Novel CAPN1 missense variants in complex hereditary spastic paraplegia with early-onset psychosis

Abstract

CAPN1-associated hereditary spastic paraplegia (SPG76) is a rare and clinically heterogenous syndrome due to loss of calpain-1 function. Here we illustrate a translational approach to the case of an 18-year-old patient who first presented with psychiatric symptoms followed by spastic gait, intention tremor, and neurogenic bladder dysfunction, consistent with a complex form of HSP. Exome sequencing showed compound-heterozygous missense variants in CAPN1 (NM_001198868.2: c.1712A>G (p.Asn571Ser)/c.1991C>T (p.Ser664Leu)) and a previously reported heterozygous stop-gain variant in RCL1. In silico analyses of the CAPN1 variants predicted a deleterious effect and in vitro functional studies confirmed reduced calpain-1 activity and dysregulated downstream signaling. These findings support a diagnosis of SPG76 and highlight that the psychiatric symptoms can precede the motor symptoms in HSP. Our results also suggest that multiple genes can potentially contribute to complex neuropsychiatric diseases.

Figure 1

Calpain‐1 structure with novel variants, variant distribution, and CADD PHRED scores. (A) The sites of predicted amino acid exchange in the PEF domain in the proband compared to wildtype. Mutated amino acids are colored in light red, interaction partners in light green, and hydrogen bonds in yellow. (B, upper panel) Schematic of the calpain‐1 primary protein structure. Disease‐associated variants identified in the literature are annotated along the protein with colored dots representing coding impacts. Novel variants identified in this report are labeled in red. (B, lower panel) CADD PHRED scores for all possible missense variants aligned to the CAPN1 protein structure. The recommended cut‐off for deleteriousness (20) is depicted as a red line. C2L, C2‐like domain; PC, protease core domains; PEF, large subunit of the penta‐EF‐hand domains. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Calpain‐1 pathway and results of functional studies. (A) Schematic of the calpain‐1 and calpain‐2 pathways. The left half depicts physiological calpain signaling. The right half shows the impact of the loss of calpain‐1 function and associated dysregulation of downstream signaling in SPG76. (B) Calpain‐1 and calpain‐2 activity are measured in fibroblast lysates using a fluorogenic assay (p = 0.0004). (C) Western blot for calpain‐1 and calpain‐2 (p = 0.67, p = 0.93). (D) Western blot for calpain‐1 substrate PHLPP1 (p = 0.0087). (E) WB for calpain‐2 substrate PTEN (p > 0.99). (F) Western blot for Akt and phospho‐(Ser473)‐Akt (p = 0.004). (G) Western blot of NSC117079‐ or DMSO‐treated fibroblasts for Akt and phospho‐Akt. Whole‐cell fibroblast lysates were used for all WB experiments. Molecular weights are provided in kilodaltons (kDa). C, control; CAPN1, calpain‐1; CAPN2, calpain‐2; P, proband. Values are shown as mean ± SD, (ns = not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001; [B–F] Mann–Whitney U test; [G] two‐way ANOVA with multiple comparisons and Tukey's post hoc analysis). [Colour figure can be viewed at wileyonlinelibrary.com]