A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Combining cell-based therapy and normothermic machine perfusion for kidney graft conditioning has gone one step further.

peer reviewe

Combining Kidney Organoids and Genome Editing Technologies for a Better Understanding of Physiopathological Mechanisms of Renal Diseases: State of the Art

International audienceKidney organoids derived from pluripotent stem cells became a real alternative to the use of in vitro cellular models or in vivo animal models. Indeed, the comprehension of the key steps involved during kidney embryonic development led to the establishment of protocols enabling the differentiation of pluripotent stem cells into highly complex and organized structures, composed of various renal cell types. These organoids are linked with one major application based on iPSC technology advantage: the possibility to control iPSC genome, by selecting patients with specific disease or by genome editing tools such as CRISPR/Cas9 system. This allows the generation of kidney organoïds which recapitulate important physiopathological mechanisms such as cyst formation in renal polycystic disease for example. This review will focus on studies combining these both cutting edge technologies i.e., kidney organoid differentiation and genome editing and will describe what are the main advances performed in the comprehension of physiopathological mechanisms of renal diseases, as well as discuss remaining technical barriers and perspectives in the field

Les organoïdes rénaux

Les organoïdes rénaux dérivés de cellules souches pluripotentes sont devenus une réelle alternative à l’utilisation de modèles in vitro limités ou de modèles animaux contraignants et d’utilisation sensible. La compréhension des mécanismes clés de développement du rein a permis d’établir des protocoles permettant, à partir de cellules souches pluripotentes, d’obtenir de tels organoïdes, qui sont constitués de structures hautement complexes et organisées, contenant plusieurs types cellulaires. Ces organes miniatures permettent des applications majeures : la possibilité de contrôler le génome des iPSC (induced pluripotent stem cell), par sélection de patients atteints de pathologies spécifiques ou par édition de génome, permet d’obtenir in vitro des organoïdes rénaux qui intègrent des mécanismes physiopathologiques, comme le développement de kystes observé dans la polykystose rénale. Ces organoïdes peuvent également être utilisés pour des applications « haut-débit » afin d’accélérer la mise au point de tests de molécules néphrotoxiques ou de composés thérapeutiques. Enfin, les organoïdes rénaux présentent un intérêt majeur dans un contexte de réparation tissulaire, une application qui reste limitée actuellement et pour laquelle de nombreuses barrières restent à franchir

Urine-derived stem/progenitor cells: A focus on their characterization and potential

International audienceCell therapy, i.e., the use of cells to repair an affected tissue or organ, is at the forefront of regenerative and personalized medicine. Among the multiple cell types that have been used for this purpose [including adult stem cells such as mesenchymal stem cells or pluripotent stem cells], urine-derived stem cells (USCs) have aroused interest in the past years. USCs display classical features of mesenchymal stem cells such as differentiation capacity and immunomodulation. Importantly, they have the main advantage of being isolable from one sample of voided urine with a cheap and unpainful procedure, which is broadly applicable, whereas most adult stem cell types require invasive procedure. Moreover, USCs can be differentiated into renal cell types. This is of high interest for renal cell therapy-based regenerative approaches. This review will firstly describe the isolation and characterization of USCs. We will specifically present USC phenotype, which is not an object of consensus in the literature, as well as detail their differentiation capacity. In the second part of this review, we will present and discuss the main applications of USCs. These include use as a substrate to generate human induced pluripotent stem cells, but we will deeply focus on the use of USCs for cell therapy approaches with a detailed analysis depending on the targeted organ or system. Importantly, we will also focus on the applications that rely on the use of USC-derived products such as microvesicles including exosomes, which is a strategy being increasingly employed. In the last section, we will discuss the remaining barriers and challenges in the field of USC-based regenerative medicine

Rho GTPases in kidney physiology and diseases

International audienceRho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton, but also of cellular morphology, motility, adhesion and proliferation. The prototypic members of this family (RhoA, Rac1 and Cdc42) also contribute to the normal kidney function and play important roles in the structure and function of various kidney cells including tubular epithelial cells, mesangial cells and podocytes. The kidney’s vital filtration function depends on the structural integrity of the glomerulus, the proximal portion of the nephron. Within the glomerulus, the architecturally actin-based cytoskeleton podocyte forms the final cellular barrier to filtration. The glomerulus appears as a highly dynamic signalling hub that is capable of integrating intracellular cues from its individual structural components. Dynamic regulation of the podocyte cytoskeleton is required for efficient barrier function of the kidney. As master regulators of actin cytoskeletal dynamics, Rho GTPases are therefore of critical importance for sustained kidney barrier function. Dysregulated activities of the Rho GTPases and of their effectors are implicated in the pathogenesis of both hereditary and idiopathic forms of kidney diseases. Diabetic nephropathy is a progressive kidney disease that is caused by injury to kidney glomeruli. High glucose activates RhoA/Rho-kinase in mesangial cells, leading to excessive extracellular matrix production (glomerulosclerosis). This RhoA/Rho-kinase pathway also seems involved in the post-transplant hypertension frequently observed during treatment with calcineurin inhibitors, whereas Rac1 activation was observed in post-transplant ischaemic acute kidney injury

Use of tramadol and the risk of bleeding complications in patients on oral anticoagulants: A systematic review and meta-analysis

International audiencePurpose This systematic review and meta-analysis aimed to determine whether tramadol intake increases the risk of bleeding in patients receiving oral anticoagulants. Methods This systematic review was registered on PROSPERO, CRD42022327230. We searched Pubmed and Embase up to 14 April 2022 and references and citations of included studies were screened. Comparative and non-comparative studies exploring bleeding complications among adult patients on oral anticoagulants and tramadol were included. Risk of bias was assessed using an adaptation of the Drug Interaction Probability Scale for case reports and case series, and the Newcastle-Ottawa Scale for comparative studies. A metaanalysis was performed for the risk of serious bleeding (leading to hospitalisation or death) associated with tramadol in patients on vitamin K antagonists. Results A total of 17 studies were included: 1 case series, 12 case reports, 2 case-control studies, and 2 cohort studies. Most of the studies described tramadol-vitamin K antagonists concomitant use; one case-control study also assessed dabigatran and rivaroxaban; one case report involved dabigatran. Among case reports/series, a total of 33 patients had a bleeding complication while using tramadol and an oral anticoagulant. The 4 comparative studies reported an increased bleeding risk during tramadol and vitamin K antagonist intake which was statistically significant in one study. The pooled risk ratio of serious bleeding was 2.68 [95% CI: 1.45 to 4.96; p < 0.001]. Conclusion This systematic review confirms an association between tramadol use and risk of bleeding in patients on vitamin K antagonists. Evidence is too limited to assess whether this risk extends to patients on direct oral anticoagulants and further studies are needed

Successful Derivation of Hepatoblasts, Cholangiocytes and Hepatocytes from Simian Induced Pluripotent Stem Cells

The use of primary cells in human liver therapy is limited by a lack of cells. Induced pluripotent stem cells (iPSCs) represent an alternative to primary cells as they are infinitely expandable and can be differentiated into different liver cell types. The aim of our work was to demonstrate that simian iPSCs (siPSCs) could be used as a new source of liver cells to be used as a large animal model for preclinical studies. We first differentiated siPSCs into a homogenous population of hepatoblasts (siHBs). We then separately differentiated them into hepatocytes (siHeps) and cholangiocytes (siChols) expressing respective specific markers and displaying epithelial polarity. Moreover, we showed that polarized siChols can self-organize into 3D structures. These results should facilitate the deciphering of liver development and open the way to exploring co-culture systems that could be assessed during preclinical studies, including in autologous monkey donors, for regenerative medicine purposes