Epidemiological, Clinical and Genetic Study of Hypophosphatasia in A Spanish Population: Identification of Two Novel Mutations in The Alpl Gene

Hypophosphatasia (HPP) is a genetic disease caused by one or several mutations in ALPL gene encoding the tissue-nonspecific alkaline phosphatase affecting the mineralization process. Due to its low prevalence and lack of recognition, this metabolic disorder is generally confused with other more frequent bone disorders. An assessment of serum total alkaline phosphatase (ALP) levels was performed in 78,590 subjects. Pyridoxal-5′-phosphate (PLP) concentrations were determined and ALPL gene was sequenced in patients potentially affected by HPP. Functional validation of the novel mutations found was performed using a cell-based assay. Our results showed persistently low serum ALP levels in 0.12% of subjects. Among the studied subjects, 40% presented with HPP-related symptoms. Nine of them (~28%) had a history of fractures, 5 (~16%) subjects showed chondrocalcinosis and 4 (~13%) subjects presented with dental abnormalities. Eleven subjects showed increased PLP concentrations. Seven of them showed ALPL gene mutations (2 of the mutations corresponded to novel genetic variants). In summary, we identified two novel ALPL gene mutations associated with adult HPP. Using this protocol, almost half of the studied patients were diagnosed with HPP. Based on these results, the estimated prevalence of mild HPP in Spain could be up to double than previously reported.Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (with support from NIH P41-GM103311)grants from Alexion and FEIOMM, by Instituto de Salud Carlos III (grants PI18-00803 and PI18-01235)co-funding from FEDER and by Junta de Andalucía (grant PI-0207-2016)GM-N is supported by the predoctoral program from Instituto de Salud Carlos III (FI17/00178) and by the Research Initiation Grants for Official Master Students program from the University of Granada (2017)PJR is a Ramon y Cajal Researcher from the MINECO (RYC-2015-18383) at GENyO and University of Granada

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patientsThis work was supported by the Ramon y Cajal (RYC-2015-18382) to PJR founded by the Ministry of Economy and Competitiveness; the Instituto de Salud Carlos III-FEDER (CP12/03175 and CPII17/00032) to V.R-M., (PI17/01311) to M.L.L and J.R., (PI17/01966; Fundación Mutua Madrileña AP172142019; Premio Lopez Borrasca SETH 2019; GRS2061/A/19) to J.M.B. and (CPII15/00018 and PI16/01340) to PJR; by the Chair "Doctors Galera-Requena in cancer stem cell research" (CMC-CTS963) to J.A.M. and C.G-L

Adelante / Endavant

Séptimo desafío por la erradicación de la violencia contra las mujeres del Institut Universitari d’Estudis Feministes i de Gènere "Purificación Escribano" de la Universitat Jaume

Development of gene-therapy tools for bernard-soulier syndrome type c treatment

Las plaquetas son fragmentos celulares anucleados liberados de los megacariocitos, sus células precursoras, que residen en la médula ósea. Las plaquetas participan en una gran variedad de procesos, como la cicatrización de heridas, el papel en el cáncer y la respuesta inmunitaria, entre otros. Sin embargo, su función principal y mejor definida es la regulación de la hemostasia. Cuando se produce una lesión vascular, las células endoteliales se desprenden y queda expuesta la membrana basal y, más concretamente, las fibras de colágeno. El factor von Willebrand (VWF) circulante se adhiere a estas fibras formando un complejo que es reconocido por el receptor GPIb- V-IX de la superficie de las plaquetas. Esta interacción induce un efecto de rodamiento de las plaquetas circulantes sobre la zona lesionada, hasta detenerlas. Este proceso induce la activación, reclutamiento y agregación de las plaquetas circundantes sobre la zona afectada, formando un tapón plaquetario y poniendo fin a la hemorragia al interaccionar con los factores de coagulación. El complejo GPIb-V-IX resulta del ensamblaje de cuatro subunidades, GPIbα, GPIbβ, GPIX y GPV. Las variantes patogénicas que afectan a sus genes codificantes, excepto GP5, dan lugar a la manifestación de un trastorno plaquetario hereditario, el síndrome de Bernard-Soulier (SBS). Según el gen afectado podemos distinguir el SBS Tipo A1 cuando la variante aparece en GP1BA, Tipo B (GP1BB) y Tipo C (GP9). Significativamente, el GP9 presenta un mayor número de variantes patogénicas que conducen al SBS. Como resultado de estas variantes patogénicas, el receptor GPIb-V-IX es incapaz de exteriorizarse completamente en la superficie plaquetaria. En consecuencia, esto genera plaquetas no funcionales que son incapaces de reconocer las zonas de lesión y continuar con la cascada de coagulación. Los pacientes que padecen SBS se caracterizan por frecuentes episodios de sangrados mucocutáneos y hemorragias graves. Además, experimentan macrotrombocitopenia, es decir, un recuento reducido de plaquetas gigantes. La ausencia del receptor explica el fenotipo hemorrágico observado. Estos pacientes sólo pueden recurrir a medidas de prevención para evitar los episodios hemorrágicos, y beneficiarse de tratamientos paliativos para disminuir la gravedad de la hemorragia. Sin embargo, siguen enfrentándose a una enfermedad de por vida que merma la calidad de ésta. En casos excepcionales, el trasplante de células madre hematopoyéticas (CMH) entre hermanos afectados y no afectados por el SBS ha conseguido revertir la enfermedad. Sin embargo, el número limitado de donantes HLA compatibles y el riesgo de aloinmunización limitan su uso frecuente como abordaje terapéutico. Utilizando la revolucionaria tecnología CRISPR-Cas9, desarrollamos knockouts para cada gen codificador de subunidad en dos líneas celulares megacarioblásticas que expresan constitutivamente el receptor GPIb-V-IX. Estos knockouts informaron sobre la importancia que cada subunidad tiene para el ensamblaje de todo el receptor en un microambiente humano fisiológico y replicaron la mayoría de los fenotipos descritos en la literatura, estos son, ausencia o alteración de la externalización de GPIb-V-IX para genes inductores de SBS. En cuanto a GP5-KOs, confirmamos que este gen no impide la externalización del receptor GPIb-IX pero disminuye la presencia de GPIbβ. Este hecho dentro del ensamblaje del receptor no impidió su funcionalidad uniendo VWF pero se redujo ligeramente. A medida que avanza la investigación, la terapia génica adquiere cada vez más importancia en el tratamiento de las enfermedades monogénicas. Concretamente, la terapia génica que utiliza vectores integradores en las CMH, que son la fuente de todos los componentes celulares de la sangre, está cobrando importancia en el campo de la hematología como enfoque curativo para abordar las neoplasias malignas en su origen. De hecho, actualmente se están investigando numerosas enfermedades en ensayos clínicos, empleando diversos tratamientos basados en vectores lentivirales autoinactivantes. Estos ensayos pretenden corregir deficiencias primarias observadas en afecciones como el síndrome de Wiskott-Aldrich, SCID-X1 o ADA-SCID. Además, a finales de 2022, la “Food and Drug Administration” ya ha concedido la aprobación para la comercialización y distribución de dos terapias génicas basadas en la tecnología de vectores lentivirales. Estas terapias están dirigidas al tratamiento de la β-Talasemia y al de la Adrenoleucodistrofia Cerebral, marcando avances significativos en este campo. En cuanto al SBS, algunos estudios han aportado pruebas convincentes de la reversión fenotípica y la restauración de la funcionalidad de las plaquetas mediante la generación de plaquetas producidas in vitro a partir de células madre pluripotentes inducidas derivadas de pacientes con mutaciones en GP1BA y GP1BB. Además, la transducción de CMHs con vectores lentivirales que expresan GP1BA y GP1BB, seguida de su infusión en modelos murinos in vivo carentes de sus respectivos genes (Gp1banull y Gp1bbnull), ha demostrado de forma consistente una recuperación de los tiempos de sangrado y un alivio significativo de la macrotrombocitopenia. Estos hallazgos nos han inspirado para investigar la idoneidad potencial del SBS tipo C como candidato a ser corregido mediante un enfoque similar. La estrategia terapéutica propuesta consiste en tratar a los pacientes con SBS tipo C que albergan variantes patogénicas de GP9 con un vector lentiviral autoinactivante que sobreexpresa GP9 en las CMH del paciente. Estas CMH se tratarían ex vivo y posteriormente se reinfundirían en el paciente una vez corregidas. Para lograr una expresión de GPIX estable y específica de tejido, es crucial la integración de nuestro casete terapéutico en las CMH del paciente. Estas CMH son responsables de la producción de megacariocitos (MK) y plaquetas anormales en individuos con EVC. Sin embargo, debido a la limitada disponibilidad de pacientes y a la singularidad de sus características hemorrágicas, puede que no sea factible aislar las CMH como herramienta donde optimizar nuestras herramientas de terapia génica. Por lo tanto, son necesarios modelos preclínicos alternativos para evaluar la eficacia de nuestras estrategias de sustitución génica antes de aplicarlas a nuestras células diana finales. Además, también desarrollamos otro modelo de enfermedad de SBS tipo C mediante la eliminación de GP9 en células madre pluripotentes inducidas (iPSCs). Este modelo añadió un paso más de sofisticación, ya que, mientras que el modelo de células megacarioblásticas sirvió para explorar la biología de los receptores, en este caso pudimos reproducir el desarrollo hematopoyético y megacariocítico completo, desde las CMH hasta la producción de MKs y plaquetas. Pudimos confirmar que las iPSCs carentes de GP9 generaban in vitro plaquetas gigantes similares a las plaquetas de los pacientes con SBS, convirtiéndose en una alternativa perfecta a las CMHs de los pacientes. El siguiente paso crucial de nuestra investigación fue el desarrollo de vectores lentivirales autoinactivantes (LV) que expresan GPIX bajo promotores fisiológicos, asegurando una expresión megacariocítica específica adecuada para su aplicación clínica. Los modelos de enfermedad GP9-KO previamente establecidos sirvieron como valiosas herramientas preclínicas para evaluar la funcionalidad de nuestros LVs en la restauración de la expresión de GPIX. Sorprendentemente, los GPIX-LVs transdujeron con éxito células GP9-KO, conduciendo a la recuperación de niveles de expresión de GPIX comparables a los de las células de tipo salvaje (WT). Es importante destacar que esta expresión era específica de tejido, ya que era indetectable en otras líneas celulares no megacarioblásticas. Además, la introducción exógena de GPIX facilitó la externalización de las subunidades restantes, en particular GPIbα, que actúa como subunidad funcional del complejo. Estos receptores rescatados genéticamente recuperaron su capacidad funcional, incluido el reconocimiento de VWF soluble, la aglutinación en presencia de ristocetina y la adhesión firme a recubrimientos de VWF en presencia de botrocetina. De forma similar, en nuestro modelo de enfermedad de SBS tipo C basado en iPSCs, la transducción no sólo restauró la expresión de GPIX en la membrana de los MKs sino que también recuperó su expresión en las plaquetas, lo que resultó en una reversión a su tamaño normal. En ambos casos, los niveles de expresión fueron comparables a los de las células WT. Es importante destacar que nuestras LVs exhibieron actividad funcional a lo largo de todo el proceso de diferenciación, sin estar sujetas a silenciamiento epigenético. Una vez demostrada la funcionalidad de nuestras herramientas de terapia génica, quisimos validar su eficacia recuperando la expresión de GPIX, ahora en nuestras futuras células diana, CMHs aisladas de pacientes con SBS Tipo C portadores de diferentes variantes patogénicas de GP9. A través de su aislamiento a partir de sangre periférica no movilizada, transducción y posterior diferenciación en MKs y plaquetas, validamos la reversión del fenotipo SBS ex vivo. Por lo tanto, estos resultados confirman el prometedor potencial terapéutico de nuestro enfoque de terapia génica. Por último, desarrollamos un modelo murino específicamente diseñado para evaluar la eficacia de nuestros LVs en la restauración de funciones fisiológicas normales, como el tiempo de sangrado. Este modelo tiene una gran relevancia como estudio preclínico definitivo antes de pasar a la fase clínica. No sólo nos permitirá seguir evaluando la eficacia de nuestros LVs que expresan GPIX, sino también valorar la bioseguridad integradora de nuestros LVs y evaluar la presencia de posibles autoanticuerpos contra el complejo GPIb-V-IX rescatado genéticamente. Los hallazgos previos de nuestra propia investigación, junto con descubrimientos relevantes realizados por otros investigadores en modelos murinos Gp1banull y Gp1bbnull, nos llenan de optimismo respecto a los posibles resultados al tratar modelos de ratón Gp9null SBS en un futuro próximo. En conclusión, los hallazgos significativos presentados en este trabajo de investigación establecen firmemente el síndrome de Bernard-Soulier tipo C como un trastorno hereditario que tiene el potencial de ser curado mediante estrategias de terapia génica.Platelets are anucleate cell fragments released from megakaryocytes, their precursor cells which reside within the bone marrow. Platelets participate in a wide variety of processes like wound healing, role in cancer, immune response, among others. However, their main and bestdefined function is in the regulation of hemostasia. Once a vascular injury occurs, endothelial cells become detached and the basal membrane, and more specifically, collagen fibers, become exposed. Circulating von Willebrand factor (VWF) adhere to these fibers forming a complex which is recognized by the platelet surface receptor GPIb-V-IX. This interaction induces a rolling effect of circulating platelets over the injury zone, till arresting them. This process induces the activation, recruitment, and aggregation of surrounding platelets over the affected area, forming a platelet plug and ending the hemorrhage when interacting with the coagulation factors. GPIb-V-IX complex results from the assembly of four subunits, GPIbα, GPIbβ, GPIX and GPV. Pathogenic variants affecting their coding genes except for GP5 results in the manifestation of an inherited platelet disorder, Bernard-Soulier Syndrome (BSS). Accordingly to the affected gene we can distinguish BSS Type A1 when the variant appears in GP1BA, Type B (GP1BB) and Type C (GP9). Significantly, GP9 exhibits a higher number of pathogenic variants that lead to BSS As a result of those pathogenic variants, GPIb-V-IX receptor is unable to fully externalize on the platelet surface. Consequently, this generates non-functional platelets that are incapable of recognizing injury zones and performing subsequent following necessary steps. Patients suffering for BSS are characterized by frequent mucocutaneous bleeding episodes and severe hemorrhages. Additionally, they experience macrothrombocytopenia, this is, reduced count of giant platelets. Pathogenic variants affecting the assembly of the receptor explains the observed bleeding phenotype. These patients uniquely can take advantage of prevention, to avoid the bleeding episodes, and palliative treatments in order to diminish the hemorrhage severity. However, they still face a lifetime disease which diminish their life quality. In exceptional cases, hematopoietic stem cells (HSCs) transplantation among siblings affected and nonaffected by BSS has been successful in reversing the disease. Nevertheless, the limited number of compatible HLA donors and the risk of alloimmunization limits its frequent use as a therapeutic approach. Utilizing the revolutionary CRISPR-Cas9 technology, we developed knockouts for each subunit coding gene in two megakaryoblastic cell lines that constitutively express the GPIb-V-IX receptor. These knockouts shed light on the importance that each subunit makes for the assemblage of the entire receptor in a physiological human microenvironment and replicated most of the phenotypes described in the literature, these are, absence or impaired GPIb-V-IX externalization for BSS driver genes. Regarding GP5-KOs, we confirmed that this gene does not impede GPIb-IX receptor externalization but decreases GPIbβ presence. This fact within the receptor assembly did not hinder its functionality by binding VWF but it was slightly reduced. As research progresses, gene therapy is becoming increasingly important in the treatment of monogenic diseases. Specifically, gene therapy utilizing integrative vectors on HSCs, which are the source of all blood cellular components, is gaining significance in the field of hematology as a curative approach for addressing malignancies at their origin. In fact, numerous diseases are currently being investigated in clinical trials, employing various treatments based on self-inactivating lentiviral vectors. These trials aim to correct primary deficiencies seen in conditions such as Wiskott-Aldrich Syndrome, SCID-X1, or ADA-SCID. Moreover, by the end of 2022, the Food and Drug Administration has already granted approval for the marketing and distribution of two gene therapies based on lentiviral vector technology. These therapies target β-Thalassemia and Cerebral Adrenoleukodystrophy, marking significant advancements in the field. Regarding BSS, numerous studies have provided compelling evidence of the phenotypical reversion and restoration of platelet functionality through the generation of in vitro-produced platelets from induced pluripotent stem cells derived from patients with GP1BA and GP1BB mutations. Furthermore, the transduction of HSCs with lentiviral vectors expressing GP1BA and GP1BB, followed by their infusion into in vivo murine models lacking their respective genes (Gp1banull and Gp1bbnull), has consistently demonstrated a recovery of bleeding times and a significant alleviation of macrothrombocytopenia. These findings have inspired us to investigate the potential suitability of BSS type C as candidate for being corrected using a similar approach. The proposed therapeutic strategy involves treating BSS type C patients harboring GP9 pathogenic variants with a self-inactivating lentiviral vector that overexpresses GP9 in the patient's HSCs. These HSCs would be treated ex vivo and subsequently reinfused into the patient once they have been corrected. To achieve stable and tissue-specific expression of GPIX, the integration of our therapeutic cassette into the patient's HSCs is crucial. These HSCs are responsible for the production of abnormal megakaryocytes (MKs) and platelets in individuals with BSS. However, due to the limited availability of patients and the uniqueness of their bleeding characteristics, it may not be feasible to isolate HSCs for testing our gene therapy tools. Therefore, alternative preclinical models are necessary to evaluate the effectiveness of our gene replacement strategies before applying them on our final target cells. Additionally, we also developed another BSS type C disease model by knocking-out GP9 in induced pluripotent stem cells (iPSCs). This model added one step more of sophistication, because, while megakaryoblastic cell model served to explore receptor biology, in this case we were able to reproduce whole hematopoietic and megakaryocytic development, from HSCs to produce MKs and platelets. We could confirm that iPSCs lacking GP9 generated in vitro giant platelets similar to BSS patients’ platelets, becoming a perfect alternative to patients’ HSCs. The next crucial step involved in our research was the development of self-inactivating lentiviral vectors (LV) that express GPIX under physiological promoters, ensuring megakaryocytic-specific expression suitable for clinical application. The previously established GP9-KO disease models served as valuable preclinical tools for assessing the functionality of our LVs in restoring GPIX expression. Remarkably, GPIX-LVs successfully transduced GP9-KO cells, leading to the recovery of GPIX expression levels comparable to wild-type (WT) cells. Importantly, this expression was tissue-specific, as it was undetectable in other non-megakaryoblastic cell lines. Furthermore, the introduction of exogenous GPIX facilitated the externalization of the remaining subunits, particularly GPIbα, which serves as the functional subunit of the complex. These genetically rescued receptors regained their functional capacity, including the recognition of soluble VWF, agglutination in the presence of ristocetin and firm adhesion to coated VWF in the presence of botrocetin. Similarly, in our BSS Type C disease model based on iPSCs, the transduction not only restored GPIX expression on the membrane of MKs but also recovered its expression in platelets, resulting in a reversion to their normal size. In both cases, the expression levels were comparable to those of WT cells. Importantly, our LVs exhibited functional activity throughout the entire differentiation process, without being subject to epigenetic silencing. Once proved the functionality of our gene therapy tools, we wanted to validate its efficacy by recovering the GPIX expression, now in our future target cells, HSCs isolated from BSS Type C patients carrying different GP9 pathogenic variants. Through its isolation from non-mobilized peripheral blood, transduction, and subsequent differentiation into MKs and platelets, we validated the reversal of the BSS phenotype ex vivo. Therefore, these results confirm the promising therapeutic potential of our gene therapy approach. Lastly, we developed a novel murine model specifically designed to evaluate the effectiveness of our LVs in restoring normal physiological functions, such as bleeding time. This model holds significant relevance as the definitive preclinical study before progressing to the clinical phase. It will not only allow us to further evaluate the efficacy of our GPIX-expressing LVs, but also assess the integrative biosafety of our LVs and evaluate the presence of possible autoantibodies against the genetically rescued GPIb-V-IX complex. The previous findings from our own research, along with relevant discoveries made by other researchers in Gp1banull and Gp1bbnull murine models, fill us with a sense of optimism regarding the potential outcomes when treating BSS Gp9null mouse models in the near future. In conclusion, the significant findings presented in this research work firmly establish Bernard-Soulier Syndrome type C as an inherited disorder that holds the potential to be cured through gene therapy strategies.Tesis Univ. Granada

Copper and melanin play a role in Myxococcus xanthus predation on Sinorhizobium meliloti

Myxococcus xanthus is a soil myxobacterium that exhibits a complex lifecycle with two multicellular stages: cooperative predation and development. During predation, myxobacterial cells produce a wide variety of secondary metabolites and hydrolytic enzymes to kill and consume the prey. It is known that eukaryotic predators, such as ameba and macrophages, introduce copper and other metals into the phagosomes to kill their prey by oxidative stress. However, the role of metals in bacterial predation has not yet been established. In this work, we have addressed the role of copper during predation of M. xanthus on Sinorhizobium meliloti. The use of biosensors, variable pressure scanning electron microscopy, high-resolution scanning transmission electron microscopy, and energy dispersive X ray analysis has revealed that copper accumulates in the region where predator and prey collide. This accumulation of metal up-regulates the expression of several mechanisms involved in copper detoxification in the predator (the P1B-ATPase CopA, the multicopper oxidase CuoA and the tripartite pump Cus2), and the production by the prey of copper-inducible melanin, which is a polymer with the ability to protect cells from oxidative stress. We have identified two genes in S. meliloti (encoding a tyrosinase and a multicopper oxidase) that participate in the biosynthesis of melanin. Analysis of prey survivability in the co-culture of M. xanthus and a mutant of S. meliloti in which the two genes involved in melanin biosynthesis have been deleted has revealed that this mutant is more sensitive to predation than the wild-type strain. These results indicate that copper plays a role in bacterial predation and that melanin is used by the prey to defend itself from the predator. Taking into consideration that S. meliloti is a nitrogen-fixing bacterium in symbiosis with legumes that coexists in soils with M. xanthus and that copper is a common metal found in this habitat as a consequence of several human activities, these results provide clear evidence that the accumulation of this metal in the soil may influence the microbial ecosystems by affecting bacterial predatory activities.This work has been supported by the Spanish Government grant BFU2016-75425-P to AM-M (70% funded by FEDER). FC-M and NG-T were granted with contracts (Ref. 6010 and 2811, respectively) from Programa Empleo Juvenil-Fondo Social Europeo from Junta de Andalucía

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.Health Institute Carlos III (ISCIII/FEDER, PI17/01574

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

This research was funded by the Instituto de Salud Carlos III grants (PI18-00803, PI21/01069 and PI18-01235), co-funded by the European Regional Development Fund (FEDER) and by Junta de Andalucia grant (PI-0268-2019). In addition, VC-B is supported by postdoctoral fellowship from Junta de Andalucia (RH-0141-2020) and JMV-S and SG-S are funded by predoctoral fellowships from Instituto de Salud Carlos III (CM19/00188 and FI19/00118 respectively). CG-F and RS-dT are funded by postdoctoral Sara Borrell fellowship and Research investigator grant in the framework of the youth guarantee Program from the Instituto de Salud Carlos III and the University of Granada with co-funding by FEDER respectively (CD20/00022 and 8110 grant number). GM-N is supported by the predoctoral program from Instituto de Salud Carlos III (FI17/00178) and PR is a Ramon y Cajal Researcher from the MINECO (RYC-2015-18383) at GENyO and University of Granada. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.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.Instituto de Salud Carlos III European Commission PI21/01069 PI18-01235 CM19/00188 FI19/00118 FI17/00178 PI18-00803European CommissionJunta de Andalucia PI-0268-2019 RH-0141-2020University of GranadaEuropean Commission CD20/00022 8110Spanish Government RYC-2015-1838