ABSTRACT: Fragile X syndrome (FXS) is one of the most prevalent inherited intellectual disabilities. The patients carry the expansion of over 200 CGG repeats located at the 5′ untranslated region of fragile X mental retardation 1 (FMR1). As a result, the FMR1 promoter becomes hypermethylated leading to decreased or absent expression of its encoded RNA-binding protein fragile X mental retardation protein (FMRP). We previously generated human induced pluripotent stem (iPS) cells from fibroblasts of a FXS patient (FXS-iPSC) with expanded CGG-repeats at the FMR1 promoter. In the present work, we explored the transcriptional misregulation during the embryonic neurogenesis by in-vitro differentiation of FXS-iPSC into neurons through the intermediate stages. At each differentiation stage, we collected RNA and performed RNA-seq. After an integrated analysis, we found up-regulation of many genes encoding TFs for neuronal differentiation (WNT1, BMP4, POU3F4, TFAP2C, and PAX3), down-regulation of potassium channels (KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9, and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. Furthermore, we found the cholesterol synthesis genes (e.g., SQLE, LSS, and FASN) are up-regulated during the in-vitro neuronal differentiation of FXS-iPSC, which may contribute to the obesity phenotype of FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, and also discovered many potential candidate genes for the cure of FXS symptoms such as neuronal abnormalities and obesity.
