Validation of the VACTERL Episignature and Evidence for Epigenomic Convergence Across Recurrent Constellations of Embryonic Malformations

Abstract

BackgroundRecurrent 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. MethodsGenome-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). ResultsThe 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). ConclusionsIndependent 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.