Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder

From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.Junta de Andalucía FEDER TCMR0021/06, PI246-2008Instituto de Salud Carlos III (FEDER) RD12/0019/0028, RD012/0036/0017, PI10/00964, PI11/02923, PI14/0101

Antenatal diagnosis of jejunal atresia by 3D HDlive ultrasound: case report and literature review

Objetivos: reportar el caso de una paciente con diagnóstico prenatal de atresia de yeyuno y hacer una revisión de la literatura en torno al resultado y al diagnóstico de esta entidad, implementando el uso de métodos no convencionales: ecografía tridimensional (3D) o resonancia magnética fetal (RM). Materiales y métodos: se reporta el caso de una gestante de 18 años, remitida a la Unidad de Medicina Materno-Fetal, Genética y Reproducción del Hospital Virgen del Rocío de Sevilla (España), por feto con imagen quística abdominal, peristáltica, compatible con atresia de yeyuno, la cual se confirmó con ecografía 3D modo HDlive. Se realizó una búsqueda bibliográfica en Medline/PubMed, LILACS y Google Scholar, restringiendo por tipo de idioma (inglés y español) y fecha de publicación (enero de 1995 a junio de 2020). Se incluyeron estudios primarios de reportes y series de caso, que abordaran el resultado y el diagnóstico prenatal de esta patología. Resultados: la búsqueda identificó 1.033 títulos, de los cuales cuatro estudios cumplieron con los criterios de inclusión, estos fueron reporte o series de casos. En total se reportaron doce fetos con diagnóstico prenatal de atresia de yeyuno detectada con métodos no convencionales. Una gestación finalizó con óbito a la semana 26 de gestación y en todos los demás casos, el diagnóstico se ratificó durante el periodo neonatal, requiriendo la resección del segmento comprometido. Dos neonatos desarrollaron síndrome de intestino corto como consecuencia de una resección intestinal amplia. El resto experimentó una evolución posoperatoria favorable. Conclusión: la literatura disponible en torno al diagnóstico prenatal de atresia de yeyuno utilizando métodos no convencionales es escasa y se limita a reporte o series de casos. La literatura revisada sugiere que, en presencia de dilatación intestinal, la ecografía 3D y la resonancia magnética fetal podrían tener cierta utilidad a la hora de caracterizar la porción atrésica y establecer el diagnóstico diferencial. Se requieren más estudios que evalúen la utilidad diagnóstica de estas dos alternativas.Objectives: To report the case of a patient with a prenatal diagnosis of jejunal atresia and to review the literature regarding the results and prenatal diagnosis of this entity, implementing the use of non-conventional methods (3D ultrasound or magnetic resonance imaging). Material and methods: Report of a case of an 18-year-old pregnant woman referred to the Maternal-Fetal Medicine, Genetics and Reproduction Unit of the Virgen del Rocío Hospital in Seville due to fetus with abdominal peristaltic cystic image, consistent with jejunal atresia, confirmed with 3D HDLive mode ultrasound. A bibliographic search was carried out in Medline/PubMed, Google Scholar and LILACS, restricting by type of language (English and Spanish) and date of publication (January 1995 to June 2020). Primary studies of reports and case series relating to the outcome and prenatal diagnosis of this pathology were included. Results: The search identified 1,033 titles, of which four studies met the inclusion criteria, these being reports or case series. A total of twelve fetuses with a prenatal diagnosis of jejunal atresia detected with unconventional methods were reported. In all cases, the prenatal diagnosis was confirmed during the neonatal period, which required resection of the compromised segment; one of them died and two neonates developed short bowel syndrome because of a wide bowel resection. The postoperative course in the remaining cases was favorable. Conclusion: The available literature on the prenatal diagnosis of jejunal atresia using non-conventional methods is scarce and is limited to case reports or case series. The literature reviewed suggests that, in the presence of intestinal dilation, 3D ultrasound and magnetic resonance imaging could be of some use in characterizing the atretic portion and establishing the differential diagnosis. More studies are required to evaluate the diagnostic utility of these two alternatives

Influencers on thyroid cancer onset: molecular genetic basis

Thyroid cancer, a cancerous tumor or growth located within the thyroid gland, is the most common endocrine cancer. It is one of the few cancers whereby incidence rates have increased in recent years. It occurs in all age groups, from children through to seniors. Most studies are focused on dissecting its genetic basis, since our current knowledge of the genetic background of the di erent forms of thyroid cancer is far from complete, which poses a challenge for diagnosis and prognosis of the disease. In this review, we describe prevailing advances and update our understanding of the molecular genetics of thyroid cancer, focusing on the main genes related with the pathology, including the di erent noncoding RNAs associated with the disease

Preimplantation Genetic Diagnosis in Europe

In vitro fertilisation (IVF) and preimplantation genetic diagnosis (PGD) are now well-established treatments and are provided in many European countries. However, regulations, practices, professional standards and accreditation requirements are often markedly different between Member States (MS). Differences between MS seem to be becoming especially pronounced because of the interface between assisted reproduction and genetics. To assess the extent of these differences and try to obtain an initial picture of the overall situation in Europe, in March 2005 the Institute for Prospective Technological Studies (IPTS) of the European Commission's Joint Research Centre (JRC), the European Society of Human Genetics (ESHG) and the European Society for Human Reproduction and Embryology (ESHRE) organised a workshop on the abovementioned interface . The two-day event brought together 50 experts from different specialities to review current practices in Europe and discuss potential needs in this area. The first thing that became clear was that no full picture was available of PGD practice and provision in Europe. Secondly, the lack of quality assurance for these services in general was perceived as a potential problem. The participants in the workshop unanimously agreed that European clinics should be certified or accredited and that licensing systems should be developed by professional self-regulation. Minimum quality standards should be set. The lack of common European rules and regulations to guarantee minimum standards was said to be adding to the problem. However, quality assurance and accreditation have taken on new significance in the light of the recent EU Directive 2004/23/EC on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Thirdly, as a result of the abovementioned differences between MS, patients are travelling abroad to gain access to treatment which is not available in their own country. This, in turn, sometimes requires movements of gametes (oocytes and sperm) and embryos within the EU. Whilst it is known that couples and reproductive tissue are moving around Europe, the extent is not known – especially in the new MS. Lastly, an overview of how the different regulatory frameworks are having an impact on the actual practices of PGD services was deemed necessary in order to gain a better understanding of the trans-border flows. Having pinpointed some of the needs in this area, the IPTS launched this study in an effort to address them and to obtain the missing knowledge on provision of PGD services in Europe. The aims of this study are two-fold: 1)to obtain a clear picture of current PGD practice in Europe, including the quality of the services and cross-border activities (flows of couples or reproductive tissue); 2)to carry out a comparative review of the different regulatory frameworks at MS level and identify potential gaps at European level and the impact these might have.JRC.J.5-Agriculture and Life Sciences in the Econom

Epigenetic Mechanisms in Hirschsprung Disease

Hirschsprung disease (HSCR, OMIM 142623) is due to a failure of enteric precursor cells derived from neural crest (EPCs) to proliferate, migrate, survive or differentiate during Enteric Nervous System (ENS) formation. This is a complex process which requires a strict regulation that results in an ENS specific gene expression pattern. Alterations at this level lead to the onset of neurocristopathies such as HSCR. Gene expression is regulated by different mechanisms, such as DNA modifications (at the epigenetic level), transcriptional mechanisms (transcription factors, silencers, enhancers and repressors), postranscriptional mechanisms (30UTR and ncRNA) and regulation of translation. All these mechanisms are finally implicated in cell signaling to determine the migration, proliferation, differentiation and survival processes for correct ENS development. In this review, we have performed an overview on the role of epigenetic mechanisms at transcriptional and posttranscriptional levels on these cellular events in neural crest cells (NCCs), ENS development, as well as in HSCR.Instituto de Salud Carlos III (ISCIII) PI16/01422Spanish Ministry of Economy and competitiveness PI16/01422European Union PI16/0142

Next-generation-based targeted sequencing as an efficient tool for the study of the genetic background in Hirschsprung patients

Background: The development of next-generation sequencing (NGS) technologies has a great impact in the human variation detection given their high-throughput. These techniques are particularly helpful for the evaluation of the genetic background in disorders of complex genetic etiology such as Hirschsprung disease (HSCR). The purpose of this study was the design of a panel of HSCR associated genes as a rapid and efficient tool to perform genetic screening in a series of patients. Methods: We have performed NGS-based targeted sequencing (454-GS Junior) using a panel containing 26 associated or candidate genes for HSCR in a group of 11 selected HSCR patients. Results: The average percentage of covered bases was of 97 %, the 91.4 % of the targeted bases were covered with depth above 20X and the mean coverage was 422X. In addition, we have found a total of 13 new coding variants and 11 new variants within regulatory regions among our patients. These outcomes allowed us to re-evaluate the genetic component associated to HSCR in these patients. Conclusions: Our validated NGS panel constitutes an optimum method for the identification of new variants in our patients. This approach could be used for a fast, reliable and more thorough genetic screening in future series of patients.Instituto de Salud Carlos III (ISCIII)Spanish Ministry of Economy and Competitiveness, Spain (PI13/01560)Consejería de Innovación Ciencia y Empresa de la Junta de Andalucía (CTS-7447)CIBERER is an initiative of the ISCIII, Spanish Ministry of Economy and Competitivenes

CSVS, a crowdsourcing database of the Spanish population genetic variability

The knowledge of the genetic variability of the local population is of utmost importance in personalized medicine and has been revealed as a critical factor for the discovery of new disease variants. Here, we present the Collaborative Spanish Variability Server (CSVS), which currently contains more than 2000 genomes and exomes of unrelated Spanish individuals. This database has been generated in a collaborative crowdsourcing effort collecting sequencing data produced by local genomic projects and for other purposes. Sequences have been grouped by ICD10 upper categories. A web interface allows querying the database removing one or more ICD10 categories. In this way, aggregated counts of allele frequencies of the pseudo-control Spanish population can be obtained for diseases belonging to the category removed. Interestingly, in addition to pseudo-control studies, some population studies can be made, as, for example, prevalence of pharmacogenomic variants, etc. In addition, this genomic data has been used to define the first Spanish Genome Reference Panel (SGRP1.0) for imputation. This is the first local repository of variability entirely produced by a crowdsourcing effort and constitutes an example for future initiatives to characterize local variability worldwide. CSVS is also part of the GA4GH Beacon network

Dnmt3b knock-down in enteric precursors reveals a possible mechanism by which this de novo methyltransferase is involved in the enteric nervous system development and the onset of Hirschsprung disease

Hirschsprung disease (HSCR, OMIM 142623) is a pathology that shows a lack of enteric ganglia along of the distal gastrointestinal tract. This aganglionosis is attributed to an abnormal proliferation, migration, differentiation and/or survival of enteric precursor cells (EPCs) derived from neural crest cells (NCCs) during the enteric nervous system (ENS) embryogenesis. DNMT3b de novo methyltransferase is associated with NCCs development and has been shown to be implicated in ENS formation as well as in HSCR. In this study we have aimed to elucidate the specific mechanism underlying the DNMT3b role in such processes. We have performed the knockdown of Dnmt3b expression (Dnmt3b-KD) in enteric precursor cells (EPCs) to clarify its role on these cells in vitro. Moreover, we have analyzed several signaling pathways to determine the mechanisms responsible for the effect caused by Dnmt3b- KD in EPCs. Our results seem to support that Dnmt3b-KD promotes an increase EPCs proliferation that may be mediated by P53 and P21 activity, since both proteins were observed to be down-regulated in our Dnmt3b-KD cultures. Moreover, we observed a down-regulation of P53 and P21 in HSCR patients. These results lead us to propose that DNMT3b could be involved in HSCR through P53 and P21 activity.Instituto de Salud Carlos III PI16/01422Junta de Andalucía CTS-744

The role of the interactome in the maintenance of deleterious variability in human populations

Recent genomic projects have revealed the existence of an unexpectedly large amount of deleterious variability in the human genome. Several hypotheses have been proposed to explain such an apparently high mutational load. However, the mechanisms by which deleterious mutations in some genes cause a pathological effect but are apparently innocuous in other genes remain largely unknown. This study searched for deleterious variants in the 1,000 genomes populations, as well as in a newly sequenced population of 252 healthy Spanish indi-viduals. In addition, variants causative of monogenic diseases and somatic variants from 41 chronic lymphocytic leukaemia patients were analysed. The deleterious variants found were analysed in the context of the interactome to understand the role of network topology in the maintenance of the observe

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative

CIBER (Center for Biomedical Network Research; Centro de Investigacion Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mis sion is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low prevalence diseases, in line with the International Rare Diseases Research Consor tium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this arti cle, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research