Caracterización de anomalías cromosómicas en diagnóstico prenatal y postnatal mediante técnicas de citogenética molecular

Consultable des del TDXTítol obtingut de la portada digitalitzadaLas anomalías cromosómicas están implicadas en la aparición de muchas enfermedades hereditarias, siendo también la principal causa de retraso mental y de pérdidas gestacionales. La pérdida, ganancia o reordenación de fragmentos cromosómicos de un tamaño similar, puede tener distintas consecuencias dependiendo del número y función de los genes que contengan. Los métodos clásicos de bandeo cromosómico han sido durante muchos años la única herramienta para la detección de anomalías cromosómicas tanto numéricas como estructurales. Gracias a estas técnicas se han podido identificar muchas anomalías cromosómicas asociadas a diferentes malformaciones congénitas, síndromes o cánceres colaborando eficazmente en la comprensión de la etiología de dichas patologías. No obstante, las alteraciones cromosómicas de un tamaño < 3-5 Mb o las reorganizaciones complejas son muy difíciles de identificar mediante las técnicas de bandas convencionales. Desde su inicio, hacia 1980, las técnicas de citogenética molecular basadas en la hibridación in situ fluorescente (FISH) y sus variantes tecnológicas, entre ellas la Hibridación Genómica Comparada (CGH) i la FISH-Multicolor (M-FISH), han permitido la detección e identificación precisa de un gran número de anomalías cromosómicas, como las microdeleciones, las reorganizaciones cromosómicas complejas y las reorganizaciones crípticas, que hasta ese momento pasaban desapercibidas. El objetivo principal de este trabajo ha sido profundizar en el papel que desempeñan regiones cromosómicas específicas, que al alterarse, originan fenotipos concretos mediante la aplicación conjunta de diferentes técnicas de citogenética molecular (CGH y FISH) y de citogenética convencional. Hoy en día, se conocen muy pocas regiones cromosómicas cuya alteración, por pérdida o duplicación, esté asociada a un síndrome clínico bien definido. Distintos estudios, incluido el presente, muestran que tanto la CGH, como la M-FISH, son dos técnicas que contribuyen eficazmente en la identificación de regiones cromosómicas específicas asociadas a fenotipos concretos. Esta información es de suma importancia para los biólogos moleculares como indicación de qué regiones del genoma deben considerar como «dianas» para identificar los genes implicados. En esta tesis además de la puesta a punto u optimización de las técnicas de M-FISH, CGH y HR-CGH para su aplicación al diagnóstico prenatal y postnatal, se han determinado las limitaciones tanto de las propias técnicas como de los softwares empleados. Ello nos ha permitido establecer nuevas correlaciones genotipo-fenotipo gracias a la identificación de 27 monosomías y trisomías autosómicas y gonosómicas, 15 cromosomas marcadores supernumerarios y 9 reorganizaciones complejas y/o crípticas. Al mismo tiempo, se ha establecido un protocolo para la identificación de marcadores cromosómicos mediante la utilización combinada de las técnicas de citogenética convencional, FISH y CGH. Por último, el análisis de los 77 puntos de rotura implicados en las anomalías cromosómicas estudiadas ha revelado que no se producen al azar y que fundamentalmente afectan a los cromosomas: 2, 7, 9, 15, 16, 18, X e Y. Las bandas más afectadas han sido: 9p23, 11q22.2, 14q11.2, 15q11.2, 15q15, 16p11.2, 18q22, 22q11.2, Xp22.3, Xq21.2, Yp11.3 e Yq12, evidenciando que las roturas cromosómicas se producen en bandas cromosómicas claras (80%) que corresponden a regiones del genoma con un mayor contenido genético. El análisis de estos puntos revela que coinciden en un 90% con regiones donde se ha descrito duplicaciones segmentarias. Este último hallazgo es de suma importancia y abre un nuevo campo de investigación. En resumen, esta tesis muestra la utilidad y aplicabilidad de la CGH y M-FISH, tanto en Diagnóstico Prenatal como Postnatal, al identificar tanto pequeños desequilibrios como reorganizaciones complejas y/o crípticas permitiendo establecer nuevas correlaciones genotipo-fenotipo que facilitaran en un futuro un consejo genético mucho más preciso.Chromosomal abnormalities are involved in the appearance of many hereditary diseases representing the principal factor of mental retardation as well as miscarriages. The loss, gain or redistribution of chromosomal fragments with a similar size may have different consequences depending on the number and function of the respective genes. During lots of years conventional chromosomal banding techniques had been applied as a unique tool in order to detect numerical and structural chromosomal abnormalities. Thanks to those techniques a significant number of chromosome-related abnormalities associated with congenital malformations, diverse syndromes and cancer forms could be indeed identified consolidating on this way, even a better comprehension of the etiology of these pathologies. However, the chromosomal alterations with a size of < 3-5 Mb or complex reorganizations are rather difficult to be identified by conventional banding techniques. Since 1980 molecular cytogenetic techniques based on a fluorescence in situ hybridization (FISH) with its technologic variants as for instance the Comparative Genomic Hybridization (CGH) and the multicolour-FISH (M-FISH) made possible the exact detection and identification of numerous chromosomal abnormalities as there are microdeletions, complex chromosomal reorganizations as well as cryptic reorganizations which until that moment could not be recognized. The main objective of this work consisted of focussing the specific chromosomal regions which cause in case of alteration concrete phenotypes by means of a combined application of different molecular cytogenetic and conventional techniques (CGH and FISH). Nowadays we dispose of a very limited know how about chromosomal regions of which an alteration by deletion or duplication is associated with a well defined clinical syndrome. Different studies including the present one had showed that both procedures CGH and M-FISH are techniques able to identify efficiently specific chromosomal regions associated with specific phenotypes. This information is a quite important for molecular biologists as it reveals which regions of a genome can be considered as key regions within the definition of implicated genes. With this doctoral thesis, besides the improvement and/or optimisation of M-FISH, CGH and HR-CGH techniques relevant for prenatal and postnatal diagnosis, limitations had been determined on the one hand regarding the same techniques and on the other hand with respect to the software. That circumstance allowed us indeed to establish new genotype-phenotype correlations due to the identification of 27 autosomal and gonosomal monosomies and trisomies, 15 supernumerary marker chromosomes and 9 complex and/or cryptic reorganizations. At the same time a protocol could be established for the identification of supernumerary marker chromosomes by the combined application of the conventional cytogenetic techniques FISH and CGH. Finally the analysis of the 77 breakpoints implicated in the investigated chromosomal abnormalities unveiled that they had not been brought out accidentally and that they fundamentally affect the chromosomes: 2, 7, 9, 15, 16, 18, X and Y. On the other hand the bands more affected were 9p23, 11q22.2, 14q11.2, 15q15, 16p11.2, 18q22, 22q11.2, Xp22.3, Xq21.2, Yp11.3 and Yq12, showing that chromosome breaks are mainly located on clear chromosome bands (80%) corresponding to genome regions with a bigger genetic content. The analysis of these points reveals that a 90 % of them coincide with regions in which segmental duplications had been described. This last recognition represents an outstanding circumstance and initiates in a future a new serie of investigations. Summing up this doctoral thesis shows the utility and applicability of CGH and M-FISH as prenatal and postnatal diagnosis identifying as well slighter deviations as complex and cryptic reorganisations. It will equally allow us to establish new genotypic and phenotypic correlations making possible in a future a more exact genetic assessment

Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases

Altres ajuts: Generalitat de Catalunya, Departament de Salut; Generalitat de Catalunya, Departament d'Empresa i Coneixement i CERCA Program; Ministerio de Ciencia e Innovación; Instituto Nacional de Bioinformática; ELIXIR Implementation Studies (CNAG-CRG); Centro de Investigaciones Biomédicas en Red de Enfermedades Raras; Centro de Excelencia Severo Ochoa; European Regional Development Fund (FEDER).Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%)

Advanced Optical Microscopy: Unveiling Functional Insights Regarding a Novel <i>PPP2R1A</i> Variant and Its Unreported Phenotype

The number of genes implicated in neurodevelopmental conditions is rapidly growing. Recently, variants in PPP2R1A have been associated with syndromic intellectual disability and a consistent, but still expanding, phenotype. The PPP2R1A gene encodes a protein subunit of the serine/threonine protein phosphatase 2A enzyme, which plays a critical role in cellular function. We report an individual showing pontocerebellar hypoplasia (PCH), microcephaly, optic and peripheral nerve abnormalities, and an absence of typical features like epilepsy and an abnormal corpus callosum. He bears an unreported variant in an atypical region of PPP2R1A. In silico studies, functional analysis using immunofluorescence, and super-resolution microscopy techniques were performed to investigate the pathogenicity of the variant. This analysis involved a comparative analysis of the patient’s fibroblasts with both healthy control cells and cells from an individual with the previously described phenotype. The results showed reduced expression of PPP2R1A and the presence of aberrant protein aggregates in the patient’s fibroblasts, supporting the pathogenicity of the variant. These findings suggest a potential association between PPP2R1A variants and PCH, expanding the clinical spectrum of PPP2R1A-related neurodevelopmental disorder. Further studies and descriptions of additional patients are needed to fully understand the genotype–phenotype correlation and the underlying mechanisms of this novel phenotype

SDS22 coordinates the assembly of holoenzymes from nascent protein phosphatase-1

Abstract SDS22 forms an inactive complex with nascent protein phosphatase PP1 and Inhibitor-3. SDS22:PP1:Inhibitor-3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 stabilizes nascent PP1. In the absence of SDS22, PP1 is gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. Similarly, we identify a female individual with a severe neurodevelopmental disorder bearing an unstable SDS22 mutant, associated with decreased PP1 levels. We furthermore find that SDS22 directly binds to Inhibitor-3 and that this is essential for the stable assembly of SDS22:PP1: Inhibitor-3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled Inhibitor-3 binding site co-transfers with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, through simultaneous interaction with PP1 and Inhibitor-3, integrates the major steps of PP1 holoenzyme assembly

Severe ipsilateral musculoskeletal involvement in a Cornelia de Lange patient with a novel NIPBL mutation

Cornelia de Lange Syndrome (CdLS) is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked (SMC1A and HDAC8) disorder characterized by facial dysmorphism, pre and postnatal growth retardation, developmental delay and/or intellectual disability, and multiorgan involvement. Musculoskeletal malformations are usually bilateral and affect mainly the upper limbs; the range goes from brachyclinodactyly to severe reduction defects. Instead lower extremities are usually less and mildly involved. Here, we report on a 3-year-old Senegalese boy with typical craniofacial CdLS features, pre and postnatal growth retardation, atrial septal defect, developmental delay and right ipsilateral limb malformations, consistent with oligodactyly of the 3rd and 4th fingers, tibial agenesis and fibula hypoplasia. Exome sequencing and Sanger sequencing showed a novel missense mutation in NIPBL gene (c.6647A>G; p.(Tyr2216Cys)), which affects a conserved residue located within NIPBL HEAT repeat elements. Pyrosequencing analysis of NIPBL gene, disclosed similar levels of wild-type and mutated alleles in DNA and RNA samples from all tissues analyzed (oral mucosa epithelial cells, peripheral blood leukocytes and fibroblasts). These findings indicated the absence of somatic mosaicism, despite of the segmental asymmetry of the limbs, and confirmed biallelic expression for NIPBL transcripts, respectively. Additionally, conditions like Split-hand/foot malformation with long-bone deficiency secondary to duplication of BHLHA9 gene have been ruled out by the array-CGH and MLPA analysis. To our knowledge, this is the first CdLS patient described with major ipsilateral malformations of both the upper and lower extremities, that even though this finding could be due to a random event, expands the spectrum of limb reduction defects in CdLS.Fil: Baquero Montoya, Carolina. Universidad de Zaragoza; España. Hospital Pablo Tobón Uribe; ColombiaFil: Gil Rodríguez, María Concepción. Universidad de Zaragoza; EspañaFil: Hernández Marcos, María. Universidad de Zaragoza; EspañaFil: Teresa Rodrigo, María Esperanza. Universidad de Zaragoza; EspañaFil: Vicente Gabas, Alicia. Universidad de Zaragoza; España. Hospital Clínico Universitario “Lozano Blesa”; EspañaFil: Bernal, María Luisa. Universidad de Zaragoza; EspañaFil: Casale, Cesar Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Bueno Lozano, Gloria. Hospital Clínico Universitario “Lozano Blesa”; EspañaFil: Bueno Martínez, Inés. Universidad de Zaragoza; España. Hospital Clínico Universitario “Lozano Blesa”; EspañaFil: Queralt, Ethel. Universidad de Barcelona. Hospital Duran I Reynals. Instituto de Investigación Biomédica de Bellvitge; EspañaFil: Villa, Olaya. Quantitative Genomic Medicine Laboratories; EspañaFil: Hernando Davalillo, Cristina. Quantitative Genomic Medicine Laboratories; EspañaFil: Armengol, Lluís. Quantitative Genomic Medicine Laboratories; EspañaFil: Gómez Puertas, Paulino. Centro de Biología Molecular Severo Ochoa; EspañaFil: Puisac, Beatriz. Universidad de Zaragoza; EspañaFil: Selicorni, Angelo. University of Milano-Bicocca; ItaliaFil: Ramos, Feliciano J.. Universidad de Zaragoza; España. Hospital Clínico Universitario “Lozano Blesa”; EspañaFil: Pié, Juan. Universidad de Zaragoza; Españ

Targeting HER2-AXL heterodimerization to overcome resistance to HER2 blockade in breast cancer

Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer. AXL is a prognostic biomarker and a potential therapeutic target to restore trastuzumab response in HER2 + breast cancer

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder

International audienceACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder

ACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.status: publishe