Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly.

The transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

Inferring the initiation and development of myeloproliferative neoplasms.

The developmental history of blood cancer begins with mutation acquisition and the resulting malignant clone expansion. The two most prevalent driver mutations found in myeloproliferative neoplasms-JAK2V617F and CALRm-occur in hematopoietic stem cells, which are highly complex to observe in vivo. To circumvent this difficulty, we propose a method relying on mathematical modeling and statistical inference to determine disease initiation and dynamics. Our findings suggest that CALRm mutations tend to occur later in life than JAK2V617F. Our results confirm the higher proliferative advantage of the CALRm malignant clone compared to JAK2V617F. Furthermore, we illustrate how mathematical modeling and Bayesian inference can be used for setting up early screening strategies.

Red cell mass measurement is still useful for myeloproliferative neoplasms management. Comment to "Role of red cell mass evaluation in myeloproliferative neoplasms with splanchnic vein thrombosis and normal hemoglobin value: a study of the France Intergroupe des Syndromes Myeloprolifératifs".

Not available.

Benefit of phlebotomy and low-dose aspirin in the prevention of vascular events in patients with EPOR primary familial polycythemia on the island of New Caledonia.

Not available.

CALR-mutated patients with low allele burden represent a specific subtype of essential thrombocythemia: A study on behalf of FIM and GBMHM.

A low allele burden (i.e., <20%) of the CALR driver mutation is found in 10.8% of CALR-mutated MPNs, mostly in essential thrombocythemia, and correlates with a milder phenotype and a more indolent evolution compared to patients with an allele burden ≥20%.

Heterozygosity for bisphosphoglycerate mutase deficiency expressing clinically as congenital erythrocytosis: A case series and literature review.

Erythrocytosis is associated with increased red blood cell mass and can be either congenital or acquired. Congenital secondary causes are rare and include germline variants increasing haemoglobin (Hb)-oxygen affinity (e.g., Hb or bisphosphoglycerate mutase (BPGM) variants) or affecting oxygen-sensing pathway proteins. Here, we describe five adults from three kindreds with erythrocytosis associated with heterozygosity for BPGM variants, including one novel. Functional analyses showed partial BPGM deficiency, reduced 2,3-bisphosphoglycerate levels and/or increased Hb-oxygen affinity. We also review currently known BPGM variants. This study contributes to raising awareness of BPGM variants, and in particular that heterozygosity for BPGM deficiency may already manifest clinically.

Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten 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 hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Splanchnic vein thromboses associated with myeloproliferative neoplasms: An international, retrospective study on 518 cases.

Myeloproliferative Neoplasms (MPN) course can be complicated by thrombosis involving unusual sites as the splanchnic veins (SVT). Their management is challenging, given their composite vascular risk. We performed a retrospective, cohort study in the framework of the International Working Group for MPN Research and Treatment (IWG-MRT), and AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM). A total of 518 MPN-SVT cases were collected and compared with 1628 unselected, control MPN population, matched for disease subtype. Those with MPN-SVT were younger (median 44 years) and enriched in females compared to controls; PV (37.1%) and ET (34.4%) were the most frequent diagnoses. JAK2V617F mutation was highly prevalent (90.2%), and 38.6% of cases had an additional hypercoagulable disorder. SVT recurrence rate was 1.6 per 100 patient-years. Vitamin K-antagonists (VKA) halved the incidence of recurrence (OR 0.48), unlike cytoreduction (OR 0.96), and were not associated with overall or gastrointestinal bleeding in multivariable analysis. Esophageal varices were the only independent predictor for major bleeding (OR 17.4). Among MPN-SVT, risk of subsequent vascular events was skewed towards venous thromboses compared to controls. However, MPN-SVT clinical course was overall benign: SVT were enriched in PMF with lower IPSS, resulting in significantly longer survival than controls; survival was not affected in PV and slightly reduced in ET. MPN-U with SVT (n = 55) showed a particularly indolent phenotype, with no signs of disease evolution. In the to-date largest, contemporary cohort of MPN-SVT, VKA were confirmed effective in preventing recurrence, unlike cytoreduction, and safe; the major risk factor for bleeding was esophageal varices that therefore represent a major therapeutic target.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

A retrospective analysis of the impact of treatments and blood counts on survival and the risk of vascular events during the course of polycythaemia vera.

Vascular and non-vascular complications are common in patients with polycythaemia vera. This retrospective study of 217 patients with polycythaemia vera aimed to determine whether blood counts with respect to different treatments influenced the complication rate and survival. We found that 78 (36%) patients suffered from at least one complication during follow-up. Older age and elevated lactate dehydrogenase at diagnosis were found to be risk factors for vascular complications. When the vascular complication occurred, 41% of the patients with a complication had elevated white blood cells (WBC) compared with 20% of patients without a complication (P = 0·042). Patients treated with hydroxycarbamide (HC; also termed hydroxyurea) experienced significantly fewer vascular complications (11%) than patients treated with phlebotomy only (27%) (P = 0·013). We also found a survival advantage for patients treated with HC, when adjusted for age, gender and time period of diagnosis (Hazard ratio for phlebotomy-treated patients compared to HC-treated patients at 5 years was 2·42, 95% confidence interval 1·03-5·72, P = 0·043). Concerning survival and vascular complications, HC-treated patients who needed at least one phlebotomy per year were not significantly different from HC-treated patients with a low phlebotomy requirement. We conclude that complementary phlebotomy in HC-treated patients in order to maintain the haematocrit, is safe.

Cohen syndrome is associated with major glycosylation defects.

Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS-PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal-lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.

Presence of calreticulin mutations in JAK2-negative polycythemia vera.

Calreticulin (CALR) mutations have been reported in Janus kinase 2 (JAK2)- and myeloproliferative leukemia (MPL)-negative essential thrombocythemia and primary myelofibrosis. In contrast, no CALR mutations have ever been reported in the context of polycythemia vera (PV). Here, we describe 2 JAK2(V617F)-JAK2(exon12)-negative PV patients who presented with a CALR mutation in peripheral granulocytes at the time of diagnosis. In both cases, the CALR mutation was a 52-bp deletion. Single burst-forming units-erythroid (BFU-E) from 1 patient were grown in vitro and genotyped: the same CALR del 52-bp mutation was noted in 31 of the 37 colonies examined; 30 of 31 BFU-E were heterozygous for CALR del 52 bp, and 1 of 31 BFU-E was homozygous for CALR del 52 bp. In summary, although unknown mutations leading to PV cannot be ruled out, our results suggest that CALR mutations can be associated with JAK2-negative PV.