Functional Characterization of Rare RAB12 Variants and Their Role in Musician's and Other Dystonias

Abstract

Mutations in RAB (member of the Ras superfamily) genes are increasingly recognized as cause of a variety of disorders including neurological conditions. While musician's dystonia (MD) and writer's dystonia (WD) are task-specific movement disorders, other dystonias persistently affect postures as in cervical dystonia. Little is known about the underlying etiology. Next-generation sequencing revealed a rare missense variant (c.586A>G; p.Ile196Val) in RAB12 in two of three MD/WD families. Next, we tested 916 additional dystonia patients; 512 Parkinson's disease patients; and 461 healthy controls for RAB12 variants and identified 10 additional carriers of rare missense changes among dystonia patients (1.1%) but only one carrier in non-dystonic individuals (0.1%; p = 0.005). The detected variants among index patients comprised p.Ile196Val (n = 6); p.Ala174Thr (n = 3); p.Gly13Asp; p.Ala148Thr; and p.Arg181Gln in patients with MD; cervical dystonia; or WD. Two relatives of MD patients with WD also carried p.Ile196Val. The two variants identified in MD patients (p.Ile196Val; p.Gly13Asp) were characterized on endogenous levels in patient-derived fibroblasts and in two RAB12-overexpressing cell models. The ability to hydrolyze guanosine triphosphate (GTP), so called GTPase activity, was increased in mutants compared to wildtype. Furthermore, subcellular distribution of RAB12 in mutants was altered in fibroblasts. Soluble Transferrin receptor 1 levels were reduced in the blood of all three tested p.Ile196Val carriers. In conclusion, we demonstrate an enrichment of missense changes among dystonia patients. Functional characterization revealed altered enzyme activity and lysosomal distribution in mutants suggesting a contribution of RAB12 variants to MD and other dystonias.

Keywords: GTPase; RAB12; Transferrin receptor; lysosomal degradation; musician’s dystonia.

Figure 1

Pedigrees of four musician´s dystonia (MD) families. Black symbols represent individuals with MD, gray symbols mark individuals with WD, the dotted filled symbol indicates an individual with possibly mild, generalized dystonia, and the question mark points to an individual that was initially reported to have dystonia which however was not confirmed upon re-evaluation by two neurologists. L-2329 did not agree to be neurologically examined. A photograph of L-10289 illustrating her dystonic postures is shown in Figure S1. Patients of Family A were exome sequenced at Atlas Biolabs (superscript “E”) and three patients of Families B and C underwent genome sequencing at Complete Genomics (superscript “G”). Additional exome sequencing was performed for individuals of Families A, B, and D at Centogene (superscript“+E”). Mutational status for the p.Ile196Val mutation in RAB12 is indicated: +/+ for homozygous carriers; +/− for heterozygous carriers; −/− for non-carriers. For individuals for whom DNA was available a sample ID number (L code) is shown. Dashed symbols represent deceased family members.

Figure 2

RAB12 and functionally investigated variants. (a) Schematic view of the RAB12 protein with the identified mutations p.Gly13Asp and p.Ile196Val in MD patients (red arrows), the guanosine diphosphate/guanosine triphosphate (GDP/GTP)-binding sites (red), the predicted effector region (blue), and the posttranslational modifications on amino acid positions 1 (N-acetylation), 21, 25, 106 (phosphorylations), 243, and 244 (geranylgeranylations) are indicated. RefSeq: NM_001025300.2, NP_001020471; (b) The Glycine at position 13 (left panel, red) and the Isoleucine at position 196 (right panel, red) are conserved across different species; (c) Proteins of transfected SH-SY5Y cells were incubated with GTP for 1 h and PO₄³⁻ production was measured. GTPase activity of mutant RAB12 expressing cells was normalized for GTPase activity of wild-type (WT) RAB12 expressing cells. The bars indicate the means ± standard error of mean, One-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test. ** p < 0.01, n = 4; (d) Homology model of active/GTP-bound RAB12 in ribbon representation. WT is presented in black (left model) and p.Ile196Val in blue (right model). Magnesium ions are shown as green spheres, amino acid residue 196 is marked by red points, and GTP is indicated by sticks. The figures were generated with PyMOL (PyMOL Molecular Graphics System, Version 1.7.0 Schrödinger, LLC, New York, NY, USA).

Figure 2

RAB12 and functionally investigated variants. (a) Schematic view of the RAB12 protein with the identified mutations p.Gly13Asp and p.Ile196Val in MD patients (red arrows), the guanosine diphosphate/guanosine triphosphate (GDP/GTP)-binding sites (red), the predicted effector region (blue), and the posttranslational modifications on amino acid positions 1 (N-acetylation), 21, 25, 106 (phosphorylations), 243, and 244 (geranylgeranylations) are indicated. RefSeq: NM_001025300.2, NP_001020471; (b) The Glycine at position 13 (left panel, red) and the Isoleucine at position 196 (right panel, red) are conserved across different species; (c) Proteins of transfected SH-SY5Y cells were incubated with GTP for 1 h and PO₄³⁻ production was measured. GTPase activity of mutant RAB12 expressing cells was normalized for GTPase activity of wild-type (WT) RAB12 expressing cells. The bars indicate the means ± standard error of mean, One-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test. ** p < 0.01, n = 4; (d) Homology model of active/GTP-bound RAB12 in ribbon representation. WT is presented in black (left model) and p.Ile196Val in blue (right model). Magnesium ions are shown as green spheres, amino acid residue 196 is marked by red points, and GTP is indicated by sticks. The figures were generated with PyMOL (PyMOL Molecular Graphics System, Version 1.7.0 Schrödinger, LLC, New York, NY, USA).

Figure 3

RAB12 mutations changed the subcellular localization of RAB12 proteins and lysosomes. (a) Ectopical protein expression of FLAG-RAB12 and expression of the lysosome-associated protein LAMP-1 (lysosomal associated membrane protein 1) is equal in RAB12 WT and mutant SH-SY5Y cells. All protein signals were detected on the same membrane. Blots were cropped for clarity but no bands were removed. Original blots are shown in the supplementary material (Figure S5); (b) In WT and mutated RAB12-overexpressing cells, three different patterns of RAB12 distribution were observed and categorized. Proportions of cells revealed RAB12 (red) that was evenly distributed in the cytoplasm (upper left panel), that was distributed in the cytoplasm and accumulated in close proximity to the nucleus (upper middle panel), or that accumulated exclusively in the perinuclear region of the fibroblast (upper right panel). FLAG-RAB12 and LAMP-1 (green) colocalized in the cytoplasm (lower panels). Nuclei were stained with DAPI (blue). Scale bar: 20 µm; (c) 110 randomly chosen fibroblasts with each construct were categorized and cellular localization of FLAG-RAB12 and LAMP-1 was quantified. Chi-square test: p < 0.0001; (d) 110 randomly chosen fibroblasts of two MD patients (p.Ile196Val) and two healthy controls were categorized and cellular localization of LAMP-1 was quantified. Chi-square test: p = 0.0002.