EpiSign International: Expanding Epigenomic Testing in Rare Disease

Rare diseases are conditions that affect fewer than 1 in 2,000 individuals and can be serious, chronic or life-threatening. Collectively, rare diseases affect an estimated 3.5-5.9% of the global population, corresponding to approximately 236-446 million people worldwide, with new rare disorders continuing to be identified. Prenatal environmental exposures and maternal conditions may also be considered in the differential diagnosis of rare genetic disorders, as these can affect foetal development. Such factors include exposure to drugs and medications, lifestyle factors such as alcohol consumption, infectious agents, and maternal conditions including diabetes mellitus and epilepsy.

Diagnostic assessment of patients with suspected rare disorders often includes chromosomal microarray analysis (CMA) to identify large structural copy number variations (CNVs), with an average diagnostic yield of approximately 10-15%. Where initial genomic testing is negative or inconclusive, further testing may include targeted gene panels, whole-exome sequencing (WES) or whole-genome sequencing (WGS). These approaches have substantially improved the diagnosis of rare diseases, but a significant proportion of patients remain unresolved following genomic testing. Some unresolved patients may have genetic variants of uncertain significance (VUS), while others may have disease-causing variants or disease mechanisms that are not readily identifiable using current genomic approaches.

Patients with rare diseases may experience prolonged diagnostic journeys, with delays of several years and, in some cases, decades. Recent studies suggest that approximately 25% of patients in the UK may wait between five and thirty years for a final diagnosis, while global estimates of diagnostic delay range from approximately five to twelve years, with some patients remaining undiagnosed for up to thirty years. This diagnostic odyssey can have significant consequences for patients and their families, as well as for healthcare systems. Establishing a molecular diagnosis can inform clinical management, particularly for patients with genetically heterogeneous disorders or atypical presentations. Earlier diagnosis may facilitate timely access to appropriate treatment, surveillance and support, including during critical periods of development, with the potential to improve patient outcomes.

There is therefore a need for additional cost-effective, sensitive and specific approaches that can support the diagnosis of patients who remain unresolved or have ambiguous findings following conventional genomic testing. Such approaches may be particularly valuable where genetic and environmental factors contribute to disease presentation, or where clinical features are atypical or overlap between multiple disorders.

Epigenomic approaches, and specifically DNA methylation analysis, provide one potential avenue for addressing this diagnostic gap. DNA methylation is a reversible epigenetic modification involving the addition of a methyl group to cytosine residues within CpG dinucleotides across the genome. DNA methylation has important roles in genomic imprinting, silencing of retroviral elements, regulation of tissue-specific gene expression, X-chromosome inactivation and chromatin structure. DNA methylation is also important during development, with dynamic changes in DNA methylation and demethylation contributing to the spatiotemporal regulation of gene expression, particularly during neuronal development.

Research has demonstrated that a number of rare genetic disorders and disorders associated with environmental exposures are characterised by distinctive patterns of DNA methylation at multiple genomic locations, known as episignatures. These reproducible patterns can provide additional diagnostic information and may assist in resolving ambiguous clinical cases, including cases involving VUSs in relevant genes or where a molecular diagnosis has not been established. Episignatures have been identified in association with both genetic and environmental aetiologies and can be detected in peripheral blood, an accessible and commonly used source of DNA for clinical diagnostic testing. An episignature is a recurring and reproducible DNA methylation pattern associated with a particular disease or aetiology. Although some DNA methylation changes overlap between rare disorders, the use of large reference datasets and advanced computational approaches enables the development of disease-specific episignature biomarkers with high sensitivity and specificity.

EpiSign™ is a proprietary technology developed by EpiSign Inc. that combines machine-learning approaches with reference data from the EpiSign Knowledge Database (EKD), a large and continuously expanding database of DNA methylation profiles from individuals with rare diseases. As part of a broader diagnostic assessment, EpiSign analysis may provide additional information to physicians and diagnostic laboratories when evaluating patients with suspected rare disorders. For example, a recent study of 2,399 individuals with unsolved rare disorders reported a positivity rate of 32.4% (237/732) among patients undergoing targeted assessment based on a previously identified genetic VUS or a suspected clinical diagnosis without a confirmed molecular diagnosis. Among patients undergoing comprehensive EpiSign analysis as a screening assay for known episignature disorders, 18.7% (312/1,667) had a positive result.

The range of disorders assessed using EpiSign continues to expand as novel episignatures are identified and validated. New versions of the EpiSign assay undergo validation to establish the analytical performance of individual classifiers, including assessment using reference samples from the EKD and prospective testing within the EpiSign Services Laboratory Network. Cross-laboratory external quality assessment (EQA) and sample exchange programmes within this international clinically licensed laboratory network provide additional mechanisms for monitoring analytical performance, reproducibility and quality.

This study will assess the clinical utility and health-system impact of EpiSign as part of the diagnostic assessment of patients with suspected rare diseases. The study will quantify EpiSign positivity rates and assess its contribution to molecular and clinical diagnosis, as well as its impact on subsequent clinical and diagnostic pathways and associated healthcare costs. The study will generate prospective real-world evidence to inform the implementation of EpiSign within diagnostic pathways and to identify optimal implementation parameters across different health-system settings.

The study comprises two phases. Phase 1 will assess the clinical utility and health-system impact of EpiSign analysis using the standard commercial EPIC DNA methylation microarray platform as part of the diagnostic assessment of individuals with suspected rare diseases.

Phase 2 will assess the utility of EpiSign analysis using a newly developed integrated EPIC/CNV microarray and will additionally evaluate the technical performance and analytical yield of the integrated microarray in detecting CNVs alongside disease-associated DNA methylation patterns. This phase will provide evidence on the potential utility of combining DNA methylation and CNV analysis on a single microarray platform.

Together, the two phases will provide evidence regarding the clinical utility of EpiSign analysis, the technical performance of the integrated EPIC/CNV microarray, and the potential impact of incorporating these approaches into diagnostic pathways for patients with suspected rare diseases.

Inclusion criteria

• Eligible for, or have undergone, standard-of-care first-tier genetic testing at the participating site with a negative or inconclusive result.
• No established molecular or clinical diagnosis explaining their presentation.
• No diagnostic result from subsequent/reflex genetic testing available at enrolment.
• For Phase 2 CNV-positive participants: have undergone standard-of-care CNV testing at the participating site and have a CNV reported by standard-of-care testing.
• For Phase 2 first-tier participants: meet the Phase 1 inclusion criteria.
• For Phase 2 CNV-positive participants, priority will be given to those with VUS, pathogenic/likely pathogenic, or other CNVs where EpiSign analysis may provide clinically relevant information.

Exclusion criteria

• Do not meet the applicable inclusion criteria.
• Isolated, non-syndromic autism or isolated learning disability without additional features suggestive of a syndromic rare disorder.