Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained &gt;95% target region coverage at a read depth of 20 and &gt;50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.</p

The research landscape of tuberous sclerosis complex–associated neuropsychiatric disorders (TAND)—a comprehensive scoping review

Background Tuberous sclerosis complex (TSC)–associated neuropsychiatric disorders (TAND) is an umbrella term for the behavioural, psychiatric, intellectual, academic, neuropsychological and psychosocial manifestations of TSC. Although TAND affects 90% of individuals with TSC during their lifetime, these manifestations are relatively under-assessed, under-treated and under-researched. We performed a comprehensive scoping review of all TAND research to date (a) to describe the existing TAND research landscape and (b) to identify knowledge gaps to guide future TAND research. Methods The study was conducted in accordance with stages outlined within the Arksey and O’Malley scoping review framework. Ten research questions relating to study characteristics, research design and research content of TAND levels and clusters were examined. Results Of the 2841 returned searches, 230 articles published between 1987 and 2020 were included (animal studies = 30, case studies = 47, cohort studies = 153), with more than half published since the term TAND was coined in 2012 (118/230; 51%). Cohort studies largely involved children and/or adolescents (63%) as opposed to older adults (16%). Studies were represented across 341 individual research sites from 45 countries, the majority from the USA (89/341; 26%) and the UK (50/341; 15%). Only 48 research sites (14%) were within low–middle income countries (LMICs). Animal studies and case studies were of relatively high/high quality, but cohort studies showed significant variability. Of the 153 cohort studies, only 16 (10%) included interventions. None of these were non-pharmacological, and only 13 employed remote methodologies (e.g. telephone interviews, online surveys). Of all TAND clusters, the autism spectrum disorder–like cluster was the most widely researched (138/230; 60%) and the scholastic cluster the least (53/200; 27%). Conclusions Despite the recent increase in TAND research, studies that represent participants across the lifespan, LMIC research sites and non-pharmacological interventions were identified as future priorities. The quality of cohort studies requires improvement, to which the use of standardised direct behavioural assessments may contribute. In human studies, the academic level in particular warrants further investigation. Remote technologies could help to address many of the TAND knowledge gaps identified

Increased Understanding of Stem Cell Behavior in Neurodegenerative and Neuromuscular Disorders by Use of Noninvasive Cell Imaging

Numerous neurodegenerative and neuromuscular disorders are associated with cell-specific depletion in the human body. This imbalance in tissue homeostasis is in healthy individuals repaired by the presence of endogenous stem cells that can replace the lost cell type. However, in most disorders, a genetic origin or limited presence or exhaustion of stem cells impairs correct cell replacement. During the last 30 years, methods to readily isolate and expand stem cells have been developed and this resulted in a major change in the regenerative medicine field as it generates sufficient amount of cells for human transplantation applications. Furthermore, stem cells have been shown to release cytokines with beneficial effects for several diseases. At present however, clinical stem cell transplantations studies are struggling to demonstrate clinical efficacy despite promising preclinical results. Therefore, to allow stem cell therapy to achieve its full potential, more insight in their in vivo behavior has to be achieved. Different methods to noninvasively monitor these cells have been developed and are discussed. In some cases, stem cell monitoring even reached the clinical setting. We anticipate that by further exploring these imaging possibilities and unraveling their in vivo behavior further improvement in stem cell transplantations will be achieved

Activin A Modulates CRIPTO-1/HNF4 alpha(+) Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes.status: publishe

Activin A Modulates CRIPTO-1/HNF4α+ Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes

Long-term data with idebenone on respiratory function outcomes in patients with Duchenne muscular dystrophy.

peer reviewedDecline in respiratory function in patients with DMD starts during early teenage years and leads to early morbidity and mortality. Published evidence of efficacy for idebenone on respiratory function outcomes is currently limited to 12 months of follow-up time. Here we report data collected as retrospective cohort study (SYROS) from 18 DMD patients not using glucocorticoids who were treated with idebenone (900mg/day) under Expanded Access Programs (EAPs). The objective was to assess the long-term respiratory function evolution for periods On-Idebenone compared to periods Off-Idebenone in the same patients. The mean idebenone exposure in the EAPs was 4.2 (range 2.4-6.1) years. The primary endpoint was the annual change in forced vital capacity percent of predicted (FVC%p) compared between Off-Idebenone and On-Idebenone periods. The annual rate of decline in FVC%p was reduced by approximately 50% from -7.4% (95% CI: -9.1, -5.8) for the Off-Idebenone periods to -3.8% (95% CI: -4.8, -2.8) for the On-Idebenone periods (N=11). Similarly, annual change in peak expiratory flow percent of predicted (PEF%p) was -5.9% (95% CI: -8.0, -3.9) for the Off-Idebenone periods (N=9) and reduced to -1.9% (95% CI: -3.2, -0.7) for the On-Idebenone periods during the EAPs. The reduced rates of decline in FVC%p and PEF%p were maintained for several years with possible beneficial effects on the rate of bronchopulmonary adverse events, time to 10% decline in FVC%p and risk of hospitalization due to respiratory cause. These long-term data provide Class IV evidence to further support the disease modifying treatment effect of idebenone previously observed in randomized, controlled trials

Long-term data with idebenone on respiratory function outcomes in patients with Duchenne muscular dystrophy

Decline in respiratory function in patients with DMD starts during early teenage years and leads to early morbidity and mortality. Published evidence of efficacy for idebenone on respiratory function outcomes is currently limited to 12 months of follow-up time. Here we report data collected as retrospective cohort study (SYROS) from 18 DMD patients not using glucocorticoids who were treated with idebenone (900 mg/day) under Expanded Access Programs (EAPs). The objective was to assess the long-term respiratory function evolution for periods On-Idebenone compared to periods Off-Idebenone in the same patients. The mean idebenone exposure in the EAPs was 4.2 (range 2.4-6.1) years. The primary endpoint was the annual change in forced vital capacity percent of predicted (FVC%p) compared between Off-Idebenone and On-Idebenone periods. The annual rate of decline in FVC%p was reduced by approximately 50% from -7.4% (95% CI: -9.1, -5.8) for the Off-Idebenone periods to -3.8% (95% CI: -4.8, -2.8) for the On-Idebenone periods (N = 11). Similarly, annual change in peak expiratory flow percent of predicted (PEF%p) was -5.9% (95% CI: -8.0, -3.9) for the Off-Idebenone periods (N = 9) and reduced to -1.9% (95% CI: -3.2, -0.7) for the On-Idebenone periods during the EAPs. The reduced rates of decline in FVC%p and PEF%p were maintained for several years with possible beneficial effects on the rate of bronchopulmonary adverse events, time to 10% decline in FVC%p and risk of hospitalization due to respiratory cause. These long-term data provide Class IV evidence to further support the disease modifying treatment effect of idebenone previously observed in randomized, controlled trials.status: publishe

Dystrophin deficiency leads to dysfunctional glutamate clearance in iPSC derived astrocytes

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.status: publishe

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions

: Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells