Monogenic variants in dystonia: an exome-wide sequencing study

Background Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. Methods For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. Findings We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222;excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. Interpretation In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations

CD83: Einblicke in dessen Funktion und transkriptioneller Regulation in humanen Tregs

Tregs play a critical role in immune homeostasis by suppressing other immune cells. Autoimmunity is a consequence of the breakdown in mechanisms controlling tolerance, which may be impacted by inadequate Treg function. Hence, an appealing therapeutic option is the modulation of Tregs. While CD83 is best known as a marker for mature dendritic cells, several other types of cells, including activated Tregs, express CD83. Previous studies suggest that CD83 is important for the regulatory capacity of Tregs. Furthermore, the tripartite transcriptional module regulating CD83 expression in DCs is cell type-specific while the regulatory elements at the CD83 locus in Tregs have not been identified. Insights into the function and transcriptional regulation of CD83 in human Tregs will be critical for potential Treg therapies in autoimmunity. To facilitate the further exploration of CD83 in human Tregs, we first honed an expansion protocol to generate sufficient stable and functional Tregs. These expanded FOXP3+ Tregs maintained CCR7/CD62L expression and upregulated markers characteristic for Tregs including Helios, LAP, as well as GARP upon stimulation. Furthermore, they were highly suppressive in vitro, in contrast to expanded CD4+ Tconvs. In-depth analyses of CD83 in these cells over 96 hours revealed that CD83 peaked as early as three hours after stimulation on mRNA and protein level, while at the later time points it was found in the soluble form in the supernatant, in line with the expression profile of non-expanded Tregs. Further investigations into the function of this molecule by knocking down its expression in Tregs revealed that CD83 does not affect the transcription factor FOXP3, crucial for Treg function, but GARP mRNA expression. GARP has not only been described as a safeguard of FOXP3, but also as a receptor for LAP in the latent TGF-β complex. Although CD83-specific siRNA transfected Tregs produced less active TGF-β upon activation, their suppressive capacity was not impacted. Finally, to determine the regulatory elements of the CD83 gene governing its expression in Tregs, ChIP-seq experiments were performed whereby four potential enhancer regions were identified based on their epigenetic profile. Further analyses revealed clusters of transcription factor binding sites for NFκB, as well as ABDB, SORY, HMTB and SORY as multiple organized regulatory elements, supporting the definition of these putative regions as enhancers. These findings offer insights into the role and transcriptional regulation of CD83 in highly stable and functional in vitro expanded Tregs. This provides the groundwork for transcriptionally targeting Tregs in vivo to modulate their function, which could lead to new clinical applications for the treatment of autoimmune diseases in the future.Tregs sind essentiell für die Aufrechterhaltung immunologischer Toleranz, da sie andere Immunzellen supprimieren. Autoimmunität ist auf eine Störung dieser Homöostase zurückzuführen, so dass eine gestörte Funktion von Tregs dazu beitragen kann. Eine attraktive therapeutische Option ist daher die Modulation der Tregs. Obwohl CD83 vor allem als Marker für reife dendritische Zellen bekannt ist, wird es auch von anderen Zelltypen, einschließlich aktivierter Tregs, exprimiert. Außerdem wird CD83 eine wichtige Rolle für die Funktionalität von murinen Tregs zugeschrieben. Während die zelltypspezifischen regulatorischen Elemente am CD83-Lokus in dendritischen Zellen schon charakterisiert wurden, sind die Mechanismen der transkriptionellen Regulation dieses Moleküls in Tregs noch unbekannt. Erkenntnisse hierüber sind jedoch für potenzielle Therapien bei Autoimmunität von sehr großem Interesse. Um die Erforschung des CD83-Moleküls in Tregs zu ermöglichen, wurde im Rahmen der vorliegenden Arbeit als erstes ein Protokoll zur Generierung einer großen Anzahl stabiler und funktionaler Tregs etabliert. Die anschließende Analyse dieser expandierten FOXP3+-Tregs zeigte eine stabile CCR7/CD62L-Expression und eine Hochregulation der für Tregs charakteristischen Marker wie Helios, LAP sowie GARP nach Stimulation. Darüber hinaus zeigten sie in vitro, im Gegensatz zu den ebenfalls expandierten konventionellen CD4+-T- Zellen, eine starke suppressorische Aktivität. Umfassende Analysen von CD83 in diesen Zellen über 96 Stunden ergaben, dass die CD83-Expression bereits drei Stunden nach der Stimulation auf mRNA- und Proteinebene seinen Höhepunkt erreichte, während es zu den späteren Zeitpunkten in der löslichen Form im Überstand vorlag, entsprechend dem Expressionsprofil der nicht-expandierten Tregs. Um die Rolle von CD83 hinsichtlich der Funktion in Tregs näher zu betrachten, wurde dessen Expression mittels RNA-Interferenz in den expandierten humanen Tregs unterdrückt. Hierbei zeigte die Herunterregulierung von CD83 keinen Einfluss auf die Expression des Transkriptionsfaktors FOXP3, welcher entscheidend für die Funktion von Tregs ist. Wir konnten allerdings eine Zunahme der Expression von GARP-mRNA detektieren, das zum einen als Stabilisator der FOXP3- Expression und zum anderen als Rezeptor für LAP im latenten TGF-β-Komplex dient. Infolge der Transfektion mit CD83-spezifischer siRNA wurde weniger aktives TGF-β durch die stimulierten Tregs sezerniert. Dies führte allerdings zu keiner verminderten suppressiven Kapazität. Um schließlich die regulatorischen Elemente des CD83-Gens zu bestimmen wurden ChIP-seq-Experimente durchgeführt. Hierbei wurden vier potenzielle Enhancer- Regionen anhand des epigenetischen Profils identifiziert. Weitere Analysen ergaben, dass Ansammlungen von Transkriptionsfaktorbindestellen für NFκB sowie ABDB, SORY, HMTB und SORY als mehrfach organisierte regulatorische Elemente vorliegen, was wiederum die Definition dieser Regionen als sogenannte Enhancer unterstützt. Die in der vorliegenden Arbeit gewonnenen Erkenntnisse unter Verwendung der hochstabilen und funktionellen in vitro expandierten Tregs geben Aufschluss über (a) die Rolle und (b) die transkriptionelle Regulation von CD83. Diese Erkenntnisse bilden die Grundlage für die zielgerichtete Expression therapeutischer Moleküle unter der Kontrolle des CD83-Promotors zur Modulation der Funktion von Tregs und damit zu neuen klinischen Anwendungen für die Behandlung von Autoimmunkrankheiten

Monogenic variants in dystonia: an exome-wide sequencing study

Background Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. Methods For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. Findings We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. Interpretation In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations