DYT-THAP1: exploring gene expression in fibroblasts for potential biomarker discovery.

Dystonia due to pathogenic variants in the THAP1 gene (DYT-THAP1) shows variable expressivity and reduced penetrance of ~ 50%. Since THAP1 encodes a transcription factor, modifiers influencing this variability likely operate at the gene expression level. This study aimed to assess the transferability of differentially expressed genes (DEGs) in neuronal cells related to pathogenic variants in the THAP1 gene, which were previously identified by transcriptome analyses. For this, we performed quantitative (qPCR) and Digital PCR (dPCR) in cultured fibroblasts. RNA was extracted from THAP1 manifesting (MMCs) and non-manifesting mutation carriers (NMCs) as well as from healthy controls. The expression profiles of ten of 14 known neuronal DEGs demonstrated differences in fibroblasts between these three groups. This included transcription factors and targets (ATF4, CLN3, EIF2A, RRM1, YY1), genes involved in G protein-coupled receptor signaling (BDKRB2, LPAR1), and a gene linked to apoptosis and DNA replication/repair (CRADD), which all showed higher expression levels in MMCs and NMCs than in controls. Moreover, the analysis of genes linked to neurological disorders (STXBP1, TOR1A) unveiled differences in expression patterns between MMCs and controls. Notably, the genes CUEDC2, DRD4, ECH1, and SIX2 were not statistically significantly differentially expressed in fibroblast cultures. With > 70% of the tested genes being DEGs also in fibroblasts, fibroblasts seem to be a suitable model for DYT-THAP1 research despite some restrictions. Furthermore, at least some of these DEGs may potentially also serve as biomarkers of DYT-THAP1 and influence its penetrance and expressivity.

Large-Scale Screening: Phenotypic and Mutational Spectrum in Isolated and Combined Dystonia Genes.

BACKGROUND: Pathogenic variants in several genes have been linked to genetic forms of isolated or combined dystonia. The phenotypic and genetic spectrum and the frequency of pathogenic variants in these genes have not yet been fully elucidated, neither in patients with dystonia nor with other, sometimes co-occurring movement disorders such as Parkinson's disease (PD). OBJECTIVES: To screen >2000 patients with dystonia or PD for rare variants in known dystonia-causing genes. METHODS: We screened 1207 dystonia patients from Germany (DysTract consortium), Spain, and South Korea, and 1036 PD patients from Germany for pathogenic variants using a next-generation sequencing gene panel. The impact on DNA methylation of KMT2B variants was evaluated by analyzing the gene's characteristic episignature. RESULTS: We identified 171 carriers (109 with dystonia [9.0%]; 62 with PD [6.0%]) of 131 rare variants (minor allele frequency <0.005). A total of 52 patients (48 dystonia [4.0%]; four PD [0.4%, all with GCH1 variants]) carried 33 different (likely) pathogenic variants, of which 17 were not previously reported. Pathogenic biallelic variants in PRKRA were not found. Episignature analysis of 48 KMT2B variants revealed that only two of these should be considered (likely) pathogenic. CONCLUSION: This study confirms pathogenic variants in GCH1, GNAL, KMT2B, SGCE, THAP1, and TOR1A as relevant causes in dystonia and expands the mutational spectrum. Of note, likely pathogenic variants only in GCH1 were also found among PD patients. For DYT-KMT2B, the recently described episignature served as a reliable readout to determine the functional effect of newly identified variants. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Generalized Dystonia Due to a Pathogenic THAP1 Variant Showing Sustained Response to Globus Pallidus Deep Brain Stimulation.

A 21-year-old woman of south Asian origin presented with cervical dystonia which had progressed over the previous three years. Her symptoms started as writer's cramp since the age of seven years. She did not respond to medications and needed botulinum toxin injection for generalised dystonia. Subsequent whole genome sequencing revealed a likely pathogenic c.98G>A p.(Cys33Tyr) heterozygous variant in the THAP1 gene. She underwent bilateral posteroventral globus pallidus interna (GPi) deep brain stimulation (Medtronic Activa PC) implantation at the age of thirty-one years. She responded well to the deep brain stimulation even after more than 8 years post-surgery though she needs botulinum toxin injection for her cervical dystonia.

Genetic spectrum and clinical features in a cohort of Chinese patients with isolated dystonia.

Dystonia is a genetically and phenotypically heterogeneous disorder that occurs in isolation (isolated dystonia) or in combination with other movement disorders. To determine the genetic spectrum in isolated dystonia, we enrolled 88 patients with isolated dystonia for whole-exome sequencing (WES). Seventeen mutations, including nine novel ones, were identified in 19 of the 88 patients, providing a 21.59% positive molecular diagnostic rate. Eleven distinct genes were involved, of which TOR1A and THAP1 accounted for 47.37% (9/19) of the positive cases. A novel missense variant, p.S225R in TOR1A, was found in a patient with adolescence-onset generalized dystonia. Cellular experiments revealed that p.S255R results in the abnormal aggregation of Torsin-1A encoding by TOR1A. In addition, we reviewed the clinical and genetic features of the isolated dystonia patients carrying TOR1A, THAP1, ANO3, and GNAL mutations in the Chinese population. Our results expand the genetic spectrum and clinical profiles of patients with isolated dystonia and demonstrate WES as an effective strategy for the molecular diagnosis of isolated dystonia.

Transcriptional regulatory network for neuron-glia interactions and its implication for DYT6 dystonia.

Advances in sequencing technologies have identified novel genes associated with inherited forms of dystonia, providing valuable insights into its genetic basis and revealing diverse genetic pathways and mechanisms involved in its pathophysiology. Since identifying genetic variation in the transcription factor coding THAP1 gene linked to isolated dystonia, numerous investigations have employed transcriptomic studies in DYT-THAP1 models to uncover pathogenic molecular mechanisms underlying dystonia. This review examines key findings from transcriptomic studies conducted on in vivo and in vitro DYT-THAP1 models, which demonstrate that the THAP1-regulated transcriptome is diverse and cell-specific, yet it is bound and co-regulated by a common set of proteins. Prominent among its functions, THAP1 and its co-regulatory network target molecular pathways critical for generating myelinating oligodendrocytes that ensheath axons and generate white matter in the central nervous system. Several lines of investigation have demonstrated the importance of myelination and oligodendrogenesis in motor function during development and in adults, emphasizing the non-cell autonomous contributions of glial cells to neural circuits involved in motor function. Further research on the role of myelin abnormalities in motor deficits in DYT6 models will enhance our understanding of axon-glia interactions in dystonia pathophysiology and provide potential therapeutic interventions targeting these pathways.

Novel THAP1 missense variant with incomplete penetrance in a case of generalized young onset dystonia showing good response to deep brain stimulation.

We describe a case of young onset generalized dystonia, harboring a previously unreported likely pathogenic THAP1 missense variant (c.109 G > A; p.Glu37Lys) that was inherited from her unaffected father. Moreover, we report a positive effect of deep brain stimulation, particularly on the cervical component of dystonia.

Circular RNA testis-expressed 14 overexpression induces apoptosis and suppresses migration of ox-LDL-stimulated vascular smooth muscle cells via regulating the microRNA 6509-3p/thanatos-associated domain-containing apoptosis-associated protein 1 axis.

Atherosclerosis is a severe vascular disorder causing myocardial infarction, stroke, and gangrene. Circular RNA Testis-expressed 14 (hsa_circ_0107197, CircTEX14) is a newly discovered circRNA that may have a critical role in the pathogenesis of atherosclerosis. Here, we aimed to further explore the exact role of circRNA TEX14 in the cardiovascular system. Serum samples of atherosclerosis patients (n = 48) and healthy volunteers (n = 48) were collected to assess circTEX14 expressions. Quantitative reverse transcription-PCR (qRT-PCR), cell proliferation assay, migration assay, cell necrosis assay, Annexin staining, TUNEL assays, RNA immunoprecipitation (RIP) assays, dual-luciferase reporter assays, wound healing assays, and Western blot were performed to examine the roles of circTEX14, miR-6509-3p, and thanatos-associated domain-containing apoptosis-associated protein 1 (THAP1) in ox-LDL-stimulated vascular smooth muscle cells (VSMCs). We found that circTEX14 expressions were decreased and miR-6509-3p expressions were increased in the serum samples of atherosclerosis patients and ox-LDL-stimulated VSMCs. CircTEX14 overexpression inhibited proliferation and migration and enhanced apoptosis of VSMCs. CircTEX14 suppressed miR-6509-3p expressions through direct interaction. MiR-6509-3p or THAP1 knockdown reversed the effects of circTEX14 overexpression on proliferation, migration, and apoptosis of ox-LDL-stimulated VSMCs. In conclusion, circTEX14 inhibited proliferation and enhanced apoptosis via modulating miR-6509-3p/THAP1 in ox-LDL-stimulated VSMCs and might be a useful target for atherosclerosis treatment.

DYT6 mutated THAP1 is a cell type dependent regulator of the SP1 family.

DYT6 dystonia is caused by mutations in the transcription factor THAP1. THAP1 knock-out or knock-in mouse models revealed complex gene expression changes, which are potentially responsible for the pathogenesis of DYT6 dystonia. However, how THAP1 mutations lead to these gene expression alterations and whether the gene expression changes are also reflected in the brain of THAP1 patients are still unclear. In this study we used epigenetic and transcriptomic approaches combined with multiple model systems [THAP1 patients' frontal cortex, THAP1 patients' induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons, THAP1 heterozygous knock-out rat model, and THAP1 heterozygous knock-out SH-SY5Y cell lines] to uncover a novel function of THAP1 and the potential pathogenesis of DYT6 dystonia. We observed that THAP1 targeted only a minority of differentially expressed genes caused by its mutation. THAP1 mutations lead to dysregulation of genes mainly through regulation of SP1 family members, SP1 and SP4, in a cell type dependent manner. Comparing global differentially expressed genes detected in THAP1 patients' iPSC-derived midbrain dopaminergic neurons and THAP1 heterozygous knock-out rat striatum, we observed many common dysregulated genes and 61 of them were involved in dystonic syndrome-related pathways, like synaptic transmission, nervous system development, and locomotor behaviour. Further behavioural and electrophysiological studies confirmed the involvement of these pathways in THAP1 knock-out rats. Taken together, our study characterized the function of THAP1 and contributes to the understanding of the pathogenesis of primary dystonia in humans and rats. As SP1 family members were dysregulated in some neurodegenerative diseases, our data may link THAP1 dystonia to multiple neurological diseases and may thus provide common treatment targets.

Emerging role of a systems biology approach to elucidate factors of reduced penetrance: transcriptional changes in THAP1-linked dystonia as an example.

Pathogenic variants in THAP1 can cause dystonia with a penetrance of about 50 %. The underlying mechanisms are unknown and can be considered as means of endogenous disease protection. Since THAP1 encodes a transcription factor, drivers of this variability putatively act at the transcriptome level. Several transcriptome studies tried to elucidate THAP1 function in diverse cellular and mouse models, including mutation carrier-derived cells and iPSC-derived neurons, unveiling various differentially expressed genes and affected pathways. These include nervous system development, dopamine signalling, myelination, or cell-cell adhesion. A network diffusion analysis revealed mRNA splicing, mitochondria, DNA repair, and metabolism as significant pathways that may represent potential targets for therapeutic interventions.

Dystonia-specific mutations in THAP1 alter transcription of genes associated with neurodevelopment and myelin.

Dystonia is a neurologic disorder associated with an increasingly large number of genetic variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is critical in understanding the pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations. We used this strategy to investigate the molecular consequences of genetic variation in THAP1, which encodes a transcription factor linked to neural differentiation. Multiple pathogenic mutations associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter DNA-binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight alterations in a common induced pluripotent stem cell background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling followed by joint analysis of the most robust signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. On the basis of these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.

Reduced Expression of GABA A Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABA A receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABA A receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

The dystonia gene THAP1 controls DNA double-strand break repair choice.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

Linking Penetrance and Transcription in DYT-THAP1: Insights From a Human iPSC-Derived Cortical Model.

BACKGROUND: The THAP1 gene encodes a transcription factor, and pathogenic variants cause a form of autosomal dominant, isolated dystonia (DYT-THAP1) with reduced penetrance. Factors underlying both reduced penetrance and the disease mechanism of DYT-THAP1 are largely unknown. METHODS: We performed transcriptome analysis on 29 cortical neuronal precursors derived from human-induced pluripotent stem cell lines generated from manifesting and nonmanifesting THAP1 mutation carriers and control individuals. RESULTS: Whole transcriptome analysis showed a penetrance-linked signature with expressional changes more pronounced in the group of manifesting (MMCs) than in nonmanifesting mutation carriers (NMCs) when compared to controls. A direct comparison of the transcriptomes in MMCs versus NMCs showed significant upregulation of the DRD4 gene in MMCs. A gene set enrichment analysis demonstrated alterations in various neurotransmitter release cycle pathways, extracellular matrix organization, and deoxyribonucleic acid methylation between MMCs and NMCs. When specifically considering transcription factors, the expression of YY1 and SIX2 differed in MMCs versus NMCs. Further, THAP1 was upregulated in the group of MMCs. CONCLUSIONS: To our knowledge, this is the first report systematically analyzing reduced penetrance in DYT-THAP1 in a human model using transcriptomes. Our findings indicate that transcriptional alterations during cortical development influence DYT-THAP1 pathogenesis and penetrance. We reinforce previously linked pathways including dopamine and eukaryotic translation initiation factor 2 alpha signaling in the pathogenesis of dystonia including DYT-THAP1 and suggest extracellular matrix organization and deoxyribonucleic acid methylation as mediators of disease protection. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genotype-Phenotype Relations for Isolated Dystonia Genes: MDSGene Systematic Review.

This comprehensive MDSGene review is devoted to 7 genes - TOR1A, THAP1, GNAL, ANO3, PRKRA, KMT2B, and HPCA - mutations in which may cause isolated dystonia. It followed MDSGene's standardized data extraction protocol and screened a total of ~1200 citations. Phenotypic and genotypic data on ~1200 patients with 254 different mutations were curated and analyzed. There were differences regarding age at onset, site of onset, and distribution of symptoms across mutation carriers in all 7 genes. Although carriers of TOR1A, THAP1, PRKRA, KMT2B, or HPCA mutations mostly showed childhood and adolescent onset, patients with GNAL and ANO3 mutations often developed first symptoms in adulthood. GNAL and KMT2B mutation carriers frequently have 1 predominant site of onset, that is, the neck (GNAL) or the lower limbs (KMT2B), whereas site of onset in DYT-TOR1A, DYT-THAP1, DYT-ANO3, DYT-PRKRA, and DYT-HPCA was broader. However, in most DYT-THAP1 and DYT-ANO3 patients, dystonia first manifested in the upper half of the body (upper limb, neck, and craniofacial/laryngeal), whereas onset in DYT-TOR1A, DYT-PRKRA and DYT-HPCA was frequently observed in an extremity, including both upper and lower ones. For ANO3, a segmental/multifocal distribution was typical, whereas TOR1A, PRKRA, KMT2B, and HPCA mutation carriers commonly developed generalized dystonia. THAP1 mutation carriers presented with focal, segmental/multifocal, or generalized dystonia in almost equal proportions. GNAL mutation carriers rarely showed generalization. This review provides a comprehensive overview of the current knowledge of hereditary isolated dystonia. The data are also available in an online database (http://www.mdsgene.org), which additionally offers descriptive summary statistics. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Abnormal cerebellar function and tremor in a mouse model for non-manifesting partially penetrant dystonia type 6.

KEY POINTS: Loss-of-function mutations in the Thap1 gene cause partially penetrant dystonia type 6 (DYT6). Some non-manifesting DYT6 mutation carriers have tremor and abnormal cerebello-thalamo-cortical signalling. We show that Thap1 heterozygote mice have action tremor, a reduction in cerebellar neuron number, and abnormal electrophysiological signals in the remaining neurons. These results underscore the importance of Thap1 levels for cerebellar function. These results uncover how cerebellar abnormalities contribute to different dystonia-associated motor symptoms. ABSTRACT: Loss-of-function mutations in the Thanatos-associated domain-containing apoptosis-associated protein 1 (THAP1) gene cause partially penetrant autosomal dominant dystonia type 6 (DYT6). However, the neural abnormalities that promote the resultant motor dysfunctions remain elusive. Studies in humans show that some non-manifesting DYT6 carriers have altered cerebello-thalamo-cortical function with subtle but reproducible tremor. Here, we uncover that Thap1 heterozygote mice have action tremor that rises above normal baseline values even though they do not exhibit overt dystonia-like twisting behaviour. At the neural circuit level, we show using in vivo recordings in awake Thap1+/- mice that Purkinje cells have abnormal firing patterns and that cerebellar nuclei neurons, which connect the cerebellum to the thalamus, fire at a lower frequency. Although the Thap1+/- mice have fewer Purkinje cells and cerebellar nuclei neurons, the number of long-range excitatory outflow projection neurons is unaltered. The preservation of interregional connectivity suggests that abnormal neural function rather than neuron loss instigates the network dysfunction and the tremor in Thap1+/- mice. Accordingly, we report an inverse correlation between the average firing rate of cerebellar nuclei neurons and tremor power. Our data show that cerebellar circuitry is vulnerable to Thap1 mutations and that cerebellar dysfunction may be a primary cause of tremor in non-manifesting DYT6 carriers and a trigger for the abnormal postures in manifesting patients.