Genome-wide DNA methylation analysis of patients with imprinting disorders identifies differentially methylated regions associated with novel candidate imprinted genes

Abstract

Background: Genomic imprinting is allelic restriction of gene expression potential depending on parent of origin, maintained by epigenetic mechanisms including parent of origin-specific DNA methylation. Among approximately 70 known imprinted genes are some causing disorders affecting growth, metabolism and cancer predisposition. Some imprinting disorder patients have hypomethylation of several imprinted loci (HIL) throughout the genome and may have atypically severe clinical features. Here we used array analysis in HIL patients to define patterns of aberrant methylation throughout the genome.

Design: We developed a novel informatic pipeline capable of small sample number analysis, and profiled 10 HIL patients with two clinical presentations (Beckwith-Wiedemann syndrome and neonatal diabetes) using the Illumina Infinium Human Methylation450 BeadChip array to identify candidate imprinted regions. We used robust statistical criteria to quantify DNA methylation.

Results: We detected hypomethylation at known imprinted loci, and 25 further candidate imprinted regions (nine shared between patient groups) including one in the Down syndrome critical region (WRB) and another previously associated with bipolar disorder (PPIEL). Targeted analysis of three candidate regions (NHP2L1, WRB and PPIEL) showed allelic expression, methylation patterns consistent with allelic maternal methylation and frequent hypomethylation among an additional cohort of HIL patients, including six with Silver-Russell syndrome presentations and one with pseudohypoparathyroidism 1B.

Conclusions: This study identified novel candidate imprinted genes, revealed remarkable epigenetic convergence among clinically divergent patients, and highlights the potential of epigenomic profiling to expand our understanding of the normal methylome and its disruption in human disease.

Keywords: Epigenetics; Genome-wide; Imprinting.

Figure 1

Distribution of known and candidate differentially methylated CpG sites in (A) Beckwith–Wiedemann syndrome (BWS) and (B) transient neonatal diabetes (TND). In each case, the pie chart to the left shows CpG sites compared between cases and controls (in grey), including those meeting criteria for differential methylation; the pie chart to the right highlights hypomethylated CpG sites, including those in known clinically-relevant loci (red), loci reported to be imprinted (pink) and loci not currently reported to be imprinted, that is, candidate loci (blue). (C) Chromosome ideogram showing the distribution across all autosomes of known and candidate differentially methylated loci. Black dots represent known imprinted genes that were shown to be hypomethylated in the TND patient group in this study; the green dots represent known imprinted genes shown to be hypomethylated in the BWS patient group in this study. Red and blue squares correspond to candidate imprinted loci in TND-HIL and BWS-HIL, respectively. The names of imprinted loci associated with imprinting disorders are displayed next to loci, in black, where they were detected as hypomethylated in patient samples.

Figure 2

DNA methylation and expression analysis of NHP2L1 in patients with Beckwith–Wiedemann syndrome (BWS) and transient neonatal diabetes (TND). (A) Screengrab from UCSC genome browser representing the NHP2L1 gene and imprinted locus. The subregion highlighted in (B) is marked by a red double-ended arrow. Small numbers under the screengrab denote the exon numbering as used for expression analysis in (E); red asterisk indicates the position of the SNP analysed in (E). Note that NHP2L1 is transcription from right to left with respect to genomic orientation. (B) Divergent DNA methylation between normal controls and patients, detected by methylation array. Solid lines denote M-values (left axis). Dashed lines represent p values of methylation difference between patients and controls (right axis). Black line represents normal controls; blue lines represent averaged methylation of five BWS patients; red lines represent averaged methylation of five TND patients. (C) Illustrative electropherogram from methylation-specific PCR experiment showing difference in DNA methylation between a single patient and control. Amplicons derived from methylated and unmethylated DNA are marked by red and blue lines, respectively. (D) Summary of bisulfite cloning and sequencing experiment comparing a patient with a normal control. The circles represent CpG dinucleotides within a sequence amplified after bisulfite modification, with filled and empty circles representing methylated and unmethylated DNA sequences respectively. The number to the right indicates the number of times the sequence was detected in individual clones. In no case were methylated and unmethylated CpG dinucleotides detected within a single clone. (E) Allele-specific expression analysis of NHP2L1. Top electropherogram represents genomic sequencing across rs8779 showing heterozygous SNP. Lower electropherograms represent sequencing of RT-PCR products from pancreatic cDNA, amplified from exons 1–4 (biallelic expression) and 2–4 (monoallelic).

Figure 3

DNA methylation and expression analysis of WRB in patients with Beckwith–Wiedemann syndrome (BWS) and transient neonatal diabetes (TND). (A) Screengrab from UCSC genome browser, representing the WRB gene and imprinted locus. The subregion highlighted in (B) is marked by a red double-ended arrow. Small numbers under the screengrab denote the exon numbering as used for expression analysis in (E); red asterisk indicates the position of the SNP analysed in (E). (B) Divergent DNA methylation between normal controls and patients, detected by methylation array. Solid lines denote M-values (left axis). Dashed lines represent p values of methylation difference between patients and controls (right axis). Black line represents normal controls; blue lines represent averaged methylation of five BWS patients; red lines represent averaged methylation of five TND patients. (C) Illustrative electropherogram from methylation-specific PCR experiment, showing difference in DNA methylation between a single patient and control. Amplicons derived from methylated and unmethylated DNA are marked by red and blue lines, respectively. (D) Summary of bisulfite cloning and sequencing experiment comparing a patient with a normal control. The circles represent CpG dinucleotides within a sequence amplified after bisulfite modification, with filled and empty circles representing methylated and unmethylated DNA sequences, respectively. The number to the right indicates the number of times that sequence was detected in individual clones. In no case were methylated and unmethylated CpG dinucleotides detected within a single clone. (E) Allele-specific expression analysis of WRB. Top electropherogram represents genomic sequencing across rs1060180 showing heterozygous SNP. Lower electropherograms represent sequencing of RT-PCR amplicons in human fetal tissues as stated.