Mendelian neurodevelopmental disorders customarily present with complex and overlapping symptoms, complicating the clinical diagnosis. Individuals with a growing number of the so-called rare disorders exhibit unique, disorder-specific DNA methylation patterns, consequent to the underlying gene defects. Besides providing insights to the pathophysiology and molecular biology of these disorders, we can use these epigenetic patterns as functional biomarkers for the screening and diagnosis of these conditions. This review summarizes our current understanding of DNA methylation episignatures in rare disorders and describes the underlying technology and analytical approaches. We discuss the computational parameters, including statistical and machine learning methods, used for the screening and classification of genetic variants of uncertain clinical significance. Describing the rationale and principles applied to the specific computational models that are used to develop and adapt the DNA methylation episignatures for the diagnosis of rare disorders, we highlight the opportunities and challenges in this emerging branch of diagnostic medicine.

Wiedemann–Steiner syndrome (WDSTS) is a Mendelian syndromic intellectual disability (ID) condition associated with hypertrichosis cubiti, short stature, and characteristic facies caused by pathogenic variants in the KMT2A gene. Clinical features can be inconclusive in mild and unusual WDSTS presentations with variable ID (mild to severe), facies (typical or not) and other associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Interpretation and classification of rare KMT2A variants can be challenging. A genome-wide DNA methylation episignature for KMT2A-related syndrome could allow functional classification of variants and provide insights into the pathophysiology of WDSTS. Therefore, we assessed genome-wide DNA methylation profiles in a cohort of 60 patients with clinical diagnosis for WDSTS or Kabuki and identified a unique highly sensitive and specific DNA methylation episignature as a molecular biomarker of WDSTS. WDSTS episignature enabled classification of variants of uncertain significance in the KMT2A gene as well as confirmation of diagnosis in patients with clinical presentation of WDSTS without known genetic variants. The changes in the methylation profile resulting from KMT2A mutations involve global reduction in methylation in various genes, including homeobox gene promoters. These findings provide novel insights into the molecular etiology of WDSTS and explain the broad phenotypic spectrum of the disease.

A growing number of genetic neurodevelopmental disorders are known to be associated with unique genomic DNA methylation patterns, called episignatures, which are detectable in peripheral blood. The intellectual developmental disorder, X-linked, syndromic, Armfield type (MRXSA) is caused by missense variants in FAM50A. Functional studies revealed the pathogenesis to be a spliceosomopathy that is characterized by atypical mRNA processing during development. In this study, we assessed the peripheral blood specimens in a cohort of individuals with MRXSA and detected a unique and highly specific DNA methylation episignature associated with this disorder. We used this episignature to construct a support vector machine model capable of sensitive and specific identification of individuals with pathogenic variants in FAM50A. This study contributes to the expanding number of genetic neurodevelopmental disorders with defined DNA methylation episignatures, provides an additional understanding of the associated molecular mechanisms, and further enhances our ability to diagnose patients with rare disorders.

Background: Recurrent constellations of embryonic malformations (RCEMs) comprise multiple malformation conditions with largely unexplained etiologies and no established molecular biomarkers. A shared DNA methylation episignature was recently identified in VACTERL association and oculoauriculovertebral spectrum (OAVS). We sought to validate this episignature in an independent, deeply phenotyped cohort and evaluate its detection across related RCEMs. Methods: Genome-wide DNA methylation profiling was performed on peripheral blood from 38 participants with clinically diagnosed RCEMs, including VACTERL (n=21), partial VACTERL (n=3), OAVS (n=3), and other RCEM-related conditions (n=11). Results: The Episign V5 RCEM episignature demonstrated robust sensitivity for VACTERL (18/21, 85.7% positive), while the remaining three participants showed intermediate positivity. Of three participants with partial VACTERL, one with tracheoesophageal fistula demonstrated intermediate positivity, whereas the other two were negative. Episignature positivity was also identified in oculoauriculofrontonasal dysplasia (1/1, robust) and rhomboencephalosynapsis (1/2, robust) but was limited in OAVS (1/3, intermediate) and absent in frontonasal dysplasia (0/4). Conclusions: Independent validation establishes the Episign V5 RCEM episignature as a reproducible molecular biomarker for VACTERL, a condition that remains a diagnosis of exclusion. Variable detection across related malformation conditions suggests etiologic heterogeneity, whereas overlap among selected phenotypes supports epigenomic convergence across the RCEM spectrum.