8 research outputs found
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