Prader-Willi syndrome (PWS) is caused by a loss of genes normally expressed only from the paternal chromosome 15. About 70% of PWS cases arise from Type 1 and Type 2 deletions, which are about 5 million DNA base pairs in size. Genetic mapping data from unique patients harboring smaller deletions, “microdeletions”, in the PWS region implicate the gene SNORD116@ as a candidate gene, whose absence is sufficient to cause the most salient features of PWS, including feeding difficulties in infancy, rapid weight gain after about two years of age, extreme hunger, obesity, and developmental delay. PWS affects specific brain regions at the cellular level, making stem cell technology a particularly effective method for the study of this disease. Stem cell technologies enable researchers to generate and study the cell types affected by human diseases, creating a model of the ‘disease in a dish’. In order to better understand how Prader-Willi syndrome affects brain cells, we will transform skin cells from PWS patients into pluripotent stem cells (iPSC). To specifically understand the effects of loss of SNORD116@ we will use genetic engineering to delete the SNORD116@ gene in an iPSC line from an unaffected individual. This approach allows us to study the effects of the loss of SNORD116@ without confounds of naturally occurring variations in human genetics. The neurons generated from PWS patients will allow us to characterize the molecular and cellular defects that are caused by this genetic syndrome. Understanding how PWS affects the brain at the cellular and molecular level can inform novel therapeutic targets for PWS patients. We will also study the connection between the loss of SNORD116@ and increased levels of ghrelin, the ‘hunger hormone’. A special aspect of this proposal is the inclusion of young investigators who could become the next generation of PWS scientists.
Research Outcomes: Project Summary
Our findings of a reduction in NHLH2 and PC1 at both the transcript and protein level in PWS iPSC-derived neurons (general and hypothalamic) suggest that the major neuroendocrine phenotypes of PWS are due to defects in prohormone processing (Fig. 10). Identification of defects in NHLH2 and PC1 levels in iPSC-derived neurons made from fibroblasts of a PWS microdeletion patient whose deletion region includes only the three noncoding RNA genes SNORD109A, SNORD116, and IPW, further suggests that deletion of one of the genes within this interval is sufficient to cause decreased production of NHLH2, and in turn, PC1. Mice that are functionally null for Snord116 display endocrine phenotypes similar to mice deficient in PC1, including, hyperghrelinemia, low growth hormone, stunted growth, and, as recently identified by us, impaired processing of proinsulin to insulin. In the aggregate, these data suggest that: 1) deletion of paternal SNORD116 alone is sufficient to cause decreased production of NHLH2 and PC1, and 2) in vivo deficiency of paternal SNORD116 may impair prohormone processing with consequences for both ingestive behavior and neuroendocrine function. These exciting findings lend themselves to immediate further investigation of the role of NHLH2 and PC1 in the molecular pathophysiology of PWS.
Research Outcomes: Publications
Loss of the imprinted, non-coding Snord116 gene cluster in the interval deleted in the Prader Willi syndrome results in murine neuronal and endocrine pancreatic developmental phenotypes. Burnett LC, Hubner G, LeDuc C, Morabito MV, Carli JFM, Leibel RL. Human Molecular Genetics. 2017 Sep 6.
Induced pluripotent stem cells (iPSC) created from skin fibroblasts of patients with Prader-Willi syndrome (PWS) retain the molecular signature of PWS. Burnett LC, LeDuc CA, Sulsona CR, Paull D, Eddiry S, Levy B, Salles JP, Tauber M, Driscoll DJ, Egli D, Leibel RL. Stem Cell Research. 2016 Nov; 17(3):526-530.
Determination of the half-life of circulating leptin in the mouse. Burnett LC, Skowronski AA, Rausch R, LeDuc CA, Leibel RL. International Journey of Obesity (London). 2017 Mar;41(3):355-359.
PC1/3 Deficiency Impacts Pro-opiomelanocortin Processing in Human Embryonic Stem Cell-Derived Hypothalamic Neurons. Wang L, Sui L, Panigrahi K, Meece K, Xin Y, Kim J, Gromada J, Doege CA, Wardlaw S, Egli D, Leibel RL. Stem Cell Reports. 2017 Feb 14; 8(2): 264–277.
Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome. Burnett LC, LeDuc CA, Sulsona CR, Paull D, Rausch R, Eddiry S, Carli JF, Morabito MV, Skowronski AA, Hubner G, Zimmer M, Wang L, Day R, Levy B, Fennoy I, Dubern B, Poitou C, Clement K, Butler MG, Rosenbaum M, Salles JP, Tauber M, Driscoll DJ, Egli D, Leibel RL. Journal of Clinical Investigation. 2017 Jan 3;127(1):293-305.
Efficient Generation of Hypothalamic Neurons from Human Pluripotent Stem Cells. Wang L, Egli D, Leibel RL. Current Protocols in Human Genetics. 2016 Jul 1;90:21.5.1-21.5.14.
Differentiation of hypothalamic-like neurons from human pluripotent stem cells. Wang L, Meece K, Williams DJ, Lo KA, Zimmer M, Heinrich G, Martin Carli J, Leduc CA, Sun L, Zeltser LM, Freeby M, Goland R, Tsang SH, Wardlaw SL, Egli D, Leibel RL. Journal of Clinical Investigation. 2015 Feb 2;125(2):796-808.
Rudolph Leibel, MD