Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging

Abstract

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.

Figure 1.

TBRS DNMT3A variants are associated with widespread DNA hypomethylation. (A) Simplified pedigree indicating the genotyping of individuals in the Amish family investigated: (+/−) heterozygous carriers of the DNMT3A c.2312G > A p.(Arg771Gln) variant; (+/− Mosaic) the DNMT3A c.2312G > A p.(Arg771Gln) mosaic father; (−/−) wild-type individuals. Black shading indicates individuals with a phenotype consistent with TBRS, gray shading, the father with macrocephaly and mild intellectual impairment; and white shading, unaffected individuals. Each of these samples was profiled on the Illumina 450K DNA methylation array. (B) Volcano plot showing site-specific DNA methylation differences (x-axis) and −log₁₀ P-values (y-axis) from an analysis comparing Amish DNMT3A c.2312G > A; p.(Arg771Gln) pathogenic variant carriers and wild-type family members using the Illumina 450K array. Red values indicate the 2606 differentially methylated positions (DMPs) detected at a Benjamini–Hochberg FDR < 0.05. (C) Top 20 Gene Ontology enrichment analysis categories associated with the 2606 DMPs identified in DNMT3A c.2312G > A; p.(Arg771Gln) pathogenic variant carriers versus wild-type family members. (D) Comparison of DNMT3A c.2312G > A; p.(Arg771Gln) identified DMPs (log₂ fold change) relative to other DNMT3A TBRS-associated variants assessed in this study (all variants grouped and measured relative to controls). Pearson correlation coefficient = 0.6620, P-value <2.2 × 10⁻¹⁶. (E) Boxplot illustrating the DNA methylation changes observed in association with the DNMT3A TBRS variants studied at the DMPs identified in the Amish DNMT3A c.2312G > A p.(Arg771Gln) carriers. The predicted protein consequence of each DNMT3A variant studied is indicated: Pink indicates in-frame deletion; yellow, single-nucleotide variant; cyan, duplications predicted to result in a frameshift; green, Amish c.2312G > A; p.(Arg771Gln) variant.

Figure 2.

Schematic representation of DNMT3A. The positions of the disease-associated variants included in this study are indicated relative to the protein domain architecture. PWWP, proline-tryptophan-tryptophan-proline domain; ADD, ATRX-Dnmt3-Dnmt3L domain; MTase, Methyltransferase domain; AA, amino acid.

Figure 3.

Altered epigenetic aging is observed in methyltransferase-associated human growth disorders. (A) Scatter plot comparing “DNA methylation age” derived from the Illumina 450K data (y-axis) and actual chronological age (x-axis) in DNMT3A c.2312G > A p.(Arg771Gln) pathogenic variant carriers (red) versus wild-type family members (blue). Green indicates the mosaic individual. The linear regression model is also shown. (B) Scatter plot comparing DNA methylation age versus chronological age in patients with Sotos syndrome. In-frame legend illustrates the different NSD1 pathogenic variants studied. (C) Scatter plot comparing DNA methylation age versus chronological age in patients with Kabuki syndrome. In-frame legend illustrates the different KMT2D pathogenic variants studied. (D) Boxplot comparing the epigenetic age acceleration rates found in association with TBRS DNMT3A variants, KMT2D Kabuki syndrome variants, and NSD1 Sotos syndrome variants. Each age acceleration observation is plotted as a circle. The dotted red line denotes no age acceleration.