Expanding the mutational spectrum of ReNU syndrome: insights into 5' Stem-loop variants.

A narrow spectrum of heterozygous variants in RNU4-2, encoding the small nuclear RNA (snRNA) U4, underlies ReNU syndrome, a neurodevelopmental disorder (NDD) characterized by moderate to severe developmental delay (DD), intellectual disability (ID), a distinctive facial gestalt, and multisystem involvement. Pathogenic variants have primarily been reported within an 18-nt critical region contributing to stabilizing the U4/U6 snRNA duplex and proper spliceosome assembly. By combining whole genome sequencing reanalysis and targeted direct sequencing in 190 molecularly unexplained NDD cases, we report on five affected individuals carrying pathogenic/putative pathogenic RNU4-2 variants (2.6%). Three individuals harbored the recurrent pathogenic n.64_65insT variant, while two were heterozygous for private/rare maternally inherited variants (n.30 A > T and n.43_44insT) within the 5' Stem-loop region. Deep clinical phenotyping confirmed a homogeneous constellation of features in all individuals, with global DD, ID, brain malformations, and a recognizable facial gestalt representing core findings. Based on structural homology models and available cryo-EM data, n.30 A > T and n.43_44insT were predicted to disrupt key intra- and inter-molecular interactions critical for spliceosome function. Our findings expand the mutational spectrum of ReNU syndrome, and confirm the 5' Stem-loop as a second mutational hotspot in RNU4-2. We propose that a more complex genetics likely underlies the inheritance of a subset of disease-causing RNU4-2 variants from an apparently unaffected parent. We anticipate a relatively high proportion of pathogenic RNU4-2 variants among individuals with unclassified NDD despite extensive genomic testing, and propose a set of facial gestalt core features as a clinical screening tool to prioritize patients for RNU4-2 analysis.