Whole exome sequencing of Rett syndrome-like patients reveals the mutational diversity of the clinical phenotype

Classical Rett syndrome (RTT) is a neurodevelopmental disorder where most of cases carry MECP2 mutations. Atypical RTT variants involve mutations in CDKL5 and FOXG1. However, a subset of RTT patients remains that do not carry any mutation in the described genes. Whole exome sequencing was carried out in a cohort of 21 female probands with clinical features overlapping with those of RTT, but without mutations in the customarily studied genes. Candidates were functionally validated by assessing the appearance of a neurological phenotype in Caenorhabditis elegans upon disruption of the corresponding ortholog gene. We detected pathogenic variants that accounted for the RTT-like phenotype in 14 (66.6 %) patients. Five patients were carriers of mutations in genes already known to be associated with other syndromic neurodevelopmental disorders. We determined that the other patients harbored mutations in genes that have not previously been linked to RTT or other neurodevelopmental syndromes, such as the ankyrin repeat containing protein ANKRD31 or the neuronal acetylcholine receptor subunit alpha-5 (CHRNA5). Furthermore, worm assays demonstrated that mutations in the studied candidate genes caused locomotion defects. Our findings indicate that mutations in a variety of genes contribute to the development of RTT-like phenotypes

New genes and pathways implicated in Rett syndrome: considerations and future applications

[eng] STUDY I HYPOTHESIS Genetic alterations have already been recognized as major etiological factors for ASD and ID, of which RTT is an example. In addition to the contribution of polymorphic variants that confer low or moderate risk of appearance of these neurodevelopmental defects, de novo mutations affecting genes in a number of cellular pathways have been reported to be a cause of ASD, ID and associated NDDs (Vissers et al., 2010; Ronemus et al., 2014; Gilissen et al., 2014; Deciphering Developmental Disorders Study, 2015). Although the strong correlation between NDDs and genetic factors has been long established, the exact genetic background of ASD and ID remains unclear because of the strong heterogeneity of these disabilities. Genetic research has focused on the use of unbiased genome-wide approaches, including genomic microarrays and, more recently, NGS technology with the use of extensive gene panels, the exome or the whole genome. Consequently, new ASD and ID genes are now being identified in rapid succession. Among these new candidate genes, supportive role for the Jumonji Domain Containing 1C (JMJD1C) histone demethylase in ASD and ID has been fostered by both positional cloning strategies (Castermans et al., 2007) and exome-sequencing studies (Neale et al., 2012; Iossifov et al., 2014). Thus, based on the strong genetic architecture underlying NDDs and the emerging evidences for a role of JMJD1C as potential candidate genes, in the present PhD thesis we proposed to study the involvement of JMJD1C alterations in ASD, ID and RTT and their effects on the protein function. OBJECTIVES The specific objectives of study I are as follows: 1. To investigate the occurrence of JMJD1C mutations in the mentioned disabilities, a comprehensive mutational analysis was performed in samples from ASD, ID and RTT, searching for SNVs and indels, as well as larger genetic defects. 2. To address the functional consequences of the identified de novo JMJD1C- Pro163Leu mutation, the intracellular localization of the wildtype and mutated JMJD1C protein, as well as the efficiency in demethylating a non-histone target of JMJD1C, MDC1 (mediator of DNA-damage checkpoint 1), was studied in immunofluorescence, fractionation and immunoprecipitation experiments in HEK293 cells. 3. To direct a molecular explanation for the implication of JMJD1C in Rett Syndrome, the interaction between MeCP2 and JMJD1C, in the wildtype and mutated form, was assessed in an immunoprecipitation assay. 4. To study the cellular effects of the disruption of wildtype JMJD1C on a neuronal system, we analyzed the existence of changes in dendritic branching of primary neuron cultures from neonatal mouse hippocampus. STUDY II HYPOTHESIS Mutations in MECP2 cause most of the classical or typical forms of RTT (Chahrour and Zoghbi, 2007). Approximately 8 % of classic RTT and 42 % of variant RTT patients are MECP2 mutation-negative (Monrós et al. 2001; Percy, 2008). Some of the latter group have mutations in other genes, such as that of CDKL5, which is described in individuals with an early seizure onset variant of RTT (Kalscheuer et al., 2003) or FOXG1, which is responsible for the congenital variant of RTT (Ariani et al., 2008). However, there remains a subset of patients with a clinical diagnosis of RTT who are mutation-negative for all the aforementioned genes. In the present PhD thesis, we proposed to identify new candidate genes that could explain the RTT-like phenotype of several clinical cases without mutations in MECP2, CDKL5 and FOXG1, using NGS, with the purpose of expanding the knowledge of the impaired biological pathways in RTT. OBJECTIVES The specific objectives of study II are as follows: 1. To identify previously undescribed variants potentially implicated in RTT-like phenotype, WES was performed on a cohort of 19 Spanish parent–child trios and one family with two affected daughters presenting features associated with RTT. A bioinformatics process of WES data was realized to filter and select putative pathogenic de novo variants absent or present with very low frequency in the control population, and putatively dangerous for protein function. 2. To realize a differential diagnosis among diverse neurodevelopmental disorders with overlapping phenotype, a gene-association analysis was carried out to define a list of variants previously associated with neurodevelopmental disorders and another one with undescribed new variants not previously associated with. 3. To demonstrate a neurological implication for a loss of function of detected candidate genes not previously associated with neurodevelopmental disorders, the model organism C. elegans was used to confirm a genotype-phenotype correlation by performing locomotion assays in worm mutants that carry deleterious mutations in the orthologous genes to those human genes with potentially pathogenic mutations in the patients

Optimal generation scheduling for a hybrid stand-alone power system using renewable energy sources and hydrogen storage

This paper presents a novel energy management strategy (EMS) for hybrid stand-alone power generation systems powered by renewable energy sources and equipped with batteries and a hydrogen storage section. In particular, the proposed EMS defines the optimal generation scheduling of the two storage devices to minimize operating costs and maximize system efficiency. Since the achievement of the optimal EMS for stand-alone systems powered by renewable energy sources is strongly influenced by the uncertainties of solar and wind resources, a stochastic approach was adopted to deal with these uncertainties. The proposed EMS was used to evaluate the expected annual performance of a microgrid currently under construction at the Concentrating Solar and Hydrogen From Renewable Energy Sources Laboratory, in Sardinia (Italy). The study shows that the current hydrogen storage capacity of the microgrid is insufficient to meet the annual energy requirements, especially in winter months. To highlight the benefits of including weather forecasts and operating costs in the EMS, a comparative analysis with a simpler SOC-based EMS was carried out. The results of the comparative study demonstrate that the proposed EMS leads to a decrease of almost 5-10% in the annual operating costs and energy losses, especially for high values of the hydrogen storage capacity. In particular, for a hydrogen storage capacity of the studied microgrid 10 times higher than the planned one (55 Nm3), the adoption of the proposed EMS can achieve a decrease of about 10% in the annual operating costs and about 6% in the annual energy losses with respect to a conventional SOC-based EMS

Hydrogen as a clean energy carrier: the microgrid at the “Concentrating Solar and Hydrogen from Renewable Energy Sources Laboratory"

The use of renewable energy sources for distributed power generation is the most considered alternative to the use of fossil fuels in centralized power plants. However, due to their discontinuous operation, the availability of suitable energy storage systems is required. In this field, hydrogen storage technologies are one of the most interesting options. Hydrogen production, storage and utilization technologies have a high innovation content even if several tecnical and economical problems remain to be solved. In this framework, Sardegna Ricerche has promoted the realization of the “Concentrating Solar and Hydrogen from Renewable Energy Sources” Laboratory in collaboration with the University of Cagliari. Main mission of the laboratory is implementation, testing and demonstration of the technologies related to the production, storage and use of hydrogen from renewable energy sources. For this reason, the main research activities are in the field of the electrochemical characterization of fuel cell stacks and their materials with the aim to improve performance in terms of durability and efficiency, as well as the development of hydrogen storage systems. In particular, the laboratory includes a stand-alone micro-grid powered exclusively by a photovoltaic array and a wind turbine. The microgrid is also coupled with two different energy storage systems: a battery bank and a hydrogen storage system. In particular, the latter is based on two PEM electrolyzers (1 Nm3/h each), four hydrogen storage tanks (1 m3 each) and a PEMFC fuel cell (5 kW). The main aim of this paper is to carry out a detailed analysis of the expected micro-grid performance. In particular, the focus was laid on the implementation of a novel energy management system (EMS). The proposed EMS defines the optimal generation scheduling of the two storage devices in order to minimize operating costs and maximize the efficiency of the system. The results of a comparative study with more conventional EMS demonstrate that the proposed EMS leads to a decrease of almost 5-10% of the annual operating costs and energy losses

Optimal Integration of Hydrogen-Based Energy Storage Systems in Photovoltaic Microgrids: A Techno-Economic Assessment

The feasibility and cost-effectiveness of hydrogen-based microgrids in facilities, such as public buildings and small- and medium-sized enterprises, provided by photovoltaic (PV) plants and characterized by low electric demand during weekends, were investigated in this paper. Starting from the experience of the microgrid being built at the Renewable Energy Facility of Sardegna Ricerche (Italy), which, among various energy production and storage systems, includes a hydrogen storage system, a modeling of the hydrogen-based microgrid was developed. The model was used to analyze the expected performance of the microgrid considering different load profiles and equipment sizes. Finally, the microgrid cost-effectiveness was evaluated using a preliminary economic analysis. The results demonstrate that an effective design can be achieved with a PV system sized for an annual energy production 20% higher than the annual energy requested by the user and a hydrogen generator size 60% of the PV nominal power size. This configuration leads to a self-sufficiency rate of about 80% and, without public grants, a levelized cost of energy comparable with the cost of electricity in Italy can be achieved with a reduction of at least 25–40% of the current initial costs charged for the whole plant, depending on the load profile shape

Left Atrial Appendage Closure Devices

Atrial fibrillation (AF) increases the risk for thromboembolic stroke five-fold. The left atrial appendage (LAA) has been shown to be the main source of thrombus formation in the majority of strokes associated with AF. Oral anticoagulation with warfarin and novel anticoagulants remains the standard of care; however, it has several limitations, including bleeding and poor compliance. Occlusion of the LAA has been shown to be an alternative therapeutic approach to drug therapy. The purpose of this article is to review the different techniques and devices that have emerged for the purpose of occluding this structure, with a particular emphasis on the efficacy and safety studies published to date in the medical literature

Kinematic Evaluation of the Sagittal Posture during Walking in Healthy Subjects by 3D Motion Analysis Using DB-Total Protocol

Posture can be evaluated by clinical and instrumental methods. Three-dimensional motion analysis is the gold standard for the static and dynamic postural assessment. Conventional stereophotogrammetric protocols are used to assess the posture of pelvis, hip, knee, ankle, trunk (considered as a single segment) and rarely head and upper limbs during walking. A few studies also analyzed the multi-segmental trunk and whole-body kinematics. Aim of our study was to evaluate the sagittal spine and the whole-body during walking in healthy subjects by 3D motion analysis using a new marker set. Fourteen healthy subjects were assessed by 3D-Stereophotogrammetry using the DB-Total protocol. Excursion Range, Absolute Excursion Range, Average, intra-subject Coefficient of Variation (CV) and inter-subject Standard Deviation Average (SD Average) of eighteen new kinematic parameters related to sagittal spine and whole-body posture were calculated. The analysis of the DB-Total parameters showed a high intra-subject (CV < 50%) and a high inter-subject (SD Average < 1) repeatability for the most of them. Kinematic curves and new additional values were reported. The present study introduced new postural values characterizing the sagittal spinal and whole-body alignment of healthy subjects during walking. DB-Total parameters may be useful for understanding multi-segmental body biomechanics and as a benchmark for pathological patterns

Potentially malignant and malignant lesions of the lip. Role of silver staining nucleolar organizer regions, proliferating cell nuclear antigen, p53, and c-myc in differentiation and prognosis.

The cellular changes leading to carcinoma of the lip are still not completely understood. This study was carried out on 44 malignant and potentially malignant lesions of the lower lip [30 squamous cell carcinomas (SCC), 7 actinic cheilitis, 3 leukoplakias, and 4 nodal metastases from lower lip SCC]. Silver-stained nucleolar organizer regions (AgNORs) and the immunohistochemical expression of proliferating cell nuclear antigen (PCNA), p53, and c-myc were evaluated on formalin-fixed, paraffin-embedded sections. The results indicate that the size and numbers of AgNORs and the percentage of PCNA-positive cells are sensitive parameters for discriminating between potentially malignant lesions and SCC, and for the prognostic sub-typing of lower lip SCC. Furthermore, while p53 positivity was found more frequently in high-grade carcinomas, p53-positive cellular clones were also found in some potentially malignant lesions, a finding probably related to ultraviolet-related cellular damage. These p53-positive lesions could be considered at higher risk of progression to malignancy than the p53-negative ones, although there is no evidence for this as yet. c-myc positivity was found only in some high-grade carcinomas and metastases, and appeared correlated with the later phases of lip carcinogenesis. The combined evaluation of the proliferation status, together with the changes in p53 and c-myc oncoproteins, might constitute useful markers for the prognostic evaluation of potentially malignant, as well as malignant, lesions of the lip

Mutations in JMJD1C are involved in Rett syndrome and intellectual disability

PURPOSE: Autism spectrum disorders are associated with defects in social response and communication that often occur in the context of intellectual disability. Rett syndrome is one example in which epilepsy, motor impairment, and motor disturbance may co-occur. Mutations in histone demethylases are known to occur in several of these syndromes. Herein, we aimed to identify whether mutations in the candidate histone demethylase JMJD1C (jumonji domain containing 1C) are implicated in these disorders. METHODS: We performed the mutational and functional analysis of JMJD1C in 215 cases of autism spectrum disorders, intellectual disability, and Rett syndrome without a known genetic defect. RESULTS: We found seven JMJD1C variants that were not present in any control sample ( 6,000) and caused an amino acid change involving a different functional group. From these, two de novo JMJD1C germline mutations were identified in a case of Rett syndrome and in a patient with intellectual disability. The functional study of the JMJD1C mutant Rett syndrome patient demonstrated that the altered protein had abnormal subcellular localization, diminished activity to demethylate the DNA damage-response protein MDC1, and reduced binding to MECP2. We confirmed that JMJD1C protein is widely expressed in brain regions and that its depletion compromises dendritic activity. CONCLUSIONS: Our findings indicate that mutations in JMJD1C contribute to the development of Rett syndrome and intellectual disability.Genet Med 18 1, 378-385.This study was supported by the European Community’s Seventh Framework Program (FP7/2007–2013) under grant agreement PITN-GA-2012–316758 of the EPITRAIN project and PITN-GA-2009–238242 of DISCHROM; ERC grant agreement 268626 of the EPINORC project; the E-RARE EuroRETT network (Carlos III Health Institute project PI071327); the Fondation Lejeune (France); MINECO projects SAF2011-22803 and CSD2006-00049; the Cellex Foundation; the Botín Foundation; the Catalan Association for Rett Syndrome; Fundación Alicia Koplowitz 2011 Grant AKOPLOWITZ11_006; the FIS project PI1002512; Grants PI10/01422, PI13/00285, CA10/01474, RD06/0020/1050, RD12/0036/008, and 2009-SGR293; and the Health and Science Departments of the Catalan government (Generalitat de Catalunya). K.S. and P.P. are EPITRAIN Research Fellows. M.E. is an ICREA Research Professor