Hereditary Anemias (HA) are genetic blood disorders affecting a large number of subjects and can cause important clinical complications. Correct, rapid and automatic identification of each type of anemia is pivotal for proper treatment and patient management. Digital holographic microscopy provides label-free quantitative phase measurements of red blood cells (RBCs) morphology. However, standardized and reproducible computational workflows for feature extraction and RBCs classification are still scarce. Here we present a collection of roughly 4600 holographic phase-contrast maps (PCMs) representing RBCs from healthy controls and patients affected by five different HA subtypes. Furthermore, a complete and openly available set of MATLAB scripts is released to implement an analysis workflow, taking PCMs as input. The workflow includes handcrafted feature extraction, as well as different conventional machine and deep learning models. Notably, deep learning architectures are trained directly on PCMs. By releasing the full pipeline, from raw phase maps to trained models, this work delivers an open and extensible framework that facilitates method comparison, ensures reproducibility, and supports the development of new approaches.

ABSTRACT Background The complex pathogenetic mechanisms of rare genetic diseases make the diagnostic process highly challenging. Advances in molecular genomic techniques, such as exome sequencing, have improved the identification of copy number variants (CNVs), increasing diagnostic yield. Methods We report the case of a female patient with global developmental delay, growth alterations, and dysmorphic features. Clinical exome sequencing did not reveal point mutations. CNV analysis from exome data identified a 6 Mb microdeletion in 10p15.3p14, involving the ZMYND11 gene, and a 7.6 Mb microduplication in 7p22.3p21.3; both rearrangements were subsequently confirmed by chromosomal microarray analysis. Conventional karyotyping revealed a derivative chromosome 10 [46,XX,der (10)], a finding consistent with the possibility of an unbalanced translocation involving chromosomes 7 and 10. The combined cytogenetic and molecular findings are consistent with the possibility that the duplicated 7p segment is inserted into the short arm of chromosome 10. Results ZMYND11, a dosage‐sensitive gene, has been associated with Cornelia de Lange Syndrome (CdLS)‐like phenotypes, and its haploinsufficiency is linked to 10p15.3 microdeletion syndrome. Our patient presented a complex phenotype due to the concurrent 7p duplication and 10p deletion, highlighting the importance of ZMYND11 in chromatinopathies. A review of similar cases supports considering ZMYND11 in evaluating chromatinopathy‐related features. Notably, she also exhibited unique characteristics that have not been previously described in association with either CNV. Conclusion Next‐generation sequencing, capable of detecting both single nucleotide variants and CNVs, is a critical tool for diagnosing neurodevelopmental disorders and uncovering diverse causative variants. This case emphasizes how NGS facilitates the identification of co‐occurring CNVs and expands the phenotypic spectrum associated with chromatinopathies. Detailed characterization of such complex phenotypes using NGS is essential for advancing our understanding of rare genetic conditions and improving diagnostic accuracy.

Abstract The ATP11C gene, localized on the X chromosome, encodes the major phosphatidylserine flippase in human red blood cells (RBCs). Flippases actively transport phospholipids from the outer to the inner leaflet of the lipid bilayer, establishing and maintaining phospholipid asymmetry crucial for cell survival. Variants in ATP11C have been recently associated with a novel form of X-linked congenital hemolytic anemia. In this study, we identified 10 individuals from 7 unrelated families harboring 6 rare, novel variants in the ATP11C gene. Three of the variants were further characterized and functionally validated. Of note, the first variant, p.R467C, was identified in a male neonate presenting with mild hemolytic anemia. The second variant, c.2226-1G>C, was found in a male aged 53 years who was originally suspected of hereditary hemochromatosis. The third variant, p.D609V, was detected in a female aged 68 years with mild anemia. Functional studies revealed reduced ATP11C protein expression, as well as decreased flippase activity both in vitro and in ex vivo RBCs supporting a loss-of-function mechanism. Additionally, in the female patient, we identified skewed X-chromosome inactivation, because of which the pathogenic effect of the heterozygous ATP11C variant became clinically evident. Our study expands the clinical spectrum of ATP11C-related hemolytic anemia, highlighting its association with adult-onset disease and hepatic iron overload. We emphasize the importance of including ATP11C in genetic testing for the diagnosis of hereditary hemolytic anemia and iron metabolism alterations. These findings enhance our understanding of RBCs membrane homeostasis and elucidate the critical role of ATP11C in erythropoiesis and systemic iron regulation.

Thalassemia and sickle cell disease (SCD) are among the most common monogenic disorders worldwide. They cause chronic hemolytic anemia, the consequences and prognosis of which vary considerably depending on the genetic characteristics of patients and the healthcare system in their country of residence. Both diseases are autosomal recessive in their transmission, with carriers generally being asymptomatic. Informing carriers of thalassemia or SCD about reproductive risks and choices, while taking into account cultural and religious considerations, is a priority within global strategies to improve outcomes for these diseases. The European Hematology Association (EHA)'s Topic In Focus (TIF) Hemoglobinopathies Group created a focus group of hematologists, patients, anthropologists, and an obstetrician from Europe, the Middle East, India, and Africa. The Group considered that preconceptual screening tests would correspond to tests conducted before pregnancy (screening for carriers before marriage/conception), antenatal screening referred to tests completed on pregnant women, and prenatal diagnosis referred to tests performed on the fetus. It proposed guidelines addressing optimal timing of screening, appropriate laboratory tests, and communication strategies, taking into account the great diversity of regions and cultures where thalassemia and SCD are present. A main discussion point was that no recommendations would be given for couples about reproductive decisions, and that the aim was to present the existing and available options in different countries. Eight questions were examined using available literature, leading to the formulation of seven recommendations, which were submitted to a vote using the Delphi method. Consensus agreement was obtained for all recommendations.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases originating from hematopoietic stem cell transformation, characterized by the clonal proliferation of hematopoietic progenitors. A specific subset includes myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase (TK) gene fusions, particularly involving PDGFR A or B, which are sensitive to TK inhibitor treatment. We report a case of a 21-year-old patient with a myeloproliferative/myelodysplastic neoplasm, presenting with hyperleukocytosis, anemia, thrombocytopenia, and elevated LDH. The peripheral blood smear showed hypogranular neutrophils, eosinophils, basophils, and myeloid precursors. The absence of BCR::ABL1 and mutations in JAK2, CALR, and MPL excluded common MPNs. Cytogenetic analysis revealed a rearrangement between chromosomes 5 and 14. FISH analysis confirmed an inverted insertion from chromosome 5 to chromosome 14, involving the PDGFRB gene. WGS and RNAseq identified a fusion between PDGFRB and CCDC88C, causing the constitutive activation of PDGFRB. The fusion gene was confirmed by sequencing. This allowed for targeted therapy with a tyrosine kinase inhibitor (TKI), leading to molecular remission monitored by RT-qPCR. This case highlights how a multidisciplinary approach can identify atypical transcripts in MPN, guiding targeted therapy with TK inhibitors, thus resulting in effective treatment and molecular remission.

Neonatal hypoxemia in absence of structural cardiopulmonary anomalies is a diagnostic challenge. Haemoglobin variants involving the gamma-globin gene are recognized causes of transient desaturation in the newborn period. We report a novel HBG2 mutation identified in a clinically stable, non-cyanotic neonate born to a mother with active SARS-CoV-2 infection, highlighting a previously unreported variant with potential functional consequences on haemoglobin-oxygen affinity. A term female newborn was delivered vaginally to a 39-years-old mother with gestational diabetes, thrombocytopenia, and SARS-CoV-2 infection, confirmed by real-time PCR at the time of delivery. The infant exhibited no signs of respiratory distress and normal blood gas analysis, but pulse oximetry revealed persistent arterial oxygen saturation of 88

ABSTRACT:Interpreting genetic variants in complex genes such as PIEZO1 remains challenging because of marked allelic heterogeneity, relative tolerance to missense variation, and overlapping clinical phenotypes. Gain-of-function variants in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS1, or hereditary xerocytosis), a pleiotropic syndrome characterized by anemia of variable severity and iron overload. In this study, we provided and applied an integrative framework combining the American College of Medical Genetics and Genomics guidelines, quantitative in silico predictions, structural domain annotation, and detailed patient phenotyping to classify 2565 PIEZO1 variants. A Bayesian scoring system with weighted evidence and a composite predictive score enabled reclassification of nearly 1000 variants of uncertain significance and highlighted nonrandom clustering of pathogenic variants within functionally constrained domains, particularly the anchor, inner helix, and C-terminal domains. Genotype-phenotype correlation analysis in 176 in-house DHS cases identified 3 phenotypic clusters, ranging from classical DHS1 with severe hemolysis and iron overload to atypical or subclinical presentations, reflecting domain-specific pathogenic mechanisms. Overall, this reclassification of PIEZO1 variants, integrated with genotype-phenotype correlation analysis, improves diagnostic precision, supports genotype-guided patient management, and underscores the value of integrating structural and clinical data into interpretation of rare genetic variants.

Hypoxia is a common feature of inflamed and ischemic tissues and represents an important regulatory signal for innate immune cells. The master regulator of this response is hypoxia-inducible factor-1α (HIF-1α), a transcription factor whose stabilization and activity are tightly regulated by the presence of oxygen, inflammatory signaling, and cellular metabolism. Monocytes, key players in innate immunity, rapidly sense oxygen deprivation and display specific responses during acute hypoxia, primarily aimed at adapting and maintaining cellular homeostasis. Unlike macrophages, in which HIF-1α activity is known, the mechanisms regulating HIF-1α stabilization, subcellular localization, and transcriptional activity in circulating monocytes remain incompletely elucidated. Recent studies indicate that acute hypoxia primarily triggers post-translational stabilization of HIF-1α, calcium- and PKC-dependent signaling, metabolic reprogramming, and early inflammatory responses, while transcriptional activation of HIF-1α may require additional inflammatory or stress-related signals. Furthermore, extensive crosstalk between HIF-1α and NF-κB integrates hypoxic and inflammatory signals, modulating cytokine production, cell migration, and survival. Epigenetic regulators can also modulate these responses and contribute to hypoxia-induced trained immunity. In this review, we summarize current knowledge of the mechanisms controlling the stabilization, localization, and function of HIF-1α in human monocytes and monocyte–macrophages during acute hypoxia, highlighting the key differences between these cell types and discussing their implications for inflammation, tissue homeostasis, and disease.

Hereditary anemias encompass a genetically heterogeneous spectrum of disorders, often involving multi-locus inheritance, which can complicate clinical management and worsen disease severity. This study investigates the impact of the co-inheritance of SEC23B loss-of-function pathogenic variants, which lead to congenital dyserythropoietic anemia type II (CDA II), and PIEZO1 gain-of-function pathogenic variants, associated with dehydrated hereditary stomatocytosis type I (DHS1), on hematological parameters and iron metabolism. Among 583 patients with suspected hereditary anemia, 13 were found to carry both SEC23B and PIEZO1 variants, leading to a dual diagnosis of CDA II and DHS1. Compared to those with isolated CDA II, these patients exhibited a significantly higher absolute reticulocyte count and bone marrow responsiveness index, alongside an increased prevalence of elevated ferritin levels. Functional studies in Hep3B human hepatoma cells confirmed that SEC23B knockdown combined with PIEZO1 gain-of-function led to marked ferritin accumulation and reduced hepcidin expression, driven by altered BMP/SMAD signaling and ERK1/2 MAPK pathway. These findings demonstrate how multi-locus inheritance can modify disease severity, particularly by exacerbating iron overload. Our results underscore the clinical relevance of comprehensive genetic testing for enhanced risk stratification and personalized management of hereditary anemias.

PURPOSE:Pathogenic variants in the POU3F3 gene are responsible for an ultra-rare neurogenetic disorder, characterized by a combination of neuropsychiatric and systemic manifestations. Epilepsy is observed in approximately 15 % of affected individuals, ranging from focal non-motor sensitive seizures (gelastic and dacristic) to generalized motor and non-motor seizures including either tonic-clonic, tonic, myoclonic, atonic or atypical absences. To date, no cases of infantile epileptic spasms have been associated with this neurogenetic condition. METHODS:We report on a 20 months male patient presenting at 4 months of life with epileptic spasms, encephalopathic pattern on EEG, confirmed by BASED score, severe hypotonia, microcephaly, cerebral malformation and hyperkinetic movement disorder, consisting of stereotypies and dyskinesias. Epileptic spasms relapsed after ACTH cycle and responded to treatment with vigabatrin. RESULTS:Exome analysis revealed a novel missense de novo pathogenic variant (c.1071G>C; p.Gln337His) in the POU3F3 gene. CONCLUSIONS:This case expands the epileptic phenotype of the POU3F3-related neurodevelopment disorder and contributes to the identification of a novel potential gene, involved in the genetic etiology of Infantile Epileptic Spasm Syndrome.

Purpose of reviewCongenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and by distinct morphological abnormalities of erythroblasts in the bone marrow. Characteristic morphological aberrations were the cornerstone of diagnosis, but following the identification of several causative genes, the molecular approach could represent a rapid tool for the identification of these conditions. This review presents advances in diagnosis and classification of CDAs.Recent findingsThe classification of CDAs has long been based on morphological features. Now, the discovery of some of the responsible genes allows reconsideration of part of the classification. The first CDA partly accounted for genetically has been CDA 1, through the discovery in 2002 of the gene responsible, CDAN1, encoding codanin-1. Recently, the dramatic identification of the genes responsible for CDA II, SEC23B, and for a hitherto unnamed CDA, KLF1, took place. SEC23B encodes SEC23B which is a component of the coated vesicles transiting from the endoplasmic reticulum to the cis compartment of the Golgi apparatus. A unique mutation in KLF1, which encodes the erythroid transcription factor KLF1, causes major ultrastructural abnormalities, the persistence of embryonic and fetal hemoglobins, and the absence of some red cell membrane proteins.SummaryStudies of genotype-phenotype relationship, as has already been done for CDA II, will allow a more accurate prognosis. Identification of the responsible genes has opened new vistas for research on CDAs.

ABSTRACT:Hereditary stomatocytosis represents a heterogeneous group of inherited erythrocyte membrane defects characterized by hemolytic anemia of variable degree, with alterations in cellular salt and water, ranging from dehydration to overhydration, and the presence of stomatocytes on peripheral blood smear. This condition encompasses various subtypes, each with distinct clinical and genetic features. The pathophysiology underlying these conditions involves altered red blood cell membrane properties, leading to impaired deformability and alterations in cation permeability and volume, causing increased susceptibility to hemolysis. Advancements in genetic testing have enabled the identification of some causative genes in the last years, such as PIEZO1, KCNN4, and ABCB6. These genetic discoveries have facilitated a deeper understanding of the molecular mechanisms underlying the pathogenesis and have paved the way for improved diagnostic accuracy and genetic counseling. This review provides an overview of the clinical presentation, pathophysiology, molecular genetics, diagnosis, and management strategies of hereditary stomatocytosis, highlighting recent advancements in the field of dehydrated hereditary stomatocytosis (DHS), or hereditary xerocytosis, and hepatic iron overload. This latter is directly associated with the physiological role of PIEZO1, the causative gene of DHS, at hepatic and macrophagic levels. Particularly, gain-of-function mutations in PIEZO1 account for a pleiotropic syndrome characterized by different phenotypes depending on the expression of PIEZO1 in multiple cells and tissues.

The innate immune system is the first player involved in the recognition/interaction with nanomaterials. Still, it is not the only system involved. The co-evolution of the microbiota with the innate immune system built an interdependence regulating immune homeostasis that is poorly studied. Herein, the simultaneous interaction of iron-oxide nanoparticles (Fe-oxide NPs), immune cells, and the microbiota associated with the blood of the sea urchin Paracentrotus lividus was explored by using a microbiota/immune cell model in vitro-ex vivo and a battery of complementary tools, including Raman spectroscopy, 16S Next-Generation Sequencing, high-content imaging, NanoString nCounter. Our findings highlight the P. lividus immune cells and microbiota dynamics in response to Fe-oxide NPs, including i) morphological rearrangement and immune cell health status maintenance (intracellular trafficking increasing, no phenotypic alterations or caspase 3/7 activation), ii) transcriptomic reprogramming in immune cells (Smad6, Lmo2, Univin, suPaxB, Frizzled-7, Fgfr2, Gp96 upregulation), iii) immune signaling unchanged (e.g., P-p38 MAPK, P-ERK, TLR4, IL-6 protein level unchanged), iv) enrichment in extracellular vesicle released in the co-culture medium, and v) a shift in the composition of microbial groups mainly in favor of Gram-positive bacteria (e.g., Firmicutes, Actinobacteria),. Our findings suggest that Fe-oxide NPs induce a multi-level immune cell-microbiota response restoring homeostasis.

BACKGROUND:Despite several publications covering patients from multiple centers, no international registry covered all patients with red blood cell diseases (RBCD) affected by COVID- 19. The ERN-EuroBloodNet's registry provided real-time registration of SARS-CoV- 2 patients with RBCD, promoting timely disease-specific knowledge sharing during the pandemic's early stages. PROCEDURES:The study evaluated patient distribution, the infection across different RBCDs, and severity risk factors across similar healthcare systems, using data collected from the ERN-EuroBloodNet's REDCap platform. RESULTS:From April 2020 to April 2023, 681 infections were recorded among 663 patients, of which 373 had transfusion-dependent thalassemia or non-transfusion-dependent thalassemia (TDT/NTDT), and 269 had sickle cell disease (SCD). SCD patients had a higher incidence of COVID- 19 than those with TDT/NTDT (10.5 vs. 4.8 COVID/100 patients). Notably, 92% of the cases were mild, with neither age nor the specific RBCD affecting severity. The number of comorbidities, notably obesity and hypertension, that patients had prior to infection was associated with more severe COVID- 19. During the infection, the presence of vaso-occlusive crises, acute chest syndrome, kidney failure, and ground-glass opacities on chest tomography scans were associated with a more severe clinical picture. The vaccination rate (32%) mirrored that of the general population and showed a protective effect against severe COVID- 19. The observed mortality rate was 0.7%, aligning with Europe's general population. CONCLUSION:SARS-CoV- 2 infection in SCD and TDT/NTDT patients is mild and without higher mortality than the general population. The ERN-Eurobloodnet's registry collaborative structure exemplifies the power of international cooperation in tackling rare diseases, especially during health emergencies.