DNA methylation in PRDM8 is indicative for dyskeratosis congenita

Abstract

Dyskeratosis congenita (DKC) is associated with impaired telomere maintenance and with clinical features of premature aging. In this study, we analysed global DNA methylation (DNAm) profiles of DKC patients. Age-associated DNAm changes were not generally accelerated in DKC, but there were significant differences to DNAm patterns of healthy controls, particularly in CpG sites related to an internal promoter region of PR domain containing 8 (PRDM8). Notably, the same genomic region was also hypermethylated in aplastic anemia (AA) - another bone marrow failure syndrome. Site-specific analysis of DNAm level in PRDM8 with pyrosequencing and MassARRAY validated aberrant hypermethylation in 11 DKC patients and 27 AA patients. Telomere length, measured by flow-FISH, did not directly correlate with DNAm in PRDM8. Therefore the two methods may be complementary to also identify patients with still normal telomere length. In conclusion, blood of DKC patients reveals aberrant DNAm patterns, albeit age-associated DNAm patterns are not generally accelerated. Aberrant hypermethylation is particularly observed in PRDM8 and this may support identification and classification of bone marrow failure syndromes.

Keywords: DNA methylation; Gerotarget; aplastic anemia; bone marrow failure; dyskeratosis congenita; epigenetic.

Figure 1. Epigenetic age-predictions of DKC samples

Biological age was estimated in DNAm profiles of four DKC samples (red; whole blood) and healthy control samples (blue; here 14 samples are exemplarily depicted from GSE32148) [33] using a model for epigenetic age-predictions by Horvath A. [13], or our previously published model based on 99 age-associated CpGs B. [12, 14] [46]. The mean absolute deviation of predicted and chronological age (MAD) is indicated for the control samples. Heatmaps of the 353 age-associated CpGs of the Horvath model C. and the 99 age-related CpGs of our age-prediction model D. demonstrate similar DNAm patterns of DKCs patients and controls. Although DKC is considered to be a premature aging syndrome these broader signatures did not reveal the epigenetic age-acceleration in DKC that we previously described for three age-associated CpGs [12].

Figure 2. Dyskeratosis congenita is associated with hypermethylation in PRDM8

A. Scatter plots of mean DNAm levels (four DKC patients as compared to four age and gender matched controls) depict 764 hypo- and 1,369 hyper-methylated CpGs in DKC (adjusted P value < 0.05; relevant CpGs of PRDM8 are indicated in red). B. DNAm levels (beta-values) of CpGs associated with PRDM8 reflect hypermethylation at an internal promoter region in DKC patients (as compared to DNAm profiles of normal blood). The positions of two relevant CpGs are indicated (cg19409579 and cg27242132). C. Boxplots represent distributions of beta-values at cg27242132 in four DKC samples and 4,131 DNAm profiles of blood samples of 16 different studies (see also supplemental Figure 3). The 99 percentile of controls is indicated as dotted line (DNAm level of 52%). D. Quantitative RT-PCR reveals moderate down-regulation of PRDM8 expression in DKC patients (n = 5; two-sided Student's t-test; P = 0.0197). E. MassARRAY analysis of DNAm at cg27242132 (PRDM8) revealed consistent DNAm levels in neighboring CpGs (controls: n = 10; AA: n = 27; DKC: n = 11). F. In comparison to normal controls the DNAm levels at the CpG site cg27242132 were significantly higher in AA (P = 1.49E-05) and particularly in DKC patients (P = 2.51E-06).

Figure 3. DNAm in PRDM8 is indicative for DKC and AA

A. Telomere length of lymphocytes was measured by flow-FISH for 114 healthy individuals (grey and blue) [12], 11 dyskeratosis congenital patients (DKC; red) and 27 patients with aplastic anemia (AA; yellow). B. Delta telomere length - in relation to age-adjusted mean telomere length in 104 healthy controls - revealed telomere attrition particularly in DKC patients (P = 3.07E-08), but overall also in AA patients (P = 0.0232). C. Delta telomere length was plotted against DNAm in PRDM8 for DKC and AA samples. Cutoffs for 1 percentile in telomere length and 99 percentile in DNAm of PRDM8 are indicated by areas shaded in blue.