Hereditary erythrocytosis (HE) is a rare hematological disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2160 ...patients with erythrocytosis sequenced in 10 different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of the Hypoxia-Inducible Factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: in silico study of localization, conservation, and deleterious effects; analysis of hematological parameters of carriers identified in the UK Biobank; functional studies of the protein activity and stability; and comprehensive study of PHD2 splicing. Altogether, this study allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extented to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences with the variants of unknown significance regarding the severity of the developed disease (hematological parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare pathology to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematological diseases.
Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant ...hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (-38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (-89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19 ± 0.77-fold in HLC-S127R compared to 1.38 ± 0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 ...technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time.
For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).
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•Generation of knockout and knockin edits in hiPSCs using the CRISPR-Cas9 RNP system•FACS-assisted genome editing of hiPSCs•An optimized approach for culturing and genotyping hiPSC clones
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time.
Pediatric pineoblastomas (PBs) are rare and aggressive tumors of grade IV histology. Although some oncogenic drivers are characterized, including germline mutations in RB1 and DICER1, the role of ...epigenetic deregulation and
-regulatory regions in PB pathogenesis and progression is largely unknown. Here, we generated genome-wide gene expression, chromatin accessibility, and H3K27ac profiles covering key time points of PB initiation and progression from pineal tissues of a mouse model of
-driven PB. We identified PB-specific enhancers and super-enhancers, and found that in some cases, the accessible genome dynamics precede transcriptomic changes, a characteristic that is underexplored in tumor progression. During progression of PB, newly acquired open chromatin regions lacking H3K27ac signal become enriched for repressive state elements and harbor motifs of repressor transcription factors like HINFP, GLI2, and YY1. Copy number variant analysis identified deletion events specific to the tumorigenic stage, affecting, among others, the histone gene cluster and
, the growth arrest specific gene. Gene set enrichment analysis and gene expression signatures positioned the model used here close to human PB samples, showing the potential of our findings for exploring new avenues in PB management and therapy. Overall, this study reports the first temporal and in vivo
-regulatory, expression, and accessibility maps in PB.
Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in ...hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) β-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.
•PCSK9 is highly expressed in undifferentiated hiPSCs•PCSK9 regulates hiPSC proliferation•PCSK9 controls NODAL signaling and SMAD2 phosphorylation in hiPSCs•PCSK9 controls TGFβ-R1 expression, potentially through its interaction with DACT2
In this article, Si-Tayeb and colleagues described that proprotein convertase PCSK9 controls the NODAL signaling pathway through a regulation of SMAD2 phosphorylation in hiPSCs. The endogenous inhibitor of the type 1 TGFβ receptor DACT2 was found as a potential target for PCSK9. These results highlight a new role for PCSK9 in cell proliferation and development.
PCSK9 est un régulateur clé du métabolisme du cholestérol par le foie à travers la dégradation lysosomiale du récepteur aux LDL (low-density lipoprotein). Alors que les mutations gain de fonction ...(GOF) de PCSK9 induisent une hypercholestérolémie autosomique dominante, les mutations pertes de fonctions (LOF) entraînent un taux spontanément bas de LDL-cholestérol, ainsi qu’un protection cardiovasculaire. Du fait des limitations inhérentes aux modèles d’études, tels que les lignées cellulaires transfectées ou des animaux transgéniques, les fonctions de PCSK9 restent encore mal connues. Ainsi, nous avons utiliser des cellules souches pluripotentes induites (hiPSC) spécifiques de patients pour les différencier en hépatocytes et modéliser la physiopathologie liée aux mutations de PCSK9 GOF-S127R et LOF-R104C/V114A. Nous avons démontré que les hépatocytes obtenus récapitulaient la physiopathologie liés aux mutations de PCSK9. De plus, les cellules portant la mutation S127R ont montré une importante réponse au traitement par les statines, qui est corrélée à la réponse clinique des patients portant cette même mutation. Enfin, notre étude nous a permis de mettre à jour une fonction inattendue de PCSK9 dans les hiPSC et pendant leur différenciation. Elle montre que PCSK9 affecterait la prolifération des hiPSC ainsi qu’une voie de signalisation clé du développement régulée par NODAL. Cette régulation se ferait à travers une interaction directe entre PCSK9 et DACT2, un régulateur intracellulaire de la voie de signalisation de NODAL. En conclusion, les hiPSC s’avèrent être un modèle cellulaire translationnel pertinent pour mettre à jour de nouvelles fonctions hépatiques de PCSK9.
PCSK9 has been identified as a key regulator of cholesterol metabolism by the liver through inducing lysosomal degradation of the low-density lipoprotein receptor (LDLR). While PCSK9 gain-of-function (GOF) mutations induced autosomal dominant hypercholesterolemia and increased cardiovascular risk, loss-of-function (LOF) mutations are associated with low LDL-cholesterol levels and cardiovascular protection. Due to limitations inherent to current models including animal and human cells lines transfected with DNA constructs or transgenic animal models, PCSK9 functions are not fully understood. Therefore, we took advantage of patient related somatic cells reprogramming intoinduced pluripotent stem cells (hiPSC) to generate hepatocyte-like cells (HLC) and model the pathophysiology of PCSK9 mutations in dyslipidemia through focusing on two intracellular mutation forms; GOF (S127R) and LOF (R104C/V114A). We showed that HLC could recapitulate the key pathophysiological features of PCSK9 mutations. Moreover, HLC with the S127R mutation displayed an increased uptake of LDL upon statin treatment, which was correlated with the original patient clinical response. In parallel, this model enabled us to unravel a new unexpected role of PCSK9 in hiPSC and during differentiation. PCSK9 was found to affect the proliferation of hiPSC and regulate a key developmental signaling pathway mediated by NODAL. This regulation might occur by a direct interaction between PCSK9 and DACT2, an intracellular attenuator of NODAL signaling pathway. In conclusion, hiPSC provide a pertinent translational model to decipher PCSK9 hepatic functions and a novel cellular environment to highlight new functions.
IntroductionWe previously showed that patient-derived induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes is a relevant model to study the impact of PCSK9 gain-of-function (GOF) ...and loss-of-function (LOF) mutations on cholesterol metabolism regulation. The hiPSCs hepatic differentiation process reproduces the major steps of mammalian liver development. Upon PCSK9 gene expression analysis, we discovered that PCSK9 expression is tightly regulated during hiPSCs differentiation, with the highest expression level in undifferentiated hiPSCs.HypothesisUndifferentiated hiPSCs and their early differentiation will provide a novel approach to highlight new PCSK9 functions.MethodsControl hiPSCs or carrying the S127R GOF or R104C/V114A LOF mutations, and hiPSCs silenced for PCSK9 expression with the use of a shRNA, were analyzed in undifferentiated hiPSCs (gene expression and protein) and during their endoderm (hepatocytes) and mesoderm (cardiomyocytes) differentiation (gene expression). The impacts of LOF mutations and shRNA-mediated PCSK9 expression inhibition on hiSPCs proliferation were also evaluated. Finally, differential PCSK9 expression during early mouse embryonic development has been elucidated by immunofluorescent staining.ResultsWe detected a differential expression of several genes of a cell signaling pathway involved in hiPSCs pluripotency and differentiation. The impact on protein effectors has been verified and we identified a new potential intracellular target of PCSK9. While we did not observe an effect during endoderm differentiation of hiPSCs, we detected an abnormal expression of the endodermal transcription factor FOXA2 during the mesoderm differentiation whenever PCSK9 activity or expressions were modified. Finally, our study showed that the R104C/V114A PCSK9 LOF mutations or PCSK9 silencing with shRNA significantly reduced hiPSCs proliferation. PCSK9 expression during early mouse development and throughout hiPSCs hepatic and cardiac differentiation will be discussed.ConclusionsWe described for the first time the expression of PCSK9 in pluripotent stem cells, and the use of patient-specific hiPSCs allowed us to highlight a new PCSK9 function in developmental processes.
All countries are required to implement International Health Regulations (IHR) through development and implementation of multi-year National Action Plans for Health Security (NAPHS). IHR ...implementation requires annual operational planning which involves several tools such as NAPHS, State Party Annual Report (SPAR), Joint External Evaluation (JEE) and WHO IHR Benchmarks tool. Sierra Leone has successfully improved IHR capacities across the years through successful annual operational planning using the above tools. We conducted a study to document and share the country's unique approach to implementation of NAPHS.
This was an observational study where the process of implementing and monitoring NAPHS in Sierra Leone was observed at the national level from 2018 to 2021. Data was obtained through review and analysis of NAPHS annual operational plans, quarterly review reports and annual IHR assessment reports. Available data was supplemented by information from key informants. Qualitative data was captured as notes and analysed for various themes while quantitative data was analyzed mainly for means and proportions.
The overall national IHR Joint External Evaluation self-assessment score for human health improved from 44% in 2018 to 51% in 2019 and 57% in 2020. The score for the animal sector improved from 32% in 2018 to 43% in 2019 and 52% in 2020. A new JEE tool with new indicators was used in 2021 and the score for both human and animal sectors declined slightly to 51%. Key enablers of success included strong political commitment, whole-of-government approach, annual assessments using JEE tool, annual operational planning using WHO IHR Benchmarks tool and real time online monitoring of progress. Key challenges included disruption created by COVID-19 response, poor health infrastructure, low funding and inadequate health workforce.
IHR annual operational planning and implementation using evidence-based data and tools can facilitate strengthening of IHR capacity and should be encouraged.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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