Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome

Abstract

Introduction and ResultsImmunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues.ConclusionsIn conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing

“…Methylome studies carried out in ICF1 B-LCLs suggested that DNA methylation defects might be more extensive than what supposed until now (45,46). However, these studies did not explore which DNA methylation defects are dependent on mutant-DNMT3B, how they functionally impact on gene expression regulation, and whether this effect occurs influencing other epigenetic marks.…”

DNA Methylation in the Diagnosis of Monogenic Diseases

“…However, at least in a cohort of TBRS patients, the observed epi-signatures were not significantly influenced by cell-type variation [40]. Moreover, for some CRDs, reproducible epi-signatures have been obtained in both peripheral blood leukocytes and fibroblasts, after filtering out tissue-specific differences [41,43].…”

Genetics meets DNA methylation in rare diseases

“…Characterization of DNAme defects in ICF patients shows that, in addition to Sat‐2 and Sat‐3 repeats, reduced DNAme markedly affects non‐satellite NBL2 (now called SST1) and D4Z4 DNA repeats, subtelomeric regions, CpG islands of the inactive X chromosome in females, and a subset of germ line gene promoters . Hypomethylation of these germ line genes, which are maintained silent in all normal somatic cells, represents a specific pathological signature of DNMT3B dysfunction that could serve as a powerful biomarker for diagnosis, although the extent of its contribution to disease phenotype is unclear .…”

“…24 37 subtelomeric regions, 38 CpG islands of the inactive X chromosome in females, 39 and a subset of germ line gene promoters. [40][41][42][43] Hypomethylation of these germ line genes, which are maintained silent in all normal somatic cells, represents a specific pathological signature of DNMT3B dysfunction that could serve as a powerful biomarker for diagnosis, although the extent of its contribution to disease phenotype is unclear. 42 However, studies analyzing transcriptional defects as downstream consequences of DNAme defects have failed to provide a clear explanation for the immunological defects that characterize ICF patients, 32,44,45 and rather suggest that the perturbed gene expression in ICF cells is probably the consequence, rather than the cause, of defects in the maturation of patients' B-cells.…”