estimated using the Rotamers tool with standard parameters. The modelling in (d), (e), and (f) was performed using ChimeraX-1.5 on the spastin (PDB ID: 6PEN) crystal structure.
case_images: CRIG2023-4553365.002.jpg
case_images: PMC10771913_02_CRIG2023-4553365.002.jpg
case_images: fig2
case_images: NGS panel was based on Genomics England PanelApp (https://panelapp.genomicsengland.co.uk/) and included red and amber genes from the following panels: hereditary spastic paraplegia (version 1.276), hereditary spastic paraplegia:adult onset (version 1.88), and hereditary spastic paraplegia:childhood onset (version 2.121). NGS revealed a heterozygous variant in SPAST, c.1751A > G p.(Asp584Gly) (NM_014946.4) (Figure 2(a)). The p.(Asp584Gly) substitution targets an evolutionarily conserved residue in orthologues (Figure 2(b)) and paralogues (Figure 2(c)). The variant is localized to the AAA cassette domain (Figure 2(c)), a hot spot for pathogenic variation. In silico modelling of the spastin homohexamer, based on the spastin (Protein Data Bank (PDB) ID: 6PEN) crystal structure using ChimeraX-1.5, showed that the p.(Asp584Gly) is situated in the outer part of the protein (Figure 2(d)). The substitution from aspartic acid to glycine results in a side chain alteration from a negative charge to a single hydrogen atom predicted to disrupt hydrogen bonds with Arg578 and Arg581, potentially resulting in structural and/or functional effects (Figures 2(e), 2(f)).