Mutations in GNAL cause primary torsion dystonia

Abstract

Dystonia is a movement disorder characterized by repetitive twisting muscle contractions and postures. Its molecular pathophysiology is poorly understood, in part owing to limited knowledge of the genetic basis of the disorder. Only three genes for primary torsion dystonia (PTD), TOR1A (DYT1), THAP1 (DYT6) and CIZ1 (ref. 5), have been identified. Using exome sequencing in two families with PTD, we identified a new causative gene, GNAL, with a nonsense mutation encoding p.Ser293* resulting in a premature stop codon in one family and a missense mutation encoding p.Val137Met in the other. Screening of GNAL in 39 families with PTD identified 6 additional new mutations in this gene. Impaired function of several of the mutants was shown by bioluminescence resonance energy transfer (BRET) assays.

Figure 1

Mutations identified in GNAL in PTD patients. a. Schematic representation of exon:intron structure of the short isoform of GNAL (NM_001142339) with mutations indicated. Missense mutations are depicted in pink, in-frame deletion is in blue, nonsense mutations are in green, frame-shift mutations are in yellow and the tentative splice mutation is in grey. b. Protein sequence alignment of Gαolf orthologs from vertebrate species. Protein sequences were obtained from RefSeq database and aligned using ClustalW. The regions of alignment corresponding to the in-frame deletion and missense mutations are shown. The mutations are colored as in panel a. RefSeq accession numbers are indicated.

Figure 2

Effect of mutations on Gαolf coupling to D1R. a. Schematics of the assay design. Stimulation of the D1R by dopamine results in the dissociation of Gαolf from the heterotrimer. Released Gβγ subunits tagged with Venus become available for the interaction with Rluc8-tagged GRK reporter producing the BRET signal which is determined by the change in the emission ratio at wavelengths 535nm and 480nm. b. Time course of the BRET signal change upon stimulation of cells with dopamine and subsequent deactivation by haloperidol. c. Basal BRET ratios calculated before the application of dopamine that reflect the extent of the Gαolf-Gβγ heterotrimer formation. d. Changes in the BRET ratio from basal signal to maximal response that reflect the amplitude of the response. e. Analysis of the expression levels of Gαolf and Gβγ (detected by anti-GFP antibodies) subunits by Western blotting. Ponceau S staining of total cell lysates was used as a loading control. Results represent the mean of quadruplicate wells from a typical experiment. Similar results were seen in two independent experiments. Error bars represent the standard error of the mean. One way ANOVA followed by the Holm–Sidak method were performed to determine statistically significant differences. Asterisks indicate statistical significance from wild type control: ***, p < 0.001. The Gαolf vectors were based on transcript NM_001142339.