2060-P: RFX6, a Causative Gene of Mitchell-Riley Syndrome, Regulates ParaHox Genes in Early Endodermal Differentiation

Background: RFX6 was identified as a causative gene of Mitchell-Riley syndrome (MRS), an autosomal recessive disease associated with neonatal diabetes and morphological disorders in development of the endodermal lineage such as pancreatic hypoplasia and intestinal atresia. However, little is known of RFX6 regulation of other genes, especially in the early developmental stage of the endoderm. Aim: To clarify the mechanism of human early development for clues to understanding the pathophysiological features of MRS. Method: We established human induced pluripotent stem cell (hiPSC) reporter lines, RFX6 +/eGFP and RFX6 eGFP/eGFP, by homologous recombination using a bacterial artificial chromosome vector containing eGFP-pA-loxP-Pgk-NeoR-loxP cassette following 5’UTR of RFX6. Their locus specificity was validated by fluorescent in situ hybridization. We then compared gene expression at the primitive gut stage using the in-vitro differentiation method. Results: We compared bulk gene expression between RFX6 +/+ with RFX6 eGFP/eGFP using RNA-seq. Among downregulated genes in the latter, we focused on PDX1 and CDX2, which are marker genes of posterior foregut and mid-hindgut, respectively. mRNA expression of these genes was significantly decreased in RFX6 eGFP/eGFP compared to those in RFX6 +/eGFP. Immunocytochemistry and FACS studies showed that most PDX1+ cells were GFP+ in RFX6 +/eGFP; however, the number of PDX1+GFP+ cells was highly decreased in RFX6 eGFP/eGFP while GFP+ cells were observed similarly to those in RFX6 +/eGFP. Regarding CDX2, most CDX2+ cells were GFP+ in RFX6 +/eGFP; however, CDX2+GFP+ cells were observed less frequently in RFX6eGFP/eGFP than in RFX6+/eGFP. Conclusion: Our present findings show that RFX6 deficiency results in downregulation of the major ParaHox genes, PDX1 and CDX2, in early endodermal differentiation. These findings may underlie the association between the genetic and pathophysiological features of MRS. Disclosure T. Nakamura: None. J. Fujikura: None. R. Ito: None. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Drawbridge Health, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Life Scan Japan, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Pharma Ltd.,, Sanofi K.K., Sanwa Kagaku Kenkyusho, Terumo Medical Corporation. Speaker’s Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.