Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Formin-mediated nuclear actin at androgen receptors promotes transcription.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic and behavioural functions, and is involved in various conditions including androgen insensitivity syndrome and prostate cancer1. Here we identified functional mutations in the formin and actin nucleator DAAM2 in patients with androgen insensitivity syndrome. DAAM2 was enriched in the nucleus, where its localization correlated with that of the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2 AR droplets ranged from 0.02 to 0.06 µm3 in size and associated with active RNA polymerase II. DAAM2 polymerized actin directly at the AR to promote droplet coalescence in a highly dynamic manner, and nuclear actin polymerization is required for prostate-specific antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

STIGMA: Single-cell tissue-specific gene prioritization using machine learning.

Clinical exome and genome sequencing have revolutionized the understanding of human disease genetics. Yet many genes remain functionally uncharacterized, complicating the establishment of causal disease links for genetic variants. While several scoring methods have been devised to prioritize these candidate genes, these methods fall short of capturing the expression heterogeneity across cell subpopulations within tissues. Here, we introduce single-cell tissue-specific gene prioritization using machine learning (STIGMA), an approach that leverages single-cell RNA-seq (scRNA-seq) data to prioritize candidate genes associated with rare congenital diseases. STIGMA prioritizes genes by learning the temporal dynamics of gene expression across cell types during healthy organogenesis. To assess the efficacy of our framework, we applied STIGMA to mouse limb and human fetal heart scRNA-seq datasets. In a cohort of individuals with congenital limb malformation, STIGMA prioritized 469 variants in 345 genes, with UBA2 as a notable example. For congenital heart defects, we detected 34 genes harboring nonsynonymous de novo variants (nsDNVs) in two or more individuals from a set of 7,958 individuals, including the ortholog of Prdm1, which is associated with hypoplastic left ventricle and hypoplastic aortic arch. Overall, our findings demonstrate that STIGMA effectively prioritizes tissue-specific candidate genes by utilizing single-cell transcriptome data. The ability to capture the heterogeneity of gene expression across cell populations makes STIGMA a powerful tool for the discovery of disease-associated genes and facilitates the identification of causal variants underlying human genetic disorders.

Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator.

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.

Single-cell RNA-based phenotyping reveals a pivotal role of thyroid hormone receptor alpha for hypothalamic development.

Thyroid hormone and its receptor TRα1 play an important role in brain development. Several animal models have been used to investigate this function, including mice heterozygous for the TRα1R384C mutation, which confers receptor-mediated hypothyroidism. These mice display abnormalities in several autonomic functions, which was partially attributed to a developmental defect in hypothalamic parvalbumin neurons. However, whether other cell types in the hypothalamus are similarly affected remains unknown. Here, we used single-nucleus RNA sequencing to obtain an unbiased view on the importance of TRα1 for hypothalamic development and cellular diversity. Our data show that defective TRα1 signaling has surprisingly little effect on the development of hypothalamic neuronal populations, but it heavily affects hypothalamic oligodendrocytes. Using selective reactivation of the mutant TRα1 during specific developmental periods, we find that early postnatal thyroid hormone action seems to be crucial for proper hypothalamic oligodendrocyte maturation. Taken together, our findings underline the well-known importance of postnatal thyroid health for brain development and provide an unbiased roadmap for the identification of cellular targets of TRα1 action in mouse hypothalamic development.

Structural Variants at the LMNB1 Locus: Deciphering Pathomechanisms in Autosomal Dominant Adult-Onset Demyelinating Leukodystrophy.

OBJECTIVE: We aimed to elucidate the pathogenic mechanisms underlying autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), and to understand the genotype/phenotype correlation of structural variants (SVs) in the LMNB1 locus. BACKGROUND: Since the discovery of 3D genome architectures and topologically associating domains (TADs), new pathomechanisms have been postulated for SVs, regardless of gene dosage changes. ADLD is a rare genetic disease associated with duplications (classical ADLD) or noncoding deletions (atypical ADLD) in the LMNB1 locus. METHODS: High-throughput chromosome conformation capture, RNA sequencing, histopathological analyses of postmortem brain tissues, and clinical and neuroradiological investigations were performed. RESULTS: We collected data from >20 families worldwide carrying SVs in the LMNB1 locus and reported strong clinical variability, even among patients carrying duplications of the entire LMNB1 gene, ranging from classical and atypical ADLD to asymptomatic carriers. We showed that patients with classic ADLD always carried intra-TAD duplications, resulting in a simple gene dose gain. Atypical ADLD was caused by LMNB1 forebrain-specific misexpression due to inter-TAD deletions or duplications. The inter-TAD duplication, which extends centromerically and crosses the 2 TAD boundaries, did not cause ADLD. Our results provide evidence that astrocytes are key players in ADLD pathology. INTERPRETATION: Our study sheds light on the 3D genome and TAD structural changes associated with SVs in the LMNB1 locus, and shows that a duplication encompassing LMNB1 is not sufficient per se to diagnose ADLD, thereby strongly affecting genetic counseling. Our study supports breaking TADs as an emerging pathogenic mechanism that should be considered when studying brain diseases. ANN NEUROL 2024;96:855-870.

The single-cell transcriptional landscape of mammalian organogenesis.

Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution. Using single-cell combinatorial indexing, we profiled the transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting 'mouse organogenesis cell atlas' (MOCA) provides a global view of developmental processes during this critical window. We use Monocle 3 to identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. We explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle.

Position effects at the FGF8 locus are associated with femoral hypoplasia.

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.

Multi-gene panel sequencing in highly consanguineous families and patients with congenital forms of skeletal dysplasias.

Skeletal dysplasias (SKDs) are a heterogeneous group of more than 750 genetic disorders characterized by abnormal development, growth, and maintenance of bones or cartilage in the human skeleton. SKDs are often caused by variants in early patterning genes and in many cases part of multiple malformation syndromes and occur in combination with non-skeletal phenotypes. The aim of this study was to investigate the underlying genetic cause of congenital SKDs in highly consanguineous Pakistani families, as well as in sporadic and familial SKD cases from India using multigene panel sequencing analysis. Therefore, we performed panel sequencing of 386 bone-related genes in 7 highly consanguineous families from Pakistan and 27 cases from India affected with SKDs. In the highly consanguineous families, we were able to identify the underlying genetic cause in five out of seven families, resulting in a diagnostic yield of 71%. Whereas, in the sporadic and familial SKD cases, we identified 12 causative variants, corresponding to a diagnostic yield of 44%. The genetic heterogeneity in our cohorts was very high and we were able to detect various types of variants, including missense, nonsense, and frameshift variants, across multiple genes known to cause different types of SKDs. In conclusion, panel sequencing proved to be a highly effective way to decipher the genetic basis of SKDs in highly consanguineous families as well as sporadic and or familial cases from South Asia. Furthermore, our findings expand the allelic spectrum of skeletal dysplasias.

Structural variation in the 3D genome.

Structural and quantitative chromosomal rearrangements, collectively referred to as structural variation (SV), contribute to a large extent to the genetic diversity of the human genome and thus are of high relevance for cancer genetics, rare diseases and evolutionary genetics. Recent studies have shown that SVs can not only affect gene dosage but also modulate basic mechanisms of gene regulation. SVs can alter the copy number of regulatory elements or modify the 3D genome by disrupting higher-order chromatin organization such as topologically associating domains. As a result of these position effects, SVs can influence the expression of genes distant from the SV breakpoints, thereby causing disease. The impact of SVs on the 3D genome and on gene expression regulation has to be considered when interpreting the pathogenic potential of these variant types.

Disruption of the topologically associated domain at Xp21.2 is related to 46,XY gonadal dysgenesis.

BACKGROUND: Duplications at the Xp21.2 locus have previously been linked to 46,XY gonadal dysgenesis (GD), which is thought to result from gene dosage effects of NR0B1 (DAX1), but the exact disease mechanism remains unknown. METHODS: Patients with 46,XY GD were analysed by whole genome sequencing. Identified structural variants were confirmed by array CGH and analysed by high-throughput chromosome conformation capture (Hi-C). RESULTS: We identified two unrelated patients: one showing a complex rearrangement upstream of NR0B1 and a second harbouring a 1.2 Mb triplication, including NR0B1. Whole genome sequencing and Hi-C analysis revealed the rewiring of a topological-associated domain (TAD) boundary close to NR0B1 associated with neo-TAD formation and may cause enhancer hijacking and ectopic NR0B1 expression. Modelling of previous Xp21.2 structural variations associated with isolated GD support our hypothesis and predict similar neo-TAD formation as well as TAD fusion. CONCLUSION: Here we present a general mechanism how deletions, duplications or inversions at the NR0B1 locus can lead to partial or complete GD by disrupting the cognate TAD in the vicinity of NR0B1. This model not only allows better diagnosis of GD with copy number variations (CNVs) at Xp21.2, but also gives deeper insight on how spatiotemporal activation of developmental genes can be disrupted by reorganised TADs causing impairment of gonadal development.

Hi-C Identifies Complex Genomic Rearrangements and TAD-Shuffling in Developmental Diseases.

Genome-wide analysis methods, such as array comparative genomic hybridization (CGH) and whole-genome sequencing (WGS), have greatly advanced the identification of structural variants (SVs) in the human genome. However, even with standard high-throughput sequencing techniques, complex rearrangements with multiple breakpoints are often difficult to resolve, and predicting their effects on gene expression and phenotype remains a challenge. Here, we address these problems by using high-throughput chromosome conformation capture (Hi-C) generated from cultured cells of nine individuals with developmental disorders (DDs). Three individuals had previously been identified as harboring duplications at the SOX9 locus and six had been identified with translocations. Hi-C resolved the positions of the duplications and was instructive in interpreting their distinct pathogenic effects, including the formation of new topologically associating domains (neo-TADs). Hi-C was very sensitive in detecting translocations, and it revealed previously unrecognized complex rearrangements at the breakpoints. In several cases, we observed the formation of fused-TADs promoting ectopic enhancer-promoter interactions that were likely to be involved in the disease pathology. In summary, we show that Hi-C is a sensible method for the detection of complex SVs in a clinical setting. The results help interpret the possible pathogenic effects of the SVs in individuals with DDs.

Precision medicine in rare diseases: What is next?

Molecular diagnostics is a cornerstone of modern precision medicine, broadly understood as tailoring an individual's treatment, follow-up, and care based on molecular data. In rare diseases (RDs), molecular diagnoses reveal valuable information about the cause of symptoms, disease progression, familial risk, and in certain cases, unlock access to targeted therapies. Due to decreasing DNA sequencing costs, genome sequencing (GS) is emerging as the primary method for precision diagnostics in RDs. Several ongoing European initiatives for precision medicine have chosen GS as their method of choice. Recent research supports the role for GS as first-line genetic investigation in individuals with suspected RD, due to its improved diagnostic yield compared to other methods. Moreover, GS can detect a broad range of genetic aberrations including those in noncoding regions, producing comprehensive data that can be periodically reanalyzed for years to come when further evidence emerges. Indeed, targeted drug development and repurposing of medicines can be accelerated as more individuals with RDs receive a molecular diagnosis. Multidisciplinary teams in which clinical specialists collaborate with geneticists, genomics education of professionals and the public, and dialogue with patient advocacy groups are essential elements for the integration of precision medicine into clinical practice worldwide. It is also paramount that large research projects share genetic data and leverage novel technologies to fully diagnose individuals with RDs. In conclusion, GS increases diagnostic yields and is a crucial step toward precision medicine for RDs. Its clinical implementation will enable better patient management, unlock targeted therapies, and guide the development of innovative treatments.

Comparative single-cell analysis of the adult heart and coronary vasculature.

The structure and function of the circulatory system, including the heart, have undergone substantial changes with the vertebrate evolution. Although the basic function of the heart is to pump blood through the body, its size, shape, speed, regeneration capacity, etc. vary considerably across species. Here, we address the differences among vertebrate hearts using a single-cell transcriptomics approach. Published datasets of macaque (Macaca fascicularis), mouse, and zebrafish hearts were integrated and compared to the human heart as a reference. While the three mammalian hearts integrated well, the zebrafish heart showed very little overlap with the other species. Our analysis revealed a mouse-specific cell subpopulation of ventricular cardiomyocytes (CM), represented by strikingly different expression patterns of specific genes related to high-energy metabolism. Interestingly, the observed differences between mouse and human CM coincided with actual biological differences between the two species. Smooth muscle and endothelial cells (EC) exhibited species-specific differences in clustering and gene expression, respectively, which we attribute to the tissues selected for sequencing, given different focuses of the original studies. Finally, we compared human and zebrafish heart-specific fibroblasts (FB) and identified a distinctively high expression of genes associated with heart regeneration following injury in zebrafish. Together, our results show that integration of numerous datasets of different species and different sequencing technologies is feasible and that this approach can identify species-specific differences and similarities in the heart.

HOXD13-associated synpolydactyly: Extending and validating the genotypic and phenotypic spectrum with 38 new and 49 published families.

PURPOSE: HOXD13 is an important regulator of limb development. Pathogenic variants in HOXD13 cause synpolydactyly type 1 (SPD1). How different types and positions of HOXD13 variants contribute to genotype-phenotype correlations, penetrance, and expressivity of SPD1 remains elusive. Here, we present a novel cohort and a literature review to elucidate HOXD13 phenotype-genotype correlations. METHODS: Patients with limb anomalies suggestive of SPD1 were selected for analysis of HOXD13 by Sanger sequencing, repeat length analysis, and next-generation sequencing. Literature was reviewed for HOXD13 heterozygotes. Variants were annotated for phenotypic data. Severity was calculated, and cluster and decision-tree analyses were performed. RESULTS: We identified 98 affected members of 38 families featuring 11 different (likely) causative variants and 4 variants of uncertain significance. The most frequent (25/38) were alanine repeat expansions. Phenotypes ranged from unaffected heterozygotes to severe osseous synpolydactyly, with intra- and inter-familial heterogeneity and asymmetry. A literature review provided 160 evaluable affected members of 49 families with SPD1. Computer-aided analysis only corroborated a positive correlation between alanine repeat length and phenotype severity. CONCLUSION: Our findings support that HOXD13-protein condensation in addition to haploinsufficiency is the molecular pathomechanism of SPD1. Our data may, also, facilitate the interpretation of synpolydactyly radiographs by future automated tools.

Interaction of Mitochondrial Polygenic Score and Lifestyle Factors in LRRK2 p.Gly2019Ser Parkinsonism.

BACKGROUND: A mitochondrial polygenic score (MGS) is composed of genes related to mitochondrial function and found to be associated with Parkinson's disease (PD) risk. OBJECTIVE: To investigate the impact of the MGS and lifestyle/environment on age at onset (AAO) in LRRK2 p.Gly2019Ser parkinsonism (LRRK2-PD) and idiopathic PD (iPD). METHODS: We included N = 486 patients with LRRK2-PD and N = 9259 with iPD from the Accelerating Medicines Partnership® Parkinson's Disease Knowledge Platform (AMP-PD), Fox Insight, and a Tunisian Arab-Berber founder population. Genotyping data were used to perform the MGS analysis. Additionally, lifestyle/environmental data were obtained from the PD Risk Factor Questionnaire (PD-RFQ). Linear regression models were used to assess the relationship between MGS, lifestyle/environment, and AAO. RESULTS: Our derived MGS was significantly higher in PD cases compared with controls (P = 1.1 × 10-8 ). We observed that higher MGS was significantly associated with earlier AAO in LRRK2-PD (P = 0.047, ß = -1.40) and there was the same trend with a smaller effect size in iPD (P = 0.231, ß = 0.22). There was a correlation between MGS and AAO in LRRK2-PD patients of European descent (P = 0.049, r = -0.12) that was visibly less pronounced in Tunisians (P = 0.449, r = -0.05). We found that the MGS interacted with caffeinated soda consumption (P = 0.003, ß = -5.65) in LRRK2-PD and with tobacco use (P = 0.010, ß = 1.32) in iPD. Thus, patients with a high MGS had an earlier AAO only if they consumed caffeinated soda or were non-smokers. CONCLUSIONS: The MGS was more strongly associated with earlier AAO in LRRK2-PD compared with iPD. Caffeinated soda consumption or tobacco use interacted with MGS to predict AAO. Our study suggests gene-environment interactions as modifiers of AAO in LRRK2-PD. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state.

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease aetiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease using the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of post-mortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabelling of the same tissues. Moreover, we analysed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in idiopathic Parkinson's disease midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signalling and immunomodulatory treatments in Parkinson's disease.