De novo non-synonymous DPYSL2 (CRMP2) variants in two patients with intellectual disabilities and documentation of functional relevance through zebrafish rescue and cellular transfection experiments

Abstract

Abstract Collapsin response mediator protein 2 (Crmp2) is an evolutionarily well-conserved tubulin-binding cytosolic protein that plays critical roles in the formation of neural circuitry in model organisms including zebrafish and rodents. No clinical evidence that CRMP2 variants are responsible for monogenic neurogenic disorders in humans presently exists. Here, we describe two patients with de novo non-synonymous variants (S14R and R565C) of CRMP2 and intellectual disability associated with hypoplasia of the corpus callosum. We further performed various functional assays of CRMP2 variants using zebrafish and zebrafish Crmp2 (abbreviated as z-CRMP2 hereafter) and an antisense morpholino oligonucleotide [AMO]-based experimental system in which crmp2-morphant zebrafish exhibit the ectopic positioning of caudal primary (CaP) motor neurons. Whereas the co-injection of wild-type z-CRMP2 mRNA suppressed the ectopic positioning of CaP motor neurons in Crmp2-morphant zebrafish, the co-injection of R566C or S15R, z-CRMP2, which corresponds to R565C and S14R of human CRMP2, failed to rescue the ectopic positioning. Transfection experiments of zebrafish or rat Crmp2 using plasmid vectors in HeLa cells, with or without a proteasome inhibitor, demonstrated that the expression levels of mutant Crmp2 protein encoded by R565C and S14R CRMP2 variants were decreased, presumably because of increased degradation by proteasomes. When we compared CRMP2-tubulin interactions using co-immunoprecipitation and cellular localization studies, the R565C and S14R mutations weakened the interactions. These results collectively suggest that the CRMP2 variants detected in the present study consistently led to the loss-of-function of CRMP2 protein and support the notion that pathogenic variants in CRMP2 can cause intellectual disabilities in humans.