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

Abstract 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.


Introduction
Dystonia is clinically characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive movements, postures, or both [1].Dystonic movements are typically patterned, twisting, and may be tremulous.Dystonia is a rare disease with a prevalence of about 16 per 100,000 people [2] and may thus affect some 16,000 people in Germany.Within the national research consortium DysTract (http://dystract.cio-marburg.de/de/), information on ∼2,500 of these patients has been collected in a database and DNA biobank for research towards improved understanding of the genetic basis and to develop individualized, i. e., pathophysiology-based, treatment.Within the past 20 years, several genetic forms have been identified for isolated (dystonia as the only disease manifestation) and combined (dystonia in combination with another movement disorder) dystonia [1].The former includes the THAP1 (THAP domain-containing apoptosis-associated protein 1) gene [3].THAP1-linked dystonia, previously referred to as DYT6 dystonia, is characterized by early-onset dystonia with prominent craniocervical and upper limb muscle involvement (Figure 1) [4,5].Speech impairment due to laryngeal dystonia is common and very characteristic.However, the THAP1-linked phenotype is highly variable, ranging from unaffected carriers to severe generalized dystonia, even within a single family.The disease is inherited in an autosomal dominant fashion with a penetrance of about 50 % [4,6,7].The THAP1 gene encodes a ubiquitously expressed transcription factor consisting of 213 amino acids and is putatively regulating the expression of various target genes [8], including TOR1A [9], the gene mutated in another form of dystonia, and THAP1 itself [10].Its DNA-binding properties are associated with the N-terminal THAP domain (amino acids 1-81), including a zinc-finger structure.Towards the C-terminus, THAP1 contains a prolinerich region (amino acids 96-108) and a coiled-coil domain (amino acids  with a nuclear localization signal (NLS, amino acids 147-162) [11].To date, ∼100 missense, nonsense, and frameshift mutations in THAP1 have been described in dystonia patients of different ethnicities [4].It is believed that the mutations act in a loss-of-function mechanism [12,13].
Reduced penetrance may reflect endogenous disease protection.Therefore, understanding the underlying factors and processes might open novel therapeutic avenues.However, factors contributing to incomplete penetrance and variable expressivity as well as the disease mechanism of THAP1 dystonia are largely unknown.Despite identical variants in the THAP1 gene, disease manifestation varies considerably between individuals.Since THAP1 encodes a transcription factor, it is tempting to speculate that the drivers of this variability are acting at the transcriptomic level accompanied by alterations at the genomic, epige- netic, proteomic, metabolomic, and/or environmental levels.It can be hypothesized that identifying protective variants will enable expansion of genetic testing and thus a more sophisticated prediction of the disease course in THAP1 mutation carriers.Further, the elucidation of altered (transcriptional) networks that contribute to disease protection can unravel potential therapeutic targets by manipulating these networks in the desired direction using, for instance, small molecules.

Transcriptional studies
Initial attempts to understand the role of THAP1 in transcriptional regulation were carried out before establishing the disease link to dystonia.At that time, researchers investigated transcriptional changes in endothelial cells [8].They linked THAP1 function to cell cycle control: Retroviral-mediated gene transfer of THAP1 in primary human endothelial cells inhibited proliferation and G1/S cell-cycle progression.THAP1 overexpression downregulated > 50 genes encoding proteins associated with cellcycle/cell proliferation [8].Further, a few other differentially expressed genes (DEGs) were linked to diverse biological functions.siRNA-mediated THAP1 knock-down resulted in inhibition of S-phase DNA synthesis.Of note, this early study had already indicated downregulation of THAP1 when over-expressed [8], the mechanism of which, i. e., autoregulation, was resolved later [10].
To unravel the THAP1-mediated disease mechanism in dystonia, the search for neuronal targets and DEGs is ongoing, and different cellular and mouse models have been established.In mutant or overexpression THAP1 models, it is expected that target expression will be altered.Mean-while, several dysregulated genes have been found using these models.Table 1 summarizes relevant candidate DEGs from unbiased transcriptome studies [8,[14][15][16][17][18][19][20][21][22].
By this long list of DEGs and the diverse pathways affected by dysfunctional THAP1, the role of THAP1 seems to be manifold: Transgenic mice expressing heterozygous loss-of-function THAP1 showed alterations in the expression of genes involved in nervous system development [14].THAP1 is also necessary for the timing of myelination initiation in oligodendrocytes [16].Further, dysregulation of genes involved in the eIF2α (Eukaryotic Initiation Factor 2 alpha) signalling pathway, mitochondrial dysfunction, and neuron projection development have been observed in the brains of THAP1 +/-(ΔExon2) knockout mice [15].An essential role for THAP1 in cell survival and proliferation has been demonstrated in murine embryonic stem cells [23].Moreover, it was found that wild-type THAP1 regulates genes involved in cell growth and proliferation in neuronal cells, while mutant THAP1 leads to the dysregulation of genes related to synaptic function, a process that has been reported as a pathogenic mechanism of other subtypes of dystonia [19].In patient-derived cortical neurons, dopamine signalling seemed to be altered and involved in disease expression [17].

DNA-binding of the transcription factor THAP1
It is thought that transcriptional regulation via THAP1 is mediated by binding of THAP1 to promoter regions within the nuclear DNA.There is a significant co-binding with other transcription factors, such as HCFC1 [24] or YY1 [16], as also underlined by the ENCODE CHIP-Seq studies.YY1 encodes another transcription factor with a recognized and established role in myelination; pathogenic variants in YY1 cause a severe neurodevelopmental disorder [25] as well as early-onset dystonia [26,27].It has been shown that THAP1 modulates the DNA occupancy of YY1 in nonconditional knockout mice and that loss of THAP1 impairs myelination in the central nervous system via a cellautonomous role in decreased DNA occupancy and oligodendrocyte lineage [16].Of note, YY1 has been found to be upregulated in cortical neurons of manifesting when compared to non-manifesting mutation carriers or healthy controls [17].The low percentage (around 10 %) of overlap between RNA-Seq and ChIP-Seq datasets may highlight the role of important THAP1 co-factors like YY1 and HCFC1 and indicate a different role of THAP1 in regulating gene expression other than direct binding at DNA, as in other zinc-finger factors [23].Further, THAP1 ChIP-Seq analysis in neuronal cells overexpressing THAP1 revealed that THAP1 is able to bind and activate promoter regions of different SOD2 isoforms in SK-N-AS human neuroblastoma cells [19].Knockout of SOD2 in mice seems to impair mitochondrial enzyme activity leading to elevated reactive oxygen species (ROS) content in synaptosomes, altering synaptic function [28].This discovery is a possible means of how THAP1 mutations cause an expressional change of genes related to synaptic function.

Pathway analyses in the pathogenesis of THAP1
To put the different DEGs from the various THAP1 studies into a broader picture, gene enrichment analysis has been performed using Gene Ontology (GO) terms or KEGG pathways.Affected pathways are diverse and include peripheral nervous system development, cytoskeleton, neuron projection development, dopamine signalling, myelination, axonal guidance, long-term synaptic depression, cell-cell adhesion, gliogenesis, and muscle movement and spasm [14,15,17,29].In fact, higher brain regions such as those underlying sensorimotor function may be dysfunctional, acting jointly with abnormalities attributable to the noradrenergic system originating in the locus coeruleus of the brainstem.
The link to dopamine signalling seems particularly interesting and strong: Links between dystonia and dopamine are numerous [30], and although isolated dystonia, especially DYT-THAP1, is usually not responsive to dopaminergic treatment, recent data suggest that DRD4 expression levels may play a role in mediating penetrance in THAP1 mutation carriers [17].DRD4 is a member of the dopamine D2-like receptor family, characterized by its ability to inhibit adenylyl cyclase.DRD2 encodes a dopamine receptor expressed by striatal medium spiny neurons and plays a critical role in the indirect pathway of the basal ganglia.Recently, DRD2 was demonstrated to have a fundamental role in motor control and balance in knockout mice's medium spiny neurons and cholinergic interneurons [31].Further, DRD2 may be one of the indirect targets involved in the pathogenic pathways disrupted by THAP1 transcriptional deficit [14].Other studies also provided evidence for changes in the dopamine signalling pathway as one of the top hits among several neurotransmitter-linked pathways upregulated in dystonia [14,17].A subnetwork of differentially regulated genes connected to cell cycle regulation and neurogenesis has been identified and may provide a molecular explanation for the disrupted dopaminergic neurotransmission and neuronal biogenesis in the pathogenesis of dystonia [31].
Further, a significant downregulation of genes related to apoptosis, including CRADD (CASP2 And RIPK1 Domain Containing Adaptor with Death Domain), SIX2 (Homeobox protein SIX2), and a significant dysregulation of genes implicated in autophagy and mitochondrial homeostasis including ATF4 (Activating Transcription Factor 4), LYRM1 (LYR Motif Containing 1) and SOD2 (Superoxide dismutase 2) were observed in cortical neuronal precursors derived from human iPSC as well as in mice [14,15,17].
Most studies on differential gene expression in neuronal cells also revealed a transcriptional signature that point to THAP1 as a regulator of inflammatory responses by regulation of Interleukin-5 and Interleukin-6 production [14,16,17,21].Further, GO analyses of biological processes of upregulated genes in murine embryonic stem cells revealed terms related to embryonic pattern specification, chromosome organization, meiosis, and negative regulation of cell differentiation [23].Analysis of downregulated genes in transgenic mice and murine embryonic stem cells proved enrichment for processes involved in neuronal development like axonogenesis, differentiation of neurons, and cell projection assembly and organization [14,23].
Another pathway that repeatedly came up as being regulated by THAP1 was the eIF2α pathway in mouse and human models.Interestingly, the elF2α pathway seems to be involved in the pathogenesis of different (monogenic) forms of dystonia, such as DYT-TOR1A or DYT-PRKRA, and probably also SGCE-linked myoclonus dystonia [17,32].The most recent evidence for the role of the elF2α pathway stems from the discovery of pathogenic variants in a member of the eIF2α kinases family, EIF2AK2 (Eukaryotic translation initiation factor 2 alpha kinase 2), in early-onset generalized dystonia [32].In addition to being a key component of Endoplasmic Reticulum (ER) stress responses and synaptic plasticity, the eIF2α signalling also regulates important physiological events under homeostatic conditions like the accumulation of misfolded proteins [15].Thus, the eIF2α dysregulation may represent a point of convergence between different forms of dystonia through its influence on critical homeostatic neurodevelopmental events.Therefore, it is conceivable that eIF2α signalling is involved in the expressivity of THAP1 mutations [15].Interestingly dysregulation of the eIF2α pathway has been ob-served in cortical neurons and fibroblasts of dystonia patients as well [17].
Several of the affected pathways have recurrently been implicated in response to alterations of THAP1.To test for the functional relatedness of all the studies mentioned above and to find regulatory interactions of all the identified THAP1-associated genes and proteins in an unbiased way, we mapped the genes from Table 1 on the protein interaction network from StringDB [33] (Version 11, confidence cutoff score 0.7).This database includes both known and predicted protein-protein interactions of various organisms that are inferred from direct physical and indirect functional associations from high-and low- throughput experiments, as well as computational prediction.
In total, 45 genes were assigned to the human proteinprotein interaction network, with 33 proteins sharing 62 interactions (Figure 2).Interestingly, we would expect only 27 interactions from a network of 45 randomly picked proteins.Thus, we have significantly higher connectivity of THAP1-associated proteins (p-value = 1.02 × 10 −8 ), which indicates their functional relatedness that should be investigated further.
To further investigate the function of the differentially regulated transcripts and proteins in combination with their neighbouring interacting proteins, we performed a network diffusion on the protein-protein interaction network from StringDB using the R library diffuStats [34].Network diffusion assumes that the effect of differential gene regulation also spreads to the neighbours on a proteinprotein interaction network.The effect size is calculated by "diffusing" the magnitude of differential regulation across the protein interaction network until a steady-state is reached.Considering all affected nodes above a certain diffusion score then provides a broader view of the molecular function affected by the initial gene set.Here we considered both the significance and direction of differential regulation of the 45 mapped genes by a log 10 transform of the reported p-values and the sign according to up-or downregulation.Network diffusion was done on a regularised Laplacian kernel, and the 1 %, or respectively 169, pro-teins having the most positive or negative diffusion scores were investigated separately for pathway enrichment by a hypergeometric test [35].Among the proteins affected by upregulated transcripts, we found mRNA splicing, mitochondria, DNA repair, and metabolism as the most significant pathways, while glycosylation, axonogenesis, sphingolipid, death receptor, and TNF signalling seem to be downregulated (Figure 3).While transcriptome analyses repeatedly revealed differential gene regulation correlated with cell cycle regulation, neurogenesis, inflammatory responses, and cell death in different cell and mice models of THAP1-DYT, these overarching analyses expand the pathways that may play a role in the pathophysiology of (THAP1) dystonia and warrant further studies and validation.

Conclusions and outlook
THAP1 activities are likely due to the regulation of gene expression via its role as a transcription factor.However, THAP1 downstream targets in neurons, the mechanism via which THAP1 mutations cause disease, and the disease mechanism underlying isolated dystonia, in general, are all largely unknown.There are no reported THAP1 genotype-phenotype-predictors.A systems biology approach that not only focuses on a single isolated component of the regulatory mechanisms may help to shed further light on dysregulated pathways underlying THAP1 dystonia.These insights will probably allow predictions as to the likely clinical course in THAP1 mutation carriers and are also expected to foster the development of diseasemodifying treatments with the ultimate aim of individualized therapeutic strategies.Thus, certain systems biology patterns may be associated with an overall favorable outcome justifying a "wait and see" strategy, others may indicate the preference for a more severe clinical course necessitating more rigorous management.DYT-THAP1 is likely caused by an interplay of molecular aetiologies that are poorly understood, thereby limiting the efforts of designing functional assays that could be utilized to screen for novel therapeutics.In this context, identifying pathways impacted by THAP1 mutations as factors influencing penetrance and expressivity should be prioritized.These pathways may represent the best potential targets for therapeutic intervention, which could eventually offer widespread benefit to a broad and diverse population of dystonia patients.
Research funding: German Research Foundation (DFG, FOR 2488, TP4).The funding organization played no role in the study design; in the collection, analysis, and Interpretation of data; in the writing of the report; or in the decision to submit the report for publication.Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.Competing interests: Authors state no conflict of interest.Informed consent: Informed consent was obtained from all individuals included in this study.Ethical approval: The local Institutional Review Board deemed the study exempt from review.

Figure 1 :
Figure1: Phenotypic spectrum of reported carriers of THAP1 mutations.In the left panel, the presence (red) or absence (blue) of the indicated signs and symptoms is shown for 249 patients with THAP1 mutations.In the right panel, the age variability at onset is illustrated (modified from www.mdsgene.org[4]).

Figure 2 :
Figure 2: Protein Interaction Network related to THAP1.Interactions of the THAP1-mediated DEGs were obtained via StringDB.The colors of the nodes symbolize the affiliation to specific GO:terms, colors of the edges symbolize the type of protein interaction.The most frequent interaction among these genes is related to data mining in current studies (light yellow border).Only individual clusters are linked via coexpression (black edge) or experimental evidence.

Figure 3 :
Figure3: Hypergeometric test on Reactome pathways of proteins affected by differential gene expression in THAP1 knockdown models.The dot plot includes pathways having at least three affected proteins at a significance cutoff of p < 0.05.Red and black font colors refer to upand down-regulated pathways, respectively.The dot size corresponds to the pathway set size.

Table 1 :
Overview of THAP1 transcriptional expression and regulation in various research models.