Trisha Multhaupt-Buell

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.

Phenotypic spectrum and sex effects in eleven myoclonus‐dystonia families with ε‐sarcoglycan mutations

Myoclonus‐dystonia (M‐D) due to SGCE mutations is characterized by early onset myoclonic jerks, often associated with dystonia. Penetrance is influenced by parental sex, but other sex effects have not been established. In 42 affected individuals from 11 families with identified mutations, we found that sex was highly associated with age at onset regardless of mutation type; the median age onset for girls was 5 years versus 8 years for boys (P < 0.0097). We found no association between mutation type and phenotype.

Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells.

ABSTRACT X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containing TAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 of TAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression of TAF1 and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells, XDP fibroblasts exhibited decreased expression of TAF1 transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34′), was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. Summary: This study describes a new iPSC model of X-linked dystonia-parkinsonism (XDP), which was initially validated by demonstrating a similar transcriptional defect as has been previously reported in XDP brain tissue.

Pallidal deep brain stimulation for dystonia: a case series.

OBJECT Pallidal deep brain stimulation (DBS) is a treatment option for those with early-onset dystonia. However, there are limited data on long-term outcome and treatment complications. The authors report on the short- and long-term effects of pallidal DBS in a cohort of patients with early-onset dystonia. METHODS Fourteen consecutive pediatric patients with early-onset dystonia were systematically evaluated and treated. The duration of follow-up ranged from 16 to 84 months. RESULTS There were no immediate postoperative complications. At last follow-up, 12 of the 14 patients displayed a significant decline in the Burke-Fahn-Marsden Dystonia Rating Scale motor subscale score, with an average decrease of 62% ± 8.4%. The most common hardware complication was lead fracture (14.3%). CONCLUSIONS These data provide further evidence that DBS is a safe and effective treatment for those with earlyonset dystonia.

Mutation in 5′ upstream region of GCHI gene causes familial dopa-responsive dystonia†‡§

Dopa-responsive dystonia (DRD) is commonly caused by heterozygous mutations in the guanosine triphosphate (GTP) cyclohydrolase I gene (GCH1) [1]. In the 5’ upstream region, 3 different mutations have been identified in 2 subjects with DRD [2,3]. One subject had 2 mutations, -39C>T and -132C>T and another had a single mutation, -22 C>T, with no data available on first-degree relatives [2, 3]. We report on multiple generations of one family with DRD, in whom the -22 C>T mutation segregates with affected status. One family of Irish/French-Canadian ethnicity was studied. Ten family members, spanning 3 generations, underwent a neurologic exam and provided blood samples. The local institutional review board approved the study. All participants gave informed consent. The criterion for definite DRD was definite dystonia and a marked, sustained response to levodopa or dopamine agonist [4]. The criterion for probable DRD was definite dystonia in a subject who declined a trial of medication. A movement disorders neurologist (NS) determined the diagnosis and affected sites as described [5]. Establishment of dystonia status was made prior to genetic testing, which was done blind to clinical designation. The full GCHI gene (exons 1 -6) was analyzed by bi-directional sequencing from genomic DNA from every subject. Control samples, consisting of 46 European Caucasian samples from the CEPH collection and 7 from the discarded sample collection of the Massachusetts General Hospital Neurogenetics DNA Diagnostic Lab, were utilized. Six subjects had dystonia (III-2, III-4, IV-2, IV-5, V-2 and V-3). Four were receiving medical treatment (III-2, III-4, IV-5 and V-2). All six affected subjects experienced onset in a foot or leg during childhood. Two displayed involvement of a non-contiguous body region; the neck and right leg in subject III-2 and the neck, right leg and left hand in subject V-2. Duration of disease varied, from one year (V-3) to fifty-four years (III-2). Subject V-2, with the greatest spread in symptoms, had DRD for twelve years. Subject III-4 did not display any progression, with dystonia remaining in the right foot, and had DRD for fifty-three years. Of the affected subjects, three demonstrated a good response to relatively low therapeutic doses of carbidopa/levodopa (III-2, III-4, V-2). IV-5 became nauseous on carbidopa/levodopa but displayed a good response to a relatively low therapeutic dose of ropinirole. Subject IV-2 had taken carbidopa/levodopa in the past and saw improvement, but discontinued medication based on personal preference. Subject V-3 had relatively mild symptoms and declined medical treatment. Thus, V-3 was classified as probable DRD and the other affected subjects (III-2, III-4, IV-2, IV-5, V-2) were classified as definite DRD. We demonstrate that the -22 C>T mutation in the GCH1 gene segregates with affected status in multiple generations of a single DRD family. This mutation was not found in 53 control samples (106 normal alleles), nor in 214 clinical samples (428 alleles), about which we have no phenotypic information, that have been sequenced in the MGH Neurogenetics DNA Diagnostic Laboratory. This makes it likely that the -22 C>T mutation is pathogenic and results in DRD.

Disease onset in X-linked dystonia-parkinsonism correlates with expansion of a hexameric repeat within an SVA retrotransposon in TAF1

Significance The genetic basis of X-Linked dystonia-parkinsonism (XDP) has been difficult to unravel, in part because all patients inherit the same haplotype of seven sequence variants, none of which has ever been identified in control individuals. This study revealed that one of the haplotype markers, a retrotransposon insertion within an intron of TAF1, has a variable number of hexameric repeats among affected individuals with an increase in repeat number strongly correlated with earlier age at disease onset. These data support a contributing role for this sequence in disease pathogenesis while further suggesting that XDP may be part of a growing list of neurodegenerative disorders associated with unstable repeat expansions. X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This unique insertion coincides with six additional noncoding sequence changes in TAF1, the gene that encodes TATA-binding protein–associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n = 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)n. The number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference in TAF1 expression.

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.

Dystonia-Causing Mutations as a Contribution to the Etiology of Spasmodic Dysphonia

Objective Spasmodic dysphonia is a focal dystonia of the larynx with heterogeneous manifestations and association with familial risk factors. There are scarce data to allow precise understanding of etiology and pathophysiology. Screening for dystonia-causing genetic mutations has the potential to allow accurate diagnosis, inform about genotype-phenotype correlations, and allow a better understanding of mechanisms of disease. Study Design Cross-sectional study. Setting Tertiary academic medical center. Subjects and Methods We enrolled patients presenting with spasmodic dysphonia to the voice clinic of our academic medical center. Data included demographics, clinical features, family history, and treatments administered. The following genes with disease-causing mutations previously associated with spasmodic dysphonia were screened: TOR1A (DYT1), TUBB4 (DYT4), and THAP1 (DYT6). Results Eighty-six patients were recruited, comprising 77% females and 23% males. A definite family history of neurologic disorder was present in 15% (13 of 86). Average age (± standard deviation) of symptom onset was 42.1 ± 15.7 years. Most (99%; 85 of 86) were treated with botulinum toxin, and 12% (11 of 86) received oral medications. Genetic screening was negative in all patients for the GAG deletion in TOR1A (DYT1) and in the 5 exons currently associated with disease-causing mutations in TUBB4 (DYT4). Two patients tested positive for novel/rare variants in THAP1 (DYT6). Conclusion Genetic screening targeted at currently known disease-causing mutations in TOR1A, THAP1, and TUBB4 appears to have low diagnostic yield in sporadic spasmodic dysphonia. In our cohort, only 2 patients tested positive for novel/rare variants in THAP1. Clinicians should make use of genetic testing judiciously and in cost-effective ways.

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.