Atypical juvenile presentation of GM2 gangliosidosis AB in a patient compound-heterozygote for c.259G > T and c.164C > T mutations in the GM2A gene

Abstract

G_M2-gangliosidosis, AB variant is an extremely rare autosomal recessive inherited disorder caused by mutations in the GM2A gene that encodes G_M2 ganglioside activator protein (GM2AP). GM2AP is necessary for solubilisation of G_M2 ganglioside in endolysosomes and its presentation to β-hexosaminidase A. Conversely GM2AP deficiency impairs lysosomal catabolism of G_M2 ganglioside, leading to its storage in cells and tissues. We describe a 9-year-old child with an unusual juvenile clinical onset of G_M2-gangliosidosis AB. At the age of 3 years he presented with global developmental delay, progressive epilepsy, intellectual disability, axial hypertonia, spasticity, seizures and ataxia, but without the macular cherry-red spots typical for G_M2 gangliosidosis. Brain MRI detected a rapid onset of diffuse atrophy, whereas whole exome sequencing showed that the patient is a compound heterozygote for two mutations in GM2A: a novel nonsense mutation, c.259G > T (p.E87X) and a missense mutation c.164C > T (p.P55L) that was recently identified in homozygosity in patients of a Saudi family with a progressive chorea-dementia syndrome. Western blot analysis showed an absence of GM2AP in cultured fibroblasts from the patient, suggesting that both mutations interfere with the synthesis and/or folding of the protein. Finally, impaired catabolism of G_M2 ganglioside in the patient's fibroblasts was demonstrated by metabolic labeling with fluorescently labeled G_M1 ganglioside and by immunohistochemistry with anti-G_M2 and anti-G_M3 antibodies. Our observation expands the molecular and clinical spectrum of molecular defects linked to G_M2-gangliosidosis and suggests novel diagnostic approach by whole exome sequencing and perhaps ganglioside analysis in cultured patient's cells.

Keywords: GM2 ganglioside; GM2 ganglioside activator protein; Gangliosidosis; Lysosomal storage disease.

Fig. 1

GM2AP is not detected in the patient's cultured fibroblasts by Western blot. Either 40 or 50 μg of total protein extracted from cultured fibroblasts of the patient or normal healthy controls (N = 2) were resolved on SDS PAGE gels, transferred to nitrocellulose membrane and hybridized with anti-GM2AP antibody. A mature ~ 20 kDa GM2AP band was detected in control fibroblasts but not in the patient's fibroblasts. The two bands detected in the protein extract of Cos7 cells transfected with a plasmid encoding GM2AP correspond to the mature ~ 20 kDa protein and its ~ 22 kDa precursor. α-Tubulin was used as a loading control.

Fig. 2

Thin-layer chromatography of fluorescently labeled gangliosides reveals accumulation of G_M2 ganglioside in the patient's cultured fibroblasts. Fibroblasts of the patient or a normal healthy control subject were cultured in DMEM containing 1.45 μM of fluorescently labeled BODIPY® FL C5 G_M1 ganglioside for 72 h. Total lipids were extracted from cell pellets with 1:1 chloroform/methanol mixture and the gangliosides were separated from neutral lipids and analysed by TLC on silica plates. (A) The fluorescence image of the TLC plate acquired on G:Box Chemi XQR system. The position of fluorescent G_M1 ganglioside used as a standard is shown. (B) G_M1 and G_M2 ganglioside bands were quantified in control and patient's fibroblasts by ImageJ software.

Fig. 3

Increased lysosomal G_M2 and reduced G_M3 ganglioside in the patient's cultured fibroblasts. (A) Fibroblasts from the patient and two normal healthy controls cultured on glass coverslips were fixed and stained with monoclonal humanized anti-G_M2 and mouse anti-LAMP-2 antibodies followed by anti-human IgG Alexa 488-labeled (green) and anti-mouse IgG Alexa 555-labeled (red) secondary antibodies. Nuclei were stained with Draq5 (blue). The patient's fibroblasts show a higher intensity of G_M2 staining (green) than control fibroblasts and increased co-localization of G_M2 and LAMP-2 staining. (B) Fibroblasts from the patient and two normal healthy controls were stained with monoclonal mouse anti-G_M3 antibody followed by anti-mouse IgG Alexa-555-labeled secondary antibody (red). Nuclei were stained with Draq5 (blue). The images were acquired in a Leica SP8 confocal microscope with a 63 × objective. Bar represents 10 μM. The panels show representative images of at least 40 studied for each cell type. (C) Mean G_M2 and G_M3 staining intensity and a fraction of G_M2-positive cells in control and patient's fibroblasts. Mean staining intensities per μm² were measured with ImageJ software. Data show mean (± SEM) of individual values measured for 35 randomly selected cells. **P < 0.01, *P < 0.05 in unpaired two-tailed t-test. G_M2-positive cells were manually counted in three randomly selected microscope fields (~ 150 cells each). Data show mean values (± SEM) of 3 independent experiments. *P < 0.05 in unpaired two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)