Array Pinpoints Imprinted Genes with Potential Links to Disease

Researchers from North Carolina State University have developed an array that assesses methylation levels of genes located in imprint control regions (ICRs) within the human genome. The array represents a cost-effective, efficient method for exploring potential links between environmental exposures and epigenetic dysregulation during the early developmental origins of diseases and behavioral disorders.

ICRs regulate the expression of imprinted genes – genes where only one parental copy of the gene is active, while the other copy is silenced early in development. Imprinted genes are of special interest to epidemiologists, geneticists, and toxicologists who study the connections between environmental influences and disease because the methylation marks that control their expression are susceptible to environmental influences.

These DNA methylation modifications can be stable throughout the life of the affected individual and may even be passed on to their children. This is of particular interest in epigenetics, which is the study of heritable changes in gene expression in the absence of DNA sequence changes.

“Methylation – whether a gene is ‘off’ or ‘on’ – is the easiest thing to look at when you’re investigating epigenetic effects,” says Cathrine Hoyo, professor of biological sciences at NC State and co-corresponding author of the work. “It serves as a jumping off point for figuring out relationships between the environment and gene expression.”

While methylation arrays do exist for investigating gene expression, the ones most commonly used do not include probes specific to ICRs. Instead, scientists interested in imprinted gene regulation must sequence the entire genome of a subject, which is costly, time-consuming and impractical in large population studies.

The new array contains 22,000 fluorescent probes that are specific to 1,000 of the known 1,488 ICRs in the human genome. The probes are short DNA sequences that target specific methylation sites within these ICRs, with alternate probes binding the methylated and unmethylated versions. The alternate probes for each target site have different fluorescent signals, so that the relative amount of each probe bound can be measured, and the methylation level for each site determined from the ratio of the specific probes.

As proof of concept, the research team used DNA from a set of Alzheimer’s patients to compare ICR methylation data from the array data to the methylation results obtained by whole genome sequencing, and found a significant correlation between the two methods. Most notably for clinical application, the results were complete after one week, as compared to potentially months required for interpreting full genomes.

“In large studies we have to screen the participants,” Hoyo says. “If there are 1,000 people in a study, it simply isn’t possible to do that many complete genomic sequences in a timely and cost-effective way. It is especially wasteful when you consider that we are only interested in 22,000 sites out of millions in the genome.

“This array does the screening for us – it looks only at the sites of interest and allows us to focus time and energy on full sequencing only when necessary. Essentially it sifts the wheat from the chaff so we can focus only on the ICRs that may be involved in disease.”

The work appears in Epigenetics Communications and was supported by the National Institutes of Health under grant numbers R01ES093351, R01HD098857, R01MD011746, and R21HD093351, and by funding from NC State’s Center for Human Health and the Environment. The technology has been licensed by TruDiagnostic, Inc. Ryan Smith from TruDiagnostic is co-corresponding author. Other co-authors include first author Natalia Carreras-Gallo, Varun B. Dwaraka, and Tavis L. Mendez from TruDiagnostic; Dereje D. Jima, David A. Skaar, Antonio Planchart and Randy L. Jirtle from NC State University; and Wanding Zhou from the University of Pennsylvania.

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Note to editors: An abstract follows.

“Creation and validation of the first infinium DNA methylation array for the human imprintome”

DOI: 10.1186/s43682-024-00028-6

Authors: Dereje D. Jima, David A. Skaar, Antonio Planchart, Randy L. Jirtle, Cathrine Hoyo, North Carolina State University; Wanding Zhou, University of Pennsylvania; Ryan Smith, Natalia Carreras-Gallo, Varun B. Dwaraka, Tavis L. Mendez, TruDiagnostic, Inc.
Published: July 11, 2024 in Epigenetics Communications

Abstract:
Background: Differentially methylated imprint control regions (ICRs) regulate the monoallelic expression of imprinted genes. Their epigenetic dysregulation by environmental exposures throughout life results in the formation of common chronic diseases. Unfortunately, existing Infinium methylation arrays lack the ability to profile these regions adequately. Whole genome bisulfite sequencing (WGBS) is the unique method able to profile the ICRs. However, it is very expensive and it requires not only a high coverage, but it is also computationally intensive to assess these regions.
Findings: To address this deficiency, we developed a custom methylation array containing 22,819 probes. Among them, 10,438 are CG probes targeting unique CpG sites, with 9,757 probes successfully mapping to 1,088 out of the 1,488 candidate ICRs recently described. To assess the performance of the array, we created matched samples processed with the Human Imprintome array and WGBS, which is the current standard method for assessing the methylation of the Human Imprintome. We compared the methylation levels from the shared CpG sites, and obtained a mean R2=0.569. We also created matched samples processed with the Human Imprintome array and the Infinium Methylation EPIC v2 array, and obtained a mean R2=0.796. Furthermore, replication experiments demonstrated high reliability (ICC: 0.799–0.945).
Conclusions: Our custom array will be useful for replicable and accurate assessment, mechanistic insight, and targeted investigation of ICRs. This tool should accelerate the discovery of ICRs associated with a wide range of diseases and exposures, and advance our understanding of genomic imprinting and its relevance in development and disease formation throughout the life course.

This post was originally published in NC State News.