Genetic and neuropathological study of primary and secondary dystonic syndromes

Abstract

Dystonia 1 (DYT1) abstract Early onset primary dystonia, DYT1 is linked to a three base pair deletion (ΔGAG) mutation in the TOR1A gene. Clinical manifestation includes intermittent muscle contraction leading to twisted movements or abnormal postures. Neuropathological studies on DYT1 cases are limited, most showing no significant abnormalities. In one study, brainstem intraneuronal inclusions immunoreactive for ubiquitin, torsinA and lamin A/C were described. Using the largest cohort of DYT1 cases reported to date we aimed to identify consistent neuropathological features in the disease and determine whether we would find the same intraneuronal inclusions as previously reported. Sanger sequencing was used to screen all the cases for (ΔGAG) mutation in TOR1A gene. Using immunohistochemistry seven DYT1 cases and five age and sex matched controls were studied for the presence of ubiquitinated inclusions in the brainstem and other anatomical regions implicated in dystonia. The pathological changes of brainstem inclusions reported in DYT1 dystonia were not replicated in our cohort. Other anatomical regions implicated in dystonia showed no disease-specific pathological intracellular inclusions or evidence of more than mild neuronal loss. Our findings suggest that the intracellular inclusions described previously in DYT1 dystonia may not be a hallmark feature of the disorder. In DYT1, biochemical changes may be more relevant than the morphological changes. Dystonia 6 (DYT6) abstract Mutations in the thanatos-associated protein domain containing, apoptosis associated protein 1 gene (THAP1) are responsible for the adult-onset primary dystonia (DYT6). However, no neuropathological descriptions of genetically proven DYT6 cases have been reported to our knowledge. We report the clinical, genetic and neuropathological features of two DYT6 cases. The two DYT6 cases were genetically screened for the THAP1 gene mutations using standard Sanger polymerase chain reaction sequencing. A detailed neuropathological assessment of the cases was performed using histochemical and immunohistochemical preparations. Both DYT6 cases showed no significant neurodegeneration and no specific disease-related pathology. This is the first detailed neuropthological investigation carried out on adult-onset primary dystonia, DYT6 brains. We did not identify any neuropathological features that could be defined as hallmark features of DYT6 dystonia. Our study supports the notion that in primary dystonia there is no significant neurodegeneration or associated neuropathological lesions. Dopa-responsive dystonia (DRD) abstract Autosomal dominant hereditary dopa-responsive dystonia (DYT5) is a rare movement disorder which presents typically in childhood with lower limb dystonia and subsequent generalization. Mutations in the GCH1 gene on chromosome 14 q21.1-q22.2 are pathogenic in DYT5. The hallmark of the disease is an excellent and sustained response to small doses of levodopa, generally without the occurrence of motor fluctuations. 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 gene. To determine if noncoding mutations in the 5’ upstream region of GCH1 gene are pathogenic in DYT5 dystonia Sanger sequencing of the region was carried out in a cohort of DRD cases negative for mutation in the coding regions of the GCH1 gene. Two unrelated cases were identified with both -39C>T and -132C>T mutations in the 5’ UTR of GCH1 gene. However, one of the cases has an affected sibling without these mutations. Functional study identified negligible effect of these two mutations. Hence, in the DRD cohort studied, there is not enough evidence to conclude that the two non-coding variants underlie the disease manifestation. Spinocerebellar ataxia type 8 (SCA8) abstract SCA8 is characterized by repeat instability and presents as an ataxia with slow progression that largely spares brainstem and cerebral function. There is no anticipation observed and penetrance is reduced as the expansion can also be found in healthy individuals. Potentially pathogenic alleles contain 85 or more combined CTA/CTG repeats. However, repeats of this size are surprisingly frequent in the general population and also occur in asymptomatic relatives of ataxia individuals. This study examined the length of SCA8 repeats in English ataxia patients, English controls and in diversity (from 25 different countries) controls. In the ataxia cohort investigated, 6 out of 631 cases were in the potentially pathogenic range. However, a case out of 631 English controls and 3 cases out of 1148 individuals in diversity control cohort also fall in the same range.The significant difference (p= 0.018) in occurrence of SCA8 repeat expansions in ataxia patients compared with English controls but not with the diversity controls (p=0.192) suggests that the pathogenicity of SCA8 repeat expansion is not only dependent on the repeat size but also on the population, environment and the genetic modifiers. This study demonstrates that the presence of a SCA8 expansion cannot be used to predict whether or not an asymptomatic individual will develop ataxia as the control cases were asymptomatic at the time of study. Beta-propeller protein associated neurodegeneration (BPAN) abstract Neurodegenerative disorders with high iron in the basal ganglia encompass a single gene disorders collectively known as NBIA disorders. NBIA-5 (also known as BPAN) is linked to the mutations in the WD repeat domain 45 (WDR45) gene on chromosome Xp11. This study describes the genetic, clinical and neuropathological features of a case of BPAN. The clinical history of the case matches to other BPAN cases described which is characterized by global developmental delay and intellectual deficiency in early childhood then followed by further neurological and cognitive regression in early adulthood, with dystonia, and sometimes ocular defects and sleep perturbation. A cranial computed tomography (CT) revealed generalised brain atrophy and bilateral generalised mineralisation of the globi pallidi and substantia nigri that are likely to represent iron deposition on subsequent magnetic resonance imaging (MRI). The major pathological findings in this case were severe neuronal loss with gliosis affecting the substantia nigra, iron deposition in the substantia nigra and globus pallidus, axonal swellings in the substantia nigra, gracile nucleus and cuneate nucleus and very extensive phospho-tau deposition. Tau pathology was in the form of neurofibrillary tangles, pre-tangles and neuropil threads and histological studies indicated that many of the tau immunoreactive structures contained fibrillary protein (Gallyas silver positive and AT100 immunoreactive) and contained both 3-and 4-repeat tau isoforms similar to the tau deposits of Alzheimer’s disease. This finding was confirmed by immunoblotting of non-dephosphorylated tangle extractions which showed the classical triplet band as observed in AD cases (i.e. 3R+4R). When dephosphorylated, the tau isoform composition is almost identical to the AD case with 0N3R, 0N4R, 1N3R and 1N4R tau-isoforms. This study supports the involvement of beta–propeller protein (encoded by WDR45 gene) in autophagy as has been documented in yeast and mammalian cells. The increased accumulation of LC3-II protein in brain tissue of the BPAN case compared to the three control cases implies that the autophagosome formation is hindered. BPAN represents the first direct link between autophagy mechanism and neurodegeneration. However, the role of iron in this process is still elusive.