Cellular impact of RAI1 haploinsufficiency: RAI1 functions through specific pathways to regulate growth, metabolism, and circadian rhythm.

Smith-Magenis syndrome (SMS) is a complex syndrome characterized by a constellation of ~30 features that include sleep disturbance, craniofacial defects, neurological and behavioral anomalies, and variable systemic features. SMS is caused by a 17p11.2 deletion encompassing the retinoic acid induced 1 (RAI1) gene or by mutation of RAI1. While haploinsufficiency of RAI1, a known transcription factor, is the major cause of SMS, its exact functional pathways are not known. We hypothesized that RAI1 acts through specific cellular pathways involving several downstream targets which are altered by RAI1 haploinsufficiency. To identify genes in these RAI1-mediated pathways, we performed genome-wide gene expression analysis on cells haploinsufficient for RAI1. Human embryonic kidney (HEK293T) cells with RNA-interference-based ~50% knockdown of RAI1 and lymphoblastoid cell lines created from SMS patients were utilized. Genome-wide gene expression profiling using microarrays showed that ~60 genes were upregulated and ~200 genes were downregulated due to RAI1 haploinsufficiency. Real-time qRT-PCR confirmed the gene expression profile in knockdown HEK293T cells. Lymphoblastoid cell lines obtained from SMS patients confirmed the altered expression pattern in downstream genes that are involved in growth signaling and insulin sensitivity (INSIG1, PIK3R1, ZNF236), circadian activity (NR1D2), neuronal differentiation (ZIC1, PSEN2, RXRb, SMA4, CLN8, NF1, MLL), lipid biosynthesis and fat mobilization (LIPE, HMGCS1), skeletal development (GLI3, PSTPIP2, ANKH), behavior (SCN12A), gene expression (SPTBN1, POLDIP3, PPP1R14D, ADD3), cell cycle regulation (RUNX1T1, AKR7A3, FBLN1, ZNF236), and recombination (RAD51). Our study suggests that RAI1 functions through several genes in specific pathways regulating various biological processes, that when disrupted result in the phenotypic effects observed in Smith-Magenis syndrome.