Reduced serum levels of pro-inflammatory chemokines in fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and the most commonly identified monogenic cause of autism. Recent studies have shown that long-term pathological consequences of FXS are not solely confined to the central nervous system (CNS) but rather extend to other physiological dysfunctions in peripheral organs. To gain insights into possible immune dysfunctions in FXS, we profiled a large panel of immune-related biomarkers in the serum of FXS patients and healthy controls. METHODS: We have used a sensitive and robust Electro Chemi Luminescence (ECL)-based immunoassay to measure the levels of 52 cytokines in the serum of n = 25 FXS patients and n = 29 healthy controls. We then used univariate statistics and multivariate analysis, as well as an advanced unsupervised clustering method, to identify combinations of immune-related biomarkers that could discriminate FXS patients from healthy individuals. RESULTS: While the majority of the tested cytokines were present at similar levels in FXS patients and healthy individuals, nine chemokines, CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26 and CXCL10, were present at much lower levels in FXS patients. Using robust regression, we show that six of these biomarkers (CCL2, CCL3, CCL11, CCL22, CCL26 and CXCL10) were negatively associated with FXS diagnosis. Finally, applying the K-sparse unsupervised clustering method to the biomarker dataset allowed for the identification of two subsets of individuals, which essentially matched the FXS and healthy control categories. CONCLUSIONS: Our data show that FXS patients exhibit reduced serum levels of several chemokines and may therefore exhibit impaired immune responses. The present study also highlights the power of unsupervised clustering methods to identify combinations of biomarkers for diagnosis and prognosis in medicine.

Metformin as targeted treatment in fragile X syndrome.

BACKGROUND: Individuals with fragile X syndrome (FXS) have both behavioral and medical comorbidities and the latter include obesity in approximately 30% and the Prader-Willi Phenotype (PWP) characterized by severe hyperphagia and morbid obesity in less than 10%. Metformin is a drug used in individuals with type 2 diabetes, obesity or impaired glucose tolerance and it has a strong safety profile in children and adults. Recently published studies in the Drosophila model and the knock out mouse model of FXS treated with metformin demonstrate the rescue of multiple phenotypes of FXS. MATERIALS AND METHODS: We present 7 cases of individuals with FXS who have been treated with metformin clinically. One case with type 2 diabetes, 3 cases with the PWP, 2 adults with obesity and/or behavioral problems and, a young child with FXS. These individuals were clinically treated with metformin and monitored for behavioral changes with the Aberrant Behavior Checklist and metabolic changes with a fasting glucose and HgbA1c. RESULTS: We found consistent improvements in irritability, social responsiveness, hyperactivity, and social avoidance, in addition to comments from the family regarding improvements in language and conversational skills. No significant side-effects were noted and most patients with obesity lost weight. CONCLUSION: We recommend a controlled trial of metformin in those with FXS. Metformin appears to be an effective treatment of obesity including those with the PWP in FXS. Our study suggests that metformin may also be a targeted treatment for improving behavior and language in children and adults with FXS.

Platelets as a surrogate disease model of neurodevelopmental disorders: Insights from Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and the leading monogenic cause of autism spectrum disorders (ASD). Despite a large number of therapeutics developed in past years, there is currently no targeted treatment approved for FXS. In fact, translation of the positive and very promising preclinical findings from animal models to human subjects has so far fallen short owing in part to the low predictive validity of the Fmr1 ko mouse, an overly simplistic model of the complex human disease. This issue stresses the critical need to identify new surrogate human peripheral cell models of FXS, which may in fact allow for the identification of novel and more efficient therapies. Of all described models, blood platelets appear to be one of the most promising and appropriate disease models of FXS, in part owing to their close biochemical similarities with neurons. Noteworthy, they also recapitulate some of FXS neuron's core molecular dysregulations, such as hyperactivity of the MAPK/ERK and PI3K/Akt/mTOR pathways, elevated enzymatic activity of MMP9 and decreased production of cAMP. Platelets might therefore help furthering our understanding of FXS pathophysiology and might also lead to the identification of disease-specific biomarkers, as was shown in several psychiatric disorders such as schizophrenia and Alzheimer's disease. Moreover, there is additional evidence suggesting that platelet signaling may assist with prediction of cognitive phenotype and could represent a potent readout of drug efficacy in clinical trials. Globally, given the neurobiological overlap between different forms of intellectual disability, platelets may be a valuable window to access the molecular underpinnings of ASD and other neurodevelopmental disorders (NDD) sharing similar synaptic plasticity defects with FXS. Platelets are indeed an attractive model for unraveling pathophysiological mechanisms involved in NDD as well as to search for diagnostic and therapeutic biomarkers.

Novel Blood Biomarkers Are Associated with White Matter Lesions in Fragile X- Associated Tremor/Ataxia Syndrome.

BACKGROUND: The need for accessible cellular biomarkers of neurodegeneration in carriers of the fragile X mental retardation 1 (FMR1) premutation (PM) alleles. OBJECTIVE: To assess the mitochondrial status and respiration in blood lymphoblasts from PM carriers manifesting the fragile X-associated tremor/ataxia syndrome (FXTAS) and non-FXTAS carriers, and their relationship with the brain white matter lesions. METHODS: Oxygen consumption rates (OCR) and ATP synthesis using a Seahorse XFe24 Extracellular Flux Analyser, and steady-state parameters of mitochondrial function were assessed in cultured lymphoblasts from 16 PM males (including 11 FXTAS patients) and 9 matched controls. The regional white matter hyperintensity (WMH) scores were obtained from MRI. RESULTS: Mitochondrial respiratory activity was significantly elevated in lymphoblasts from PM carriers compared with controls, with a 2- to 3-fold increase in basal and maximum OCR attributable to complex I activity, and ATP synthesis, accompanied by unaltered mitochondrial mass and membrane potential. The changes, which were more advanced in FXTAS patients, were significantly associated with the WMH scores in the supratentorial regions. CONCLUSION: The dramatic increase in mitochondrial activity in lymphoblasts from PM carriers may represent either the early stages of disease (specific alterations in short-lived blood cells) or an activation of the lymphocytes under pathological situations. These changes may provide early, convenient blood biomarkers of clinical involvements.

Lovastatin corrects ERK pathway hyperactivation in fragile X syndrome: potential of platelet's signaling cascades as new outcome measures in clinical trials.

AIM: To establish whether platelets from fragile X syndrome (FXS) individuals recapitulate FXS mouse neurons' defects in ERK and Akt pathways, and to evaluate the effect of lovastatin on these pathways. METHODS: ERK and Akt phosphorylation (pERK, pAkt) statuses were assessed with quantitative Western blotting before and after a 12-week lovastatin trial. RESULTS: Levels of pERK and pAkt were increased in FXS platelets, and lovastatin specifically normalized ERK activity. Changes in ERK phosphorylation were correlated with clinical response to lovastatin. CONCLUSIONS: Platelets' signaling pathways provide biomarkers that can be used as treatment outcome measures in FXS clinical trials.

Plasma Levels of Leptin and Adiponectin in Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common form of familial mental retardation and one of the leading known causes of autism. The mutation responsible for FXS is a large expansion of the CGG repeats in the promoter region of the FMR1 gene, resulting in the transcriptional silencing of the gene. Leptin may be considered a cytokine-like hormone with pleiotropic actions since it may be involved in the regulation of neuroendocrine functions and the immune system response, in addition to playing a role in development. Leptin and adiponectin may act in parallel as opposing metabolic counterparts. The involvement of leptin and adiponectin in the pathophysiology of FXS was hypothesized. MATERIAL AND METHODS: Twenty-three male patients affected by FXS (full mutation in the FMR1 gene) and 24 controls were included in the study. Plasma leptin and adiponectin levels were measured by the ELISA method using commercially available kits. RESULTS: Adiponectin levels in FXS patients were significantly lower than those found in controls (p < 0.04). Leptin levels in FXS patients were significantly higher than those found in controls (p = 0.03). CONCLUSION: Adipokines may be involved in the psychiatric features observed in FXS patients. Further investigations are necessary to evaluate the role of adiponectin and leptin in FXS.

Skewed X Inactivation in Women Carrying the FMR1 Premutation and Its Relation with Fragile-X-Associated Tremor/Ataxia Syndrome.

BACKGROUND: Fragile-X-associated tremor/ataxia syndrome (FXTAS) is a late-onset multisystem neurological disorder characterized by intention tremor and cerebellar ataxia. OBJECTIVE: We hypothesized that in FMR1 premutation females with FXTAS, a normal X chromosome might more frequently be inactivated; therefore, the aim of this study was to determine the relationship between skewed X chromosome inactivation (XCI) and FXTAS. METHODS: We studied the XCI patterns of cases of FMR1 premutation in 10 women with FXTAS and 21 without FXTAS. RESULTS: The distribution of XCI patterns in the FXTAS and no-FXTAS groups showed differences regarding the allele presenting severe skewed XCI. In the FXTAS group, all cases preferentially inactivated the non-expanded X chromosome, whereas in the no-FXTAS group, all inactivated the expanded X chromosome. Nevertheless, no significant differences were found on comparing XCI frequencies among FMR1 premutation carriers with and without FXTAS. As expected, we found statistically significant differences in the skewed XCI on comparing FMR1 premutation women and controls. CONCLUSION: Although the reduced sample size and blood XCI patterns are two limitations of this study, our results suggest that the skewed XCI of the normal FMR1 allele may be a risk factor for the development of FXTAS. Furthermore, our findings also support the protective effect of the expression of a normal FMR1 allele.

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Cholesterol levels in fragile X syndrome.

Fragile X syndrome (FXS) is associated with intellectual disability and behavioral dysfunction, including anxiety, ADHD symptoms, and autistic features. Although individuals with FXS are largely considered healthy and lifespan is not thought to be reduced, very little is known about the long-term medical health of adults with FXS and no systematically collected information is available on standard laboratory measures from metabolic screens. During the course of follow up of a large cohort of patients with FXS we noted that many patients had low cholesterol and high density lipoprotein (HDL) values and thus initiated a systematic chart review of all cholesterol values present in charts from a clinic cohort of over 500 patients with FXS. Total cholesterol (TC), low density lipoprotein (LDL) and HDL were all significantly reduced in males from the FXS cohort relative to age-adjusted population normative data. This finding has relevance for health monitoring in individuals with FXS, for treatments with cholesterol-lowering agents that have been proposed to target the underlying CNS disorder in FXS based on work in animal models, and for potential biomarker development in FXS.

Anti-neuronal antibodies in patients with fragile X syndrome: is there a role of autoimmunity in its pathogenesis?

BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder with a broad spectrum of involvement, including cognitive and behavioural impairments of varying degrees with specific physical features and a strong association with autism. OBJECTIVES: In this study, the frequency of serum anti-neural antibodies was investigated in FXS patients who did and those who did not manifest autism spectrum disorders (ASD) in comparison to typically developing controls. METHODS: The study involved 23 males (mean age, 19.78 ± 6.56 years) who harboured a full mutation in the FMR1 gene. The control group comprised 19 healthy students (mean age 24.63 ± 1.89 years). Serum anti-neuronal antibodies were analyzed using Western blotting. RESULTS: Serum anti-neuronal antibodies were present in 10/23 (43.48%) FXS males. CONCLUSION: Serum anti-neuronal antibodies were found in a subgroup of FXS patients. Autistic symptoms in FXS may, in part, be caused by auto-immune factors. Further studies in larger patient and control groups are necessary to elucidate the aetiopathogenic role of anti-neuronal antibodies in FXS patients.

Early detection of fragile X syndrome: applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots.

BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: We describe methylation specific-quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG <40), 143 premutation (PM) (CGG 56-170), 197 full mutation (FM) (CGG 200-2000), and 33 CGG size and methylation mosaic samples. RESULTS: In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA. CONCLUSIONS: MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes.

Cryptic FMR1 mosaic deletion in a phenotypically normal mother of a boy with fragile X syndrome: case report.

BACKGROUND: Increasing number of case reports of mosaic mutations and deletions have better armed clinicians and geneticists with more accurate and focused prenatal diagnoses. Since mosaicism means a significant increase of recurrence risk, detailed parental profiling is essential for risk assessments. CASE PRESENTATION: We here describe a clinically unaffected mother with a son who had fragile X syndrome (FXS) caused by a large deletion that includes the entire FMR1. To assess the recurrence risk regarding her second pregnancy, a series of genetic tests were conducted to establish this mother's status. Routine single nucleotide polymorphism (SNP) array and fluorescence in situ hybridisation (FISH) analyses detected two normal FMR1 copies in her blood. However, in-depth studies across the deleted region revealed varying proportions of mosaic deletion in her somatic tissues: lowest in the blood, moderately higher in the skin, urine sediment and menstrual discharge and highest in her eyebrow. Further FISH analysis of her skin-derived fibroblasts confirmed mosaicism of 13%. CONCLUSION: To our knowledge, this is the first characterized case of a female who was mosaic for an FMR1 deletion and extensive investigation of her mosaic status provided valuable information for her reproduction choices. Our case report may also alert clinicians and geneticists that a cryptic mosaicism with somatic heterogeneity should be carefully considered in families with children having clinically defined 'de novo' mutations, to avoid a second pregnancy with identical genetic abnormalities.

Fragile X protein in newborn dried blood spots.

BACKGROUND: The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, FMRP. We previously characterized 215 dried blood spots (DBS) representing different FMR1 genotypes and ages with a Luminex-based immunoassay (qFMRP). We found variable FMRP levels in the normal samples and identified affected males by the drastic reduction of FMRP. METHODS: Here, to establish the variability of expression of FMRP in a larger random population we quantified FMRP in 2,000 anonymous fresh newborn DBS. We also evaluated the effect of long term storage on qFMRP by retrospectively assaying 74 aged newborn DBS that had been stored for 7-84 months that included normal and full mutation individuals. These analyses were performed on 3 mm DBS disks. To identify the alleles associated with the lowest FMRP levels in the fresh DBS, we analyzed the DNA in the samples that were more than two standard deviations below the mean. RESULTS: Analysis of the fresh newborn DBS revealed a broad distribution of FMRP with a mean approximately 7-fold higher than that we previously reported for fresh DBS in normal adults and no samples whose FMRP level indicated FXS. DNA analysis of the lowest FMRP DBS showed that this was the low extreme of the normal range and included a female carrying a 165 CGG repeat premutation. In the retrospective study of aged newborn DBS, the FMRP mean of the normal samples was less than 30% of the mean of the fresh DBS. Despite the degraded signal from these aged DBS, qFMRP identified the FXS individuals. CONCLUSIONS: The assay showed that newborn DBS contain high levels of FMRP that will allow identification of males and potentially females, affected by FXS. The assay is also an effective screening tool for aged DBS stored for up to four years.

Don't miss patients with atypical FMR1 mutations: dysmorphism and clinical features in a boy with a partially methylated FMR1 full mutation.

UNLABELLED: Fragile X syndrome characterized by intellectual disability (ID), facial dysmorphism, and postpubertal macroorchidism is the most common monogenic cause of ID. It is typically induced by an expansion of a CGG repeat in the fragile X mental retardation 1 (FMR1) gene on Xq27 to more than 200 repeats. Only rarely patients have atypical mutations in the FMR1 gene such as point mutations, deletions, or unmethylated/partially methylated full mutations. Most of these patients show a minor phenotype or even appear clinically healthy. Here, we report the dysmorphism and clinical features of a 17-year-old boy with a partially methylated full mutation of approximately 250 repeats. Diagnosis was made subsequently to the evaluation of a FMR1 premutation as the cause for maternal premature ovarian failure. Dysmorphic evaluation revealed no strikingly long face, no prominent forehead/frontal bossing, no prominent mandible, no macroorchidism, and a head circumference in the lower normal range. Acquisition of a driving license for mopeds and unaccompanied rides by public transport in his home province indicate rather mild ID (IQ = 58). CONCLUSION: This adolescent demonstrates that apart from only minor ID, patients with a partially methylated FMR1 full mutation present less to absent pathognomonic facial dysmorphism, thus emphasizing the impact of family history for a straightforward clinical diagnosis.

FMR1 Protein Expression Correlates with Intelligence Quotient in Both Peripheral Blood Mononuclear Cells and Fibroblasts from Individuals with an FMR1 Mutation.

Fragile X syndrome (FXS) is the most common heritable form of intellectual disability and is caused by CGG repeat expansions exceeding 200 (full mutation). Such expansions lead to hypermethylation and transcriptional silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene. As a consequence, little or no FMR1 protein (FMRP) is produced; absence of the protein, which normally is responsible for neuronal development and maintenance, causes the syndrome. Previous studies have demonstrated the causal relationship between FMRP levels and cognitive abilities in peripheral blood mononuclear cells (PBMCs) and dermal fibroblast cell lines of patients with FXS. However, it is arguable whether PBMCs or fibroblasts would be the preferred surrogate for measuring molecular markers, particularly FMRP, to represent the cognitive impairment, a core symptom of FXS. To address this concern, CGG repeats, methylation status, FMR1 mRNA, and FMRP levels were measured in both PBMCs and fibroblasts derived from 66 individuals. The findings indicated a strong association between FMR1 mRNA expression levels and CGG repeat numbers in PBMCs of premutation males after correcting for methylation status. Moreover, FMRP expression levels from both PBMCs and fibroblasts of male participants with a hypermethylated full mutation and with mosaicism demonstrated significant association between the intelligence quotient levels and FMRP levels, suggesting that PBMCs may be preferable for FXS clinical studies, because of their greater accessibility.

Correlation of FMR4 expression levels to ovarian reserve markers in FMR1 premutation carriers.

BACKGROUND: Fragile X-associated primary ovarian insufficiency (FXPOI), characterized by amenorrhea before age 40 years, occurs in 20% of female FMR1 premutation carriers. Presently, there are no molecular or biomarkers that can help predicting which FMR1 premutation women will develop FXPOI. We previously demonstrated that high FMR4 levels can discriminate between FMR1 premutation carriers with and without FXPOI. In the present study the relationship between the expression levels of FMR4 and the ovarian reserve markers was assessed in female FMR1 premutation carriers under age of 35 years. METHODS: We examined the association between FMR4 transcript levels and the measures of total antral follicle count (AFC) and serum anti-müllerian hormone (AMH) levels as markers of ovarian follicle reserve. RESULTS: Results revealed a negative association between FMR4 levels and AMH (r = 0.45) and AFC (r = 0.64). Statistically significant higher FMR4 transcript levels were found among those FMR1 premutation women with both, low AFCs and AMH levels. CONCLUSIONS: These findings reinforce previous studies supporting the association between high levels of FMR4 and the risk of developing FXPOI in FMR1 premutation carriers.

High MMP-9 activity levels in fragile X syndrome are lowered by minocycline.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by lack of the FMR1 protein, FMRP, a translational repressor. Its absence leads to up-regulation of locally translated proteins involved in synaptic transmission and plasticity, including the matrix metalloproteinase-9 (MMP-9). In the Fmr1 knock-out (KO), a mouse model of FXS, an abnormal elevated expression of MMP-9 in the brain was pharmacologically down-regulated after treatment with the tetracycline derivative minocycline. Moreover, the rescue of immature dendritic spine morphology and a significant improvement of abnormal behavior were associated with down-regulation of MMP-9. Here, we report on high plasma activity of MMP-9 in individuals with FXS. In addition, we investigate MMP-9 changes in patients with FXS who have gone through a minocycline controlled clinical trial and correlate MMP-9 activity to clinical observations. The results of this study suggest that, in humans, activity levels of MMP-9 are lowered by minocycline and that, in some cases, changes in MMP-9 activity are positively associated with improvement based on clinical measures.

Association of lipid rafts cholesterol with clinical profile in fragile X syndrome.

Fragile X syndrome (FXS) is the most prevalent monogenic cause of intellectual disability and autism spectrum disorder (ASD). Affected individuals have a high prevalence of hypocholesterolemia, however, the underlying mechanisms and the clinical significance remains unknown. We hypothesized that decrease in the plasma cholesterol levels is associated with an alteration of cholesterol content within the lipid rafts (LRs) which ultimately affects the clinical profile of FXS individuals. The platelets LRs were isolated by ultracentrifugation on sucrose gradient from 27 FXS and 25 healthy controls, followed by measurements of proteins, cholesterol, and gangliosides content. Autistic and adaptive behaviour of affected individuals were respectively assessed by the Social Communication Questionnaire and Adaptive Behavior Assessment System. Our results suggest a decrease in the cholesterol content of LRs in FXS individuals as compared to controls. As opposed to controls, LR cholesterol was significantly associated with plasma total cholesterol (r = 0.47; p = 0.042) in the FXS group. Furthermore, the correlation between LRs cholesterol and the clinical profile showed a significant association with autistic traits (r = - 0.67; p < 0.001) and adaptative behavior (r = 0.70; p < 0.001). These results support the clinical significance of LR cholesterol alterations in FXS. Further studies are warranted to investigate the implication of LRs in FXS pathophysiology and ASD.

Evidence of reactive astrocytes but not peripheral immune system activation in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is one of the few known genetic causes of autism. FXS results from the loss of Fmr1 gene function; thus, Fmr1 knockout mice provide a model to study impairments associated with FXS and autism and to test potential therapeutic interventions. The inhibitory serine phosphorylation of glycogen synthase kinase-3 (GSK3) is lower in brain regions of Fmr1 knockout mice than wild-type mice and the GSK3 inhibitor lithium rescues several behavioral impairments in Fmr1 knockout mice. Therefore, we examined if the serine phosphorylation of GSK3 in Fmr1 knockout mice also was altered outside the brain and if administration of lithium ameliorated the macroorchidism phenotype. Additionally, since GSK3 regulates numerous functions of the immune system and immune alterations have been associated with autism, we tested if immune function is altered in Fmr1 knockout mice. The inhibitory serine phosphorylation of GSK3 was significantly lower in the testis and liver of Fmr1 knockout mice than wild-type mice, and chronic lithium treatment reduced macroorchidism in Fmr1 knockout mice. No alterations in peripheral immune function were identified in Fmr1 knockout mice. However, examination of glia, the immune cells of the brain, revealed reactive astrocytes in several brain regions of Fmr1 knockout mice and treatment with lithium reduced this in the striatum and cerebellum. These results provide further evidence of the involvement of dysregulated GSK3 in FXS, and demonstrate that lithium administration reduces macroorchidism and reactive astrocytes in Fmr1 knockout mice.

Rates of protein synthesis are reduced in peripheral blood mononuclear cells (PBMCs) from fragile X individuals.

BACKGROUND: Fragile X syndrome (FXS) is the leading inherited cause of intellectual disability and is caused by the loss of expression of the Fragile X mental retardation protein (FMRP). In animal model of FXS, the absence of FMRP leads to an aberrant rate of neuronal protein synthesis, which in turn is believed to be at the origin of defects regarding spine morphology and synaptic plasticity. Normalisation of protein synthesis in these models has been associated with a rescue of FXS behavioral and biochemicals phenotype, thus establishing the rate of protein synthesis as one of the most promising monitoring biomarker for FXS. However, rate of protein synthesis alteration in fragile X individuals is not well characterized. METHOD: We applied a robust radiolabeled assay to measure rate of protein synthesis in freshly extracted peripheral blood mononuclear cells (PBMCs) and blood platelets. We ultimately settle on PBMCs to measure and compare rate of protein synthesis in 13 males with fragile X and 14 matched controls individuals. RESULTS: Using this method, we measured a 26.9% decrease (p = 0,0193) in the rate of protein synthesis in fragile X individuals PBMCs. Furthermore, the rate of protein synthesis measurements obtained were highly reproducible, highlighting the robustness of the method. CONCLUSION: Our work presents the first evidence of a diminution of the rate of protein synthesis in a human peripheral model of fragile X. Our results also support the finding of previous studies using brain PET imaging in Fragile X individuals. Since our assay only requires a simple venous puncture, it could be used in other cases of intellectual disability in order to determine if an aberrant rate of protein synthesis is a common general mechanism leading to impairment in synaptic plasticity and to intellectual disability.