Holo-Tomographic Flow Cytometry (HTFC) holds the potential to transform cellular research and clinical screening through 3D label-free quantitative phase imaging (QPI) of flowing single cells. However, it has been limited by insufficient intracellular specificity in 3D refractive index (RI) distributions, since suspended cells act as highly aberrating spherical biolenses obscuring internal structures. Here, we show the Biolens Phase Compensation (BPC), a method that corrects phase aberrations in 2D QPI projections to transform the 3D RI tomogram. Working within this new 3D pseudo-RI space demonstrates for the first time the extraction of nucleoli in HTFC. Extensive validation against 2D fluorescence flow cytometry and 3D confocal microscopy demonstrates that BPC achieves reliable intranuclear specificity. Using statistically significant single-cell analysis, we provide multiplexed quantitative 3D measurements of nested intracellular compartments (cytoplasm, nucleoplasm, nucleoli). This approach extends label-free HTFC toward capabilities of gold-standard Fluorescence Microscopy, overcoming its well-known drawbacks in intracellular and intranuclear staining. ### Competing Interest Statement The authors have declared no competing interest.

Sickle cell disease (SCD) is a globally distributed hereditary red cell disorder with still high mortality. Growing evidence indicates that sickle cell related cardiovascular disease contributes to the early death of adults with SCD. Here, we show that humanized SCD mice developed an age-dependent cardiomyopathy characterized by (i) increased circulating Th17 lymphocytes, Th17 heart infiltration associated with increased plasma IL-17; (ii) collagen deposition and activation of both platelet derived growth factor-B (PDGF-B) and transforming growth factor-β1 (TGF-β1) canonical pathways; (iii) overactivation of heart NF-κB associated with up-regulation of NLRP3 and of inflammatory vasculopathy markers. We then used colchicine (CLC) that acts as anti-inflammatory drug with immunomodulatory effects. In humanized SCD mice, we demonstrated the protective role of low-dose CLC treatment against chronic inflammation and preserving myocardial performance. Of note, we found CLC also attenuating sickle cell related lung damage, suggesting a multiorgan effect of CLC in SCD mice. Our data generate the rationale to further explore CLC as new therapeutic tool to treat early stages of sickle cell cardiomyopathy.

Mitochondrial disorders (MDs) are a diverse group of genetic conditions primarily affecting the oxidative phosphorylation (OXPHOS) system and cellular energy production. Among MDs, Linear Skin Defects with Multiple Congenital Anomalies (LSDMCA), or Microphthalmia with Linear Skin Lesions (MLS) syndrome, is a rare X-linked dominant male-lethal disorder characterized by ocular malformations, linear skin defects, and multisystem developmental anomalies. These features are associated with pathogenic variants in genes related to mitochondrial function, including HCCS, COX7B, and NDUFB11 or chromosomal rearrangements of the Xp22 region encompassing HCCS. Despite progress, genotype-phenotype correlations remain insufficiently defined. In this study, we report three novel mutations in three patients with LSDMCA, broadening the phenotypic spectrum of the disorder. Whole exome sequencing revealed pathogenic missense variants in HCCS [NM_005333.5: c.625 G > C; p.(Asp209His)] and COX7B [NM_001866.3: c.221 C > T; p.(Pro74Leu)] in two unrelated patients. Functional studies confirmed that the COX7B variant impairs mitochondrial respiratory chain (MRC) function. A third patient harbored a novel frameshift pathogenic variant in NDUFB11 [NM_001135998.3: c.145_152dup; p.(Thr52Glnfs*66)], further implicating mitochondrial dysfunction in LSDMCA pathogenesis. Notably, the COX7B variant was identified in a biological male (46, XY) without X-chromosome structural rearrangements, marking the first such reported case of LSDMCA. Our data suggest that certain missense variants, resulting in mild impairment of the gene product, may allow male survival, thereby expanding the known phenotype of this rare disorder. This report advances our understanding of genotype-phenotype correlations in LSDMCA and highlights the impact of mitochondrial dysfunction during embryonic development.

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.

Virtual staining is the current state-of-the-art computational technique to cleverly enhance intracellular specificity in unstained biological samples by using convolutional neural networks (CNNs) trained on co-registered pairs of unstained/stained images. While effective, this approach suffers from unpredictable biases inherent to fluorescence microscopy and encounters challenges when applied to flow cytometry data as it would require accurate co-registration on a huge number of images. Here, we present a novel method that exploits for the first time a Holotomography-driven learning to completely eliminate the need for co-registration. We demonstrate that training a CNN on a stain-free dataset of 3D refractive index tomograms of flowing cells unlocks stain-free intracellular specificity for the first time in quantitative phase imaging flow cytometry. This self-supervised solution, by circumventing the critical obstacle of fluorescence co-registration, opens unprecedented perspectives for label-free, high-throughput imaging flow cytometry, offering a powerful new paradigm for advanced 2D and 3D single-cell analysis.

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.

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.

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.

We demonstrate a novel biomedical application of a commercial spiral microfluidic chip (Fluidic 382), originally developed for particle sorting, by repurposing it for label-free, size-based isolation of pathological blood cells, including leukemic blasts from acute myeloid leukemia (AML) samples. For the first time, we establish and validate a streamlined protocol for cell separation using Dean-driven hydrodynamic forces in a chip not originally designed for blood analysis. Using a 9-turn, 6-outlet spiral channel configuration, we achieved high-efficiency sorting of red and white blood cells from healthy donors and selectively enriched pathological blasts from AML patient blood. The device’s performance was validated through flow cytometry and numerical simulations, demonstrating strong agreement between experimental and computational results with less than 1% relative error. With its compact footprint, reagent-free operation, and automation potential, this method represents a significant advance toward point-of-care blood diagnostics in extreme environments, particularly space missions. The chip’s ability to separate pathological cells in microgravity-compatible conditions offers a promising route for real-time astronaut health monitoring, supporting early detection and mitigation of radiation-induced haematological disorders such as AML.

VPS13A disease (chorea-acanthocytosis), is an ultra-rare autosomal recessive neurodegenerative disorder caused by mutations of the VPS13A gene encoding Vps13A. Increased serum levels of the muscle isoform of creatine kinase associated with often asymptomatic muscle pathology are among the poorly understood early clinical manifestations of VPS13A disease. Here, we carried out an integrated analysis of skeletal muscle from Vps13a-/- mice and from VPS13A disease patient muscle biopsies. The absence of Vps13A impaired autophagy, resulting in pathologic metabolic remodeling characterized by cellular energy depletion, increased protein/lipid oxidation and a hyperactivated unfolded protein response. This was associated with defects in myofibril stability and the myofibrillar regulatory proteome, with accumulation of the myocyte senescence marker, NCAM1. In Vps13a-/- mice, the impairment of autophagy was further supported by the lacking effect of starvation alone or in combination with colchicine on autophagy markers. As a proof of concept, we showed that rapamycin treatment rescued the accumulation of terminal phase autophagy markers LAMP1 and p62 as well as NCAM1, supporting a connection between impaired autophagy and accelerated aging in the absence of VPS13A. The premature senescence was also corroborated by local activation of pro-inflammatory NF-kB-related pathways in both Vps13a-/- mice and patients with VPS13A disease. Our data link for the first time impaired autophagy and inflammaging with muscle dysfunction in the absence of VPS13A. The biological relevance of our mouse findings, supported by human muscle biopsy data, shed new light on the role of VPS13A in muscle homeostasis.

Adult patients with undiagnosed genetic disorders suffer most from diagnostic delay and seldom appear in cohort studies investigating the diagnostic yield in medical genetic clinical practice. Here we present the results of the diagnostic activity performed in a referral center on 654 consecutive, unselected adult subjects presenting with molecularly unsolved conditions. More than 50% of the referred individuals were affected by syndromic or isolated intellectual disability. Different molecular approaches, including clinical/whole exome sequencing (CES/WES), chromosomal microarray analysis (CMA), and/or targeted gene or gene panel sequencing were used to analyze patients' DNA. Definitive diagnosis was obtained in over 30% of individuals. The most sensitive methodology was CES/WES, which allowed us to reach a diagnosis in over 50% of the 162 solved cases. Despite the great variety of clinical presentations, our results represent a reliable picture of the "real world" daily routine in an outpatient medical genetics clinic dedicated to diagnostic activity, and contribute to better understand the great value of a definitive molecular diagnosis in adults, either for the affected individuals and their families. This retrospective analysis demonstrates the importance of adopting a genomic-first approach within the diagnostic process for adults affected with unsolved rare conditions.

Sickle cell disease (SCD) remains associated with reduced life expectancy and poor quality of life despite improvements observed in the last decades mostly related to comprehensive care, use of hydroxycarbamide, screening to identify patients at risk of strokes, and implementation of safe transfusion protocols. The course of the disease is highly variable, making it difficult to predict severity and response to therapy. Allogeneic hematopoietic stem cell transplantation potentially provides a cure with a relatively low rate of complications, but few patients have an HLA-identical sibling. The hopes of patients and healthcare providers have been raised after the initial excellent results of gene therapy studies. However, there is a strong contrast between the high expectations of families and patients and the limited availability of the product, which is technically complex and very expensive. In light of this consideration and of the limited data available on the long-term efficacy and toxicity of different gene therapy approaches, the European Hematology Association Red Cell & Iron Specialized Working Group (EHA SWG) and the hemoglobinopathy working part of the European Blood & Marrow Transplant (EBMT) Group have prioritized the development of recommendations for selection of patients with SCD who are good candidates for gene therapy. The decision-making algorithm was developed by a panel of experts in hemoglobinopathies and/or transplantation chosen by EHA SWG and EBMT, to discuss the selection of SCD patients for gene therapy and draw notes on the related clinical problems.

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.