Chromosome 21 Scan in Down Syndrome Reveals DSCAM as a Predisposing Locus in Hirschsprung Disease

Jannot, Anne-Sophie et al.Hirschsprung disease (HSCR) genetics is a paradigm for the study and understanding of multigenic disorders. Association between Down syndrome and HSCR suggests that genetic factors that predispose to HSCR map to chromosome 21. To identify these additional factors, we performed a dose-dependent association study on chromosome 21 in Down syndrome patients with HSCR. Assessing 10,895 SNPs in 26 Caucasian cases and their parents led to identify two associated SNPs (rs2837770 and rs8134673) at chromosome-wide level. Those SNPs, which were located in intron 3 of the DSCAM gene within a 19 kb-linkage disequilibrium block region were in complete association and are consistent with DSCAM expression during enteric nervous system development. We replicated the association of HSCR with this region in an independent sample of 220 non-syndromic HSCR Caucasian patients and their parents. At last, we provide the functional rationale to the involvement of DSCAM by network analysis and assessment of SOX10 regulation. Our results reveal the involvement of DSCAM as a HSCR susceptibility locus, both in Down syndrome and HSCR isolated cases. This study further ascertains the chromosome-scan dose-dependent methodology used herein as a mean to map the genetic bases of other sub-phenotypes both in Down syndrome and other aneuploidies. © 2013 Jannot et al.This work was funded by the French National Research Agency (ANR, grants MRARE-HirGenet to SL, EvoDevoMut Grant, and ERare-HSCR Consortium to SL, IC, and SB), the Fondation pour la Recherche Médicale (FRM) to SL and JA; Fondation Jérôme Lejeune; the USA National Institutes of Health (R37 HD28088 to A.C.); the Italian Telethon (GGP04257 to IC); Fondo de Investigación Sanitaria. Instituto de Salud Carlos III (PI10/01290) and Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (CTS-2590) to S.B.; the NWO (901-04-225) Bernoulle Foundation and Ubbo Emmius Foundation to R.M.W.H.Peer Reviewe

Bases Genéticas de la enfermedad de Hirschsprung: identificación y caracterización de loci de susceptibilidad

La enfermedad de Hirschsprung (HSCR) es un trastorno congénito de expresión y penetrancia variables, con una incidencia de 1/5000 y una mayor frecuencia en varones (ratio 4:1). Se caracteriza por la ausencia de los ganglios intramurales de los plexos entéricos en una porción variable del intestino grueso, como consecuencia de un fallo de las células de la cresta neural para migrar, proliferar y/o diferenciarse durante el desarrollo embrionario del sistema nervioso entérico (SNE) (1). El principal gen asociado a HSCR es el proto-oncogén RET, que codifica para un receptor de membrana con actividad tirosín kinasa. Actualmente se acepta que en la mayoría de los casos, HSCR sigue un modelo de herencia compleja en el que deben confluir diversos eventos genéticos para que se produzca el fenotipo, y sólo el 50% de casos familiares y el 10-35% de esporádicos se relacionan con mutaciones germinales en  secuencia codificante de RET (1). Alrededor del 7% son debidos a mutaciones en otros genes que participan en rutas de señalización implicadas en migración, proliferación y diferenciación neural durante la formación del SNE (1). El mecanismo molecular causante de la enfermedad en la mayoría de los pacientes HSCR no está completamente esclarecido, lo que apoya la existencia de otros genes implicados en la patogénesis de la enfermedad. El análisis molecular de pacientes HSCR ha permitido identificar otros diferentes genes/loci relacionados con la aparición de este fenotipo. Actualmente se conocen al menos 23 genes en los que se han encontrado mutaciones asociadas a HSCR. Estudios basados en GWLA o en GWAS en familias HSCR han logrado identificar otros loci de susceptibilidad para la enfermedad en diversas regiones cromosómicas, así como nuevos pathways y Gene Ontologies (GOs) candidatos para HSCR, especialmente por su participación en procesos relacionados con el desarrollo del SNE (2). Sin embargo, estos loci sólo explicarían una parte de la complejidad genética de HSCR, de ahí la necesidad de nuevos abordajes para la identificación de genes asociados a HSCR, como la secuenciación de alto rendimiento (Next Generation Sequencing, NGS), (3-5). La aplicación de estas técnicas permitirá acelerar el laborioso proceso de búsqueda tanto de mutaciones en genes ya asociados a HSCR, como el descubrimiento de nuevos genes mediante la secuenciación de exoma completo

The NER-related gene GTF2H5 predicts survival in high-grade serous ovarian cancer patients

Gayarre, Javier et al[Objective] We aimed to evaluate the prognostic and predictive value of the nucleotide excision repair-related gene GTF2H5, which is localized at the 6q24.2-26 deletion previously reported by our group to predict longer survival of high-grade serous ovarian cancer patients.[Methods] In order to test if protein levels of GTF2H5 are associated with patients' outcome, we performed GTF2H5 immunohistochemical staining in 139 high-grade serous ovarian carcinomas included in tissue microarrays. Upon stratification of cases into high- and low-GTF2H5 staining categories (> and ≤ median staining, respectively) Kaplan-Meier and logrank test were used to estimate patients’ survival and assess statistical differences. We also evaluated the association of GTF2H5 with survival at the transcriptional level by using the on-line Kaplan-Meier plotter tool, which includes gene expression and survival data of 855 high-grade serous ovarian cancer patients from 13 different datasets. Finally, we determined whether stable short hairpin RNA-mediated GTF2H5 downregulation modulates cisplatin sensitivity in the SKOV3 and COV504 cell lines by using cytotoxicity assays.[Results] Low expression of GTF2H5 was associated with longer 5-year survival of patients at the protein (hazard ratio [HR], 0.52; 95% CI, 0.29 to 0.93; p=0.024) and transcriptional level (HR, 0.80; 95% CI, 0.65 to 0.97; p=0.023) in high-grade serous ovarian cancer patients. We confirmed the association with 5-year overall survival (HR, 0.55; 95% CI, 0.38 to 0.78; p=0.0007) and also found an association with progression-free survival (HR, 0.72; 95% CI, 0.54 to 0.96; p=0.026) in a homogenous group of 388 high-stage (stages III-IV using the International Federation of Gynecology and Obstetrics staging system), optimally debulked high-grade serous ovarian cancer patients. GTF2H5- silencing induced a decrease of the half maximal inhibitory concentration upon cisplatin treatment in GTF2H5-silenced ovarian cancer cells.[Conclusion] Low levels of GTF2H5 are associated with enhanced prognosis in high-grade serous ovarian cancer patients and may contribute to cisplatin sensitization.This study was financially supported by the Fondo de Investigación Sanitaria (FIS), Instituto de Salud Carlos III (grant PI12/01319) and by FEDER funds (2014-2020 Program). MJG is recipient of a research contract from the Instituto de Salud Carlos III of the Ministerio Español de Sanidad y Consumo (Miguel Servet tipo II Program, CPII 13/00047). JG has a contract from CIBERER and MMK was a holder of a La Caixa international PhD fellowship. LPA and JP are recipients of financial support from Red Temática de Investigación Cooperativa en Cáncer (RTICC) (grants RD12/0036/0028 and RD12/0036/0064, respectively). Gayarre, Javier et al.Peer Reviewe

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

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

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

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

Experience of Preimplantation Genetic Diagnosis with HLA Matching at the University Hospital Virgen del Rocío in Spain: Technical and Clinical Overview

Preimplantation genetic diagnosis (PGD) of genetic diseases, combined with HLA matching (PGD-HLA), is an option for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here we present the results of our PGD-HLA program at the Department of Genetics, Reproduction and Fetal Medicine of the University Hospital Virgen del Roc´ıo in Seville. Seven couples have participated in our program because of different indications. Overall, 26 cycles were performed, providing a total of 202 embryos. A conclusive molecular diagnosis and HLA-typing could be assured in 96% of the embryos. The percentage of transfers per cycle was 26.9% and the birth rate per cycle was 7.7% per transfer. Our PGD-HLA program resulted in the birth of 2 healthy babies, HLA-identical to their affected siblings, with successful subsequent haematopoietic stem cell (HSC) transplantations. Both HSC-transplanted children are currently doing well 48 and 21 months following transplantation, respectively. All the procedures, including HSCs umbilical cord transplantation, were performed in our hospital