Poseter #033, Children's Hospital Los Angeles
Characterizing Novel Arg20Trp Mutation in MODY9 iPSCs
Mentors: Marney Granados, B.S and Senta Georgia,PhD
Common diabetes conditions, such as type-1 and type-2, cause abnormal insulin production or resistance. Maturity-Onset Diabetes of the Young (MODY) is a rare form of monogenic diabetes caused by a single gene mutation that impacts typical pancreatic development and beta cell differentiation in utero. MODY is associated with 14 distinct genetic subtypes, with MODY type 9 (MODY9) associated with the PAX4 gene. PAX4 is a “master-regulator” transcription factor and is essential for beta cell differentiation through the repression of alpha cell lineage, with PAX4-null and MODY-mutant models resulting in a decrease in beta cell population. A patient at Children’s Hospital Los Angeles (CHLA) was diagnosed with MODY9 with unknown clinical significance caused by a novel, single nucleotide 58c>t mutation resulting in a Arg20TRp amino acid change. Although clinical significance is unknown, in-silico analysis suggests this mutation will likely change the structure of PAX4 protein and impact its transcriptional function. Location of the mutation is also found in the highly conserved paired-domain which is responsible for recognizing and binding transcriptional targets. We aimed to characterize the mutation using newly reprogrammed “MS-29” induced pluripotent stem cells (iPSCs) to determine if it has an impact on the transcriptional function of PAX4. Three iPSC clones (#3,#4,#7) were differentiated into beta cells alongside control H1 embryonic stem cell (ESC) lines. We analyzed differentiation efficiency during peak stages(5-6) of PAX4 expression during beta cell differentiation. To investigate the development of beta cells through these peak stages, we used ICC to visually check for specific beta cell markers present in these stages and flow cytometry to quantify these results by having a count of how many of these cells were developed at these stages. In both assays, we are expecting less expression of beta cell markers in mutant iPSCs compared to ESCs.