Identification of epigenetic signature associated with alpha thalassemia/mental retardation X-linked syndrome
- Laila C. Schenkel1,
- Kristin D. Kernohan2,
- Arran McBride2,
- Ditta Reina8, 9,
- Amanda Hodge8, 9,
- Peter J. Ainsworth1, 3, 4, 5, 6, 7,
- David I. Rodenhiser4, 5, 6, 7,
- Guillaume Pare8, 9,
- Nathalie G. Bérubé4, 5, 6, 7,
- Cindy Skinner10,
- Kym M. Boycott2,
- Charles Schwartz10 and
- Bekim Sadikovic1, 3, 7Email author
© The Author(s) 2017
Received: 7 November 2016
Accepted: 1 March 2017
Published: 10 March 2017
Alpha thalassemia/mental retardation X-linked syndrome (ATR-X) is caused by a mutation at the chromatin regulator gene ATRX. The mechanisms involved in the ATR-X pathology are not completely understood, but may involve epigenetic modifications. ATRX has been linked to the regulation of histone H3 and DNA methylation, while mutations in the ATRX gene may lead to the downstream epigenetic and transcriptional effects. Elucidating the underlying epigenetic mechanisms altered in ATR-X will provide a better understanding about the pathobiology of this disease, as well as provide novel diagnostic biomarkers.
We performed genome-wide DNA methylation assessment of the peripheral blood samples from 18 patients with ATR-X and compared it to 210 controls. We demonstrated the evidence of a unique and highly specific DNA methylation “epi-signature” in the peripheral blood of ATRX patients, which was corroborated by targeted bisulfite sequencing experiments. Although genomically represented, differentially methylated regions showed evidence of preferential clustering in pericentromeric and telometric chromosomal regions, areas where ATRX has multiple functions related to maintenance of heterochromatin and genomic integrity.
Most significant methylation changes in the 14 genomic loci provide a unique epigenetic signature for this syndrome that may be used as a highly sensitive and specific diagnostic biomarker to support the diagnosis of ATR-X, particularly in patients with phenotypic complexity and in patients with ATRX gene sequence variants of unknown significance.
KeywordsATRX DNA methylation Epi-signature Intellectual disability Biomarker
An emerging development in the field of medical genetics has been the identification of Mendelian disorders involving genes encoding the writers, erasers, readers and remodelers of the epigenetic machinery . Building on several decades of evidence regarding the functions of covalent DNA methylation [2, 3] and histone modifications  in regulating gene transcription, it is evident that mutations in the proteins responsible for creating, interpreting or removing the broad arrays of epigenetic marks can be linked to genetic conditions including cancer , imprinting disorders and/or single-gene disorders .
Along with these discoveries came the opportunity, not only for the elucidation of underlying molecular mechanisms altered in these disorders, but also for the identification of epi-signatures that can be diagnostically useful, specifically where patients express a subset of clinical manifestations associated with a phenotypic spectrum shared across more than one syndrome, making a specific clinical diagnosis difficult to reach.
Among the rapidly expanding number of proteins responsible for chromatin maintenance and remodeling related to transcription is alpha thalassemia/mental retardation X-linked (ATRX; NG_008838.2). Mutations in the ATRX gene cause alpha thalassemia/mental retardation X-linked syndrome (ATR-X, OMIM 301040), a disorder characterized by moderate to severe intellectual disability, expressive language disorder, characteristic facial gestalt during infancy, often associated with hematological signs of alpha thalassemia .
The ATRX protein functions as an agent of ATP-dependent chromatin remodeling and is a member of the SWI/SNF superfamily of proteins. The latter can have a wide variety of cellular functions, as described in detail in several recent reviews [8–10]. Briefly, ATRX protein is involved in cellular processes such as meiosis, mitosis, DNA repair and regulation of transcription through an effect on chromatin [11–15]. Disruption of these activities may contribute to developmental abnormalities associated with the ATR-X syndrome.
Within the ATRX protein, a histone-binding ATRX–DNMT3–DNMT3L (ADD) domain can sense the methylation modifications of both H3K4 and H3K9 , essentially acting as an interpreter of these histone states. ATRX is also known to associate with the transcription cofactor DAXX. ATRX–DAXX complex is responsible for deposition of histone H3.3 at the telomeric and pericentromeric heterochromatic regions within chromosomes . Loss of ATRX in ES cells leads to the loss of histone H3.3 at imprinting control regions and telomeric regions, along with the concurrent loss of H3K9me3 [18, 19]. ATRX has also been linked to DNA methylation regulation, as mutations at the ATRX gene result in DNA methylation changes at subtelomeric and repetitive regions .
The role of ATRX as a regulator of heterochromatin dynamics raises the possibility that mutations in ATRX may lead to downstream transcriptional effects across the complex of genes or repetitive regions involved in the global context of the disorder, in addition to explaining phenotypical differences in these patients. For example, ATRX mutations affect the expression of α-globin gene cluster, causing α-thalassemia . Mechanistically, α-globin cluster, among other genes, has G-rich tandem repeats (TRs) sites, which have been reported to bind ATRX resulting in H3.3 deposition and gene expression regulation. In addition, it was suggested that the differences in size of these TRs among ATR-X patients contribute to the ranges in severity of the syndrome .
The orchestrated regulation of epigenetic mechanisms, including associations between ATRX and DNA methylation [11, 12], is essential for tissue homeostasis, cell identity and proper human development. Here, we describe the findings of a genome-wide DNA methylation array (GWMA) performed on peripheral blood samples from patients with ATR-X and show the genome-wide changes in DNA methylation that occur in patients with this epigenetic syndrome. We have identified a specific epi-signature of differentially hypo- and hypermethylated genes in patients clinically diagnosed with ATR-X syndrome. Our study shows the preponderance of differentially methylated genes within, or adjacent to, pericentromeric or telomeric chromosomal regions, suggesting a major role of heterochromatin in the pathophysiology of ATR-X, linked to the disruption of ATRX function in the context of its role as a regulator of heterochromatin dynamics.
The epi-signature identified in blood samples from ATR-X patients
ATR-X methylation signature: significant regions detected by methylation array in ATR-X patients (n = 17) compared with controls (n = 210)
Distance to gene (bp)
Within CpG island
We then performed a single-patient analysis to identify possible patient-specific, as opposed to patient cohort-specific, recurring methylation changes, using statistical parameters of p < 0.01, methylation difference > ±15%, across four consecutive probes. First we observed that the cohort-specific epi-signature was absent in one of the patients (patient #12). A follow-up assessment showed that although this patient has previously been identified to carry a possible mutation in the ATRX gene, more recent data demonstrated that c.5579A>G; p.N1860S in the ATRX gene is indeed a benign polymorphism and hence this patent did not have the ATR-X syndrome. The remaining 17 patients with molecular diagnosis of ATR-X, using the above statistical cutoffs, showed an average of 9.8 significant loci from the epi-signature per individual, with the minimum of four significant loci observed in two patients (#13 and #15). To evaluate the specificity of this assay, we applied the ATR-X epi-signature in randomly selected 15 individuals, which included normal controls that were not part of the discovery cohort, as well as individuals with Fragile X syndrome, Prader–Willi syndrome, Angelman syndrome and Beckwith–Wiedemann syndrome. The majority of these individuals did not present any statistical significant changes at the ATRX epi-signature loci. Six control individuals showed significant changes at 1 or 2 ATRX epi-signature loci (POTEA and PACSIN1). These two genes had slightly higher level of variable DNA methylation and were as a result removed from the final ATRX epi-signature.
Biological pathways identified by pathway analysis of the differentially methylated genes in ATR-X
Nucleic acid metabolic process
Number of genes in group
Fisher’s exact enrichment score −ln(p value)
Fisher’s exact right-tail p value
Technical validation of the methylation array
Uneven distribution of altered methylation sites across the genome
The interplay between ATRX and DNA methylation has been evidenced by early studies in EBV-transformed cells from patients with ATR-X and controls, showing that mutations at the ATRX gene cause changes in the pattern of methylation at subtelomeric and rDNA sequences . Furthermore, loss of ATRX expression has been linked to extensive epigenomic alteration including CPG island hypermethylation observed in astrocytic tumors [31, 32]. The involvement of ATRX in the regulation of DNA methylation was further supported by the discovery that ATRX interacts with MeCP2 and cohesion subunits in the brain [33, 34]. MeCP2 is a methyl-CpG-binding domain protein with affinity for GC-rich sequences and methylated DNA which in turn facilitates the recruitment of histone modifiers and chromatin remodeling complexes . Similar to ATRX, MeCP2 is essential for neurodevelopment and mutations or duplications of the MeCP2 gene cause Rett syndrome, a neurodevelopmental disorder . In addition, cohesin proteins play a role in the regulation of chromosome organization and gene expression by binding to unmethylated CTCF-associated regions and mutations at cohesin genes are associated with the developmental defects seen in patients with Cornelia de Lange syndrome . MeCP2 was shown to recruit the helicase domain of ATRX to heterochromatic regions in a DNA methylation-dependent manner . In addition, MeCP2 has been reported to interact with DNA methyltransferase 1 in order to perform maintenance methylation in vivo , as well as with histone H3 lysine 9 methyltransferase enzymes, to reinforce a repressive chromatin state by bridging DNA methylation and histone methylation .
Both ATRX protein and de novo DNA methyltransferases DNMT3A/B/L contain a histone-binding domain (ADD) that has been shown to play a role in the establishment and maintenance of DNA methylation patterns. The ADD domain interacts with specific methylation modifications of histone lysine 4 and 9 (H3K4 and H3K9). The H3K4 methylation is associated with gene transcription and promoters DNA hypomethylation, whereas methylation of H3K9 is a heterochromatin-associated mark associated with transcriptional repression and DNA hypermethylation [16, 17]. H3K4 and H3K9 methylation is proposed to act as chromatin-based signals for regulation of DNA methylation, while ATRX–heterochromatin interaction depends on these histone methylation markers . ATRX ADD domain binds to the methylated H3K9 (H3K9me3) in conjunction with unmodified H3K4 which are commonly seen in the repressed repeat elements. Therefore, mutations that functionally disrupt the ATRX protein and result in “mis-targeting” of ATRX–heterochromatin interaction may provide a mechanism for abnormal DNA methylation patterns in patients with the ATR-X syndrome. While ATRX does not contain a DNA methyltransferase domain, we and others  have clearly shown association between ATRX mutation and abnormal patterns of DNA methylation.
The mechanism for ATRX induction of DNA methylation aberrations is not well known. Evidence has shown an overlapping function of ATRX and ATF7IP2. A genome-wide promoter DNA methylation study, using methylation-dependent immunoprecipitation–Chip assay, has demonstrated hypermethylation at ATF7IP2 gene in patients with ATRX mutation . ATF7IP2, also known as MBD1-containing chromatin-associated factor 2, is known to bind to the transcription repression domain of the methylated cytosine-binding protein MBD1, as well as to interact with the H3K9 methyltransferase SETDB1 . The overlapping protein interaction of ATRX–ATF7IP2 suggests that they form part of the same repressive chromatin complex, which involves ATF7IP2-induced H3K9me3 and ATRX binding to H3K9me3. There is also evidence for ATF7IP2 and ATRX transcriptional activation role, through SP1 and DAXX interaction, in promyelocytic leukemia nuclear bodies [28, 41, 42]. These data suggest that ATRX and ATF7IP2 have overlapping repressive/activating chromatin remodeling properties and potentially function in overlapping gene regulation pathways.
Most studies assessing the regulation of DNA methylation by ATRX have been focused on repetitive sequences and gene-specific methylation analysis. A recent study using methylation-sensitive restriction endonuclease has shown that ATRX mutations are associated with alterations in the DNA methylation profiles in highly repetitive sequences . Another study using bisulfite mutagenesis analysis in mice model has demonstrated that specific gene activation at ancestral pseudoautosomal regions, which are repetitive sequences regulated by ATRX, does not involve gene-specific changes on DNA methylation, but relies on the ATRX-dependent H3.3 deposition mechanism . However, none of these studies have analyzed global DNA methylation and/or specific gene CpG islands methylation in non-repetitive sequences. By using a high-resolution methylation array technique and a large reference cohort (controls), our study has clearly shown the existence of a pattern of DNA methylation changes, including in promoter CpG islands, telomeric and pericentromeric regions, in patients with ATR-X. Accordingly, in our study, most of the DNA methylation changes observed in patients with an ATRX gene mutation were localized at telomeric and pericentromeric regions. How the epigenetic consequences of ATRX mutations actually result in the disease phenotype is not well understood. It is possible that the methylation alterations could result in differences in transcriptional regulation. For example, hypomethylation in a gene promoter CpG island may result in increased transcription, whereas hypermethylation may result in decreased transcriptional activity . Gene pathway analysis showed that many of the genes identified in the ATR-X epi-signature are associated with DNA and RNA metabolic process, which may be involved in the regulation of specific gene expression and corroborate to the cardinal developmental processes disrupted in this rare disease; however, further research involving integrative analysis of gene expression and DNA methylation profiling to investigate the relationship between these DNA methylation changes and gene expression is warranted.
Here, we propose that the most significant and recurrent regions altered in the genomic DNA of patients with ATR-X, consisting of 14 loci, provide an epigenetic signature for this syndrome which may be used as a high sensitive and specific diagnostic biomarker to support the diagnosis of ATR-X, particularly in patients with phenotypical complexity and/or with ATRX gene sequence variants of unknown significance. Previous findings have demonstrated evidence of loss of DNA methylation in the repetitive elements . While theoretically, it would be possible to use repetitive element methylation patterns as part of a unique ATRX mutation epi-signature, routine analysis of the repetitive elements DNA methylation pattern can be challenging due to the lack of specificity for assays designed for assessment of methylation of genomic repeats. Furthermore, most array or sequencing-based bisulfate protocols are limited to targeting unique genomic sequences. Therefore, identification of a robust unique epigenetic signature across a large number of unique genetic sequences that we describe in this manuscript presents an opportunity for utilization of these findings in routine clinical diagnostics.
In addition to the ATR-X epi-signature described here, our group has recently demonstrated unique DNA methylation signatures in patients with two other conditions, including Floating–Harbor syndrome, which is caused by mutation in the SRCAP gene, as well as cerebellar ataxia, deafness and narcolepsy syndrome, which is caused by mutations in the DNMT1 gene [44, 45]. Other groups have also identified epi-signatures in patients with Sotos syndrome , and the X-linked intellectual disability caused by the KDM5C gene . Taken together, these studies demonstrate the ability of genome-wide methylation array to accurately diagnose multiple epigenetic disorders. Utilization of this technology in routine clinical practice will enable the discovery of new epigenetic biomarkers and will serve to enhance our understanding of human disease etiology. However, the identification of epigenetic variants of unknown clinical significance (E-VUS) will require delivery of testing to be performed in regulated clinical laboratories along with an adequate control cohort of normal samples, together with the development and implementation of clinical and laboratory testing guidelines, and availability and integration with pre- and posttest genetic counseling.
In conclusion, the observation of genome-wide epigenetic defects in ATR-X patients expands our understanding of the pathology of this disease, in which specific DNA methylation changes could lead directly to an aberrant expression of a number of genes in ATRX-deficient patients, particularly, but not restricted to telomeric and pericentromeric regions, thus contributing to the phenotypes associated with ATR-X syndrome. In addition, the unique epi-signature identified for ATR-X syndrome can now be used as an epigenetic biomarker to support the diagnosis of new patients using a sensitive, specific and cost-effective GWMA testing protocol.
Sample collection, DNA extraction and genotyping
Clinical and molecular characteristics of ATR-X patients referred for methylation study
ATR-X patient no.
c.7366_7367 InsA; p.M2456Nfs X42
Deletion of exon 2
The methylation array of these patients was compared with a reference cohort composed of controls and individuals previously referred for microarray with no significant methylation alteration. The reference cohort (controls) is composed of 210 male controls with average age of 7.3 years (2 m–53 years).
Methylation array and data analysis
The DNA methylation array was performed using the Infinium HumanMethylation450 BeadChip (Illumina) according to standard protocol at the Genetic and Molecular Epidemiology Laboratory at McMaster University. The array coverage includes >485,000 individual methylation sites, 99% of RefSeq genes and 96% of CpG islands. Beta and intensity values for methylation were generated using the Illumina Genome Studio Software, and .idat files were imported to Partek Genomic Suite software (Partek GS). The patient cohort was compared with the laboratory reference cohort. Statistical analysis was performed to compare ATR-X patients versus the control cohort using the ANOVA test to generate probe-level statistics, including p value (t test), F value (signal to noise) and estimate value (net methylation difference). The cutoff of estimate > ±0.20, p < 0.01, F > 50 and probes >4 was used to select the top significant regions to be included in the epi-signature. A less stringent cutoff of estimate > ±0.15, p < 0.01, F > 50 and probes > 3 was used for pathway analysis and karyogram view in order to include a larger number of regions in those analysis. Significant regions were mapped against the CpG islands and gene promoter regions. Genomic visualization of the data was performed using the karyogram view toll (Partek GS) for chromosome distribution of differentially methylated regions, and the Genomic Browser Wizard (Partek GS) for locus-specific methylation levels.
The top 45 differentially methylated genes identified using a less stringent cutoffs (Additional file 2) were assessed using the pathway analysis tool in the Partek Genomics Suite software. Briefly, statistical analysis included Fisher’s exact test and was restricted to functional groups at least two genes. Results show the enrichment p value (p value of the Fisher exact test reflective of the number of the genes in versus not in the list or functional group) and the enrichment score (negative log of the enrichment p value; a high score indicates that the genes in the functional group are overrepresented in the gene list).
Genomic DNA isolated from blood of ATR-X patients (n = 2) and controls (n = 2) was sodium bisulfite treated using the EZ DNA Methylation-Direct Kit (Zymo Research) according to manufacturer’s instructions. DNA was amplified by nested PCR and the resulting products ligated into the pGEM-T Easy vector using a TOPO-TA cloning kit (Invitrogen). Positive clones were sequenced with Applied Biosystem 3730xl DNA Analyzer technology (Center for Applied Genomics, McGill University). Clones were accepted at ≥95% conversion. Non-converted cytosine residues and mismatched base pairs were used to ensure all clones originated from unique template DNA.
alpha thalassemia/mental retardation X-linked protein
alpha thalassemia/mental retardation X-linked syndrome
death domain-associated protein
- ES cells:
embryonic stem cells
epigenetic variants of unknown clinical significance
genome-wide DNA methylation array
histone 3 lysine 4
histone 3 lysine 9
lysine (K)-specific demethylase 5C
methyl CpG-binding protein 2
- Partek GS:
Partek Genomic Suite software
polymerase chain reaction
Snf2-related CREBBP activator protein
SWItch/sucrose non-fermentable family
LCS analyzed the methylation data and was a major contributor in writing the manuscript. KDK and AM performed bisulfite methylation analysis. DR and AH performed the methylation microarray. PJA, NGB and DIR contributed to data interpretation and manuscript writing. KMB, GP and BS supervised the methylation study and contributed to manuscript writing. C Schwartz and C Skinner provided the samples and clinical data for the study. All authors read and approved the final manuscript.
We thank the patients and their families for their participation in studies conducted by the Greenwood Genetic Center. Dedicated to the memory of Ethan Francis Schwartz (1996–1998).
The authors declare that they have no competing interests.
Availability of data and materials
Methylation data can be provided upon request.
Ethical approval and consent to participate
The Self Regional Healthcare Institutional Review Board approved the project (IRB #26). All patients provided a written informed consent before their inclusion in the study in accordance with the Declaration of Helsinki.
This project was supported in part by a Grant from the South Carolina Department of Disabilities and Special Needs (SC DDSN). This work was also supported by the Care4Rare Canada Consortium (Enhanced Care for Rare Genetic Diseases in Canada) funded by Genome Canada, the Canadian Institutes of Health Research, the Ontario Genomics Institute, Ontario Research Fund, Genome Quebec and Children’s Hospital of Eastern Ontario Foundation.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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