Endocannabinoid dysfunction in neurological disease: neuro-ocular DAGLA-related syndrome

Abstract

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.

Keywords: developmental delay; endocannabinoid; episodic ataxia; nystagmus; paroxysmal tonic upgaze.

Figure 1

DAGLA variants and domains. Schematic of DAGLA (bottom box) with exon–exon boundaries (dotted lines) and codon position (top). Frameshift and nonsense variants [orange (C1,3,7,8) and red (C2, 4, 5, 6, 9) lollipops, respectively] disrupt HOMER motif (green, ‘H’) and phosphorylation sites (purple ‘P’) but leave the active domain (red ‘A’) intact.

Figure 2

Immunofluorescence studies. Representative photomicrographs of immunofluorescence for DAGLA in HEK293T cells transfected with either wild-type (WT) or mutant (Mut) DAGLA cDNA (A) and in patient-derived fibroblasts (P) and control (C) cells from an unaffected individual (B). DAPI was used for nuclear counterstain. Scale bars = 50 µm. Accompanying bar graphs represent quantification of the number of cells with perinuclear (perinucl) aggregation (arrows) or membrane-bound (membr) DAGLA. Graphs: bars indicate the mean and error bars the standard deviation. Two-tailed Student’s t-test determined P-values.