High Bone Mass Disorders: New Insights from Connecting the Clinic and the Bench

Monogenic high bone mass (HBM) disorders are characterized by an increased amount of bone in general, or at specific sites in the skeleton. Here, we describe 59 HBM disorders with 50 known disease-causing genes from the literature, and we provide an overview of the signaling pathways and mechanisms involved in the pathogenesis of these disorders. Based on this, we classify the known HBM genes into HBM (sub)groups according to uniform Gene Ontology (GO) terminology. This classification system may aid in hypothesis generation, for both wet lab experimental design and clinical genetic screening strategies. We discuss how functional genomics can shape discovery of novel HBM genes and/or mechanisms in the future, through implementation of omics assessments in existing and future model systems. Finally, we address strategies to improve gene identification in unsolved HBM cases and highlight the importance for cross-laboratory collaborations encompassing multidisciplinary efforts to transfer knowledge generated at the bench to the clinic.Acknowledgements: This publication is initiated upon work from the European Cooperation for Science and Technology (COST) Action GEMSTONE, supported by COST. COST is a funding agency for research and innovation networks. Our Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation (www.cost.eu). We therefore thank current and former members of the COST GEMSTONE Working Group 3 (https://cost-gemstone.eu/working-groups/wg3-monogenic-conditions-human-ko-models/) for discussions and support during manuscript preparation. All figures in this manuscript were created with BioRender.com

A human embryonic limb cell atlas resolved in space and time

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species

Targeting Cancer Cell Signaling Using Precision Oncology Towards a Holistic Approach to Cancer Therapeutics

Cancer is a complex disease having a number of composite problems to be considered including cancer immune evasion, therapy resistance, and recurrence for a cure. Fundamentally, it remains a genetic disease as diverse aspects of the complexity of tumor growth and cancer development relate to its genetic machinery and require addressing the problems at the level of genome and epigenome. Importantly, patients with the same cancer types respond differently to cancer therapies indicating the need for patient-specific treatment options. Precision oncology is a form of cancer therapy that focuses on the genetic profiling of tumors to identify molecular alterations involved in cancer development for custom-tailored personalized treatment of the deadly disease. This article aims to briefly explain the foundations and frontiers of precision oncology in the context of ongoing technological advances in this regard to assess its scope and importance in the realization of a proper cure for cancer.Comment: Pictures and other related data have been taken from sources freely available for reuse or permission for the same can be obtained upon request. Pictures no. 1 and 4 have been added to the text with permission from Elsevier. (Order No. 5521991271884, dated 4th April 2023). 62 Pages, 4 figures and 2 table

Three decades of advancements in osteoarthritis research: insights from transcriptomic, proteomic, and metabolomic studies.

Osteoarthritis (OA) is a complex disease involving contributions from both local joint tissues and systemic sources. Patient characteristics, encompassing sociodemographic and clinical variables, are intricately linked with OA rendering its understanding challenging. Technological advancements have allowed for a comprehensive analysis of transcripts, proteomes and metabolomes in OA tissues/fluids through omic analyses. The objective of this review is to highlight the advancements achieved by omic studies in enhancing our understanding of OA pathogenesis over the last three decades. We conducted an extensive literature search focusing on transcriptomics, proteomics and metabolomics within the context of OA. Specifically, we explore how these technologies have identified individual transcripts, proteins, and metabolites, as well as distinctive endotype signatures from various body tissues or fluids of OA patients, including insights at the single-cell level, to advance our understanding of this highly complex disease. Omic studies reveal the description of numerous individual molecules and molecular patterns within OA-associated tissues and fluids. This includes the identification of specific cell (sub)types and associated pathways that contribute to disease mechanisms. However, there remains a necessity to further advance these technologies to delineate the spatial organization of cellular subtypes and molecular patterns within OA-afflicted tissues. Leveraging a multi-omics approach that integrates datasets from diverse molecular detection technologies, combined with patients' clinical and sociodemographic features, and molecular and regulatory networks, holds promise for identifying unique patient endophenotypes. This holistic approach can illuminate the heterogeneity among OA patients and, in turn, facilitate the development of tailored therapeutic interventions

Multiomic Atlasing of Human Mesenchyme Development

Development of human connective tissue, termed ‘mesenchyme’, is a dispersed process in utero involving a diversity of cell lineages across organ systems. Diseases of these cell lineages, including fibroblasts and cartilage, affect numerous organ systems throughout human lifespan, manifesting as genetic, inflammatory, malignant and degenerative conditions. In turn, these contribute to a significant proportion of global disability burden, particularly through conditions of the developing and aging musculoskeletal system. The human developmental cell atlas consortium has begun to characterise these lineages transcriptionally in the early embryonic limb. However, there remains a paucity of studies applying emerging spatial and multiomic technologies to deeply map mesenchyme development in the human embryo in spatial and epigenetic contexts. Understanding the basis of cell fate decisions that drive the development of progenitors into numerous mesenchymal lineages can shed light on disease mechanisms. The underlying motivation of my thesis was therefore to address this recognised gap in knowledge. In this thesis I first applied paired single-cell RNA and ATAC sequencing, along with spatial transcriptomics to create an atlas of the cell states within the developing human appendicular and cranial skeleton in the first trimester. I then catalogued progenitors, as structures of the synovial joints emerge, and shed new light on the taxonomy of fibroblast and chondrocyte development. I proceeded to study signals involved in the development of joint diseases and reveal disease-specific enrichment in developmental cell states. Extending on this work, I study osteogenesis by examining the cells of the cranial sutures and limbs, revealing niches that contribute to cranial and appendicular bone formation. This enabled a first transcriptional characterisation of cranial bone cells and the cell states affected by congenital conditions of the human cranium. Lastly, I profiled the developing meninges, the mesenchyme layers that overlies the brain, across the first and second trimesters, exploring transcriptional links between fibroblast development and meningioma formation. I also studied meninges affected by trisomy 21, and explored altered cell types that underlie this condition. There were numerous biological insights derived from this work. For example, I gathered evidence of human cross-lineage chondrocyte development from Schwann cell progenitors and clarified stepwise organisation of fibroblast progenitors in the first trimester. I also uncovered cellular mechanisms of bone and meninges formation in the cranium across developmental time at single-cell resolution, contributing a data resource to the Human Cell Atlas and broader research community

The role of BMP signalling in multiple myeloma

Multiple myeloma is an incurable, bone marrow-dwelling malignancy of terminally differentiated plasma cells. It disrupts bone homeostasis causing lytic lesions, skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. In this project, I performed transcriptomic profiling of isolated endosteal cell types from a murine myeloma model, which had not previously been described. I found bone morphogenetic protein (BMP) signalling to be upregulated in mesenchymal progenitor cells of the bone lining niche in myeloma-bearing mice. BMP signalling is not previously reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule Type 1 BMP receptor antagonist (LDN193189) or a solubilized Alk3-Fc (Bmpr1a-Fc) receptor ligand trap prevented trabecular and cortical bone volume loss caused by myeloma, without altering tumour burden. These inhibitors directly reduced osteoclast formation and activity, increased osteoblast number and new bone formation, and suppressed bone marrow levels of sclerostin, a Wnt inhibitor implicated in mediating the failure of bone repair in myeloma sufferers. Conversely, specific blockade of BMP6 in myeloma-bearing mice decreased levels of the iron regulatory hormone hepcidin and improved iron availability and haemoglobin, but did not alter bone disease or tumour burden, indicating this myeloma-expressed BMP is not responsible for altered bone homeostasis. In summary this work describes a novel technique in the investigation of myeloma-niche interactions, shows its use in revealing a novel pathway involved in the pathogenesis of myeloma bone disease, and demonstrates that the therapeutic targeting of the BMP pathway has both anti-resorptive and anabolic benefits to bone metabolism, which could have a role alongside anti-tumour treatments

On the underpinnings of human RNA splicing: a critical survey

RNA splicing is an essential step in mammalian gene transcription. The alternative splicing of RNA to produce multiple transcript isoforms per gene is becoming apparent as a key mechanism that regulates a vast array of key cellular processes. There remains several unanswered questions and inconsistencies in human splicing biology, which this thesis aims to address. Firstly, terminology and designations of alternative splicing are not standardised, resulting in disjointed naming schemes and identifiers for splicing, rendering scientific communication difficult. To address this, we present a standardised system of nomenclature suited for describing alternative splicing, called the Extended Delta Notation (EDN). We then describe the EDN Suite, a web app that facilitates interconversion between genomic co-ordinates and EDN expressions and visualisation of how the EDN works. Secondly, although the involvement of alternative splicing in cell identity specification has been characterised in a few cell types, its role in others remains unexplored. For this, we present a multiomic analysis of alternative splicing in the differentiation of human mesenchymal stem cells to osteoblasts, which revealed a distinct multiphasic differentiation program with distinct biological processes regulated by alternative splicing and gene expression at both the transcriptomic and proteomic level. Thirdly, it is unclear as to the extent which alternative splicing patterns reflect cell identity and/or phenotype. We address this question through a comparative alternative splicing and gene expression analysis of the RNA Atlas, an ultra-deep total RNA and polyA capture sequencing data set of 300 human cell types and tissue samples. We uncover global differences between the landscapes of alternative splicing and expression, finding that alternative splicing can often be a better indicator of cell identity. We show that the alternative splicing landscape is fundamentally different in structure to that of gene expression, with distinct cell and tissue types often marked by unique markers of splicing. We conclude that alternative splicing represents an independent layer of cellular information and that its sparse data structure could inform better methods and models for data handling

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

VenCode – a versatile entry code for post-DNA delivery identification of target cells

RESUMO: O corpo humano é feito de centenas, talvez milhares de tipos e estados celulares distintos, a maioria atualmente inacessíveis através de ferramentas genéticas. A acessibilidade genética traz consigo um potencial terapêutico e de diagnostico significante, ao permitir a entrega seletiva de mensagens genéticas, ou terapias, diretamente às células. Trabalhos em organismos modelo mostram que a atividade de um só elemento regulatório (ER) é raramente específica para um tipo celular, o que limita o seu uso a sistemas genéticos desenhados para controlar a expressão genica após a sua entrega nas células. Abordagens de genética interseccional podem, em teoria, aumentar o número de células acessíveis sem esta restrição, mas o âmbito e a segurança dessas abordagens para o organismo humano não foram ainda sistematicamente estudados devido a uma falta de bases de dados de ERs para um extenso número de tipos celulares, e de métodos para as explorar. Um típico método interseccional funciona como uma porta logica AND ao converter a informação de dois ou mais ERs ativos num só sinal de saída, que se torna único para a célula analisada. Neste trabalho, estudamos sistematicamente o panorama da genética intersecional no organismo humano, usando um grupo de células selecionado a partir de um atlas de atividade de ERs obtido através do sequenciamento “Cap analysis of Gene Expression” (CAGE-seq) de centenas de células primárias e de cancro (o atlas do consórcio FANTOM5). Desenvolvemos algoritmos e heurísticas para encontrar e recolher intersecções do tipo porta AND e em seguida para determinar a sua qualidade. Descobrimos que mais de 90% dos 154 tipos celulares primários estudados podem ser distinguidos uns dos outros com apenas 3 ou 4 ERs ativos, com segurança e robustez. Chamamos de “Versatile Entry Codes” (VEnCodes) a esse número mínimo de intersecções de ERs ativos com potencial de distinção celular. Cada uma das 158 células cancerígenas estudadas poderam ser distinguidas do grupo de células saudáveis com VEnCodes de poucos ERs, a maioria dos quais são altamente robustos à variação intra- e inter-individual. Finalmente, fornecemos métodos para a validação dos VEnCodes obtidos e para a obtenção de VEnCodes a partir de bases de dados de sequenciamento ao nível de célula-a-célula. O nosso trabalho oferece uma visão sistemática do panorama da genética interseccional no organismo humano e demonstra o potencial dessas abordagens para tecnologias futuras de terapia genética.ABSTRACT: The human body is made up of hundreds, perhaps thousands of cell types and states, most of which are currently inaccessible genetically. Genetic accessibility carries significant diagnostic and therapeutic potential by allowing the selective delivery of genetic messages or cures to cells. Research in model organisms has shown that single regulatory element (RE) activities are seldom cell type specific, limiting their usage in genetic systems designed to restrict gene expression posteriorly to their delivery to cells. Intersectional genetic approaches can theoretically increase the number of genetically accessible cells, but the scope and safety of these approaches to human have not been systematically assessed due primarily to the lack of suitable thorough RE activity databases and methods to explore them. A typical intersectional method acts like an AND logic gate by converting the input of two or more active REs into a single synthetic output, which becomes unique for that cell. Here, we systematically assessed the intersectional genetics landscape of human using a curated subset of cells from a large RE usage atlas obtained by Cap Analysis of Gene Expression sequencing (CAGE-seq) of thousands of primary and cancer cells (the FANTOM5 consortium atlas). We developed the heuristics and algorithms to retrieve AND gate intersections and quality-rank them intra- and interindividually. We find that >90% of the 154 primary cell types surveyed can be distinguished from each other with as little as 3 to 4 active REs, with quantifiable safety and robustness. We call these minimal intersections of active REs with cell-type diagnostic potential "Versatile Entry Codes" (VEnCodes). Each of the 158 cancer cell types surveyed could also be distinguished from the healthy primary cell types with small VEnCodes, most of which were highly robust to intra- and interindividual variation. Finally, we provide methods for the cross-validation of CAGE-seq-derived VEnCodes and for the extraction of VEnCodes from pooled single cell sequencing data. Our work provides a systematic view of the intersectional genetics landscape in human and demonstrates the potential of these approaches for future gene delivery technologies in human

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin