Dopa-Responsive Dystonia: Functional Analysis of Single Nucleotide Substitutions within the 5’ Untranslated GCH1 Region

Background: Mutations in the GCH1 gene are associated with childhood onset, dopa-responsive dystonia (DRD). Correct diagnosis of DRD is crucial, given the potential for complete recovery once treated with L-dopa. The majority of DRD associated mutations lie within the coding region of the GCH1 gene, but three additional single nucleotide sequence substitutions have been reported within the 5’ untranslated (5’UTR) region of the mRNA. The biologic significance of these 5’UTR GCH1 sequence substitutions has not been analyzed. Methodology/Principal Findings Luciferase reporter assays, quantitative real time PCR and RNA decay assays, combined with bioinformatics, revealed a pathogenic 5’UTR GCH1 substitution. The +142C>T single nucleotide 5’UTR substitution that segregates with affected status in DRD patients, substantially attenuates translation without altering RNA expression levels or stability. The +142C>T substitution disrupts translation most likely by creating an upstream initiation start codon (uAUG) and an upstream open reading frame (uORF). Conclusions/Significance: This is the first GCH1 regulatory substitution reported to act at a post-transcriptional level, increasing the list of genetic diseases caused by abnormal translation and reaffirming the importance of investigating potential regulatory substitutions in genetic diseases

Evidence for Altered Basal Ganglia-Brainstem Connections in Cervical Dystonia

Background: There has been increasing interest in the interaction of the basal ganglia with the cerebellum and the brainstem in motor control and movement disorders. In addition, it has been suggested that these subcortical connections with the basal ganglia may help to coordinate a network of regions involved in mediating posture and stabilization. While studies in animal models support a role for this circuitry in the pathophysiology of the movement disorder dystonia, thus far, there is only indirect evidence for this in humans with dystonia. Methodology/Principal Findings: In the current study we investigated probabilistic diffusion tractography in DYT1-negative patients with cervical dystonia and matched healthy control subjects, with the goal of showing that patients exhibit altered microstructure in the connectivity between the pallidum and brainstem. The brainstem regions investigated included nuclei that are known to exhibit strong connections with the cerebellum. We observed large clusters of tractography differences in patients relative to healthy controls, between the pallidum and the brainstem. Tractography was decreased in the left hemisphere and increased in the right hemisphere in patients, suggesting a potential basis for the left/right white matter asymmetry we previously observed in focal dystonia patients. Conclusions/Significance: These findings support the hypothesis that connections between the basal ganglia and brainstem play a role in the pathophysiology of dystonia

White Matter Changes in Cervical Dystonia Relate to Clinical Effectiveness of Botulinum Toxin Treatment

In a previous report showing white matter microstructural hemispheric asymmetries medial to the pallidum in focal dystonias, we showed preliminary evidence that this abnormality was reduced 4 weeks after botulinum toxin (BTX) injections. In the current study we report the completed treatment study in a full-size cohort of CD patients (n = 14). In addition to showing a shift toward normalization of the hemispheric asymmetry, we evaluated clinical relevance of these findings by relating white matter changes to degree of symptom improvement. We also evaluated whether the magnitude of the white matter asymmetry before treatment was related to severity, laterality, duration of dystonia, and/or number of previous BTX injections. Our results confirm the findings of our preliminary report: we observed significant fractional anisotropy (FA) changes medial to the pallidum 4 weeks after BTX in CD participants that were not observed in controls scanned at the same interval. There was a significant relationship between magnitude of hemispheric asymmetry and dystonia symptom improvement, as measured by percent reduction in dystonia scale scores. There was also a trend toward a relationship between magnitude of pre-injection white matter asymmetry and symptom severity, but not symptom laterality, disorder duration, or number of previous BTX injections. Post-hoc analyses suggested the FA changes at least partially reflected changes in pathophysiology, but a dissociation between patient perception of benefit from injections and FA changes suggested the changes did not reflect changes to the primary “driver” of the dystonia. In contrast, there were no changes or group differences in DTI diffusivity measures, suggesting the hemispheric asymmetry in CD does not reflect irreversible white matter tissue loss. These findings support the hypothesis that central nervous system white matter changes are involved in the mechanism by which BTX exerts clinical benefit

Thalamic Volume Is Reduced in Cervical and Laryngeal Dystonias

Background: Dystonia, a debilitating movement disorder characterized by abnormal fixed positions and/or twisting postures, is associated with dysfunction of motor control networks. While gross brain lesions can produce secondary dystonias, advanced neuroimaging techniques have been required to identify network abnormalities in primary dystonias. Prior neuroimaging studies have provided valuable insights into the pathophysiology of dystonia, but few directly assessed the gross volume of motor control regions, and to our knowledge, none identified abnormalities common to multiple types of idiopathic focal dystonia. Methods: We used two gross volumetric segmentation techniques and one voxelwise volumetric technique (voxel based morphometry, VBM) to compare regional volume between matched healthy controls and patients with idiopathic primary focal dystonia (cervical, n = 17, laryngeal, n = 7). We used (1) automated gross volume measures of eight motor control regions using the FreeSurfer analysis package; (2) blinded, anatomist-supervised manual segmentation of the whole thalamus (also gross volume); and (3) voxel based morphometry, which measures local T1-weighted signal intensity and estimates gray matter density or volume at the level of single voxels, for both whole-brain and thalamus. Results: Using both automated and manual gross volumetry, we found a significant volume decrease only in the thalamus in two focal dystonias. Decreases in whole-thalamic volume were independent of head and brain size, laterality of symptoms, and duration. VBM measures did not differ between dystonia and control groups in any motor control region. Conclusions: Reduced thalamic gross volume, detected in two independent analyses, suggests a common anatomical abnormality in cervical dystonia and spasmodic dysphonia. Defining the structural underpinnings of dystonia may require such complementary approaches

Voxel based morphometry in cervical dystonia.

<p>Voxel based morphometry demonstrated reduced gray matter local tissue volume in the posterior cingulate (A and B, blue voxels, shown at two significance thresholds, presented as family-wise uncorrected p-values), but no differences in the thalamus, in cervical dystonia (family-wise error corrected p = 0.9996). When the analysis was restricted to only those voxels in a thalamic mask (to minimize the loss of statistical power by multiple-comparisons correction; C, green voxels), no significant differences in local tissue volume were noted (p = 0.34). Significant voxels (A, B) and thalamic mask (C) overlie the mean gray matter structural image. Note that identical structural scans were used in VBM analyses and segmentation analyses (i.e., scans used in this figure were the same as those used for data in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155302#pone.0155302.g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155302#pone.0155302.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155302#pone.0155302.g005" target="_blank">5</a>). VBM results were corrected using threshold-free cluster enhancement (TFCE). All axial and coronal views are from a single plane, indicated in MNI Talairach coordinates. Color bars at bottom indicate TFCE-corrected p-values for the images above. Abbreviations: pat = patients; ctrl = controls. R = Right hemisphere; L = Left hemisphere.</p

Regional automated and manual gross volume measures.

<p>A reduction in thalamic volume, not seen in other regions involved in the control of movement, was seen in both cervical dystonia and spasmodic dysphonia. Total volume (i.e., number of voxels in left plus right hemispheres) is shown for each region of interest (mean ± standard error of the mean). Given the large differences in volume between brain regions, the axis has been adjusted to focus on each cluster of values. Breaks in the y-axis are indicated by hashed horizontal bars. All p-values corrected for multiple comparisons (Bonferroni corrected significance threshold, p = 0.00625); * p≤0.0060; ** p = 0.00020. Abbreviation: BA6 = Brodmann Area 6; Thal auto = automated thalamic segmentation.</p

Relationship of clinical measures to gross thalamic volume in CD patients.

<p>The relationship between individual gross thalamic volume (manually segmented, in mm³) and clinical measures suggests that reduced gross thalamic volume is a risk factor for dystonia, and is not a secondary effect of dystonia symptoms. Data are shown for all variables, even if excluded from regression models, so raw data for CD and SD can be viewed and compared. Both CD patients and controls (A) showed declining volume with age. Patient:control gross thalamic volume showed a qualitative divergence with age between CD and controls, but the divergence was not statistically significant. (B) There was no relationship between volume and age in the SD cohort (for either SD or controls), presumably reflecting the smaller age range in this cohort. Likewise, there was no divergence of slopes with age between SD and controls. Age at CD onset (C) appeared to correlate with gross thalamic volume, but this was likely driven by the relationship between age at scan and age of onset; age at SD onset (D) was not correlated with volume. Gross thalamic volume did not correlate significantly with duration for either CD (E); this relationship was not evaluated statistically for SD due to collinearity with other variables, but the positive slope suggests no indication of a decline in volume with increasing duration (F). Thalamic volume also did not correlate with severity of dystonia for either CD or SD in the multiple regression model, as measured by the Tsui scale for CD (G) or the voice-related quality of life score for SD (H), although the SD relationship to severity showed a trend toward significance (V-RQOL, p = 0.056), and appeared significant when evaluated post hoc as a single variable (p = 0.012). The asymmetry of muscles affected with cervical dystonia (as gauged by laterality of units of botulinum toxin injected) did not correlate with asymmetries in thalamic volume (I, p = 0.89). Note that for (C) and (D), thalamic volumes for control subjects are plotted vs. the age at onset for the matched patient, as control subjects do not have an age at onset. Volume for control subjects is included in C and D as a reference only (designated by Ω), to illustrate that patient:control differences persist (and in fact are more robust) when demographics (including age) are matched: with the exception of a single CD/control dyad, every patient showed lower volume than his/her matched control.</p