Autosomal dominant transmission of complicated hereditary spastic paraplegia due to a dominant negative mutation of KIF1A, SPG30 gene

Abstract

KIF1A is a brain-specific anterograde motor protein that transports cargoes towards the plus-ends of microtubules. Many variants of the KIF1A gene have been associated with neurodegenerative diseases and developmental delay. Homozygous mutations of KIF1A have been identified in a recessive subtype of hereditary spastic paraplegia (HSP), SPG30. In addition, KIF1A mutations have been found in pure HSP with autosomal dominant inheritance. Here we report the first case of familial complicated HSP with a KIF1A mutation transmitted in autosomal dominant inheritance. A heterozygous p.T258M mutation in KIF1A was found in a Korean family through targeted exome sequencing. They displayed phenotypes of mild intellectual disability with language delay, epilepsy, optic nerve atrophy, thinning of corpus callosum, periventricular white matter lesion, and microcephaly. A structural modeling revealed that the p.T258M mutation disrupted the binding of KIF1A motor domain to microtubules and its movement along microtubules. Assays of peripheral accumulation and proximal distribution of KIF1A motor indicated that the KIF1A motor domain with p.T258M mutation has reduced motor activity and exerts a dominant negative effect on wild-type KIF1A. These results suggest that the p.T258M mutation suppresses KIF1A motor activity and induces complicated HSP accompanying intellectual disability transmitted in autosomal dominant inheritance.

Figure 1

Familial segregation of the KIF1A variant. Pedigree structure of a family with c.773C > T (p.Thr258Met) variant in KIF1A. Black symbols represent patients with KIF1A. Arrow indicates proband. Only participants in the study for whom DNA is available for analysis are numbered.

Figure 2

Axial T2-weighted magnetic resonance image of patients. (A, Patient II.1; B, Patient II.2; C, Patient II.3) MRI shows thinning of corpus callosum on axial T2- weighted image which is shown in yellow arrow.

Figure 3

Fluid-attenuated inversion recovery (FLAIR) magnetic resonance image of patients. (A, Patient II.1; B, Patient II.2; C, Patient II.3) FLAIR imaging shows ill-defined lesion of periventricular white matter which is shown in yellow circle.

Figure 4

Pathogenic mutation identified in KIF1A. (A) Direct sequencing of the proband and her family shows a novel heterozygous mutation of KIF1A gene, c.773C > T (p.Thr258Met). (B) The p.T258 amino acid is completely conserved across available different species which is shown in black box. Multiple amino acid sequence alignment was generated using Cluster Omega (http://www.ebi.ac.uk/Tools/msa/clustalo) and includes human (Homo sapiens; Q12756; UniprotKB), mouse (Mus musculus; E9QAN4; UniprotKB), chicken (Gallus gallus; F1NXK6; UniprotKB), Guinea pig (Cavia porcellus; H0VGT5; UniprotKB), Japanese rice fish (Oryzias latipes; H2L6W3; UniprotKB), and Mosquito (Anopheles sinensis; A0A084WAX1; UniprotKB).

Figure 5

Structural modeling of the KIF1A motor domain with p.T258M mutation. (A) Modelled structure of the KIF1A motor domain mutant (p.Thr258Met). The crystal structure of the motor domain from WT KIF1A in complex with ADP [PDB ID: 1I5S] is depicted as a cartoon representation. The ATP binding loop (P-loop), switch-1, and switch-2 are shown in green, deep blue, and purple, respectively. L11 and L12 (unmodelled) in switch-2 are also indicated. The ADP molecule is shown by the ball-and-stick model. The red box indicates a close-up view of the boxed region. The structure of the modelled mutant (p.Thr258Met) motor domain of KIF1A is shown in yellow. The conformational change in L11 predicted to be induced by the p.Thr258Met mutation is indicated by a red arrow, together with the structure of WT L11 shown in grey. (B,C) Electrostatic charge distribution patterns in the WT (B) and modelled mutant p.Thr258Met (C) motor domains of KIF1A, as shown by the surface model. Negative and positive charges are shown in red and blue, respectively. The regions of charge changes (L11) are indicated by red dotted circles.

Figure 6

Functional impact of p.T258M mutation on KIF1A motor activity. (A) KIF1A-MD-EGFP constructs were expressed in cultured hippocampal neurons and visualized by immunofluorescence staining with anti-EGFP antibody. WT KIF1A-MD accumulated in distal regions of the axons (arrow heads), but KIF1A-MD-T258M showed a considerably reduced peripheral accumulation. The neuronal cell bodies (arrow) and dendrites were visualized by immunofluorescence staining for MAP2. (B) Quantification of the peripheral accumulation of KIF1A-MD (mean ± s.e.m.). The distal distribution of KIF1A-MD was analyzed as described in the methods section. Significant decreases are indicated (***p < 0.0001: **p < 0.001: *p < 0.05). (C) Quantification of the proximal distribution of KIF1A-MD (mean ± s.e.m.). The proximal distribution of KIF1A-MD was analyzed by measuring the brightness of the proximal neurites (blue asterisk) and the soma (red asterisk) as described in the methods section. Significant decreases are indicated (***p < 0.0001: *p < 0.05).

Figure 7

Dominant negative effect of p.T258M mutation on KIF1A motor activity. (A) WT KIF1A-MD-EGFP showed considerably reduced peripheral accumulation when co-expressed with KIF1A-MD-T258M (arrow heads). Neuronal cell bodies (arrow) and dendrites were visualized by immunofluorescence staining for MAP2. (B) Quantification of the peripheral accumulation of KIF1A-MD proteins (mean ± s.e.m.). The distal distribution of KIF1A-MD was analyzed as described in Methods. Significant decreases are indicated (***p < 0.0001: *p < 0.05).