Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. RESULTS: We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. CONCLUSIONS: We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification

Endothelial overexpression of LOX-1 increases plaque formation and promotes atherosclerosis in vivo

Aims Lectin-like oxLDL receptor-1 (LOX-1) mediates the uptake of oxidized low-density lipoprotein (oxLDL) in endothelial cells and macrophages. However, the different atherogenic potential of LOX-1-mediated endothelial and macrophage oxLDL uptake remains unclear. The present study was designed to investigate the in vivo role of endothelial LOX-1 in atherogenesis. Methods and results Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). Oxidized low-density lipoprotein uptake was enhanced in cultured endothelial cells, but not in macrophages of LOX-1TG mice. Six-week-old male LOX-1TG and wild-type (WT) mice were fed a high-cholesterol diet (HCD) for 30 weeks. Increased reactive oxygen species production, impaired endothelial nitric oxide synthase activity and endothelial dysfunction were observed in LOX-1TG mice as compared with WT littermates. LOX-1 overexpression led to p38 phosphorylation, increased nuclear factor κB activity and subsequent up-regulation of vascular cell adhesion molecule-1, thereby favouring macrophage accumulation and aortic fatty streaks. Consistently, HCD-fed double-mutant LOX-1TG/ApoE−/− displayed oxidative stress and vascular inflammation with higher aortic plaques than ApoE−/− controls. Finally, bone marrow transplantation experiments showed that endothelial LOX-1 was sufficient for atherosclerosis development in vivo. Conclusions Endothelial-specific LOX-1 overexpression enhanced aortic oxLDL levels, thereby favouring endothelial dysfunction, vascular inflammation and plaque formation. Thus, LOX-1 may serve as a novel therapeutic target for atherosclerosi

Deletion of L-Selectin Increases Atherosclerosis Development in ApoE−/− Mice

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel−/−) animals were crossed with mice lacking Apolipoprotein E (ApoE−/−). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE−/− L-sel−/−) mice and the corresponding ApoE−/− controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE−/− L-sel−/− mice as compared to ApoE−/− controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE−/− L-sel−/− animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE−/− L-sel−/− and ApoE−/− mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE−/− L-sel−/− as compared to ApoE−/− mice. Leukocyte rolling on lesions in the aorta was similar in ApoE−/− L-sel−/− and ApoE−/− animals. ApoE−/− L-sel−/− mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE−/− controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis

First-in-human study demonstrating the safety and clinical efficacy of novel anti-IL-17A monoclonal antibody CJM112 in moderate to severe plaque psoriasis

Background and objective: Anti-IL-17A IgG/κ monoclonal antibody CJM112 binds both IL-17A and IL-17AF. The purpose of this First-in-Human study was to assess CJM112 effects on safety and efficacy in patients with moderate to severe plaque psoriasis. Methods: This study had two parts: single ascending doses of 5–450 mg subcutaneous (s.c.) CJM112 (SAD) and multi-dose parallel groups of CJM112 15 mg, 50 mg and 150 mg s.c. low frequency or high frequency (MD). SAD/MD were double-blind, randomized and placebo-controlled; MD also included a secukinumab 150 mg s.c. arm as an active comparator. Patients 18–65 years with moderate to severe psoriasis were included in this study. The efficacy outcome was the change in Psoriasis Area Severity Index (PASI) from baseline to Week 4 in the SAD part of the study, and from baseline to Week 12 in the MD part. Results: 96 patients were enrolled in this study (SAD, n = 42; MD, n = 54). In SAD, CJM112 doses from 15 mg and above demonstrated higher PASI responses compared with placebo at Week 12. CJM112 450 mg did not add further efficacy, but efficacy duration was prolonged compared with CJM112 150 mg. CJM112 MD resulted in a dose-dependent decrease in PASI over time to Week 12. CJM112 150 mg high frequency did not exceed the effect of CJM112 150 mg low frequency and had similar efficacy to secukinumab 150 mg. The safety profile of CJM112 was as expected for an antibody targeting IL-17A/IL-17AF. Conclusions: CJM112 had clinical efficacy in moderate to severe psoriasis and was generally safe and well tolerated in the doses tested. Additional neutralization of IL-17AF did not translate to increased clinical efficacy compared with secukinumab

Altered and normal levels of protein synthesis in fragile X syndrome, studies in humans and mice.

Fragile X syndrome (FXS) is the most common monogenic form of inherited intellectual disability and autism spectrum disorder. Two decades of intense basic research on the function of FMRP show that its absence in FXS causes aberrant signaling leading to upregulated mRNA translation and protein synthesis and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies in mice and flies have shown that several pharmacological interventions can partially or fully restore those deficits and it was postulated that altered protein synthesis and synaptic architecture mediate the cognitive and behavioral deficits observed in patients with FXS. This led to one of the most comprehensive drug development program undertaken thus far for a genetically defined neurodevelopmental disorder but this effort yielded negative results. Our aim was to characterize the distribution of de novo rates of protein synthesis in patients with FXS and its relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts of 32 individuals with FXS, 17 controls as well as in fibroblasts and primary neurons of 30 Fmr1 KO mice, in two different isogenic backgrounds. Our results show that levels of protein synthesis are clearly increased in fibroblasts of individuals with fragile X. However, this “cellular phenotype” displays a broad distribution and a significant proportion of fragile X individuals and fmr1 KO mice have measures in the normal range. To test the validity of our study conducted in peripheral tissue, we used the fmr1 KO mouse model to show that measures in fibroblasts predict those in neurons of the same animals. Finally, we show that protein synthesis but not FMRP explains a significant proportion of the variance in adaptive skills measured in individuals with FXS. Our study offers a potential explanation for the negative trials and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis

IL-17A is a pertinent therapeutic target for moderate-to-severe hidradenitis suppurativa: Combined results from a pre-clinical and phase II proof-of-concept study

Hidradenitis Suppurativa (HS) is a chronic, recurrent, inflammatory, follicular skin disease whose pathology is complex and not fully understood. The objective of this study was to elucidate the role of IL-17A in moderate-to-severe HS. Transcriptomic and histological analyses were conducted on ex vivo HS (n = 19; lesional and non-lesional) and healthy control (n = 8) skin biopsies. Further, a Phase II exploratory, randomized, double-blind, placebo-controlled study was carried out in moderate-to-severe HS patients. Patients were treated with either CJM112 300 mg (n = 33), a fully human anti-IL-17A IgG1/κ monoclonal antibody, or placebo (n = 33). The main outcome of the translational analyses was to identify IL-17A-producing cells and indications of IL-17A activity in HS lesional skin. The primary objective of the clinical study was to determine the efficacy of CJM112 in moderate-to-severe HS patients by HS-Physician Global Assessment (HS-PGA) responder rate at Week 16. Transcriptomic and histopathologic analyses revealed the presence of heterogeneous cell types in HS lesional skin; IL-17A gene signatures were increased in HS lesional vs non-lesional or healthy skin. High expression of IL-17A was localized to T cells, neutrophils, and mast cells, confirming the transcriptional data. Clinically, the proportion of Week 16 HS-PGA responders was significantly higher (p = 0.03) in the CJM112 group vs placebo (32.3% vs 12.5%). This study elucidated the role of the IL-17A pathway in HS pathogenesis and clinically validated the IL-17A pathway in moderate-to-severe HS patients in a proof-of-concept study using the anti-IL-17A-specific antibody CJM112

Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in Fragile X syndrome

Background. The molecular pathways associated with FMR1 epigenetic silencing in Fragile X syndrome (FXS) patients are still elusive and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. Results. Here we have deployed customized epigenomic profiling assays on blood-derived samples from FXS patients and healthy volunteers to comprehensively map the FMR1 locus chromatin landscape. Using a methylated DNA immunocapture (MeDIP) assay combined with DNA microarrays covering the entire FMR1 locus, we identified novel regions of altered DNA methylation (5-methylcytosine, 5mC) within FXS patients. Changes in 5mC were accompanied by unprecedented, reciprocal, changes in DNA hydroxymethylation (5-hydroxymethylcytosine, 5hmC), representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 locus histone post-translational epigenetic marks, together DNA methylation and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 gene body 5hmC levels. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. Conclusions. We identify novel FMR1 locus-specific and cell-type specific epigenetic marks in FXS patient cells and propose that the combined profiling of 5mC and 5hmC may enhance the molecular and functional characterization and clinical stratification of FXS patients

Protein synthesis levels are increased in a subset of individuals with fragile X syndrome

Fragile X syndrome (FXS) is a monogenic form of intellectual disability and autism spectrum disorder caused by the absence of the fragile X mental retardation protein (FMRP). In biological models for the disease, this leads to upregulated mRNA translation and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here, we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here, we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those in neurons we suggest the validity of this peripheral biomarker. Our study offers a potential explanation for the comprehensive drug development program undertaken thus far yielding negative results and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis

Protein synthesis levels are increased in a subset of individuals with fragile X syndrome.

This article initially published with incomplete supplementary material. This error has now been corrected, and the correct supplementary material is published. The authors regret the error

Protein synthesis levels are increased in a subset of individuals with fragile X syndrome.

Fragile X syndrome (FXS) is a monogenic form of intellectual disability and autism spectrum disorder caused by the absence of the fragile X mental retardation protein (FMRP). In biological models for the disease, this leads to upregulated mRNA translation and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here, we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here, we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those in neurons we suggest the validity of this peripheral biomarker. Our study offers a potential explanation for the comprehensive drug development program undertaken thus far yielding negative results and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis