Autonomous Underwater Vehicles: Identifying Critical Issues and Future Perspectives in Image Acquisition.

Underwater imaging has been present for many decades due to its relevance in vision and navigation systems. In recent years, advances in robotics have led to the availability of autonomous or unmanned underwater vehicles (AUVs, UUVs). Despite the rapid development of new studies and promising algorithms in this field, there is currently a lack of research toward standardized, general-approach proposals. This issue has been stated in the literature as a limiting factor to be addressed in the future. The key starting point of this work is to identify a synergistic effect between professional photography and scientific fields by analyzing image acquisition issues. Subsequently, we discuss underwater image enhancement and quality assessment, image mosaicking and algorithmic concerns as the last processing step. In this line, statistics about 120 AUV articles fro recent decades have been analyzed, with a special focus on state-of-the-art papers from recent years. Therefore, the aim of this paper is to identify critical issues in autonomous underwater vehicles encompassing the entire process, starting from optical issues in image sensing and ending with some issues related to algorithmic processing. In addition, a global underwater workflow is proposed, extracting future requirements, outcome effects and new perspectives in this context.

Analysis of Connectome Graphs Based on Boundary Scale.

The purpose of this work is to advance in the computational study of connectome graphs from a topological point of view. Specifically, starting from a sequence of hypergraphs associated to a brain graph (obtained using the Boundary Scale model, BS2), we analyze the resulting scale-space representation using classical topological features, such as Betti numbers and average node and edge degrees. In this way, the topological information that can be extracted from the original graph is substantially enriched, thus providing an insightful description of the graph from a clinical perspective. To assess the qualitative and quantitative topological information gain of the BS2 model, we carried out an empirical analysis of neuroimaging data using a dataset that contains the connectomes of 96 healthy subjects, 52 women and 44 men, generated from MRI scans in the Human Connectome Project. The results obtained shed light on the differences between these two classes of subjects in terms of neural connectivity.

CDrift: An Algorithm to Correct Linear Drift From A Single High-Resolution STEM Image.

In this work, a new method to determine and correct the linear drift for any crystalline orientation in a single-column-resolved high-resolution scanning transmission electron microscopy (HR-STEM) image, which is based on angle measurements in the Fourier space, is presented. This proposal supposes a generalization and the improvement of a previous work that needs the presence of two symmetrical planes in the crystalline orientation to be applicable. Now, a mathematical derivation of the drift effect on two families of asymmetric planes in the reciprocal space is inferred. However, though it was not possible to find an analytical solution for all conditions, a simple formula was derived to calculate the drift effect that is exact for three specific rotation angles. Taking this into account, an iterative algorithm based on successive rotation/drift correction steps is devised to remove drift distortions in HR-STEM images. The procedure has been evaluated using a simulated micrograph of a monoclinic material in an orientation where all the reciprocal lattice vectors are different. The algorithm only needs four iterations to resolve a 15° drift angle in the image.

Enhanced hemato-endothelial specification during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions.

The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

HOXA9 promotes hematopoietic commitment of human embryonic stem cells.

The molecular determinants regulating the specification of human embryonic stem cells (hESCs) into hematopoietic cells remain elusive. HOXA9 plays a relevant role in leukemogenesis and hematopoiesis. It is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and is downregulated upon differentiation. Hoxa9-deficient mice display impaired hematopoietic development, and deregulation of HOXA9 expression is frequently associated with acute leukemia. Analysis of the genes differentially expressed in cord blood HSPCs vs hESC-derived HSPCs identified HOXA9 as the most downregulated gene in hESC-derived HSPCs, suggesting that expression levels of HOXA9 may be crucial for hematopoietic differentiation of hESC. Here we show that during hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development, but is restricted to the hemogenic precursors (HEP) (CD31(+)CD34(+)CD45(-)), and diminishes as HEPs differentiate into blood cells (CD45(+)). Different gain-of-function and loss-of-function studies reveal that HOXA9 enhances hematopoietic differentiation of hESCs by specifically promoting the commitment of HEPs into primitive and total CD45(+) blood cells. Gene expression analysis suggests that nuclear factor-κB signaling could be collaborating with HOXA9 to increase hematopoietic commitment. However, HOXA9 on its own is not sufficient to confer in vivo long-term engraftment potential to hESC-hematopoietic derivatives, reinforcing the idea that additional molecular regulators are needed for the generation of definitive in vivo functional HSPCs from hESC.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

A parallel Homological Spanning Forest framework for 2D topological image analysis.

In [14], a topologically consistent framework to support parallel topological analysis and recognition for 2D digital objects was introduced. Based on this theoretical work, we focus on the problem of finding efficient algorithmic solutions for topological interrogation of a 2D digital object of interest D of a presegmented digital image I, using 4-adjacency between pixels of D. In order to maximize the degree of parallelization of the topological processes, we use as many elementary unit processing as pixels the image I has. The mathematical model underlying this framework is an appropriate extension of the classical concept of abstract cell complex: a primal-dual abstract cell complex (pACC for short). This versatile data structure encompasses the notion of Homological Spanning Forest fostered in [14,15]. Starting from a symmetric pACC associated with I, the modus operandi is to construct via combinatorial operations another asymmetric one presenting the maximal number of non-null primal elementary interactions between the cells of D. The fundamental topological tools have been transformed so as to promote an efficient parallel implementation in any parallel-oriented architecture (GPUs, multi-threaded computers, SIMD kernels and so on). A software prototype modeling such a parallel framework is built.

Detection of the mandibular canal in orthopantomography using a Gabor-filtered anisotropic generalized Hough transform.

In this paper, we explore the possibility of applying the anisotropic generalized Hough transform (AGHT) enhanced with a Gabor based time-frequency filtering (GTF) for the determination of the mandibular canal in digital dental panoramic radiographs. The proposed method is based on template matching using the fact that the shape of the mandibular canal is usually the same, followed by a filtering of the accumulator space in the Gabor domain for a precise detection of the position. The proposed procedure consists of a detailed description of the shape of the canal in its canonical form and on preserving Gabor filtering information for sorting the hierarchy of location candidates after applying anisotropically the extraction algorithm. The experimental results show that the proposed procedure is robust to recognition under occlusion and under the presence of additional structures e.g. teeth, projection errors.

FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs.

Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits.

Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia.

Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.

Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells.

Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.

SCL/TAL1 regulates hematopoietic specification from human embryonic stem cells.

Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C.

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbß, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS type C.

The pediatric leukemia oncoprotein NUP98-KDM5A induces genomic instability that may facilitate malignant transformation.

Pediatric Acute Myeloid Leukemia (AML) is a rare and heterogeneous disease characterized by a high prevalence of gene fusions as driver mutations. Despite the improvement of survival in the last years, about 50% of patients still experience a relapse. It is not possible to improve prognosis only with further intensification of chemotherapy, as come with a severe cost to the health of patients, often resulting in treatment-related death or long-term sequels. To design more effective and less toxic therapies we need a better understanding of pediatric AML biology. The NUP98-KDM5A chimeric protein is exclusively found in a particular subgroup of young pediatric AML patients with complex karyotypes and poor prognosis. In this study, we investigated the impact of NUP98-KDM5A expression on cellular processes in human Pluripotent Stem Cell models and a patient-derived cell line. We found that NUP98-KDM5A generates genomic instability through two complementary mechanisms that involve accumulation of DNA damage and direct interference of RAE1 activity during mitosis. Overall, our data support that NUP98-KDM5A promotes genomic instability and likely contributes to malignant transformation.

Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease.

Human ESCs provide access to the earliest stages of human development and may serve as an unlimited source of functional cells for future cell therapies. The optimization of methods directing the differentiation of human embryonic stem cells (hESCs) into tissue-specific precursors becomes crucial. We report an efficient enrichment of mesenchymal stem cells (MSCs) from hESCs through specific inhibition of SMAD-2/3 signaling. Human ESC-derived MSCs (hESC-MSCs) emerged as a population of fibroblastoid cells expressing a MSC phenotype: CD73+ CD90+ CD105+ CD44+ CD166+ CD45- CD34- CD14- CD19- human leucocyte antigen-DR (HLA-DR)-. After 28 days of SMAD-2/3 inhibition, hESC cultures were enriched (>42%) in multipotent MSCs. CD73+CD90+ hESC-MSCs were fluorescence activated cell sorting (FACS)-isolated and long-term cultures were established and maintained for many passages displaying a faster growth than somatic tissue-derived MSCs while maintaining MSC morphology and phenotype. They displayed osteogenic, adipogenic, and chondrocytic differentiation potential and exhibited potent immunosuppressive and anti-inflammatory properties in vitro and in vivo, where hESC-MSCs were capable of protecting against an experimental model of inflammatory bowel disease. Interestingly, the efficient enrichment of hESCs into MSCs through inhibition of SMAD-2/3 signaling was not reproducible with distinct induced pluripotent stem cell lines. Our findings provide mechanistic insights into the differentiation of hESCs into immunosuppressive and anti-inflammatory multipotent MSCs with potential future clinical applications.

Proximity labeling identifies a repertoire of site-specific R-loop modulators.

R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

Generation of a H9 Clonal Cell Line With Inducible Expression of NUP98-KDM5A Fusion Gene in the AAVS1 Safe Harbor Locus.

Pediatric acute myeloid leukemia (AML) is a rare and heterogeneous disease that remains the major cause of mortality in children with leukemia. To improve the outcome of pediatric AML we need to gain knowledge on the biological bases of this disease. NUP98-KDM5A (NK5A) fusion protein is present in a particular subgroup of young pediatric patients with poor outcome. We report the generation and characterization of human Embryonic Stem Cell (hESC) clonal lines with inducible expression of NK5A. Temporal control of NK5A expression during hematopoietic differentiation from hESC will be critical for elucidating its participation during the leukemogenic process.

Characterization of Genetic Variants of Uncertain Significance for the ALPL Gene in Patients With Adult Hypophosphatasia.

Hypophosphatasia (HPP) a rare disease caused by mutations in the ALPL gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ALPL results in high clinical heterogeneity, which makes it difficult to establish a specific HPP symptomatology. In the present study, three variants of ALPL gene with uncertain significance and no previously described (p.Del Glu23_Lys24, p.Pro292Leu and p.His379Asn) were identified in heterozygosis in patients diagnosed with HPP. These variants were characterized at phenotypic, functional and structural levels. All genetic variants showed significantly lower in vitro ALP activity than the wild-type (WT) genotype (p-value <0.001). Structurally, p.His379Asn variant resulted in the loss of two Zn2+ binding sites in the protein dimer which may greatly affect ALP activity. In summary, we identified three novel ALPL gene mutations associated with adult HPP. The correct identification and characterization of new variants and the subsequent study of their phenotype will allow the establishment of genotype-phenotype relationships that facilitate the management of the disease as well as making it possible to individualize treatment for each specific patient. This would allow the therapeutic approach to HPP to be personalized according to the unique genetic characteristics and clinical manifestations of each patient.