Espectro de sobrecrecimientos asociados a PIK3CA: expansión de fenotipo y genotipo, y desarrollo de un protocolo diagnóstico

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 23-05-2018El sobrecrecimiento segmentario y las malformaciones vasculares son dos características clínicas prevalentes en los síndromes causados por mutaciones germinales y/o somáticas en diferentes genes de la ruta PI3K/AKT/mTOR: Megalencefalia-polimicrogiriapolidactilia-hidrocefalia (AKT3 y PIK3R2), Espectro de Sobrecrecimientos Asociados a PIK3CA (PROS; PIK3CA) o síndrome de Smith-Kingsmore (mTOR), entre otros. En el caso de PROS, debido a que las mutaciones en PIK3CA ocurren como un evento post-cigótico, causando mosaicismo somático, el espectro fenotípico dentro de este grupo varía ampliamente en severidad y localización dependiendo del tipo celular afectado y del tiempo durante el desarrollo embrionario en el que surgió la mutación. Las características clínicas no siempre son constantes entre los diferentes síndromes incluidos en PROS, existiendo además un gran solapamiento fenotípico, dificultando el diagnóstico clínico y molecular. En esta Tesis Doctoral se estudió clínica y molecularmente una cohorte de 96 pacientes con características clínicas compatibles con el espectro PROS, incluyendo pacientes con diagnóstico de Macrocefalia-Malformación Capilar, síndrome CLOVES, macrodactilia y otros con fenotipo difuso, así como una serie de pacientes con DCMO, una entidad clínica, de causa desconocida y con características similares al espectro PROS. El objetivo fue ampliar el fenotipo y el genotipo del espectro PROS, y establecer un protocolo que permita un mejor diagnóstico clínico y molecular de los pacientes y una mejor comprensión de las patologías incluidas en el espectro. Para ello se utilizaron tres paneles de genes diferentes de secuenciación masiva (NGS) para analizar el ADN extraído de muestras de linfocitos de sangre periférica, saliva y/o tejido afectado de 85 de estos pacientes. Las variantes candidatas se validaron utilizando tres metodologías alternativas y se realizaron estudios funcionales en variantes no identificadas previamente. Se detectaron 23 variantes diferentes en PIK3CA en pacientes con PROS. Además de en este gen, se detectaron variantes en otros tres genes incluidos en la ruta PI3K/AKT/mTOR en seis pacientes, cuatro de ellos con variantes en mTOR (tres en línea germinal y uno en mosaico somático), un paciente con una variante en mosaico en AKT3 y el último con una variante en PTEN. Asimismo, se identificó al primer caso de DCMO con una variante probablemente patogénica en PIK3CA. La combinación de los estudios clínicos y moleculares presentados en esta Tesis Doctoral amplía el conocimiento fenotípico y genotípico del espectro PROS, y establece un protocolo experimental y bioinformático para el diagnóstico de estos pacientes mediante el uso de NGS con grandes profundidades de lecturasSegmental overgrowth and vascular malformations are two prevalent clinical features among different syndromes caused by germline or somatic mutations in different genes in the PI3K/AKT/mTOR pathway: Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (AKT3 and PIK3R2), PIK3CA-Related Overgrowth Spectrum (PROS; PIK3CA) or SmithKingsmore syndrome (mTOR), among others. In the case of PROS, as mutations in PIK3CA occurs as a post-zigotic event, causing somatic mosaicism, the phenotypic spectrum within this group varies greatly in severity and location depending on the affected cellular type and the time of the embryonic development in which the mutation arose. Clinical characteristics are not always consistent between the different syndromes included in PROS, and there is also phenotypic overlap, making the clinical and molecular diagnosis difficult. In this Doctoral Thesis, a cohort of 96 patients with clinical characteristics compatible with the PROS spectrum was studied clinically and molecularly, including patients with a diagnosis of Macrocephaly-Capillary Malformation, CLOVES syndrome, macrodactyly and others with diffuse phenotype, as well as a series of patients with DCMO, a clinical entity of unknown cause and with similar characteristics to PROS spectrum. The objective was to expand the phenotype and genotype of PROS spectrum, and to establish a protocol that allows a better clinical and molecular diagnosis of the patients, and a better understanding of the pathologies included in the spectrum. For this, three different massive sequencing (NGS) genes panels were used to analyze the DNA extracted from blood, saliva and/or affected tissue from 85 of these patients. Candidate variants were validated using three alternative methodologies and functional studies were performed on mutations not previously reported. We detected 23 different PIK3CA variants in PROS. In addition to this gene, variants were detected in three other genes included in the PI3K/AKT/mTOR pathway in six patients referred previously as MCAP or PROS: four of them with mTOR variants (three germline and one somatic), one patient with a mosaic AKT3 variant, and the last one with a PTEN variant. Also, we identified the first patient with clinical diagnosis of DCMO and a post-zigotic variant in PIK3CA. The combination of the clinical and molecular studies presented in this doctoral thesis broadens the phenotypic and genotypic knowledge of the PROS spectrum, and establishes an experimental and bioinformatic protocol for the diagnosis of these patients through the use of NGS with great reading depth

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.Ministerio de Salud EspañaComunidad de Madri

Mindfulness dirigido a estudiantes de Educación Social: programa de formación y su impacto en entidades socio-educativas

Mindfulness conocida también como “atención plena” o “conciencia plena” (e.g. Baer, 2003; Kabat-Zinn, 1990, 2003a, 2003b; Mañas, 2009; Miró, 2006; Simón, 2006; Vallejo, 2006) es una técnica de meditación “centrada en el presente, no elaborativa ni condenatoria, en la cual cada pensamiento, sentimiento o sensación que surge en el campo atencional es reconocido y aceptado tal y como es” (e.g. Kabat-Zinn, 2003a, 2003b; Shapiro y Schwartz, 2000; Segal et al., 2002). Así la meditación tiene como componente principal la concentración y la capacidad de mantener la atención de manera consciente en la mente, de ahí, que su práctica continua permita al cuerpo adquirir altos niveles de relajación. El proyecto de innovación se ha diseñado bajo dos objetivos generales dirigidos principalmente a estudiantes de Educación Social. El primero, centrado en la formación y el segundo, en la aplicación del Mindfulness en las prácticas curriculares que realizan dichos estudiantes: 1.Diseñar y evaluar la eficacia de un programa formativo introductorio de entrenamiento en Mindfulness dirigido a estudiantes de Educación Social, para reducir el nivel de estrés percibido y aumentar el bienestar psicológico, la atención plena y la compasión. 2.Valorar intervenciones en Mindfulness llevadas a cabo por estudiantes de Educación Social quienes han recibido formación en atención plena, en las entidades en donde realizan sus prácticas. Los resultados del programa señalan una disminución del estrés percibido y un incremento en el bienestar bienestar, atención plena y compasión

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications

ESRETNET Study Group, The ERDC Study Group, The Associated Clinical Study Group.Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER), the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006)

Hypoinsulinaemic, hypoketotic hypoglycaemia due to mosaic genetic activation of PI3-kinase.

OBJECTIVE: Genetic activation of the insulin signal-transducing kinase AKT2 causes syndromic hypoketotic hypoglycaemia without elevated insulin. Mosaic activating mutations in class 1A phospatidylinositol-3-kinase (PI3K), upstream from AKT2 in insulin signalling, are known to cause segmental overgrowth, but the metabolic consequences have not been systematically reported. We assess the metabolic phenotype of 22 patients with mosaic activating mutations affecting PI3K, thereby providing new insight into the metabolic function of this complex node in insulin signal transduction. METHODS: Three patients with megalencephaly, diffuse asymmetric overgrowth, hypoketotic, hypoinsulinaemic hypoglycaemia and no AKT2 mutation underwent further genetic, clinical and metabolic investigation. Signalling in dermal fibroblasts from one patient and efficacy of the mTOR inhibitor Sirolimus on pathway activation were examined. Finally, the metabolic profile of a cohort of 19 further patients with mosaic activating mutations in PI3K was assessed. RESULTS: In the first three patients, mosaic mutations in PIK3CA (p.Gly118Asp or p.Glu726Lys) or PIK3R2 (p.Gly373Arg) were found. In different tissue samples available from one patient, the PIK3CA p.Glu726Lys mutation was present at burdens from 24% to 42%, with the highest level in the liver. Dermal fibroblasts showed increased basal AKT phosphorylation which was potently suppressed by Sirolimus. Nineteen further patients with mosaic mutations in PIK3CA had neither clinical nor biochemical evidence of hypoglycaemia. CONCLUSIONS: Mosaic mutations activating class 1A PI3K cause severe non-ketotic hypoglycaemia in a subset of patients, with the metabolic phenotype presumably related to the extent of mosaicism within the liver. mTOR or PI3K inhibitors offer the prospect for future therapy

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER), the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006). The funders played no role in study design, data collection, data analysis, manuscript preparation and/or publication decisions

Topographic correlation and asymmetry analysis of ganglion cell layer thinning and the retinal nerve fiber layer with localized visual field defects.

PurposeTo evaluate the accuracy of the measurement of the ganglion cell layer (GCL) of the posterior pole analysis (PPA) software of the Spectralis spectral-domain (SD) optical coherence tomography (OCT) device (Heidelberg Engineering, Inc., Heidelberg, Germany), the asymmetry of paired GCL sectors, the total retinal thickness asymmetry (RTA), and the peripapillary retinal nerve fiber layer (pRNFL) test to discriminate between healthy, early and advanced glaucoma eyes.MethodsThree hundred eighteen eyes of 161 individuals with reliable visual fields (VF) were enrolled in this study. All participants were examined using the standard posterior pole and the pRNFL protocols of the Spectralis OCT device. VF impairment was graded in hemifields, and the GCL sectors were correlated with this damage. Thicknesses of each GCL, the GCL map deviation asymmetry and the pRNFL were compared between control and glaucomatous eyes. The area under the receiver operating characteristic curve (AUC) of these analyses was assessed.ResultsFourteen of the 16 sectors of the GCL and pRNFL were significantly thinner in eyes with glaucoma than in control eyes (p0.823, p0.708, pConclusionsAlthough 16 central sectors of the GCL observed with PPA showed good correlation with VF damage, the pRNFL and the GCL map deviation were more effective for discrimination of glaucomatous damage