Abstract
Cornelia de Lange syndrome (CdLS) is a complex disorder with multiple structural and developmental defects caused by mutations in structural and regulatory proteins involved in the cohesin complex. NIPBL, a cohesin regulatory protein, has been identified as a critical protein responsible for the orchestration of transcriptomic regulatory networks necessary for embryonic development. Mutations in NIPBL are responsible for the majority of cases of CdLS. Through RNA-sequencing of human induced pluripotent stem cells and in vitro-derived cardiomyocytes, we identified hundreds of mRNAs, pseudogenes, and non-coding RNAs with altered expression in NIPBL+/− patient-derived cells. We demonstrate that NIPBL haploinsufficiency leads to upregulation of gene sets identified in functions related to nucleosome, chromatin assembly, RNA modification and downregulation of Wnt signaling, cholesterol biosynthesis and vesicular transport in iPSC and cardiomyocytes. Mutations in NIPBL result in the dysregulation of many genes responsible for normal heart development likely resulting in the variety of structural cardiac defects observed in the CdLS population.
Highlights
Cornelia de Lange syndrome (CdLS) is one of a family of disorders known as cohesinopathies, or more broadly, disorders of transcriptional regulation (DTRs)
To investigate the impact of NIPBL mutations in CdLS patient samples, we focused our work on the role they play in cultured human cells, both in the undifferentiated and cardiomyocyte states
The reduced expression of NIPBL (60–75% of normal) resulted in no difference in reprogramming efficiency, loss of genomic stability, or alterations in cell morphology, pluripotency markers or proliferation. This is in contrast to Kagey et al (2010), which showed that reduction of NIPBL using shRNA resulted in loss of pluripotent stem cells morphology, and reduction of key pluripotency mRNAs (Oct3/4, SOX2, and Nanog)
Summary
Development of an in vitro iPSC cardiac model for CdLS. To investigate the impact of NIPBL mutations in CdLS patient samples, we focused our work on the role they play in cultured human cells, both in the undifferentiated and cardiomyocyte states. We found that twelve were significantly dysregulated at a p-value < 0.05, but extending the confidence level to p-value < 0.15 an additional 9 genes were identified to be altered in NIPBL+/− cardiomyocytes This showed that 21/53 CHD-associated genes were differentially expressed in our NIPBL+/− patient-derived cardiomyocytes (Table 2). Only 16 of the 86 genes previously shown to be associated with the stress responses pathways identified in fibroblast and LCLs from CdLS patient samples were correlated to our data in NIPBL+/−-iPSC or CMs (Fig. 4C)[17] Data showed similar pattern of expression for epigenetics modifiers, ECM-interactors and adhesion molecules, and CHD-genes as those generated through RNASeq analysis (Supplemental Fig. 2) Taken together, this data confirms and expands the catalogue of gene and pathways currently identified to be regulated by the cohesin complex (primarily or downstream) as a result of NIPBL haploinsufficiency in iPSC and CMs
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