Estudi del gen UBE3A en la Síndrome d'Angelman i del centre d'impressió en les síndromes de Prader-Willi i d'Angelman

Consultable des del TDXTítol obtingut de la portada digitalitzadaLes síndromes de Prader-Willi (SPW) i d'Angelman (SA) són causades per diferents anomalies genètiques que afecten la regió 15q11-q13 regulada per impressió genètica. La manca d'expressió de gens paterns causa la SPW i anomalies que afecten la còpia materna del gen UBE3A causa la SA. El 70-75% del casos SPW i SA s'originen per delecions de la regió cromosòmica 15q11-q13 en el cromosoma patern o matern respectivament. Un 20-25% dels casos SPW són deguts a disomies uniparentals (DUP) maternes i el 2-5% dels casos SA són causats per DUP paternes. Entre l'1 i el 5% dels casos són causats per un defecte en la impressió (DI) en ambdues síndromes. En la SA la segona causa més freqüent (10-15%) són mutacions puntuals en el gen UBE3A i un percentatge de casos similar a l'anterior presenten una clínica consistent de la SA però es desconeix la causa genètica. Les delecions i DUP tenen un risc de recurrència molt baix. Aproximadament el 85% del casos de DI no presenten delecions ni anomalies en la seqüència del centre regulador de la impressió genètica (CI) i són considerats errors epigenètics amb un risc de recurrència molt baix. En el 15% restant el DI és originat per una deleció en el CI que pot ser familiar comportant un risc del 50% o bé pot ser de novo. L'objectiu principal de la present tesi doctoral va ser millorar les tècniques diagnòstiques de la SPW i la SA i aprofundir en la etiologia d'ambdues síndromes. Per tal d'assolir aquest objectiu es va realitzar la posta a punt de la tècnica M-PCR, per analitzar el patró de metilació del CI que permet el diagnòstic de les síndromes quan la causa és una deleció de la regió 15q11-q13, o una DUP o un DI. Tanmateix s'ha analitzat la seqüència del gen UBE3A en 30 pacients SA amb patró de metilació normal i clínica consistent, i s'ha realitzat l'estudi del CI en els casos SPW i SA amb un DI. Com resultat d'aquests estudis s'han identificat cinc mutacions en el gen UBE3A i una deleció de novo en el centre d'impressió en un pacient SPW. De les cinc mutacions en el gen UBE3A tres han estat de novo i dues familiars. A més d'aquestes cinc mutacions tres no havien estat descrites amb anterioritat. En la resta de pacients amb DI s'ha descartat la presència de deleció i de mutacions puntuals pel que el defecte d'impressió ha estat causat per un error epigenètic. Aquests resultats ens han permès l'assessorament genètic als familiars de risc i oferir un diagnòstic prenatal així com valorar les correlacions fenotip-genotip en la SPW i SA.Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by different genetic abnormalities affecting the 15q11-q13 region regulated by imprinting. Loss of paternal expression genes causes PWS and genetic abnormalities in the maternal UBE3A gene cause AS. Paternal or maternal 15q11-q13 deletions cause 70-75% of PWS and AS cases, respectively. Maternal uniparental disomy (UPD) cause 20-25% of PWS cases and 2-5% of AS cases are caused by paternal UPD. The range 1-5% of cases is due to imprinting defects (ID) in both syndromes. The second cause of AS (10-15%) is an UBE3A gene mutation and in the same percentage of AS patients the genetic cause is unknown. Deletions and UPD have a low recurrence risk. The 85% of ID do not have abnormalities in the imprinting center (IC) and are considered epigenetic defects with a low recurrence risk. The 15% of ID are caused by an IC deletion that could be hereditary with a 50% of recurrence risk or could be sporadic. The aim of this thesis was to improve the PWS and AS diagnostic techniques and to study in depth the etiology of both syndromes. In order to achieve this aim we optimized the M-PCR technique to analyze the IC methylation patern for 15q11-q13 deletions, UPD and ID diagnosis. Moreover, UBE3A sequence analysis was done in 30 AS patients with normal methylation patern and IC study was done in 2 PWS and 4 AS cases with an ID. As a result of these studies we identified five UBE3A mutations and a de novo IC deletion in a PWS ID patient. Out of the five UBE3A mutations three were sporadic and two hereditary. Moreover, three of these five UBE3A mutations have not been previously described. In the other ID patients we conclude the cause is an epimutation since the presence of a deletion or mutations in the IC was rule out. These results have allowed to offer recurrence risk and prenatal diagnosis and to assess phenotype-genotype correlations

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locu

Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop

Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an similar to 1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions similar to 5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3' untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms

Detection of norovirus in saliva samples from acute gastroenteritis cases and asymptomatic subjects : Association with age and higher shedding in stool

Norovirus infections are a leading cause of acute gastroenteritis outbreaks worldwide and across all age groups, with two main genogroups (GI and GII) infecting humans. The aim of our study was to investigate the occurrence of norovirus in saliva samples from individuals involved in outbreaks of acute gastroenteritis in closed and semiclosed institutions, and its relationship with the virus strain, virus shedding in stool, the occurrence of symptoms, age, and the secretor status of the individual. Epidemiological and clinical information was gathered from norovirus outbreaks occurring in Catalonia, Spain during 2017-2018, and stool and saliva samples were collected from affected and exposed resident individuals and workers. A total of 347 saliva specimens from 25 outbreaks were analyzed. Further, 84% of individuals also provided a paired stool sample. For GII infections, norovirus was detected in 17.9% of saliva samples from symptomatic cases and 5.2% of asymptomatic individuals. Positivity in saliva occurred in both secretors and nonsecretors. None of the individuals infected by norovirus GI was positive for the virus in saliva. Saliva positivity did not correlate with any of the studied symptoms but did correlate with age ≥ 65 years old. Individuals who were positive in saliva showed higher levels of virus shedding in stool. Mean viral load in positive saliva was 3.16 ± 1.08 log10 genome copies/mL, and the predominance of encapsidated genomes was confirmed by propidium monoazide (PMA)xx-viability RTqPCR assay. The detection of norovirus in saliva raises the possibility of oral-to-oral norovirus transmission during the symptomatic phase and, although to a lesser extent, even in cases of asymptomatic infections

Breastfeeding during the COVID-19 pandemic: analysis of the breastmilk antibodies, neutralization capacity and microbiota profile from infected and vaccinated wome

Resumen del póster presentado a las III Jornadas Científicas PTI+ Salud Global, celebradas en el Centro de Ciencias Humanas y Sociales (CCHS), CSIC (Madrid) del 20 al 22 de noviembre de 2023.[Background] Breastmilk is considered the gold standard in infant nutrition and provides bioactive compounds to the neonate, among them antibodies and microbiota. In the context of the COVID- 19 pandemics, there were great concerns about a possible mother-to-infant transfer of SARS-CoV-2, since limited knowledge about the safety of breastfeeding after natural infection or vaccination, as well as the transfer of protective antibodies and their neutralization capacity, was available. Additionally, there are concerns about potential short- and long-term adverse effects of SARS-CoV-2 infection and vaccine-induced changes to the breastmilk microbiome composition, which contributes in shaping the early-life microbiome.[Methods] This study included 60 mothers which had a confirmed SARS-CoV-2 infection and also, 86 mothers vaccinated with mRNA-based (Comirnaty, mRNA-1273) and adenoviral-vectored vaccines (ChAdOx1 nCoV-19) were recruited and breastmilk samples were collected longitudinally from baseline up to 30 days after the second dose at seven or eight time points (depending on vaccine type). In COVID-19 lactating mothers, the presence of SARS-CoV-2 was assessed by RT-qPCR targeting the N1 region of the nucleocapsid gene and the envelope (E) gene. In both studies, the levels of SARS-CoV-2 RBD-specific IgA, IgM and IgG were determined by ELISA. The neutralization capacity was tested using pseudotyped vesicular stomatitis virus carrying either the Wuhan-Hu-1, Delta, or BA.1 Omicron spike proteins. To assess the microbiome composition, DNA from breastmilk samples was extracted and the V3-V4 region of the 16S rRNA gene was sequenced using the MiSeq system of Illumina.[Results] After SARS-CoV-2 infection, no virus-specific RNA was detected in breastmilk samples. Determination of antibody levels in mothers with confirmed SARS-CoV-2 infection showed that 82.9% (58 of 70) of milk samples were positive for at least one of the three tested antibody isotypes. Vaccination elicited also a strong induction of SARS-CoV-2-specific antibodies, which was higher in IgG when compared to COVID-19 convalescent women and was strongly increased after the 2nd dose. mRNA-based vaccines induced higher IgG and IgA levels when compared to the adenovirus- vectored vaccine, and women with previous virus exposure increased their IgG antibodies levels after the first dose to a similar level observed in vaccinated women after the second dose. When assessing the neutralization capacity, natural infection resulted in higher neutralizing titers that correlated positively with levels of SARS-CoV-2-specific immunoglobulin A in breastmilk. Breastmilk samples from COVID-19 convalescent mothers infected during the first wave (Wuhan-Hu-1 strain) neutralized less effectively Omicron BA.1 than the Wuhan-Hu-1 variant. In addition, significant differences in the capacity to produce neutralizing antibodies were observed between both mRNA- based vaccines and the adenovirus-vectored ChAdOx1 COVID-19 vaccine. First results of the analysis of the breastmilk microbiome found no significant differences in the mean diversity of species (alpha-diversity) after natural SARS-CoV-2 infection, whereas some specific bacterial groups were increased (e.g. Enterobacteriaceae).[Conclusions] Overall, our results indicate that breastmilk from naturally infected women or those vaccinated with mRNA-based vaccines contain SARS-CoV-2 neutralizing antibodies that could potentially provide protection to breastfed infants from infection.Peer reviewe

INNOVASONORA: gestión de archivos sonoros, sonido inmersivo y realidad virtual en plataformas LMS

Memoria del proyecto de innovación docente "Innovasonora" (Curso 2021-2022

Çédille, revista de estudios franceses

Presentació

Power Plant Construction Projects Risk Assessment: A Proposed Method for Temporary Systems of Commissioning

The identification of hazards and risk assessment are key factors in the safety of the industries, including power plants. This paper contains an original risk analysis method that increases the level of safety in commissioning and start-up operations. With the proposed method, which has been tested in real industrial facilities, it has been possible to increase the safety of the system and reduce the likelihood of incidents and accidents in one of the most dangerous stages of project construction activities. This paper also gives an overview of the processes and procedures used in the construction of power plants compared to other industry sectors, proposing some areas for potential improvement. It has been verified that temporary systems used during commissioning apply risk evaluation techniques that do not consider some aspects related to process hazards, something that can be important for the prevention of accidents that historically happen at this stage. Analysis of the data has determined that, in these stages, there have been incidents and accidents, some of them severe. Thus, in this paper, a methodology is proposed. The method allows addressing the particularities of the execution of temporary systems in a safe way by putting into practice an agile and flexible method that can be applied to these particular systems, so that the risk levels can be reduced. The method was applied to one real application representative of this kind of system and yielded excellent results. The proposed methodology is highly recommended as an improvement for the power industry

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locu

The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locu