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Spearmanâ s correlation analysis among RTT clinical data. (DOCX 14Â kb)

Bacterial taxa which relative abundances were significantly different (p

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)-a major secondary product of lipid peroxidation- and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS. Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications. The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients.

Correlation plots of clinical data from RTT subjects. Significant positive correlations were observed among age, IgA and ESR. (PDF 37Â kb)

Multiple-rarefaction PCoA plots. Each PCoA replicate was optimally superimposed by Procrustes analysis on the master PCoA scatter plot (used in the main text). Points represent the average location of 100 rarefaction replicates. Ellipses show the 95 % confidence region assuming a multivariate normal distribution. a) PCoA plots of bacterial beta-diversity based on the unweighted and weighted UniFrac distances and the Bray-Curtis dissimilarity analysed according to individuals’ health status; b) PCoA plots of fungal beta-diversity based on the unweighted and weighted UniFrac distances and the Bray-Curtis dissimilarity analysed according to individuals’ health status. Constipated Rett syndrome subjects (RTT-C), non-constipated Rett syndrome subjects (RTT-NC) and healthy controls (HC) are coloured in red, orange or green, respectively. (PDF 185 kb)

Alpha-diversity rarefaction curves. The plot shows the alpha-diversity for HC, RTT-C and RTT-NC averaged over 100 independent rarefactions as a function of the rarefaction depth for a) the bacterial gut microbiota and b) the fungal gut microbiota. The points in the curves are the averages, while the whiskers represent the standard deviations. (PDF 47Â kb)

Measures of bacterial diversity. a) Alpha-diversity estimated on the Chao1 estimator and the Shannon entropy; ***, p

Statistics of the significantly different metabolic pathways (KEGG categories) inferred with PICRUSt in the gut microbiota of healthy controls (HC) and Rett syndrome (RTT) subjects (Welch’s t test, p

PERMANOVA p values distribution. For each rarefaction replicate (n = 100), a PERMANOVA test was conducted to assess the robustness of the results over the rarefaction. a) PERMANOVA p values distributions of bacterial beta-diversity based on the unweighted and weighted UniFrac distances and the Bray-Curtis dissimilarity analysed according to individuals’ health status; b) PERMANOVA p values distributions of fungal beta-diversity based on the unweighted and weighted UniFrac distances and the Bray-Curtis dissimilarity analysed according to individuals’ health status. (PDF 27 kb)

Wilcoxon rank-sum test comparison of bacterial relative abundances at phylum and genus levels. (DOCX 22Â kb)

Background: Autism Spectrum Disorders (ASD) and Intellectual Disability (ID) represent lifelong conditions with severe impact on behavior and lifestyle of patients and their families. Array comparative genomic hybridization (array-CGH) has clarified the underlying genetic causes of ASD and ID by CNVs identification in several chromosomal regions with susceptibility to different levels of severity of ASD or ID in up to 1% of patients. Methods: Using oligo array-CGH we analyzed 476 unrelated subjects with ASD or ID, thoroughly investigated by both child neuropsychiatrists and clinical geneticists. The inheritance of the CNV were tested in the majority of cases (82% of positive cases). Results: A total of 198 rearrangements was identified in 154 cases. CNVs were classified in three groups: i- CNVs previously known to be associated with ASD or ID (28/198, 14%), including 16p11.2, 15q13.3, 17p12 and 17q12; ii- CNVs including genes known to be associated with either ASD or ID (9/198, 4.5%); iii- CNVs of unknown significance (161/198, 81.3%). Conclusions: Our study confirmed that array-CGH analysis is able to detect the underlying genetic cause in about 18% of ASD or ID patients, highlighting it as an essential diagnostic tool for patients assessment. Overall, a prevalence of duplications with respect to deletions was observed (62% and 38% respectively) but among the deleted cases an enrichment of microdeletions in ASD cases (p=0.03) is present. Furthermore, we shown a prevalence of multiple CNVs in ASD cases compared to ID (p=0.05), pointing out the complex nature of ASD.

A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment. (C) 2015 Elsevier B.V. All rights reserved.

A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet. By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM). Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1β and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs. In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder.

In this review, we summarize the current evidence on the erythrocyte as a previously unrecognized target cell in Rett syndrome, a rare (1:10 000 females) and devastating neurodevelopmental disorder caused by loss-of-function mutations in a single gene (i.e.MeCP2,CDKL5, or rarelyFOXG1). In particular, we focus on morphological changes, membrane oxidative damage, altered membrane fatty acid profile, and aberrant skeletal organization in erythrocytes from patients with typical Rett syndrome andMeCP2gene mutations. The beneficial effects of ω-3 polyunsaturated fatty acids (PUFAs) are also summarized for this condition to be considered as a ‘model’ condition for autism spectrum disorders.

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' β-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

CDKL5 mutation is associated with an atypical Rett syndrome (RTT) variant. Recently, cholesterol homeostasis perturbation and oxidative-mediated loss of the high-density lipoprotein receptor SRB1 in typical RTT have been suggested. Here, we demonstrate an altered lipid serum profile also in CDKL5 patients with decreased levels of SRB1 and impaired activation of the defensive system Nrf2. In addition, CDKL5 fibroblasts showed an increase in 4-hydroxy-2-nonenal– and nitrotyrosine–SRB1 adducts that lead to its ubi-quitination and probable degradation. This study highlights a possible common denominator between two different RTT variants (MECP2 and CDKL5) and a possible common future therapeutic target.

Objective Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene, and characterized by cognitive and communicative regression, loss of hand use, and midline hand stereotypies. Epilepsy is a core symptom, but literature is controversial regarding genotype–phenotype correlation. Analysis of data from a large cohort should overcome this shortcoming. Methods Data from the Rett Syndrome Networked Database on 1,248 female patients were included. Data on phenotypic and genotypic parameters, age of onset, severity of epilepsy, and type of seizures were collected. Statistical analysis was done using the IBM SPSS Version 21 software, logistic regression, and Kaplan-Meier survival curves. Results Epilepsy was present in 68.1% of the patients, with uncontrolled seizures in 32.6% of the patients with epilepsy. Mean age of onset of epilepsy was 4.68 ± (standard deviation) 3.5 years. Younger age of onset was correlated to severity of epilepsy (Spearman correlation r = 0.668, p < 0.01). Patients with late truncating deletions had lower prevalence of epilepsy. Compared to them, the p.R133C mutation, associated with a milder Rett phenotype, increased the risk for epilepsy (odds ratio [OR] 2.46, confidence interval [CI] 95% 1.3–4.66), but not for severe epilepsy. The p.R255X mutation conferred an increased risk for epilepsy (OR 2.07, CI 95% 1.2–3.59) as well as for severe epilepsy (OR 3.4, CI 95% 1.6–7.3). The p.T158M and p.C306C mutations relatively increased the risk for severe epilepsy (OR 3.09 and 2.69, CI 95% 1.48–6.4 and 1.19–6.05, respectively), but not for epilepsy occurrence. Significance Various mutations in the MECP2 gene have a different influence on epilepsy, unrelated to the severity of the general Rett phenotype. This might suggest a site-specific effect of MeCp2 on epileptic pathways. Further investigation of these mechanisms should promote better understanding of epileptogenesis in Rett syndrome.

The Autistic Spectrum Disorder (ASD) refers to a range of conditions classified as neurodevelopmental disorders characteristic of the early childhood. Over the last 50 years, considerable efforts have been made to understand pathogenesis of ASD: it is generally accepted that a lot of causes could trigger the disease. Since growing evidences described an association between oxidative stress and mitochondrial dysfunction in ASD, we investigated the possible defects of mitochondrial quality control pathway as contributing factors to the altered redox homeostasis. Using freshly skin fibroblasts isolated from ASD patients and healthy subjects, we demonstrated in autistic cells an increased levels of 4HNE protein adducts - a major secondary product of lipid peroxidation - and catalase, one of the main enzymes of the cellular defensive system. Transmission electron microscope (TEM) analysis demonstrated an altered mitochondrial morphology in autistic fibroblasts. An increased expression of the genes that encode for molecules involved in mitochondrial fission/fusion processes, was also detected. In addition, overregulation of PINK1, but decreased Parkin expression, both associated to mitophagy process, were observed. Taken together our findings provide new insights on the possible contribution of the altered mitochondrial quality control pathway in the redox imbalance of ASD patients.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder with a 300-fold increased risk rate for sudden cardiac death. A subclinical myocardial biventricular dysfunction has been recently reported in RTT by our group and found to be associated with an enhanced oxidative stress (OS) status. Here, we tested the effects of the naturally occurring antioxidantsω-3 polyunsaturated fatty acids (ω-3 PUFAs) on echocardiographic parameters and systemic OS markers in a population of RTT patients with the typical clinical form. A total of 66 RTT girls were evaluated, half of whom being treated for 12 months with a dietary supplementation ofω-3 PUFAs at high dosage (docosahexaenoic acid ~71.9 ± 13.9 mg/kg b.w./day plus eicosapentaenoic acid ~115.5 ± 22.4 mg/kg b.w./day) versus the remaining half untreated population. Echocardiographic systolic longitudinal parameters of both ventricles, but not biventricular diastolic measures, improved followingω-3 PUFAs supplementation, with a parallel decrease in the OS markers levels. No significant changes in the examined echocardiographic parameters nor in the OS markers were detectable in the untreated RTT population. Our data indicate thatω-3 PUFAs are able to improve the biventricular myocardial systolic function in RTT and that this functional gain is partially mediated through a regulation of the redox balance.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n=53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n=82) and healthy age-matched controls (n=29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a syntheticN-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P=0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n= 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells,that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly linked to mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Respiratory dysfunction, historically credited to brainstem immaturity, represents a major challenge in RTT. Our aim was to characterize the relationships between pulmonary gas exchange abnormality (GEA), upper airway obstruction, and redox status in patients with typical RTT (n = 228) and to examine lung histology in a Mecp2-null mouse model of the disease. GEA was detectable in 80% (184/228) of patients versus 18% of healthy controls, with "high" (39.8%) and "low" (34.8%) patterns dominating over "mixed" (19.6%) and "simple mismatch" (5.9%) types. Increased plasma levels of non-protein-bound iron (NPBI), F2- isoprostanes (F2-IsoPs), intraerythrocyte NPBI (IE-NPBI), and reduced and oxidized glutathione (i.e., GSH and GSSG) were evidenced in RTT with consequently decreased GSH/GSSG ratios. Apnea frequency/severity was positively correlated with IE-NPBI, F2-IsoPs, and GSSG and negatively with GSH/GSSG ratio. A diffuse inflammatory infiltrate of the terminal bronchioles and alveoli was evidenced in half of the examined Mecp2-mutant mice, well fitting with the radiological findings previously observed in RTT patients. Our findings indicate that GEA is a key feature of RTT and that terminal bronchioles are a likely major target of the disease. © 2014 Claudio De Felice et al. Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly linked to mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Respiratory dysfunction, historically credited to brainstem immaturity, represents a major challenge in RTT. Our aim was to characterize the relationships between pulmonary gas exchange abnormality (GEA), upper airway obstruction, and redox status in patients with typical RTT (n = 228) and to examine lung histology in a Mecp2-null mouse model of the disease. GEA was detectable in similar to 80% (184/228) of patients versus similar to 18% of healthy controls, with "high" (39.8%) and "low" (34.8%) patterns dominating over "mixed" (19.6%) and "simple mismatch" (5.9%) types. Increased plasma levels of nonprotein-bound iron (NPBI), F-2-isoprostanes (F-2-IsoPs), intraerythrocyte NPBI (IE-NPBI), and reduced and oxidized glutathione (i.e., GSH and GSSG) were evidenced in RTT with consequently decreased GSH/GSSG ratios. Apnea frequency/severity was positively correlated with IE-NPBI, F-2-IsoPs, and GSSG and negatively with GSH/GSSG ratio. A diffuse inflammatory infiltrate of the terminal bronchioles and alveoli was evidenced in half of the examined Mecp2-mutant mice, well fitting with the radiological findings previously observed in RTT patients. Our findings indicate that GEA is a key feature of RTT and that terminal bronchioles are a likely major target of the disease.

Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT) and methyl CpG binding protein 2 (MECP2) gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs), drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: −1.82±0.15, −2.15±0.06, and −2.59±0.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs) as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.

Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused byde novoloss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., “pseudo-autistic”) RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four majorMECP2gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h,P<0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P=0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n=6spots) or negative (n=9spots), and to a lesser extent as proteins involved in the immune system (n=2spots), with some proteins having overlapping functions on metabolism (n=7spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the “pseudo-autistic” phase of RTT, which is related to the severity carried by theMECP2gene mutation.

Rett (RTT) syndrome is a severe neurological disorder that affects almost exclusively females. Several detectable mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are responsible for the onset of the disease. MeCP2 is a key transcription regulator involved in gene silencing via methylation-dependent remodeling of chromatin. Recent data highlight that lipid metabolism is perturbed in brains and livers of MECP2-null male mice. In addition, altered plasma lipid profile in RTT patients has been observed. Thus, the aim of the work is to investigate the protein network involved in cholesterol homeostasis maintenance on freshly isolated fibroblasts and plasma from both RTT and healthy donors. To this end, protein expression of 3-hydroxy-3methyl glutaryl Coenzyme A reductase (HMGR), sterol regulatory element binding proteins (SREBPs), low density lipoprotein receptor (LDLr) and scavenger receptor B-1 (SRB-1) was assessed in cultured skin fibroblasts from unaffected individuals and RTT patients. In addition, lipid profile and the abundance of proprotein convertase subtilisin/kexin type 9 (PCSK9) were analyzed on plasma samples. The obtained results demonstrate that the main proteins belonging to cholesterol regulatory network are altered in RTT female patients, providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology.

This study mainly aims at examining the erythrocyte membrane fatty acid (FAs) profile in Rett syndrome (RTT), a genetically determined neurodevelopmental disease. Early reports suggest a beneficial effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on disease severity in RTT. A total of 24 RTT patients were assigned to ω-3 PUFAs-containing fish oil for 12 months in a randomized controlled study (average DHA and EPA doses of 72.9, and 117.1mg/kgb.w./day, respectively). A distinctly altered FAs profile was detectable in RTT, with deficient ω-6 PUFAs, increased saturated FAs and reduced trans 20:4 FAs. FAs changes were found to be related to redox imbalance, subclinical inflammation, and decreased bone density. Supplementation with ω-3 PUFAs led to improved ω-6/ω-3 ratio and serum plasma lipid profile, decreased PUFAs peroxidation end-products, normalization of biochemical markers of inflammation, and reduction of bone hypodensity as compared to the untreated RTT group. Our data indicate that a significant FAs abnormality is detectable in the RTT erythrocyte membranes and is partially rescued by ω-3 PUFAs.

The mechanism of action of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) is only partially known. Prior reports suggest a partial rescue of clinical symptoms and oxidative stress (OS) alterations followingω-3 PUFAs supplementation in patients with Rett syndrome (RTT), a devastating neurodevelopmental disorder with transient autistic features, affecting almost exclusively females and mainly caused by sporadic mutations in the gene encoding the methyl CpG binding protein 2 (MeCP2) protein. Here, we tested the hypothesis thatω-3 PUFAs may modify the plasma proteome profile in typical RTT patients withMECP2mutations and classic phenotype. A total of 24 RTT girls at different clinical stages were supplemented withω-3 PUFAs as fish oil for 12 months and compared to matched healthy controls. The expression of 16 proteins, mainly related to acute phase response (APR), was changed at the baseline in the untreated patients. Followingω-3 PUFAs supplementation, the detected APR was partially rescued, with the expression of 10 out of 16 (62%) proteins being normalized.ω-3 PUFAs have a major impact on the modulation of the APR in RTT, thus providing new insights into the role of inflammation in autistic disorders and paving the way for novel therapeutic strategies.

Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6–26 years), nonautistic neurodevelopmental disorders (i.e., “positive controls”), and healthy controls (i.e., “negative controls”). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membraneβ-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, andβ-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.

The modulation of the HDL receptor scavenger receptor B1 (SRB1) was evaluated in skin fibroblasts isolated from patients with Rett syndrome (RTT), a genetic form of infantile autism. Patients showed an altered plasma lipid profile, while their skin fibroblasts showed a dramatic reduction in SRB1 (immunogold, Western blot and immunohistochemistry). The decreased SRB1 levels were demonstrated to be the consequence of its binding with 4-hydroxy-2-nonenal (4HNE), a product of lipid peroxidation, and its increased ubiquitination. Our findings show for the first time a loss of SRB1 in RTT cells and its relationship with a chronic oxidative stress status.

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive development disorder mainly caused up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. We have recently synthesized F(2)-Dihomo-isoprostanes (F(2)-Dihomo-IsoP), peroxidation products from adrenic acid (C22:4 n - 6, AdA), a known component of myelin, and tested the potential value of F(2)-Dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation, and disease progression. F(2)-Dihomo-IsoPs were determined by a gas chromatography/negative ion chemical ionization tandem mass spectrometry. The ent-7(RS)-F(2t)-Dihomo-IsoP and 17-F(2t)-Dihomo-IsoP were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M - 181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-Dihomo-IsoP and 17-F(2t)-Dihomo-IsoP. Average plasma F(2)-Dihomo-IsoP levels in RTT were about 1 order of magnitude higher than in healthy controls, being higher in typical RTT as compared to RTT variants, with a remarkable increase of about 2 orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. These data indicate for the first time that quantification of F(2)-Dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.

International audience; Autism spectrum disorders (ASDs) are epidemically explosive clinical entities, but their pathogenesis is still unclear and a definitive cure does not yet exist. Rett syndrome (RTT) is a rare genetically determined cause of autism linked to mutations in the X-linked MeCP2 gene or, more rarely, in CDKL5 or FOXG1. A wide phenotypical heterogeneity is a known feature of the disease. Although several studies have focused on the molecular genetics and possible protein changes at different levels, to date very little attention has been paid to fatty acids in this disease, which could be con- sidered as a natural paradigm for the ASDs. To this regard, a quite enigmatic feature of the disease is the evidence in the affected patients of an extensive peroxidation of polyunsaturated fatty acids (arachidonic acid, AA, docosaexahenoic acid, DHA, adrenic acid, AdA and, to a lesser extent, eicosapentaenoic acid, EPA), in contrast with amelioration of the redox changes and phenotypical severity following the supplementation of some of those same fatty acids (DHA + EPA). Therefore, fatty acids may represent a kind of Janus Bifrons in the particular context of RTT. Here, we propose a rational explanation for this apparent "fatty acid paradox" in RTT. A better understanding of this paradox could also be of help to get a better insight into the complex mechanism of action for polyunsaturated fatty acids in health and dis- ease.

Lipid peroxidation, a process known to induce oxidative damage to key cellular components, has been implicated in several diseases. Following three decades of explorations mainly onin vitromodels reproducible in the laboratories, lipid peroxidation has become increasingly relevant for the interpretation of a wide range of pathophysiological mechanisms in the clinical setting. This cumulative effort has led to the identification of several lipid peroxidation end-products meeting the needs of thein vivoevaluation. Among these different molecules, isoprostanes and 4-hydroxy-2-nonenal protein adducts appear to be particularly interesting. This review shows how specific oxidation products, deriving from polyunsaturated fatty acids precursors, are strictly related to the clinical manifestations and the natural history of Rett syndrome, a genetically determined neurodevelopmental pathology, currently classified among the autism spectrum disorders. In our experience, Rett syndrome offers a unique setting for physicians, biologists, and chemists to explore the borders of the lipid mediators concept.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the sameMECP2gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease.

A link between oxidative stress and autism spectrum disorders (ASDs) remains controversial with opposing views on its role in the pathogenesis of the disease. We investigated for the first time the levels of non-protein-bound iron (NPBI), a pro-oxidant factor, and 4-hydroxynonenal protein adducts (4-HNE PAs), as a marker of lipid peroxidation-induced protein damage, in classic autism. Patients with classic autism (n = 20, mean age 12.0 ± 6.2 years) and healthy controls (n = 18, mean age 11.7 ± 6.5 years) were examined. Intraerythrocyte and plasma NPBI were measured by high performance liquid chromatography (HPLC), and 4-HNE PAs in erythrocyte membranes and plasma were detected by Western blotting. The antioxidant defences were evaluated as erythrocyte glutathione (GSH) levels using a spectrophotometric assay. Intraerythrocyte and plasma NPBI levels were significantly increased (1.98- and 3.56-folds) in autistic patients, as compared to controls (p = 0.0019 and p < 0.0001, respectively); likewise, 4-HNE PAs were significantly higher in erythrocyte membranes and in plasma (1.58- and 1.6-folds, respectively) from autistic patients than controls (p = 0.0043 and p = 0.0001, respectively). Erythrocyte GSH was slightly decreased (−10.34%) in patients compared to controls (p = 0.0215). Our findings indicate an impairment of the redox status in classic autism patients, with a consequent imbalance between oxidative stress and antioxidant defences. Increased levels of NPBI could contribute to lipid peroxidation and, consequently, to increased plasma and erythrocyte membranes 4-HNE PAs thus amplifying the oxidative damage, potentially contributing to the autistic phenotype.

Rett syndrome (OMIM#312750) is a monogenic disorder that may manifest as a large variety of phenotypes ranging from very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005) mutation and balanced X-inactivation, one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome), while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant). In addition to the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system, exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.

International audience; The main cause of Rett syndrome (RTT), a pervasive development disorder almost exclusively affecting females, is a mutation in the methyl-CpG binding protein 2 (MeCP2) gene. To date, no cure for RTT exists, although disease reversibility has been demonstrated in animal models. Emerging evidence from our and other laboratories indicates a potential role of oxidative stress (OS) in RTT. This review examines the current state of the knowledge on the role of OS in explaining the natural history, genotype-phenotype correlation, and clinical heterogeneity of the human disease. Biochemical evidence of OS appears to be related to neurological symptom severity, mutation type, and clinical presentation. These findings pave the way for potential new genetic downstream therapeutic strategies aimed at improving patient quality of life. Further efforts in the near future are needed for investigating the yet unexplored "black box" between the MeCP2 gene mutation and subsequent OS derangement.

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of omega-3 polyunsaturated fatty acids (omega-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with omega-3 PUFAs-containing fish oil (DHA: 72.9 +/- A 8.1 mg/kg b.w./day; EPA: 117.1 +/- A 13.1 mg/kg b.w./day; total omega-3 PUFAs: 246.0 +/- A 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F-2-dihomo-isoprostanes, F-3-isoprostanes, F-4-neuroprostanes, and F-2-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the omega-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Aims Rett syndrome (RTT) is a rare neurodevelopmental disorder frequently linked to methyl-CpG-binding protein 2 ( MeCP2 ) gene mutations. RTT is associated with a 300-fold increased risk of sudden cardiac death. Rhythm abnormalities and cardiac dysautonomia do not to fully account for cardiac mortality. Conversely, heart function in RTT has not been explored to date. Recent data indicate a previously unrecognized role of MeCP2 in cardiomyocytes development. Besides, increased oxidative stress markers (OS) have been found in RTT. We hypothesized that (i) RTT patients present a subclinical biventricular dysfunction and (ii) the myocardial dysfunction correlate with OS. Methods and results We evaluated typical ( n = 72) and atypical ( n = 20) RTT female and healthy controls ( n = 92). Main outcome measurements were (i) echocardiographic biventricular systo-diastolic parameters; (ii) correlation between echocardiographic measures and OS levels, i.e. plasma and intra-erythrocyte non-protein-bound iron (NPBI) and plasma F2-Isoprostanes (F2-IsoPs). A significant reduction in longitudinal biventricular function (tricuspid annular plane systolic excursion, mitral annular plane systolic excursion, S′ of lateral and septal mitral annulus, S′ of tricuspidal annulus) was evidenced in RTT patients vs. controls. No significant changes in the LV ejection fraction were found. Peak-early filling parameters (E, E′ of lateral mitral annulus, E′ of tricuspidal annulus) and right ventricular systolic pressure were reduced. A-wave, E/A, and E/E′ were normal. OS markers were increased, but only F2-IsoPs correlated to LV systolic dysfunction. Conclusion These data indicate a previously unrecognized subclinical systo-diastolic biventricular myocardial dysfunction in typical and atypical RTT patients. A reduced preload is evidenced. The biventricular dysfunction is partially related to OS damage.

Background Hypoxemia and increased oxidative stress (OS) have been reported in Rett Syndrome (RTT), a genetical neurodevelopmental disorder. Although OS and hypoxemia can lead to red blood cells (RBCs) shape abnormalities, no information on RBCs morphology in RTT exists. Here, RBCs shape was evaluated in RTT patients and healthy subjects as a function of OS markers, blood oxygenation, pulmonary gas exchange, and cardio-respiratory parameters. Methods RBCs morphology was evaluated by Scanning Electron Microscopy. Intraerythrocyte and plasma non protein-bound iron (NPBI), esterified F2-Isoprostanes (F2-IsoPs), 4-HNE protein adducts (4-HNE PAs) were measured. Pulmonary oxygen gradients and PaO2 were evaluated by gas analyzers and cardiopulmonary variables by pulse oximetry. In RTT patients these parameters were assessed before and after ω-3 polyunsaturated fatty acids (ω-3 PUFAs) administration. Results Altered RBCs shapes (leptocytes) and increased NPBI were present in RTT, together with increased erythrocyte membrane esterified F2-IsoPs and 4-HNE PAs. Abnormal erythrocyte shapes were related to OS markers levels, pulmonary gas exchange, PaO2 and cardio-respiratory variables. After ω-3 PUFAs, a decrease of leptocytes was accompanied by a progressive increase in reversible forms of RBCs. This partial RBCs morphology rescue was related to decreased OS damage markers, improved pulmonary oxygen exchange, and cardiopulmonary physiology. Conclusions These findings indicate that in RTT 1) RBCs shape is altered; 2) the OS-hypoxia diad is critical in generating altered RBCs shape and membrane damage; 3) ω-3 PUFAs are able to partially rescue RBCs morphology and the OS-derived damage. General significance RBCs morphology is an important biosensor for OS imbalance and chronic hypoxemia.

Rett syndrome (RTT) is a pervasive development disorder, mainly caused by mutations in the methyl-CpG binding protein 2 (MeCP2) gene. No reliable biochemical markers of the disease are available. Here we assess F₄-neuroprostanes (F₄-NeuroPs), lipid peroxidation products of the docosahexaenoic acid, as a novel disease marker in RTT and correlate it with clinical presentation, MeCP2 mutation type, and disease progression. In addition, we investigate on the impact of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) supplementation on F₄-NeuroPs levels.A case-control study design was used. A cohort of RTT patients (n=144) exhibiting different clinical presentations, disease stages, and MeCP2 gene mutations were evaluated. F₄-NeuroPs were measured in free form using a GC/NICI-MS/MS technique. Plasma F₄-NeuroPs levels in patients were compared to healthy controls and related to RTT forms, disease progression, and response to ω-3 PUFAs supplementation.Plasma F₄-NeuroPs levels were i) higher in RTT than in controls; ii) increased with the severity of neurological symptoms; iii) significantly elevated during the typical disease progression; iv) higher in MeCP2-nonsense as compared to missense mutation carriers; v) higher in typical RTT as compared to RTT variants; and vi) decreased in response to 12 months ω-3 PUFAs oral supplementation.Quantification of plasma F₄-NeuroPs provides a novel RTT marker, related to neurological symptoms severity, mutation type and clinical presentation.