Identifying key underlying regulatory networks and predicting targets of orphan C/D box snoRNAs in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder characterized principally by initial symptoms of neonatal hypotonia and failure-to-thrive in infancy, followed by hyperphagia and obesity. It is well established that PWS is caused by loss of paternal expression of the imprinted region on chromosome 15q11-q13. While most PWS cases exhibit megabase-scale deletions of the paternal chromosome 15q11-q13 allele, several PWS patients have been identified harboring a much smaller deletion encompassing primarily . This finding suggests is a direct driver of PWS phenotypes. The gene cluster is composed of 30 copies of individual C/D box small nucleolar RNAs (snoRNAs). Many C/D box snoRNAs have been shown to guide chemical modifications of other RNA molecules, often ribosomal RNA (rRNA). However, snoRNAs are termed 'orphans' because no verified targets have been identified and their sequences show no significant complementarity to rRNA. It is crucial to identify the targets and functions of snoRNAs because all reported PWS cases lack their expression. To address this, we engineered two different deletions modelling PWS in two distinct human embryonic stem cell (hESC) lines to control for effects of genetic background. Utilizing an inducible expression system enabled quick, reproducible differentiation of these lines into neurons. Systematic comparisons of neuronal gene expression across deletion types and genetic backgrounds revealed a novel list of 42 consistently dysregulated genes. Employing the recently described computational tool snoGloBe, we discovered these dysregulated genes are significantly enriched for predicted targeting versus multiple control analyses. Importantly, our results showed it is critical to use multiple isogenic cell line pairs, as this eliminated many spuriously differentially expressed genes. Our results indicate a novel gene regulatory network controlled by is likely perturbed in PWS patients.