Identification and Glycerol-Induced Correction of Misfolding Mutations in the X-Linked Mental Retardation Gene CASK

Abstract

The overwhelming amount of available genomic sequence variation information demands a streamlined approach to examine known pathogenic mutations of any given protein. Here we seek to outline a strategy to easily classify pathogenic missense mutations that cause protein misfolding and are thus good candidates for chaperone-based therapeutic strategies, using previously identified mutations in the gene CASK. We applied a battery of bioinformatics algorithms designed to predict potential impact on protein structure to five pathogenic missense mutations in the protein CASK that have been shown to underlie pathologies ranging from X-linked mental retardation to autism spectrum disorder. A successful classification of the mutations as damaging was not consistently achieved despite the known pathogenicity. In addition to the bioinformatics analyses, we performed molecular modeling and phylogenetic comparisons. Finally, we developed a simple high-throughput imaging assay to measure the misfolding propensity of the CASK mutants in situ. Our data suggests that a phylogenetic analysis may be a robust method for predicting structurally damaging mutations in CASK. Mutations in two evolutionarily invariant residues (Y728C and W919R) exhibited a strong propensity to misfold and form visible aggregates in the cytosolic milieu. The remaining mutations (R28L, Y268H, and P396S) showed no evidence of aggregation and maintained their interactions with known CASK binding partners liprin-α3 Mint-1, and Veli, indicating an intact structure. Intriguingly, the protein aggregation caused by the Y728C and W919R mutations was reversed by treating the cells with a chemical chaperone (glycerol), providing a possible therapeutic strategy for treating structural mutations in CASK in the future.

Comprehensive investigation of CASK mutations and other genetic etiologies in 41 patients with intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH)

“…Recent studies have shown that CASK mutations in male patients lead to a broad spectrum of phenotypes including MICPCH. While missense mutations corresponding to functional change of the CASK protein causes non-syndromic intellectual disability [ 17 – 19 ] or FG syndrome [ 20 ], a decrease in the normal expression appears to correlate with the manifestation of MICPCH [ 8 ]. The complete loss of CASK expression was associated with a severe MICPCH phenotype and likely caused the reduced viability or in utero lethality [ 4 , 8 ], which is consistent with the neonatal lethality of Cask knockout mice [ 21 ].…”

Phenotypic and molecular insights into CASK-related disorders in males

“…In general, hemizygous CASK variants in males of phenotype group (iii) have been suggested to represent hypomorphic alleles [ 10 , 11 ], altering one or more of the multiple CASK functions while preserving others. An impact on protein structure and/or function has been demonstrated for the pathogenic CASK alterations p.Y728C and p.W919R, while the nature of the deleterious impact on CASK function remains to be determined for p.R28L, p.Y268H and p.P396S [ 25 , 26 ]. The two splice mutations c.2129A > G and c.2521-2A > T likely lead to the production of CASK proteins lacking several amino acid residues [ 10 , 11 ].…”

Presynaptic dysfunction in CASK-related neurodevelopmental disorders

“…At the presynapse, CASK regulates the synaptic vesicle exocytosis and neuronal cell adhesion through a tripartite complex with VELI1 (LIN7A) and MINT1 (APBA1) and direct interaction with NRXN1 (NRXN1) (Butz et al, 1998;LaConte et al, 2016;Sudhof, 2012). The tripartite protein complex VELI1-MINT1-CASK is unaffected by XL-ID and MICPCH missense mutations (LaConte et al, 2014;LaConte et al, 2018). Instead, the CASK-NRXN1 interaction is disrupted, suggesting that the absence of this interaction is associated with the MICPCH phenotype.…”