Reanalysis of Undiagnosed Neurodevelopmental Disorder Cases: From RNU4-2 Variants to Clinical Phenotypes

Abstract

Background and objectives: Neurodevelopmental disorders (NDDs) are often the results of genetic factors, whose identification and key role in their etiology may be elusive. Despite advancements in genetic testing, many cases remain unsolved. Single-nucleotide variants in a critical 18-bp region of the noncoding gene RNU4-2, a crucial component of the spliceosome complex, have been recently recognized as being responsible for ReNU syndrome, also known as NDD with hypotonia, brain anomalies, distinctive facies, and absent language (NEDHAFA). The aim of this study was to investigate the prevalence of RNU4-2 variants within a cohort of unsolved patients exhibiting NDDs from the Telethon Undiagnosed Disease Program (TUDP).

Methods: We conducted a reanalysis of genomic data using bioinformatic tools, followed by direct sequencing to identify variants in the RNU4-2 critical region.

Results: Causative variants were detected in 11 of 375 tested individuals, allowing us to diagnose 2.93% of our unsolved cohort. All heterozygous variants in RNU4-2 occurred de novo, including 10 with the recurrent n.64_65insT insertion and 1 with n.77_78insT. Structural modeling suggested that these variants disrupt the U4/U6 snRNA interaction, potentially impairing spliceosome function.

Discussion: Our findings reinforce the critical role of RNU4-2 variants in syndromic NDDs and underscore the importance of noncoding regions in genetic diagnoses. These findings show that RNU4-2 variants account for approximately 2.9% of the patients with TUDP in this study and highlight the need for integrating advanced molecular techniques and data sharing to refine diagnoses and enhance our understanding of rare genetic disorders.

Figure 1. Prevalence of HPO Terms

Bar plots illustrate the prevalence of HPO terms observed in the TUDP unsolved cohort. HPO terms are listed on the y-axis in the order of decreasing prevalence while the x-axis represents the percentage of patients exhibiting each term. Orange bars highlight the HPO features that are observed in our ReNU cohort. HPO = Human Phenotype Ontology; TUDP = Telethon Undiagnosed Disease Program.

Figure 2. Modeling of RNU4-2 Wild-Type and Pathogenic Variants

In each panel, predicted 3D structures of the U4/U6 di-snRNP complex are shown. RNU4-2 is shown in orange, with the critical region highlighted in red; RNU6-1 is shown in cyan with the 5′-ACAGAGA sequence in magenta, Prp31 in green, Prp3 in blue, Prp4 in pink, and Snu13 in violet. The critical region is highlighted with a red dotted square. The left panels display the full complex while the right ones zoom in on the critical region. (A) The U4/U6 complex with wild-type RNU4-2 shows the physiologic intramolecular and intermolecular interactions. (B) n.64_65insT shows mispairing in the critical region and stem-loop misorientation. (C) n.77_78insT shows misfolding in the critical region, bringing aberrant structure bending.

Figure 3. Clinical Features of Individuals With Pathogenic Variants in RNU4-2

The bar graphs illustrate the percentage of patients with pathogenic RNU4-2 variants exhibiting each of the clinical conditions represented, arranged by prevalence (data presented as percentages). ASD = autism spectrum disorder; SDS = SD score.

Figure 4. Clinical Photographs Illustrating the Facial Features of Individuals With Pathogenic Variants in RNU4-2

Panels A–I show 9 children with de novo pathogenic variants in RNU4-2, highlighting the distinct facial characteristics.

Figure 5. Brain MRI Findings of Individuals With Pathogenic Variants in RNU4-2

Panel (A) shows sagittal (left) and axial (right) images of patient 4 from eTable 1. The axial image demonstrates mild enlargement of the subarachnoid space in the left middle fossa (white arrow), attributed to a small arachnoid cyst, along with a slightly hypoplastic appearance of the ipsilateral temporal pole. Panel (B) presents sagittal (left) and axial (right) images of patient 2 from eTable 1. The sagittal image reveals hypoplasia of the corpus callosum (white arrow) while the axial image highlights periventricular gray matter heterotopia (white arrow). Additional features include thinning of the white matter, hypoplasia of the olfactory bulbs, hypoplasia of the optic nerves, and an arachnoid cyst (not shown). Panel (C) displays sagittal (left) and coronal (right) images of patient 1 from eTable 1. The sagittal image illustrates hypoplasia of the corpus callosum (white arrow) while the coronal image shows reduced periventricular white matter volume and ventricular asymmetry (white arrows).