Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages. ...Recent advances in the biology of MPNs have greatly facilitated their molecular diagnosis since most patients present with mutation(s) in the JAK2, MPL, or CALR genes. Yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of MPNs are not fully elucidated. Importantly, chronic inflammation has long been associated with MPN disease and some of the symptoms and complications can be linked to inflammation. Moreover, the JAK inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the JAK2-V617F mutant load. These observations suggested that part of the inflammation observed in patients with JAK2-mutated MPNs may not be the consequence of JAK2 mutation. The aim of this paper is to review the different aspects of inflammation in MPNs, the molecular mechanisms involved, the role of specific genetic defects, and the evidence that increased production of certain cytokines depends or not on MPN-associated mutations, and to discuss possible nongenetic causes of inflammation.
Clear cell renal cell carcinomas (RCC) frequently display inactivation of von Hippel-Lindau (VHL) gene leading to increased level of hypoxia-inducible factors (HIF). In this study, we investigated ...the potential role of HIF2α in regulating RCC susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the RCC cell line 786-0 with mutated VHL was resistant to NK-mediated lysis as compared with the VHL-corrected cell line (WT7). This resistance was found to require HIF2α stabilization. On the basis of global gene expression profiling and chromatin immunoprecipitation assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a direct novel target of HIF2α and that targeting ITPR1 significantly increased susceptibility of 786-0 cells to NK-mediated lysis. Mechanistically, HIF2α in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK-mediated killing through the activation of autophagy in target cells by NK-derived signal. Interestingly, both ITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increased granzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediated tumor regression. Our data provide insight into the link between HIF2α, the ITPR1-related pathway, and natural immunity and strongly suggest a role for the HIF2α/ITPR1 axis in regulating RCC cell survival.
Background: The definition of polycythemia, whether primary or secondary, is based on direct measurement of red cell mass (RCM) by isotope labelling method. Because of the lack of availability of ...isotopes, the use of hematocrit and hemoglobin values defined by the WHO recommendations has overtaken the use of the RCM, which can result in misdiagnosis of polycythemia.This determination is also useful for patients with myeloproliferative neoplasm (MPN) to separate two clinical entities: essential thrombocythemia and “masked” Polycythemia Vera, both of which result in high blood counts. These two conditions have different prognoses and therapeutic management. RCM measurement is also sometimes used in the follow-up of MPN with splanchnic thrombosis.The CO-rebreathing method is mainly used in sports to assess the RCM. It is minimally invasive, fast (<30 min) and its accuracy appears to be equivalent to the gold standard’s.To date, no study has compared RCM values obtained with CO-Rebreathing and with the state-of-the-art technique, i.e. isotopic labeling, in the diagnosis of polycythemia.Aims: Here, we present the result of a prospective bi-centric study comparing RCM obtained by CO-rebreathing and by isotopic measurement in a population referred to hematologists for suspicion of polycythemia.Methods: Forty-two patients were initially recruited for simultaneous RCM determination by Co-rebreathing and isotope labelling method. All patients signed an informed consent form. During the course of the study, two patients could not benefit from the Co-rebreathing measurement due to non-compliance with the inclusion criteria (smoking just before the examination) or due to the ergonomic complexity of using the spirometer. On the other hand, the isotope labelling method could not be performed for another patient. This prospective non-randomized study therefore included 39 patients (35 men and 4 women) with a median age of 57 years (range 19-91 years); three of them had Polycythemia Vera with a V617F JAK2 mutation.The isotope labelling method was performed according to the recommendations of the French Society of Radiopharmacy (SoFRa) in a nuclear medicine department using the labelling of the patient’s red blood cells (RBC) with technetium 99m.Co-rebreathing measurement was performed in a pneumology department immediately after the respiratory functional explorations and the determination of baseline HbCO. This method consists of labelling Hb with inspired carbon monoxide (CO), resulting in a temporary increase of COHb.Results: True polycythemia was defined by an increased red cell mass (RCM) above 125% of the theoretical one, for both techniques. All results were expressed as a percentage of the theoretical value.Comparing the RCM results obtained with these two methods, the two techniques were consistent for 31 patients (21 true polycythemia, 10 false polycythemia). For four patients, the CO-rebreathing measurement was underestimated, while for four others, the value obtained by CO-rebreathing was overestimated. Overall, the method yielded a sensitivity of 84% and a specificity of 71%.Summary/Conclusion: This study, carried out on a large series of patients, allows the validation of a simple, non-invasive and reliable tool for the measurement of RCM based on the use of CO-rebreathing, and which is well correlated with the reference isotopic method. It has the advantage of being more accessible to patients, for whom a hospital hosting a nuclear medicine department is too far.In addition, it will allow the measurement of RCM in countries where the isotopic method is no longer available, such as most European and North American countries.
High oxygen affinity hemoglobin (HOAH) is the main cause of constitutional erythrocytosis. Mutations in the genes coding the alpha and beta globin chains (
,
and
) strengthen the binding of oxygen to ...hemoglobin (Hb), bringing about tissue hypoxia and a secondary erythrocytosis. The diagnosis of HOAH is based upon the identification of a mutation in
,
or
in specialized laboratories. Phenotypic studies of Hb are also useful, but electrophoretic analysis can be normal in 1/3 of cases. The establishment of the dissociation curve of Hb can be used as another screening test, a shift to the left indicating an increased affinity for Hb. The direct measurement of venous P50 using a Hemox Analyzer is of great importance, but due to specific analytic conditions, it is only available in a few specialized laboratories. Alternatively, an estimated measurement of the P50 can be obtained in most of the blood gas analyzers on venous blood. The aim of our study was therefore to determine whether a normal venous P50 value could rule out HOAH. We sequenced the
,
and
genes of 75 patients with idiopathic erythrocytosis. Patients had previously undergone an exhaustive medical check-up after which the venous P50 value was defined as normal. Surprisingly, sequencing detected HOAH in three patients (Hb Olympia in two patients, and Hb St Nazaire in another). A careful retrospective examination of their medical files revealed that (i) one of the P50 samples was arterial; (ii) there was some air in another sample; and (iii) the P50 measurement was not actually done in one of the patients. Our study shows that in real life conditions, due to pre-analytical contingencies, a venous P50 value that is classified as being normal may not be sufficient to rule out a diagnosis of HOAH. Therefore, we recommend the systematic sequencing of the
,
and
genes in the exploration of idiopathic erythrocytosis.
The discovery in 2005 of the
V617F gain-of-function mutation in myeloproliferative neoplasms and more particularly in polycythemia vera has deeply changed the diagnostic and therapeutic approaches to ...polycythemia. More recently, the use of NGS in routine practice has revealed a large number of variants, although it is not always possible to classify them as pathogenic. This is notably the case for the
E846D variant for which for which questions remain unanswered. In a large French national cohort of 650 patients with well-characterized erythrocytosis, an isolated germline heterozygous
E846D substitution was observed in only two cases. For one of the patients, a family study could be performed, without segregation of the variant with the erythrocytosis phenotype. On the other hand, based on the large UK Biobank resource cohort including more than half a million UK participants, the
E846D variant was found in 760 individuals, associated with a moderate increase in hemoglobin and hematocrit values, but with no significant difference to the mean values of the rest of the studied population. Altogether, our data as well as UK Biobank cohort analyses suggest that the occurrence of an absolute polycythemia cannot be attributed to the sole demonstration of an isolated
E846D variant. However, it must be accompanied by other stimuli or favoring factors in order to generate absolute erythrocytosis.
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.
Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia ...signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.
Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively.
This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category.
As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.
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