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McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.

Objectives: Since the results of the RECOVERY trial, WHO recommendations about the use of corticosteroids (CTs) in COVID-19 have changed. The aim of the study is to analyse the evolutive use of CTs in Spain during the pandemic to assess the potential influence of new recommendations. Material and methods: A retrospective, descriptive, and observational study was conducted on adults hospitalised due to COVID-19 in Spain who were included in the SEMI-COVID-19 Registry from March to November 2020. Results: CTs were used in 6053 (36.21%) of the included patients. The patients were older (mean (SD)) (69.6 (14.6) vs. 66.0 (16.8) years; p < 0.001), with hypertension (57.0% vs. 47.7%; p < 0.001), obesity (26.4% vs. 19.3%; p < 0.0001), and multimorbidity prevalence (20.6% vs. 16.1%; p < 0.001). These patients had higher values (mean (95% CI)) of C-reactive protein (CRP) (86 (32.7–160) vs. 49.3 (16–109) mg/dL; p < 0.001), ferritin (791 (393–1534) vs. 470 (236–996) µg/dL; p < 0.001), D dimer (750 (430–1400) vs. 617 (345–1180) µg/dL; p < 0.001), and lower Sp02/Fi02 (266 (91.1) vs. 301 (101); p < 0.001). Since June 2020, there was an increment in the use of CTs (March vs. September; p < 0.001). Overall, 20% did not receive steroids, and 40% received less than 200 mg accumulated prednisone equivalent dose (APED). Severe patients are treated with higher doses. The mortality benefit was observed in patients with oxygen saturation

[ES] Las distrofias hereditarias de la retina (DHR) son un conjunto de trastornos que se caracteriza por la muerte progresiva de los fotorreceptores, lo que conlleva una pérdida de la función visual, pudiendo llegar a la ceguera legal. Se estima que afectan a alrededor de 1 de cada 3.000 individuos, por lo que, las DHR se engloban dentro de las enfermedades raras. El diagnóstico genético de las DHR es fundamental para la confirmación de la sospecha clínica, ofrecer un correcto asesoramiento genético y poder incluir a los pacientes en ensayos clínicos basados en terapias dirigidas a genotipos concretos. En este trabajo, hemos establecido el diagnóstico genético de una cohorte de pacientes con una sospecha clínica de DHR mediante secuenciación masiva dirigida. El panel de genes consta de la región codificante de 114 genes, sus regiones intrónicas flanqueantes y de varias regiones intrónicas profundas en las que previamente se han descrito mutaciones. Mediante esta estrategia se ha logrado el diagnóstico genético del 55% de los pacientes incluidos en la cohorte en consonancia con las ratios diagnósticas reportadas previamente en la bibliografía. Por otra parte, en los probandos P2 y P4 se detectó variantes patogénicas o potencialmente patogénicas en más de un gen. El hallazgo de mutaciones deletéreas en múltiples genes puede impactar significativamente sobre las elecciones reproductivas de los pacientes y sobre su elegibilidad para recibir un tratamiento genético específico para un gen. En los pacientes cuyo diagnóstico estaba completo, las mutaciones causantes de la patología se distribuían a lo largo de 8 genes distintos: 3 genes con herencia autosómica recesiva, 3 genes con herencia autosómico dominante y 2 genes con herencia ligada al X. Se identificó un total de 12 variantes patogénicas o probablemente patogénicas, incluyendo: 5 missense, 2 nonsense, 1 frameshift y 4 variantes que alteran el splicing. Por otra parte, se describieron 5 variantes noveles, incluyendo, [EN] Inherited retinal dystrophies (IRD) are a group of diseases characterised by the progressive death of photoreceptors, leading to a visual function loss and even legal blindness. It is estimated that around 1 in 3000 individuals is affected, as a result, IRD are considered among rare diseases. Genetic diagnostic plays a key role in confirming clinical suspicions, providing proper genetic assessment and including patients in clinical trials based on therapies directed to certain genotypes. In this work, we have established the genetic diagnosis of a patient cohort with an IRD clinical suspicion through directed sequencing. The gene panel is constituted by 114 genes, their corresponding flanking intronic regions and several deep intronic regions where mutations were previously reported. This approach has yielded a genetic diagnosis for 55% of the patients included in the cohort in accordance with the diagnostic ratios which had been previously reported in the literature. Furthermore, in probands P2 and P4 likely pathogenic or pathogenic variants were reported in several genes. Finding deleterious mutations in multiple genes can significantly impact patients¿ reproductive choices and eligibility for receiving a gene-specific genetic treatment. In patients with a molecular diagnostic, mutations responsible for the disease were distributed across 8 different genes: 3 genes with an autosomal recessive inheritance, 3 genes with an autosomal dominant inheritance and 2 genes with X-linked inheritance. A total of 12 likely pathogenic or pathogenic mutations were identified, including: 5 missenses, 2 nonsenses, 1 frameshift and 4 splicing altering mutations. Moreover, we described 5 novel variants, including a nonsense variant in CHM, a missense variant in BEST1 and 3 splicing variants in CACNA2D4, OFD1 y HK1. In order to determine the pathogenicity of the splicing variant in vitro assays with minigenes were carried out, concluding that c.2489-2A>G in OFD1 leads to the del

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner’s corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner’s corpuscles. Pathogenic USH2A mutations cause Usher’s syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner’s corpuscles, recorded from Ush2a−/− mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.

[ES] Las distrofias hereditarias de la retina (DHR) son un conjunto de trastornos caracterizados por la muerte, generalmente, progresiva de fotorreceptores y células del epitelio pigmentario retiniano. Las DHR afectan a más de 2 millones de personas en el mundo, produciendo una pérdida parcial y, en muchos casos, total de la visión, para la cual no existe ningún tratamiento que revierta o frene la progresión de la enfermedad. Se caracterizan por una elevada heterogeneidad clínica y genética, identificándose hasta la fecha mutaciones en más de 250 genes responsable de algún tipo de DHR. Esta tesis doctoral se ha centrado en el diagnóstico genético de 208 pacientes afectados de DHR no sindrómica (DHR-NS), mediante secuenciación masiva dirigida a un panel de 117 genes y cinco regiones intrónicas profundas en las que previamente se habían descrito mutaciones. Las variantes identificadas en los pacientes se priorizaron de acuerdo a la mínima frecuencia alélica y se clasificaron según los criterios de la guía del American College of Medical Genetics and Genomics. Las variantes de número de copias identificadas mediante la secuenciación del panel se validaron utilizando multiplex ligation-dependent probe amplification o array genómico. A todos los pacientes se les realizó un estudio oftalmológico, más o menos exhaustivo, incluyendo pruebas electrofisiológicas. Además, se realizaron ensayos in vitro con minigenes para comprobar la repercusión de ciertas variantes sobre el procesamiento del ARNm. Para ello, se empleó el plásmido pSPL3 en el que se introdujo la mutación a estudio, la transformación se realizó en bacterias E. coli electrocompetentes y la transfección en células HEK-293. Mediante la secuenciación del panel de genes, se obtuvo una tasa diagnóstica del 60%. Las mutaciones en los pacientes resueltos se distribuyeron en 30 genes diferentes, de las cuales 38 se describen por primera vez en este trabajo. Los genes mutados con mayor frecuencia en estos pacientes fuero, [CA] Les distròfies hereditàries de la retina (DHR) són un conjunt de trastorns caracteritzats per la mort, generalment, progressiva de fotoreceptors i cèl¿lules de l'epiteli pigmentari retiniano. Les DHR afecten més de 2 milions de persones al món, produint una pèrdua parcial i, en molts casos, total de la visió, per la qual no hi ha cap tractament que revertisca o frene la progressió de la malaltia. Es caracteritzen per una elevada heterogeneïtat clínica i genètica, identificant-se fins a la data mutacions en més de 250 gens responsable d'algun tipus de DHR. Esta tesi doctoral s'ha centrat en el diagnòstic genètic de 208 pacients afectats de DHR no sindròmica (DHR-NS), per mitjà de seqüenciació massiva dirigida a un panell de 117 gens i cinc regions intròniques profundes en les que prèviament s'havien descrit mutacions. Les variants identificades en els pacients es van prioritzar d'acord a la mínima freqüència allèlica i es van classificar segons els criteris de la guia de l'American College of Medical Genetics and Genomics. Les variants de nombre de còpies identificades per mitjà de la seqüenciació del panell es van validar utilitzant multiplex ligation-dependent probe amplification o array genòmic. A tots els pacients se'ls va realitzar un estudi oftalmològic, més o menys exhaustiu, incloent proves electrofisiològiques. A més, es van realitzar assajos in vitro amb minigens per comprovar la repercussió de certes variants sobre el processament de l'ARNm. Per a això, es va emprar el plasmidi pSPL3 en què es va introduir la mutació a estudi, la transformació es va realitzar en bacteris E. coli electrocompetentes i la transfecció en cèl-lules HEK-293. Per mitjà de la seqüenciació del panell de gens, es va obtindre una taxa diagnòstica del 60%. Les mutacions en els pacients resolts es van distribuir en 30 gens diferents, de les quals 38 es descriuen per primera vegada en este treball. Els gens mutats amb major freqüència en estos pacients van ser ABCA4, USH2A, RPGR i, [EN] Inherited retinal dystrophies (IRD) are a group of disorders generally characterized by the progressive death of photoreceptors and retinal pigment epithelial cells. IRD affect more than 2 million people in the world, producing a partial and, in many cases, total loss of vision, for which there is no treatment that reverses or slows the progression of the disease. They are characterized by a high clinical and genetic heterogeneity, identifying mutations in more than 250 genes responsible for some type of IRD to date. This doctoral thesis has focused on the genetic diagnosis of 208 patients affected by non-syndromic IRD (NS-IRD), through targeted genomic sequencing focuses on a panel of 117 genes and five deep intronic regions in which mutations have previously been identified. The identified variants in patients are prioritized according to the minimum allelic frequency and are classified according to the criteria of the American College of Medical Genetics and Genomics guide. The copy number variants identified by panel sequencing were validated using multiplex ligation-dependent probe amplification or genomic array. All patients underwent an ophthalmological study, more or less comprehensive, including electrophysiological tests. In addition, in vitro assays with minigenes were performed to verify the impact of certain variants on mRNA processing. To do this, pSPL3 plasmid in which the mutation was introduced was used, the transformation was performed in electrocompetent E. coli bacteria and transfection in HEK-293 cells. By the panel sequencing, a diagnostic rate of 60% was obtained. The mutations in the resolved patients were distributed in 30 different genes, of which 38 were described for the first time in this work. The most frequently mutated genes in these patients were ABCA4, USH2A, RPGR and PRPH2. In addition, eight copy number variants were identified in the genes EYS, ABCA4, PRPF31, MERTK, CRB1 and ROM1, that demonstrated the enormous potential of

[ES] El síndrome de Usher (USH) es una enfermedad rara que sigue un patrón de herencia autosómico recesivo. Se caracteriza por asociar en pacientes hipoacusia neurosensorial, retinosis pigmentaria (RP) y, en algunos casos, alteración vestibular. El síndrome de Usher comprende más del 50% de los casos de sordoceguera y el 15-20% de los casos de RP, siendo así el trastorno genético más común que combina deterioro visual y auditivo, con una prevalencia que se sitúa entre 3,2 y 6,2: 100.000, dependiendo de la población estudiada. No obstante, actualmente no existe cura más allá de la implantación coclear y los audífonos para el problema auditivo. Según la severidad, el momento de aparición de los síntomas y la progresión de la enfermedad se distinguen 3 tipos clínicos de USH, siendo el tipo I la forma más severa en las que se manifiesta la enfermedad, el tipo II la más común y el tipo III la más heterogénea. Además de la heterogeneidad fenotípica, el USH también tiene heterogeneidad genética pues hasta la fecha se han descrito 16 genes relacionados con USH, 10 de ellos asociados a alguno de los subtipos clínicos. Además, existen múltiples mutaciones posibles en un mismo gen que causan la enfermedad, lo que se conoce como heterogeneidad alélica. Como consecuencia de los efectos de la enfermedad, el diagnóstico genético se convierte en una necesidad de los pacientes, permitiendo implantar medidas que posibiliten su comunicación a largo plazo de la forma más temprana posible, cuando los síntomas de la enfermedad se encuentran en su estadio inicial. Además, el diagnóstico molecular será vital en un futuro para la aplicación de terapias génicas tales como el sistema de edición CRISPR, que permitirá realizar tratamientos personalizados condicionados por las mutaciones presentes en los pacientes. Con este propósito, el objetivo de este trabajo final de grado ha sido la caracterización genética de una cohorte de pacientes con sospecha clínica de USH que no habían sido diagnosti, [EN] Usher syndrome (USH) is a rare disease that follows an autosomal recessive inheritance pattern. It is characterized by the association of sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular alteration. Usher syndrome comprises more than 50% of deafblind cases and 15-20% of RP cases, being the most common genetic disorder combining visual and auditory impairment, with a prevalence ranging from 3.2 to 6.2: 100,000, depending on the population studied. However, there is currently no cure beyond cochlear implantation and hearing aids for the hearing problem. Depending on the severity, onset of symptoms and progression of the disease, three clinical types of USH can be distinguished, with type I being the most severe form, type II the most common and type II the most heterogeneous. In addition to phenotypic heterogeneity, USH also has genetic heterogeneity. To date, 16 genes related to USH have been described, 10 of which are associated with one of the clinical subtypes. In addition, high number of mutations in the same gene can cause the disease, known this as allelic heterogeneity. As a consequence of the effects of the disease, genetic diagnosis becomes a need for patients, allowing the implementation of measures that enable their long-term communication as early as possible, when the symptoms of the disease are in their initial stage. In addition, molecular diagnosis will be vital in the future for the application of gene therapies such as the CRISPR editing system, which will allow personalized treatments conditioned by the mutations present in patients. With this purpose, the objective of this TFG has been the genetic characterization of a cohort of patients with clinical suspicion of USH that had not been molecularly diagnosed. For this purpose, and taking into account the genetic variability of the disease, next generation sequencing tools have been used and have allowed the sequencing of, on the one hand, a selection of gene

[Purpose]: The aim of the present work is the molecular diagnosis of three patients with deafness and retinal degeneration. [Methods]: Three patients from two unrelated families were initially analyzed with custom gene panels for Usher genes, non-syndromic hearing loss, or inherited syndromic retinopathies and further investigated by means of clinical or whole exome sequencing. [Results]: The study allowed us to detect likely pathogenic variants in PEX6, a gene typically involved in peroxisomal biogenesis disorders (PBDs). Beside deaf–blindness, both families showed additional features: Siblings from Family 1 showed enamel alteration and abnormal peroxisome. In addition, the brother had mild neurodevelopmental delay and nephrolithiasis. The case II:1 from Family 2 showed intellectual disability, enamel alteration, and dysmorphism. [Conclusions]: We have reported three new cases with pathogenic variants in PEX6 presenting with milder forms of the Zellweger spectrum disorders (ZSD). The three cases showed distinct clinical features. Thus, expanding the phenotypic spectrum of PBDs and ascertaining exome sequencing is an effective strategy for an accurate diagnosis of clinically overlapping and genetically heterogeneous disorders such as deafness–blindness association.