Genomic imprinting is orchestrated by epigenetic modifications, most notably DNA methylation, which are established during gametogenesis by the de novo methyltransferase DNMT3A and its cofactor DNMT3L, then maintained throughout life by DNMT1. Imprinted genes typically cluster within genomic loci governed by imprinting control regions, or ICRs, which display roughly fifty percent DNA methylation, reflecting parent-of-origin dependent regulation. Zinc finger proteins such as ZFP57 and ZNF445 tightly regulate the establishment of these methylation marks, and during early embryogenesis, when the epigenome is largely reset, ICRs are protected from demethylation by ZFP57 and the PGC7/STELLA complex. Histone modifications, including H3K9me3 and H3K27me3 on the silenced allele and H3K4me3 and H3K27ac on the active allele, reinforce this imbalanced expression pattern. Non-coding RNAs, such as the lncRNA AIRN, which silences the IGF2 receptor gene in mice, and insulator binding by CTCF, which coordinates expression across larger imprinted regions like the IGF2/H19 locus on chromosome 11p15.5, add further layers of regulation.

The most widely accepted evolutionary explanation for imprinting is the parental conflict hypothesis. Paternally expressed genes promote offspring growth during early development, while maternally expressed genes counterbalance this growth stimulation to preserve maternal health. This framework makes imprinting disruption particularly relevant to cancer, as many imprinted genes regulate cell growth, differentiation, and apoptosis. When imprinting is lost, either through reactivation of a normally silenced allele or silencing of a normally active allele, growth-promoting genes can become biallelically overexpressed while growth-limiting genes become completely silenced, directly fuelling tumorigenesis.

The clinical consequences of imprinting dysregulation extend well beyond cancer. Congenital syndromes such as Beckwith-Wiedemann, Prader-Willi, Angelman, Silver-Russell, Temple, and Kagami-Ogata all arise from disrupted imprinting at specific loci, often through DNA methylation defects, uniparental disomy, point mutations, or chromosomal aberrations. Beckwith-Wiedemann syndrome, characterised by excessive foetal growth and elevated childhood cancer risk, stems from dysregulated imprinting at the IGF2/H19 locus. Beyond congenital disorders, altered imprinting has been linked to cardiovascular conditions, including hypertension and atherosclerosis, where the DLK1-MEG3 domain is extensively hypomethylated in plaques, as well as neuropsychiatric disorders, with parent-of-origin effects implicated in autism spectrum disorder and schizophrenia.

In cancer, LOI at the IGF2/H19 locus is the most extensively documented imprinting alteration. Re-expression of the normally maternally silenced IGF2 allele and silencing of the maternally expressed H19 non-coding RNA are considered growth-promoting events. LOI at this locus has been detected in nearly thirty percent of colorectal cancer patients’ normal colonic mucosa, and its presence in blood greatly increases the odds of both colorectal adenoma and carcinoma. IGF2/H19 LOI has also been described in breast, liver, lung, and Wilms tumour, where it recapitulates foetal-program proliferation patterns and leads to poorer treatment response and adverse clinical outcomes, making it a putative predictive and prognostic biomarker. Other imprinted loci, including DLK1 in gliomas and neuroblastomas, PEG3 in gliomas, CDKN1C in several cancer types, HM13 in breast, kidney, and lung tumours, and MEST in breast, ovarian, and lung carcinomas, also undergo LOI or aberrant methylation in cancer.

Studying imprinting in humans presents formidable technical challenges. In model organisms like mice, imprinting can be characterised cost-efficiently by mating parents from different inbred lines and profiling the largely heterozygous progeny. However, imprinting is often species-specific, making human-specific experiments necessary for clinical studies. The retinoblastoma gene RB1, for instance, is imprinted in humans but not in mice, and attempts to generate an imprinted mouse model were unsuccessful. Similarly, the synteny of WT1 and IGF2 on chromosome 11p in humans, which allows a single complex genetic event to produce both aberrations in Wilms tumour, does not exist in mice, making murine models less representative of the human disease.

The gold standard for human imprinting research combines genomics and transcriptomics in a parent-offspring trio setting. By genotyping the offspring, heterozygous loci are identified, upon which RNA sequencing of the tissue under study reveals whether only a single allele is expressed. Comparison with parental genomics data then determines from which parent the expressed allele is inherited. However, this strategy is limited by the need for heterozygosity to discriminate between alleles, requiring sufficiently large numbers of trios and expensive designs combining both genomics and transcriptomics data. Case-control studies that first screen for candidate imprinted loci in controls, followed by analysis in cases, abolish the requirement for trio data but often lead to false positives, particularly due to allele-specific expression effects independent of parental origin, such as random monoallelic expression of human leukocyte antigen genes.

Epigenetics-based strategies, focusing on DNA methylation rather than expression, offer an alternative approach. Whole-genome bisulfite sequencing provides base-resolution DNA methylation profiling by converting unmethylated cytosines to uracil, allowing evaluation of whether methylation at ICRs deviates from the anticipated fifty percent. However, bisulfite treatment induces DNA degradation, and the requirement for large, high-quality, high-purity tumour samples poses significant challenges in cancer research. Enzymatic methylation sequencing (EM-seq) has emerged as a gentler alternative, while reduced representation bisulfite sequencing and targeted capture strategies reduce cost. DNA methylation arrays, particularly the Illumina Infinium MethylationEPIC platform, offer a cost-effective genome-scale option, though they lack allele-specific read-out, making it unclear whether methylation alterations reflect dysregulated imprinting or altered cell-type composition. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), which combines copy number and methylation assessment, remains the most widely used methodology for diagnosing imprinting syndromes.

Three major methodological challenges complicate the interpretation of imprinting analyses in cancer. First, most studies rely on bulk tumour samples that may contain substantial fractions of non-tumour cells, such as tumour-associated fibroblasts or infiltrating immune cells, reaching up to fifty percent in renal cancer. If admixing cell types feature different imprinting patterns, incorrect LOI calls may result. Second, the need for heterozygosity to discriminate paternal from maternal alleles means that the power to detect LOI varies across the genome as a function of genotype frequency, a major bottleneck for clinical assays. Third, transcript-specific imprinting, observed for loci such as MEST and IGF2, can lead to inconsistent LOI calls across a gene’s SNPs, while widespread copy number alterations in cancer can cause both false-positive LOI calls and genuine LOI events through deletion or gain of an originally silenced allele.

Emerging technologies promise to address these challenges. Single-cell RNA sequencing resolves tumour heterogeneity by profiling individual tumour cells, though many platforms miss the SNPs required to separate alleles, and single-cell data sparsity limits genome-wide character. Pseudobulk strategies, combining data from tumour cells or subpopulations, mitigate this resolution problem. Spatial transcriptomics and targeted methods like Quantitative Chromogenic Imprinted Gene In Situ Hybridization (QCIGISH) enable allele-specific visualisation even in homozygous backgrounds, addressing problems with tumour purity, copy number alterations, and heterozygosity. QCIGISH has been validated in cancer patient cohorts, achieving near-perfect sensitivity and specificity for early-stage lung cancer detection and enabling cervical cancer risk stratification. Long-read sequencing technologies offer the possibility to resolve transcript-level imprinting patterns and provide direct DNA methylation read-out during genome sequencing, avoiding bisulfite-related problems. The authors argue that integrating single-cell, spatial, and long-read strategies into multi-omics approaches will largely resolve remaining issues with non-cancer cells in bulk tissues and the need for heterozygosity and transcript-level resolution, ultimately guiding the development of optimised clinical tests and even epigenome-editing therapies for cancer.

Subject of Research: Genomic imprinting and its dysregulation in cancer

Article Title: Challenges and emerging strategies for genome-wide evaluation of loss of imprinting in cancer

Article References: Challenges and emerging strategies for genome-wide evaluation of loss of imprinting in cancer. (n.d.). https://doi.org/10.3389/bjbs.2026.16709

Image Credits: AI Generated

DOI: 10.3389/bjbs.2026.16709

Keywords: genomic imprinting, loss of imprinting, cancer, epigenetics, DNA methylation, biomarkers, single-cell sequencing, long-read sequencing, Challenges, emerging, strategies, genome-wide