Written by Eli Sward PhD.
DNA methylation represents an important epigenetic modification and stands as a defining feature in gene regulation. Traditionally, whole genome bisulfite sequencing with short reads has been the gold standard for examining methylation patterns. However, the landscape is evolving rapidly, with long-read sequencing platforms providing DNA methylation simultaneously with variant calling, offering rapid data generation and distinctive advantages. In this blog, we will delve into the notable benefits of methylation analysis, explore its clinical relevance, and offer insights into the ways this invaluable information can be harnessed for viewing and analysis.
DNA Methylation and Its Impact on Gene Regulation
At a high level, epigenetic modifications serve as molecular signatures capable of influencing gene activity without perturbing the fundamental DNA sequence. DNA methylation is an epigenetic modification that entails the addition of a methyl group to the fifth carbon position of cytosine bases, a modification denoted as 5mC. This methylation event frequently occurs at cytosine-guanine (CpG) dinucleotides. Within the human genome there are approximately 28 million CpG-rich regions, spanning roughly 1 to 2 kilobases each, and are known as CpG islands (1). Notably, CpG islands are frequently located within gene promoters and near transcriptional start sites. In their unaltered state within normal cells, these CpG islands remain unmethylated, a condition that permits the binding of critical transcription factors that can lead to subsequent gene activation.
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DNA Methylation and the Phenomenon of Hypermethylation
However, when high levels of DNA methylation are observed within CpG islands, a phenomenon termed hypermethylation, the consequences are invariably linked to the repression or silencing of gene transcription (1). Given its potent regulatory influence over genes, DNA methylation has emerged as a focal point of scientific inquiry, profoundly impacting our understanding of processes ranging from development to cancer etiology and rare genetic disorders.
Within the realm of DNA methylation, a particularly intriguing avenue of investigation revolves around genomic imprinting. Genomic imprinting constitutes an epigenetic modification in the parental lineage, leading to differential expression of the two alleles of a gene in the progeny. Imprinting predominantly centers on differentially methylated regions (DMRs), a subset of which is known as imprinting control regions (ICRs)(2). These ICRs wield the remarkable ability to govern gene expression within imprinted domains, and distantly impact gene regulation.
Notably, perturbations of ICRs can lead to the loss of imprinting or the erosion of essential epigenetic marks. Several well-documented syndromes, including Beckwith-Wiedemann syndrome, Prader-Willi syndrome, and Angelman syndrome, to name a few (2), underscore the profound consequences of such perturbations. Take, for instance, Angelman syndrome, a condition associated with intellectual disability. In this syndrome, a single gene, UBE3A, which exhibits maternal expression, is lost. Imprinting aberrations in Angelman syndrome encompass the loss of maternal DNA methylation or maternal ICR deletion. These instances underscore the pivotal role played by DNA methylation and ICR homeostasis in shaping the clinical outcomes of imprinting disorders.
The Role of DNA Methylation in Fragile X Syndrome
DNA methylation also assumes significant importance in the context of rare diseases, with Fragile X syndrome serving as a prominent illustration. Individuals afflicted by Fragile X syndrome manifest delayed developmental milestones, intellectual disability, neurodivergent phenotypes, and notably, it stands as the most prevalent contributor to autism (3).
The underlying cause of this syndrome lies in a CGG trinucleotide repeat expansion, wherein the promoter region of the FMR1 gene harbors an excessive number of repeats, typically exceeding 200 copies (in contrast to the usual 6-50 copies). The presence of CpG-rich repeats within this region results in hypermethylation of the FMR1 promoter, leading to the transcriptional silencing of a critical RNA binding protein essential for the development of neuronal dendrites. This example underscores the pivotal role of CpG regions in the intricate landscape of DNA methylation, particularly in the context of rare diseases like Fragile X syndrome.
Advanced Technology for DNA Methylation Analysis
Having established the pivotal role of DNA methylation, our focus now shifts toward the domain of epigenetic sequencing and analysis. Within this realm, PacBio and Oxford Nanopore Technologies emerge as third-generation sequencing platforms that not only deliver standard variant calls but also unveil a unique capability: the provision of haplotype-specific . The DMR analysis is enabled due to the ability to call methylation.
To briefly discuss technicalities, the BAM files generated by these pipelines incorporate MM and ML tags. The MM tag serves the purpose of pinpointing the strand and position where the methylation modification was detected, while the ML tag offers insights into the probability associated with the presence of each methylation modification. The synergy of these tags empowers the creation of haplotype-resolved methylation profiles, whereby the color intensity mirrors the methylation levels. With this foundation laid, let us use an illustrative example.
In a recent study conducted on a cohort of individuals affected by rare diseases, the primary objective revolved around the utilization of HiFi sequencing to achieve genome-wide profiling of hypermethylation outliers. A key facet of this endeavor was the precise mapping of hypermethylation regions to regulatory elements (RE). Through this meticulous approach, the researchers uncovered a significant revelation: numerous instances of hypermethylation events within CpG regions were intricately linked to rare genetic variants situated in cis, with these variants manifesting either in proximity or at a distance from their target genes (4).
Illustrating this discovery, they identified a rare 2.6-kilobase insertion in cis, which was found to be responsible for inducing proximal RE hypermethylation at the DDB2 disease locus (refer to Figure 1). In Figure 1, the insertion is visually captured by the solid line, residing on haplotype 2, and its consequence—a notable hypermethylation signal—is depicted in striking red along the same allele, as illustrated by the dashed outline. This phenomenon, in essence, culminated in the transcriptional silencing of DDB2, a gene encoding a crucial protein engaged in the realm of DNA repair and maintenance (4).
It’s imperative to highlight that this investigation incorporated a comparative control, a fundamental requirement in the context of methylation analysis, to validate and contextualize their findings.
Figure 1: 2.6kb insertion (solid line) leading to hypermethylation (dashed line) on the DDB2 gene (4).
In the realm of DNA methylation analysis, the inclusion of case-control studies or parental methylation profiles is very important, depending on the research objectives and questions being addressed. However, beyond these crucial considerations, two additional factors demand attention: tissue specificity and time sensitivity.
The concept of tissue specificity underscores the realization that patterns of DNA methylation can exhibit significant variation among diverse cell types and tissues within the body. To illustrate, it’s worth noting that, except for the thymus, the brain has the highest overall level of DNA methylation (1). Consequently, it becomes imperative to exercise caution when extrapolating findings from one specific tissue or from blood to a broader global methylation profile.
Furthermore, the dynamic nature of DNA methylation patterns during development is increased by the importance of time sensitivity in DNA methylation analysis. This phenomenon is evident in the epigenetic clock—a concept that hinges on the accrual of DNA hypermethylation at specific CpG sites. Remarkably, these epigenetic signatures stand as some of the most robust biological correlates of aging in humans.
Given the multifaceted nature of DNA methylation, researchers have established numerous databases aimed at enhancing the precision and comprehensiveness of DNA methylation analysis. These resources are instrumental in navigating the intricacies posed by tissue specificity, temporal dynamics, and the manifold challenges in deciphering the epigenetic code.
Prominent Resources and Trends in DNA Methylation Analysis
A multitude of databases stand ready to facilitate the analysis and interpretation of methylation and epigenetic profiles. Among these valuable resources, the Encyclopedia of DNA Elements (ENCODE) and the NIH Roadmap Epigenomics Project emerge as indispensable sources of epigenetic insights, encompassing a diverse array of cell types and tissues within the human body. Likewise, the International Human Epigenome Consortium (IHEC) has a mission to map and comprehend the intricate landscape of epigenetic modifications across the human genome.
As the integration of third-generation sequencing techniques such as long-read sequencing data analysis, gains traction within the clinical domain, one can anticipate the emergence of an expanding array of information repositories and databases, purpose-built to streamline the identification and analysis of DNA methylation profiles. The evolving landscape promises enhanced precision and accessibility in the realm of epigenetic research and its clinical applications.
Collectively, methylation analysis is on a trajectory to offer unprecedented insights into genetic regulation, and its exploration within the clinical sphere is advancing at a rapid pace. From a broad perspective, I trust that this blog has provided valuable insights into the realm of methylation analysis and the intricate world of epigenetic modifications. Epigenetics, as a discipline, stands as an endlessly fascinating domain, likely to make substantial contributions to the expanding realm of multi-omics research and, critically, to the resolution of currently undiagnosed diseases.
References:
- Melanie Ehrlich (2019) DNA hypermethylation in disease: mechanisms and clinical relevance Epigenetics, 14:12, 11141-1163, DOI: 10.1080/155912294.2019.1638701
- Robertson, K. D. (2005). DNA Methylation and Human Disease. Nature Reviews, Volume(6), 597-610.
Meet Eli Sward, PhD, the Associate Director of Field Applications Support at Geneyx. Eli is a communications expert in genomics software. In his leisure time, he derives immense joy from savoring precious moments with his family, immersing himself in the beauty of the great outdoors, and engaging in a diverse range of sports activities, including tennis, basketball, golf, rock climbing, and hockey.
