Prediction analysis of networks and transcription factors regulating <i>IKBKAP</i> and <i>IKBKAP</i> co-regulated functional candidate genes.

<p>“String” analysis of protein networks showing the potential interaction between the majority of the <i>IKBKAP</i> co-regulated functional candidate genes. (B) Prediction of transcription factors (TF) related to <i>IKBKAP</i> and co-regulated functional candidate genes. (C) Shows relative quantification levels represented as mean ± s.d. of TF gene candidates taken from the total cDNA microarray analysis showing difference between WT and FD-hESC derived PNS neurons. * P<0.05 **P<0.01, Non statistical significance (NS).</p

GO analysis of differential gene expression between hESC-derived PNS neurons and embryo brains of WT and FD origins.

<p>The major enriched GO analysis groups showing processes (A) and gene functions (B) from the two in vivo and in vitro cDNA microarray chip data. The enrichment groups are arranged by number of downregulated and upregulated genes detected in each group. Only genes above 2-fold change are included. FDR q-value (adjusted p-values of result significance), Enrichment = (number intersection genes / input genes) / (total number of genes of specific GO term / background genes).</p

Comparative analysis of expression differences in WT and FD hESC derived PNS differentiation process.

<p>(A) Venn diagram comparing the overall number of genes that showed > 2 fold expression differences between hNP and fully differentiated hESC-derived neurons in WT (in blue) and FD (in yellow) backgrounds. (B) The normalized relative quantification expression values are shown as mean ± s.d. of several developmental PNS markers and transcription factors in WT and FD hESC-derived neurons as obtained in the transcriptome cDNA chip analysis. * P<0.05 **P<0.01.</p

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by <i>IKBKAP</i> Downregulation

<div><p>A splicing mutation in the <i>IKBKAP</i> gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that <i>IKBKAP</i> downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by <i>IKBKAP</i> downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects <i>IKBKAP</i> alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.</p></div