Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome
© Brasa et al. 2016
Received: 27 November 2015
Accepted: 24 January 2016
Published: 5 February 2016
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.
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.
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.
KeywordsFragile X syndrome (FXS) Chromatin profiling Epigenetic silencing, FMR1 5-hydroxymethylation (5hmC) Clinical biomarker
Fragile X syndrome (FXS) is the most common inherited form of mental retardation and autism in males [1, 2]. The syndrome is commonly associated with the expansion of cytosine-guanine-guanine (CGG) trinucleotide repeats in the 5′ untranslated region (5′UTR) of the human fragile X mental retardation 1 (FMR1) gene. In patients with more than 200 CGG repeats, the aberrant, CGG-repeat expanded FMR1 messenger ribonucleic acid (mRNA) mediates FMR1 gene silencing  resulting in the absence of fragile X mental retardation protein (FMRP) expression, a translational regulator involved in neurotransmitter mediated synaptic maturation and plasticity . Premutation carriers of FXS display a varying number of CGG repeats (55–200) associated with either normal or mild deficits in the expression of the FMR1 gene [4, 5]. Despite two decades of studying the (epi)genetic dynamics of the FMR1 locus, little is known about the molecular events and pathways that lead to FMR1 silencing in individuals carrying a full-mutated (>200 CGG repeats) allele. Aberrant deoxyribonucleic acid (DNA) hypermethylation of the FMR1 promoter CpG island and CGG repeats is strongly associated with FMR1 gene silencing and represents a molecular hallmark of full-mutation FXS patients [6–11]. The acquisition of DNA methylation at the FMR1 CpG island is accompanied by hypoacetylation of associated histones and acquisition of repressive histone post-translational modifications such as the methylation of the lysine 9 of histone H3 (H3K9me) and chromatin condensation, all characteristics of a transcriptionally inactive gene [12–15]. The methylation status of FMR1 in full-mutation patients can vary across cell types (methylation mosaicism) and is significantly associated with the clinical phenotype of FXS patients [5, 16–20]. The methylation observed at the FMR1 CpG island extends beyond the FMR1 promoter and spreads into the first intron [21, 22]. A small number of full-mutation, unmethylated individuals have also been reported . These patients displayed a FMR1 promoter epigenetic pattern comparable to that of normal controls, in accordance with normal transcription levels and consistent with a euchromatic configuration. The mechanisms preventing the initial methylation and protecting against a repressive chromatin configuration are unknown, and their identification may help understand the key pathways affected in the majority of full-mutation patients and might ultimately lead to new therapeutic opportunities for restoring normal FMR1 expression levels in FXS patients [12, 24].
DNA hydroxymethylation (5-hydroxymethylcytosine (5hmC)), a DNA methylation (5-methylcytosine (5mC)) derivative was rediscovered in mouse Purkinje cells and granule neurons [25, 26]. These newly characterized epigenetic marks are catalyzed by a group of enzymes belonging to the ten-eleven translocation methylcytosine dioxygenase (TET) family (TET 1, 2, and 3) . The 5hmC mark is distributed over the promoter and bodies of transcriptionally active genes as well as enhancer elements [28–30] and may both represent an epigenetic modification in its own right and also an intermediate product in an active DNA demethylation pathway, accounting for the maintenance of DNA demethylation at CpG-rich promoters [27, 31]. 5hmC is particularly enriched in the central nervous system where it may play specific and dynamic functions in the regulation of gene expression  including regulation of alternative splicing as well as synaptic function in the brain . The distribution of 5hmC is dynamically regulated during neurodevelopment, it may play a role in a number of neurodegenerative diseases [34, 35] and is also perturbed in an animal model of Rett syndrome, a neurodevelopmental disorder caused by a mutation in the MeCP2 gene which encodes a 5mC and a 5hmC binding protein which targets transcriptional activation or repression functions to its binding sites [32, 36, 37].
In the present study, we have used broad epigenomic profiling of the FMR1 locus (beyond the well-characterized promoter/CpG island regions) to identify novel epigenetic marks and mechanisms that may contribute to FMR1 silencing in blood-derived FXS full-mutation patients samples. We have integrated FMR1 locus-specific methylation (5mC and 5hmC), histone post-translational modifications, and gene expression with clinical scores of aberrant behavior in a group of eight FXS patients. Our data reveal novel molecular-clinical phenotype associations that may provide novel diagnostic tools for the prediction and stratification of FXS disease severity.
(hydroxy)-MeDIP profiling identifies novel regions of 5mC and 5hmC changes in the FMR1 locus of FXS patient blood samples
Chromosomal coordinates of regions of FMR1 epigenetic variability in FXS cells. The major regions or FMR1 epigenetic changes identified in FXS PBMCs and fibroblasts (Figs. 1 and 3). Each region is indicated with a unique ID. The functional location (relative to the transcriptional start site (TSS)/untranslated regions (UTR)), chromosome coordinates, and estimated size of the region of perturbation are indicated
Functional genomic location
TSS −12 kb/−7.5 kb
TSS −3.5 kb/+0.3 kb
TSS +0.3 kb/+10.2 kb
Gene body—introns 12–16
TSS +24.5 kb/34.5 kb
3′UTR +6.1 kb/+15.3 kb
The profiling of hydroxymethylated DNA in PBMC samples revealed broad 5hmC perturbations in FXS patient samples. Hydroxymethylated CpGs are typically found at transcribed gene bodies, active enhancers, and at a limited cohort of annotated transcriptional start site (TSS) [28–30]. Interestingly, our analyses revealed 5hmC enrichment in control over FXS samples within four FMR1 locus genomic regions located (1) upstream of FMR1, (2) at the TSS, (3) throughout the first intron, and (4) in a region located between introns 13 and 16 (Fig. 1c, Table 1), highlighting FMR1 hypo-hydroxymethylation as a novel features of fragile X syndrome. To validate the specificity of FMR1 DNA (hydroxy)methylation perturbations, additional control genomic regions were investigated from the array data and validated by (h)MeDIP-PCR and did not display differential 5mC/5hmC distribution or enrichment in healthy controls and FXS samples (Fig. 1b, Additional file 2: Figure S1C and Additional file 3: Figure S2C).
Overall, our analyses identify novel regions of 5mC changes along FMR1 and beyond the regions already described outside of the promoter/first intron regions . Our data also describe for the first time broad changes in FMR1 5hmC levels and distribution in FXS PBMCs, highlighting novel molecular features that may be associated with FMR1 epigenetic regulation in fragile X syndrome patient cells.
Inter-individual variations in FMR1 5mC and 5hmC changes in FXS PBMC
Changes in 5mC and 5hmC associate with broad changes in the FMR1 locus histone post-translational modification landscape in FXS patients
To validate the microarray-based epigenetic landscape data obtained by ChIP-array, quantitative polymerase chain reaction (qPCR) assays were run over the region surrounding the TSS using ChIP and input material used for arrays. The results, illustrated in Additional file 4: Figure S3 confirm reduced enrichment for active marks (H3K4me2, H3K9ac, H4K16ac) throughout the TSS region and show increased association of several repressive marks (H4K20me1, H4K20me3, H3K9me3), overall recapitulating array-based data.
Combined 5mC, 5hmC, and histone PTM profiling enhances the molecular and functional characterization of FXS patient samples
FMR1 methylation and hydroxymethylation are significantly correlated with ABC scores in male FXS patients
We report the identification of novel FMR1 regions of chromatin and (hydroxy)-methylation (5mC and 5hmC) perturbations associated with FMR1 epigenetic silencing in FXS patient blood-derived (PBMC) samples. The genomic regions of these epigenetic changes were consistent across individual patient samples, strengthening their functional relevance for FMR1 gene regulation. FMR1 locus-specific assays for selected epigenetic marks revealed inter-individual quantitative differences in FXS patients that may be related to FMR1 locus expression and disease severity. We have integrated histone post-translational modifications, DNA (hydroxy)-methylation, FMR1 gene expression data, and clinical severity data to identify new molecular and functional links between molecular and clinical features. In particular, we have established 5hmC and H4K20me1 as novel epigenetic marks/pathways whose loss is associated with FMR1 epigenetic silencing in FXS cells, providing new opportunities for enhanced molecular stratification of FXS patients beyond the existing CGG repeat length and DNA methylation assessments.
FMR1 locus regulation, beyond the transcription start site region
While the region surrounding the FMR1 TSS (promoter/CpG island and upstream part of the first intron) was previously reported to show epigenetic changes in FXS , we report the identification of several new regions of epigenetic changes within and flanking the FMR1 coding region. The identification of epigenetic changes outside of the FMR1 coding region suggests the presence of novel structural or regulatory regions (e.g., enhancer) regulating the expression of either FMR1 or non-coding RNA variants within the FMR1 locus, an observation that warrants further functional characterization. Among the broad changes taking place in FMR1 gene body, three regions are of particular interest. The region surrounding the FMR1 TSS where DNA hypermethylation is accompanied by a heterochromatin switch with a decrease of the active histones marks H3K4me2/H4K20me1 and an enrichment of the repressive marks, mainly H3K9me3 and H3K9me2. The 5′ part of the first intron of FMR1 is of interest as it is characterized by a lower CpG density as compared to the promoter and as previously reported by Godler et al. ; it is likely participating to the suppression of FMR1 gene expression as a consequence of CGG repeat expansion. The genomic region surrounding the referenced FMR1 AS antisense RNA TSS and the alternative-splicing rich region located between introns 13 and 16 (reported to contribute the diversity of the FMR1 isoforms [40–42]) represent two additional sites of robust epigenetic changes. Interestingly, both regions represent sites of decreased 5mC levels, particularly evident from fibroblast profiling data. Although it is known that the methylation of DNA at the promoter suppresses gene expression, the role of DNA methylation in gene bodies is unclear . Recent data support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies . Hypermethylation of FMR1 gene body in control fibroblasts may thus ensure the integrity of the FMR1 mRNA transcript(s) and avoid spurious transcription of this transcriptionally active locus. In the absence of transcription in FXS fibroblast cells, the normal regulation of FMR1 is perturbed and intragenic regions not labeled for hypermethylation, accounting for differential methylation in control and FXS fibroblasts. Overall, FMR1 flanking and intragenic regions of differential methylation/chromatin structure highlight novel regulatory features of FMR1 regulation, providing new insights into FMR1 gene regulation in normal, healthy control cells.
Novel epigenetic pathways associated to FMR1 epigenetic regulation
We report using hMeDIP-array/PCR that the promoter/intron 1 region of active FMR1 is enriched in 5hmC. Previous bisulfite sequencing analyses of the FMR1 promoter CpG island in normal individuals have consistently reported largely unmethylated DNA sequences spanning from the promoter region up to a boundary region located at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene . As bisulfite sequencing does not distinguish 5mC and 5hmC, we propose that only selected cytosines may be hydroxymethylated within healthy control FMR1 promoter. 5hmC is catalyzed by the TET family of proteins  suggesting a role for this pathway in the normal regulation of FMR1. 5hmC is believed to both represent an epigenetic mark of its own as well as contribute to active DNA demethylation, possibly providing protection against aberrant FMR1 promoter, CpG island and CGG repeats methylation in healthy individuals (5-50 CGG repeats). MeCP2 was one of the first proteins identified to bind to 5hmC . While MeCP2 is traditionally associated with gene repression through its binding to 5mC and recruitment of a co-repressor complex , it was shown to regulate gene activation in the brain upon binding to 5hmC, an interaction lost through MeCP2 point mutations in Rett syndrome patients [32, 45]. Differential 5hmC levels detected by epigenomic profiling suggest a role for this mark in the normal regulation of FMR1 gene expression in healthy cells. Interestingly, changes in 5hmC were previously observed during cerebellum development at genes regulated by the FMRP protein as well as at many genes linked to autism , thus reinforcing the importance of a TET-mediated 5hmC epigenetic pathway in normal and pathological regulation of FMR1.
Histone H4 Lys 20 mono-methylation (H4K20me1) is also affected in FXS cells. This epigenetic mark has been implicated in the regulation of diverse processes ranging from the DNA damage response, mitotic condensation, and DNA replication to gene regulation. PR-Set7/Set8/KMT5a is the sole enzyme that catalyzes H4K20me1 [47, 48]. Together, the identification of 5hmC and H4K20me1 enrichment at FMR1 in normal individuals highlights novel pathways whose deregulation upon CGG repeat expansion might contribute to FMR1 silencing and FXS physiopathology. The analyses presented here were performed in FXS fibroblasts and PBMC samples, both representing surrogate tissues for this neurological disorder. PBMCs contain an array of different blood cell types; the fibroblasts used in this study also originate from different tissues and from patients of different ages, all representing potential confounding factors when investigating epigenetic differences at the analyzed loci. An additional potential limitation relates to the relatively small sample size in these analyses (blood n = 4/8 controls/cases and fibroblasts n = 1/3 controls/cases). We thus cannot formally exclude cell-type and age-specific contributions to blood- and fibroblast-derived chromatin profiling data and future validation of key loci in sorted/purified cell populations from larger cohorts of healthy volunteer, and patient samples may be warranted to further explore the specificity of novel DNA methylation markers for FXS. In addition, to better understand the mechanisms associated to FMR1 silencing in the target tissue and cells, it will also be important to investigate the epigenetic landscape of the FMR1 locus in healthy and FXS brain samples.
Novel potential biomarkers for FXS diagnosis and drug response
The methylation status of FMR1 promoter/upstream intron 1 has been significantly correlated with the clinical phenotype of FXS patients [5, 16–19]. We hypothesized that additional epigenetic biomarkers within the FMR1 locus may enhance the development of novel clinical biomarkers for FXS disease states as well as support the discovery of novel mechanism-based therapeutic targets. Whilst 5mC measurement at the FMR1 promoter can detect full-mutated/hypermethylated FMR1 alleles in a mosaic cell population, we propose that the measurement of 5hmC (and other novel epigenetic features of FMR1 epigenetic activation) may help detect the unmethylated (pre- and full-mutated) alleles within a mosaic population.
This in-depth analysis of the FMR1 locus epigenetic landscape in full-mutation FXS patient samples identifies unprecedented regions of chromatin modifications that are characteristic of a broad FMR1 repressive epigenetic switch. Importantly, decreased levels of 5-hydroxymethylation (5hmC), a recently rediscovered epigenetic mark, correlate with FXS patient disease status. We propose that the combined measurement of 5mC and 5hmC from single patient individuals may provide novel diagnostic and therapeutic opportunities for FXS syndrome.
For this study, purified PBMC samples from eight fragile X patients were used. Subjects were male, aged 12–45 years (inclusive), with a confirmed diagnosis of FXS based on genetic sequencing results (full mutation, >200 CGG repeats). They were required to have a Clinical Global Impressions of Severity (CGI-S) score of ≥4 (moderately ill) and a score of ≥20 on the ABC-C scale (at screening). The study protocol and all amendments were reviewed by the Independent Ethics Committee for the study center. The study was conducted according to the ethical principles of the Declaration of Helsinki. Informed written consent was obtained from each patient or parent/legal guardian before randomization. PBMCs were purified from blood using 8 mL capacity PBMC separator tubes (BD Vacutainer CPT, BD). Healthy control samples were obtained from Bioreclamation (n = 4, for hMeDIP) and from consented voluntary donors (n = 3, for ChIP). GM05848 fibroblasts from a 4-year-old fragile X patient, GM07072 fetal lung fibroblasts from 22-week-old fetus with a fragile mutation, and GM09497 fibroblasts from a 28-year-old fragile X patient, from Coriell Institute for Medical Research were grown in D-MEM supplemented with 15 % FBS, penicillin/streptomycin, 2-mercaptoethanol (0.1 mM), and sodium pyruvate. The ATCC BJ1 neonatal fibroblast cell line used as control was cultured in the same condition.
FMR1 gene expression assays
FMR1 mRNA expression levels in the blood were measured by quantitative real-time polymerase chain reaction (qRT-PCR); 500 ng of total RNA isolated from the blood samples collected in PAXgene tubes was reverse transcribed to cDNA using random hexamers and the high-capacity cDNA reverse transcription kit with RNAse inhibitor according to the manufacturer’s procedure (Applied Biosystems, Foster City, CA). qPCR was performed using the ABI PRISM® 7900HT Sequence Detection System (Applied Biosystem). The following TaqMan assays obtained from Applied Biosystems were used: FMR1: Hs00924544_m1; actin B (ACTB): Hs99999903_m1; beta-glucuronidase (GUSB): Hs99999908_m1. All samples were processed in triplicate with a 25-ng cDNA (total RNA equivalent) for FMR1 and 10-ng cDNA (total RNA equivalent) for reference gene assays (ACTB, GUSB). The qPCR consisted of one step at 50 °C for 2 min, one denaturing step at 95 °C for 10 min followed by 40 cycles of melting (15 s at 95 °C), and annealing/extension (1 min at 60 °C). To correct for any variation in mRNA content and enzymatic efficiencies, FMR1 gene expression levels were normalized to the values of the most stable reference genes, ACTB (actin beta) and GUSB (glucuronidase beta). The data is presented as normalized relative quantity (NRQ). A Cq (Ct) value >38 was considered to be the background of the assay.
(Hydroxy)methylated DNA immunoprecipitation
Genomic DNA was prepared by overnight proteinase K (pK) treatment in lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 100 mM NaCl, 0.5 % SDS), phenol-chloroform extraction, ethanol precipitation, and RNaseA digestion. Genomic DNA was sonicated (Bioruptor, Diagenode) to produce random fragments ranging in size from 300 to 1000 bp and 2.5 μg of fragmented DNA was used for a standard hMeDIP assay. DNA was denatured for 10 min at 95 °C and immunoprecipitated for 3 h at 4 °C with 15 μl of monoclonal antibody against 5-methylcytidine (BI-MECY-1000, Eurogentec) (MeDIP) or with 1 μl of a rabbit polyclonal antibody against 5-hydroxymethylcytosine (#39769, active Motif) (hMeDIP) in a final volume of 500 μl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05 % Triton X-100). The mixture was incubated with 40 μl magnetic beads (MeDIP: Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen) for 2 h at 4 °C/hMeDIP: Dynabeads Protein G (#100.03D, Invitrogen) for 1 h at 4 °C) and washed three times with 1 ml of IP buffer. Beads were subsequently treated with proteinase K for 3 h at 50 °C and the methylated DNA recovered by phenol-chloroform extraction followed by ethanol precipitation. For microarray analysis, 50 ng of input DNA and 1/2 (h)MeDIP-enriched DNA was amplified using WGA2: GenomePlex Complete Whole Genome Amplification kit (Sigma). Amplified DNA was used for real-time qPCR quantification and sent to Roche Nimblegen (Madison, USA) for Cy3 and Cy5 labeling and hybridization on 12 × 135 k NimbleGen custom arrays.
Native chromatin immunoprecipitation (N-ChIP) protocol was based on a published protocol  with some modifications. Aliquots of 5 million (10 million for Fibroblasts) cells were thawed on ice and resuspended in 150 μl (250 μl fibroblasts) of buffer 1 (0.3 M sucrose, 15 mM Tris (pH 7.5), 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA); 150 μl (250 μl fibroblasts) of buffer 1 with detergent (buffer1 including 0.5 mM DTT, 0.5 % Igepal, and 1 % DOC) were added followed by an incubation on ice for 10 min, and 300 μl (500 μl fibroblasts) of MNase buffer (0.3 M Sucrose, 85 mM Tris, 3 mM MgCl2, 2 mM CaCl2) containing 0.4 U MNase (0.64 U fibroblasts) (Sigma) were added to each tube. Digestion mixes were incubated at 28 °C using a thermomixer (Eppendorf) for 8 min (20 min fibroblasts) shaking at 500 rpm. Digestion was stopped by adding EDTA to a final concentration of 5 mM and tubes were left at room temperature for 5 min. Non-soluble fractions were removed by centrifugation at 18,000g for 10 min and collecting the supernatant. The pellet was discarded; 5 to 10 μg of chromatin was used for the immunoprecipitation with each antibody: H3K4me2 (07-030, Millipore), H3K9me2 (39239, Active Motif), H3K9me3 (9754S, Cell Signaling), H4K20me1 (39727, Active Motif), H4K20me3 (07-749, Millipore), H3K36me3 (Ab5090), H3K9ac (07-352, Millipore), H4K16ac (07-329, Millipore), and H3K27me3 (07-449, Millipore). The immunoprecipitation, washes, and DNA purification were done with Magna ChIP™ A Chromatin Immunoprecipitation Kit (Millipore #17-610) following manufacturer’s protocol. For microarray analysis, input DNA and entire ChIP DNA was amplified using WGA2: GenomePlex Complete Whole Genome Amplification kit (Sigma) according to . Amplified DNA was used for real-time qPCR quantification and sent to Roche Nimblegen (Madison, USA) for Cy3 and Cy5 labeling and hybridization on 12 × 135 k NimbleGen custom arrays.
Real-time PCR was carried out using SYBR Green PCR Master Mix (Applied Biosystems) and using an ABI PRISM SDS 7900HT machine (Applied Biosystems). Primers are listed in Additional file 9: Table S2.
FMR1 locus microarray design and data analyses
The custom Nimblegen array consists of 27,656 different targeted (with known genomic location) 50-mer probes and 30,039 random probes (no matches in the human hg19 genome); 4117 probes cover the broader FMR1 locus, a 247-kbp region encompassing FMR1 (chrX:146,993,469-147,032,647), FMR1NB (chrX: 147,062,849-147,108,187), and up to 100 kb of flanking genomic DNA sequence (chrX: 146,911,760–147,159,387). The array design is available upon request. M values (log2 (IP-channel/input-channel)) were calculated per targeted probe and normalized for each chip using Loess to account for non-linear dye bias. Arrays of the same IP antibody were then normalized across arrays by scaling to the same median absolute value. Targeted probes are present four times on the array (with different location), and these were summarized by averaging after normalization. All pre-processing was performed in the R-programming language; the limma package of Bioconductor was used for normalization.
Using the EZ DNA MethylationTM Kit (ZYMO Research), 200–300 ng of genomic DNA was bisulfite treated according to the manufacturer’s protocol and eluted in 30 μl. Pyrosequencing probes were designed with the Pyromark Design 2.0 software package (QIAGEN). Primers for PCR amplification and sequencing as well as the sequence covered by each assay are indicated in Additional file 10: Table S3; 2 μl of converted DNA were used as input for PCR amplification using the AmpliTaq Gold DNA Polymerase (Applied Biosystems, N8080247), with one of two primers biotinylated. The temperature profile of the cycles was DNA polymerase activation at 95 °C for 15 min, denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, and extension at 72 °C for 1 min for the first cycle. For the next 19 cycles, the annealing temperature was decreased by 0.5 °C per cycle. Then, 36 cycles of amplification were performed at 53 °C, the final annealing temperature. The program was finished by a final elongation step at 72 °C for 10 min. Biotinylated PCR product were then purified and immobilized onto streptavidin-coated Sepharose beads (GE Healthcare). Pyrosequencing was performed on the PyroMark Q96 MD (QIAGEN) following the manufacturer’s instructions. Pyro QCpG 1.0.9 (QIAGEN) was used to quantify DNA methylation at single CpGs.
Data integration and statistical analyses
Percentage of methylation per CpG obtained by pyrosequencing was summarized by averaging the value of all CpGs per assay, 0 % being unmethylated and 100 % fully methylated. 5hmC MeDIP and ChIP real-time PCR data were first normalized using the efficacy of each qPCR assay. The ratio IP/input was calculated as percent of input and values were log base 2 transformed (adding a constant of 0.01 prior transformation) to meet the test assumptions. Significance levels of the mean difference in control and FXS PBMCs is indicated by “***” (p ≤ 0.001), “**” (p ≤ 0.01), “*” (p ≤ 0.05), or no star (p > 0.05) using a t test with unequal variance. The relationship between the different molecular endpoints (5mC, 5hmC, histones PTMs) to the clinical score (ABC-C score)  was assessed via ordinary linear regression analysis (including p value and R 2 for goodness of fit).
Aberrant Behavior Checklist Community Scale
- FMR1 :
fragile X Mental Retardation 1
fragile X Syndrome
methylated DNA Immunoprecipitation
messenger ribonucleic acid
peripheral blood mononuclear cells
quantitative polymerase chain reaction
transcriptional start site
We wish to thank L. Manzella, T. Zollinger, J. Decker, M. Larbaoui, E. Bertrand, and M. Marcellin (Novartis Institutes for Biomedical Research, Basel, Switzerland) for technical and analytical assistance, K. Johnson for general support, and F Pognan and A Vitobello for critical review of the manuscript. This work was supported by Novartis Pharma AG, Basel, Switzerland.
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