Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57

Abstract

Background: Transient neonatal diabetes mellitus 1 (TNDM1) is a rare imprinting disorder characterized by intrautering growth retardation and diabetes mellitus usually presenting within the first six weeks of life and resolves by the age of 18 months. However, patients have an increased risk of developing diabetes mellitus type 2 later in life. Transient neonatal diabetes mellitus 1 is caused by overexpression of the maternally imprinted genes PLAGL1 and HYMAI on chromosome 6q24. One of the mechanisms leading to overexpression of the locus is hypomethylation of the maternal allele of PLAGL1 and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers.

Methods: Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound heterozygous ZFP57 mutations, three relatives with heterozygous ZFP57 mutations and five controls. Methylation status of selected regions showing aberrant methylation in the patients was verified using bisulfite-sequencing.

Results: We found large variability among the patients concerning the number and identity of the differentially methylated regions, but more than 60 regions were aberrantly methylated in two or more patients and a novel region within PPP1R13L was found to be hypomethylated in all the patients. The hypomethylated regions in common between the patients are enriched for the ZFP57 DNA binding motif.

Conclusions: We have expanded the epimutational spectrum of TNDM1 associated with ZFP57 mutations and found one novel region within PPP1R13L which is hypomethylated in all TNDM1 patients included in this study. Functional studies of the locus might provide further insight into the etiology of the disease.

Keywords: DNA methylation; Imprinting disorder; Next-generation sequencing; Transient neonatal diabetes.

Fig. 1

MBD-seq results of previously identified hypomethylated DMRs. Bars show methylation levels relative to controls. Error bars show 1X standard deviation. ^a: Letters T, P and N below bars indicate methylation results from previous studies [4, 5]. T: Total loss of methylation; P: Partial loss of methylation; N: Normal methylation. Stars indicate level of significance of MBD-seq results: *:P(adjusted) < 0.05; **: P(adjusted) < 0.005; ***: P(adjusted) < 0.0005

Fig. 2

Verification of MBD-seq results by bisulfite sequencing. The plot shows log2 ratios of methylation levels in patients and ZFP57 heterozygotes versus controls obtained by MBD-seq and BS-seq

Fig. 3

Venn diagram of overlapping aberantly methylated regions in the patients. Up-arrow: hypermethylated regions. Down-arrow: hypomethylated regions. The three hypomethylated regions in common of the four patients are PLAGL1:alt-TSS-DMR, GRB10:alt-TSS-DMR and PPP1R13L

Fig. 4

Hypomethylation of PPP1R13L in the TNDM1 patients. UCSC Genome Browser screenshot with bedgraph tracks showing MBD-seq reads from the patients (TNDM1) and controls. All patients show hypomethylation of the CpG island indicated by red dots, whereas no changes in methylation is detected elsewhere in the gene. Genomic positions of ZFP57 binding motifs (TGCCGC) are indicated below the CpG islands

Fig. 5

Enrichment of the ZFP57 binding sequence in the hypomethylated regions. The number of occurrences of the TGCCGC sequence were counted in the 52 hypomethylated regions shared between at least two patients (orange bars) and in the 17,123 methylated regions detected in any of the samples (blue bars)