Analysis and comparisons of gene expression changes in patient- derived neurons from ROHHAD, CCHS, and PWS

Abstract

BackgroundRapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) syndrome is an ultra-rare neurocristopathy with no known genetic or environmental etiology. Rapid-onset obesity over a 3–12 month period with onset between ages 1.5–7 years of age is followed by an unfolding constellation of symptoms including severe hypoventilation that can lead to cardiorespiratory arrest in previously healthy children if not identified early and intervention provided. Congenital Central Hypoventilation syndrome (CCHS) and Prader-Willi syndrome (PWS) have overlapping clinical features with ROHHAD and known genetic etiologies. Here we compare patient neurons from three pediatric syndromes (ROHHAD, CCHS, and PWS) and neurotypical control subjects to identify molecular overlap that may explain the clinical similarities.MethodsDental pulp stem cells (DPSC) from neurotypical control, ROHHAD, and CCHS subjects were differentiated into neuronal cultures for RNA sequencing (RNAseq). Differential expression analysis identified transcripts variably regulated in ROHHAD and CCHS vs. neurotypical control neurons. In addition, we used previously published PWS transcript data to compare both groups to PWS patient-derived DPSC neurons. Enrichment analysis was performed on RNAseq data and downstream protein expression analysis was performed using immunoblotting.ResultsWe identified three transcripts differentially regulated in all three syndromes vs. neurotypical control subjects. Gene ontology analysis on the ROHHAD dataset revealed enrichments in several molecular pathways that may contribute to disease pathology. Importantly, we found 58 transcripts differentially expressed in both ROHHAD and CCHS patient neurons vs. control neurons. Finally, we validated transcript level changes in expression of ADORA2A, a gene encoding for an adenosine receptor, at the protein level in CCHS neurons and found variable, although significant, changes in ROHHAD neurons.ConclusionsThe molecular overlap between CCHS and ROHHAD neurons suggests that the clinical phenotypes in these syndromes likely arise from or affect similar transcriptional pathways. Further, gene ontology analysis identified enrichments in ATPase transmembrane transporters, acetylglucosaminyltransferases, and phagocytic vesicle membrane proteins that may contribute to the ROHHAD phenotype. Finally, our data imply that the rapid-onset obesity seen in both ROHHAD and PWS likely arise from different molecular mechanisms. The data presented here describes important preliminary findings that warrant further validation.

High-Titer Anti-ZSCAN1 Antibodies in a Toddler Clinically Diagnosed with Apparent Rapid-Onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation Syndrome

“…Candidate genes for ROHHAD syndrome have been reviewed comprehensively elsewhere [14,34]. A study using neurons derived from patients with ROHHAD syndrome, Prader-Willi syndrome, or CCHS found a larger variability in the gene expression in ROHHAD syndrome than in the other two syndromes [35]. In contrast, autoimmune etiology appears to be more plausible, especially in the context of the detection of autoantibodies and oligoclonal bands in the CSF of ROHHAD syndrome patients The present patient had CSF oligoclonal bands and high titers of serum anti-ZSCAN1 antibodies.…”

Antibodies Against ZSCAN1 in Pediatric and Adult Patients With Non-Paraneoplastic ROHHAD Syndrome

“…1 The diagnosis can be challenging because of the clinical similarities with other pediatric disorders caused by genetic alterations such as congenital central hypoventilation syndrome caused by mutations in the PHOX2B gene, and Prader-Willi syndrome produced by a loss of function of genes on chromosome 15. [3][4][5][6] The pathogenesis of ROHHAD syndrome has been suggested to be immune-mediated because of the frequent coexistence with neuroblastic tumors such as neuroblastoma, ganglioneuroblastoma, or ganglioneuroma, which can potentially trigger the neurologic syndrome through paraneoplastic mechanisms. 7 Antibodies targeting the zinc finger and SCAN domaincontaining protein 1 (ZSCAN1) were recently identified in 7 of 9 pediatric patients with both ROHHAD syndrome and an underlying neuroblastic tumor, suggesting these antibodies as distinct biomarkers of paraneoplastic ROHHAD syndrome in children.…”