Unraveling Molecular Pathways Altered in MeCP2-Related Syndromes, in the Search for New Potential Avenues for Therapy

Methyl-CpG-binding protein 2 (MeCP2) is an X-linked epigenetic modulator whose dosage is critical for neural development and function. Loss-of-function mutations in MECP2 cause Rett Syndrome (RTT, OMIM #312750) while duplications in the Xq28 locus containing MECP2 and Interleukin-1 receptor-associated kinase 1 (IRAK1) cause MECP2 duplication syndrome (MDS, OMIM #300260). Both are rare neurodevelopmental disorders that share clinical symptoms, including intellectual disability, loss of speech, hand stereotypies, vasomotor deficits and seizures. The main objective of this exploratory study is to identify novel signaling pathways and potential quantitative biomarkers that could aid early diagnosis and/or the monitoring of disease progression in clinical trials. We analyzed by RT-PCR gene expression in whole blood and microRNA (miRNA) expression in plasma, in a cohort of 20 females with Rett syndrome, 2 males with MECP2 duplication syndrome and 28 healthy controls, and correlated RNA expression with disease and clinical parameters. We have identified a set of potential biomarker panels for RTT diagnostic and disease stratification of patients with microcephaly and vasomotor deficits. Our study sets the basis for larger studies leading to the identification of specific miRNA signatures for early RTT detection, stratification, disease progression and segregation from other neurodevelopmental disorders. Nevertheless, these data will require verification and validation in further studies with larger sample size including a whole range of ages

IPSC‐based modeling of THD recapitulates disease phenotypes and reveals neuronal malformation

Tyrosine hydroxylase deficiency (THD) is a rare genetic disorder leading to dopaminergic depletion and early-onset Parkinsonism. Affected children present with either a severe form that does not respond to L-Dopa treatment (THD-B) or a milder L-Dopa responsive form (THD-A). We generated induced pluripotent stem cells (iPSCs) from THD patients that were differentiated into dopaminergic neurons (DAn) and compared with control-DAn from healthy individuals and gene-corrected isogenic controls. Consistent with patients, THD iPSC-DAn displayed lower levels of DA metabolites and reduced TH expression, when compared to controls. Moreover, THD iPSC-DAn showed abnormal morphology, including reduced total neurite length and neurite arborization defects, which were not evident in DAn differentiated from control-iPSC. Treatment of THD-iPSC-DAn with L-Dopa rescued the neuronal defects and disease phenotype only in THDA-DAn. Interestingly, L-Dopa treatment at the stage of neuronal precursors could prevent the alterations in THDB-iPSC-DAn, thus suggesting the existence of a critical developmental window in THD. Our iPSC-based model recapitulates THD disease phenotypes and response to treatment, representing a promising tool for investigating pathogenic mechanisms, drug screening, and personalized management

Discovery of biomarker panels for neural dysfunction in inborn errors of amino acid metabolism.

Patients with inborn errors of amino acid metabolism frequently show neuropsychiatric symptoms despite accurate metabolic control. This study aimed to gain insight into the underlying mechanisms of neural dysfunction. Here we analyzed the expression of brain-derived neurotrophic factor (BDNF) and 10 genes required for correct brain functioning in plasma and blood of patients with Urea Cycle Disorders (UCD), Maple Syrup Urine Disease (MSUD) and controls. Receiver-operating characteristic (ROC) analysis was used to evaluate sensitivity and specificity of potential biomarkers. CACNA2D2 (α2δ2 subunit of voltage-gated calcium channels) and MECP2 (methyl-CpG binding protein 2) mRNA and protein showed an excellent neural function biomarker signature (AUC ≥ 0,925) for recognition of MSUD. THBS3 (thrombospondin 3) mRNA and AABA gave a very good biomarker signature (AUC 0,911) for executive-attention deficits. THBS3, LIN28A mRNA, and alanine showed a perfect biomarker signature (AUC 1) for behavioral and mood disorders. Finally, a panel of BDNF protein and at least two large neural AAs showed a perfect biomarker signature (AUC 1) for recognition of psychomotor delay, pointing to excessive protein restriction as central causative of psychomotor delay. To conclude, our study has identified promising biomarker panels for neural function evaluation, providing a base for future studies with larger samples

Tyrosine Hydroxylase Deficiency: Studies in patient samples and in a cellular model

[eng] Monoamine Neurotransmitter diseases are a rare group of inherited disorders of metabolism that encompass 12 different genetic defects leading to abnormal dopamine and/or serotonin brain homeostasis. They correspond to enzymatic deficiencies involved in the biosynthesis, catabolism and transport of dopamine and serotonine. From a clinical point of view, they can appear at any age and manifest diverse clinical features. However, movement disorders ranging from dopa-responsive dystonia to severe parkinsonism, associated to variable degrees of cognitive impairment is the most common form of presentation. The pathophysiology underlying the wide spectrum of clinical phenotypes (from mild to severe) and response to neurotransmitter precursors (L-Dopa+carbidopa, BH4 and other dopaminergic enhancers) has not been studied in detail. Here we aim to address this important issue through different approaches: i) the study of a particular disease of neurotransmitters, Tyrosine Hydroxylase deficiency (THD), as a model of dopaminergic deficiency. Tyrosine hydroxylase (TH) enzyme catalyses the rate-limiting step in the biosynthesis of dopamine (DA). THD exhibits a wide spectrum of clinical manifestations that have been grouped according to the severity in two clinical phenotypes: “Type A” tends to present as L-Dopa responsive parkinsonism-dystonia whereas “Type B” produces a severe encephalopathy of early-onset with sub-optimal L-Dopa response. ii) The study of a large cohort of patients with neurotransmitter defects thanks to an international collaboration (I-NTD group). We have used both patients’ samples and an iPSc model to address these questions. A) Patients’ samples: A.1) We studied one THD B phenotype postmortem brain and we observed that the expression of key synaptic proteins and neurodevelopmental markers were altered: TH, VMAT 1 and 2 and dopamine receptors, especially D2DR were decreased. GABAergic and glutamatergic proteins such as GABAVT, NMDAR1 and calbindin were also altered. Finally, developmental markers for synapses, axons and dendrites were decreased, whereas markers of neuronal volume were preserved. A.2) 94 CSF samples of patients with neurotransmitter defects from 9 centres belonging to different countries/continents were collected in an international collaboration through the I-NTD working group. The proteomic study showed that the main category of overrepresented proteins was related to nervous system development. Moreover, different proteins were detected that could be useful biomarkers for severity prognosis and response to treatment that are specific of disorders. Four of them were correctly validated with an ELISA analysis: APOD, COL6A3, APOH and OMGP. These proteins are involved in diverse important biological functions such as myelination, phospholipid and other lipid related processes. B) iPSC model of THD: iPSC lines from Type A and B patients, controls and an isogenic corrected iPSC line were generated. Upon Dopaminergic differentiation, THD A and B neurons reproduced the disease-associated phenotype: decreased TH Protein, reduced enzyme activity and alteration on DA genes expression. A new neuronal phenotype was also described: less TH-immunoreactive cells and fiber density in both mutant TH+ neurons, Type A and B DAn presented altered morphology (reduced neuronal arborisation only in THD-Type B) and a reduced axonal TH localization was observed in THD-Type A. We were also able to test therapeutic approaches such as L-Dopa + carbidopa. To conclude, we have performed extensive and novel studies using different approaches and techniques to better characterize the pathophysiology underlying the spectrum of severity and response to the current pharmacological treatments of neurotransmitter defects. We have provided new information pointing towards a dysregulation of multiple neurodevelopmental functions in these diseases, and biomarkers of clinical severity that could be explored in the future as therapeutic targets. Additionally, an iPSC model for THD has been developed for the first time that introduces mechanistic and therapeutic insights in this early-parkinsonism model.[spa] Los errores congénitos de las monoaminas (ECMM) son defectos en la síntesis, degradación o transporte de catecolaminas y serotonina. Las manifestaciones neurológicas incluyen trastornos del movimiento, retraso mental y encefalopatías. La Deficiencia de la TIrosina Hidroxilasa (THD) es un ECMM causado por un defecto en el enzima TH que cataliza el paso limitante en la biosíntesis de Dopamina (DA). Dos fenotipos clínicos se han descrito: el “A” que tiende a responder a la L-Dopa y el “B” que produce una encefalopatía grave de inicio temprano con mala respuesta a la L-Dopa. Para elucidar la gran variabilidad en cuanto a fenotipo y respuesta al tratamiento de estas enfermedades hemos realizado diferentes aproximaciones en muestras de pacientes y en un modelo celular. A) Pacientes: A.1) Estudiamos un cerebro postmortem THD de fenotipo B y observamos que la expresión de proteínas sinápticas clave y marcadores de neurodesarrollo estaban alteradas: reducción en la expresión de TH, VMAT 1 y 2 y los receptores de dopamina, especialmente D2DR. También se detectaron proteínas GABAérgicas y glutamatérgicas (GABAVT, NMDAR1 y calbindina) alteradas. Finalmente, los marcadores de desarrollo para sinapsis, axones y dendritas estaban disminuidos, mientras que los marcadores de volumen neuronal estaban preservados. A.2) 94 muestras de LCR de pacientes con ECMM fueron recogidas. El estudio de proteómica mostró que la categoría principal de proteínas sobrerrepresentada estaba relacionada con el desarrollo del sistema nervioso. Además, se detectaron diferentes proteínas que podrían ser biomarcadores útiles para el pronóstico de severidad y respuesta al tratamiento. Cuatro se validaron mediante un análisis ELISA: APOD, COL6A3, APOH y OMGP. B) Modelo celular Generamos líneas iPSC a partir de pacientes A y B, controles y una línea iPSC corregida. Tras la diferenciación DAn, las neuronas THD A y B reproducen el fenotipo de la enfermedad: proteína TH y actividad enzimática disminuidas y alteración en la expresión de genes DA. También se describió un nuevo fenotipo neuronal: menos células TH-inmunorreactivas, menor densidad fibrilar y alteraciones morfológicas en las nDA de ambos. Una reducción de la arborización neuronal en el B y la reducción de la localización axonal de TH en el A . Pudimos probar diferentes aproximaciones terapéuticas

Novel Protein Biomarkers of Monoamine Metabolism Defects Correlate with Disease Severity

Background Genetic defects of monoamine neurotransmitters are rare neurological diseases amenable to treatment with variable response. They are major causes of early parkinsonism and other spectrum of movement disorders including dopa‐responsive dystonia. Objectives The objective of this study was to conduct proteomic studies in cerebrospinal fluid (CSF) samples of patients with monoamine defects to detect biomarkers involved in pathophysiology, clinical phenotypes, and treatment response. Methods A total of 90 patients from diverse centers of the International Working Group on Neurotransmitter Related Disorders were included in the study (37 untreated before CSF collection, 48 treated and 5 unknown at the collection time). Clinical and molecular metadata were related to the protein abundances in the CSF. Results Concentrations of 4 proteins were significantly altered, detected by mass spectrometry, and confirmed by immunoassays. First, decreased levels of apolipoprotein D were found in severe cases of aromatic L‐amino acid decarboxylase deficiency. Second, low levels of apolipoprotein H were observed in patients with the severe phenotype of tyrosine hydroxylase deficiency, whereas increased concentrations of oligodendrocyte myelin glycoprotein were found in the same subset of patients with tyrosine hydroxylase deficiency. Third, decreased levels of collagen6A3 were observed in treated patients with tetrahydrobiopterin deficiency. Conclusion This study with the largest cohort of patients with monoamine defects studied so far reports the proteomic characterization of CSF and identifies 4 novel biomarkers that bring new insights into the consequences of early dopaminergic deprivation in the developing brain. They open new possibilities to understand their role in the pathophysiology of these disorders, and they may serve as potential predictors of disease severity and therapies. © 2020 International Parkinson and Movement Disorder Societ

Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials

DataSheet1_Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.pdf

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.</p

Table1_Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.xlsx

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.</p

Table2_Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.XLSX

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.</p