Triple A syndrome (AAAS) is a rare, incurable, recessive disorder, characterised by achalasia, alacrima, adrenal failure and a neurodegenerative phenotype. The AAAS gene encodes ALADIN, is a nuclear pore complex (NPC) protein necessary for nuclear import of DNA protective molecules, important for redox homeostasis. ALADINs role is not fully characterised: its discovery at the centrosome and the endoplasmic reticulum suggests a role outside the NPC. To date, the interrogation of ALADINs function has been limited by suboptimal disease models: knock-out mice do not exhibit an analogous phenotype, patient fibroblast cultures do not originate from the affected tissue-type, and knock-down experiments with immortalised cell lines achieve at best an 80% reduction in ALADIN expression.
Aim: To generate cellular models of AAAS with isogenic controls and undertake characterisation.
Method: We have developed induced pluripotent stem cell (iPSC) models of AAAS using CRISPR-Cas9 gene-editing: (i) Bi-allelic exon 2 deletion (AAAS-KO) and (ii) AAAS homozygous patient mutation: a splice donor hotspot mutation p.G14fs (c.43C>A, exon 1) (AAAS-mutant). These are paired with the original healthy wild-type (WT) iPSC line and mono-allelic exon 2 deletion (AAAS-het) as isogenic controls.
Results: Immunoblotting did not detect ALADIN in AAAS-KO or AAAS-mutant cells. There was no difference in cellular proliferation after 24 hours between AAAS-KO compared to WT by cell counting (P value 0.24). Immunofluorescence with Ki67 antibody confirmed there were no significant changes in cellular proliferation (WT: 100% of cells exhibit Ki67 compared to 88.46% of AAAS-KO cells, P value 0.40). RNA sequencing was performed to identify differences in the transcriptome of the iPSC lines, comparing WT to AAAS-KO, AAAS-mutant and a mono-allelic exon 2 deletion (AAAS-het) (n=3). This identified 9 genes with significantly altered transcription (LogFC values >1.1 and <−1.1) with preliminary analysis suggesting an impact of AAAS deficiency on genes involved in oxidative stress. We demonstrated that AAAS-KO and AAAS-mutant cells will differentiate along a neurocortical lineage, expressing neuronal transcription factors OTX2 PAX6 and Nestin.
Conclusion: We present a viable alternative iPSC model for the study of ALADIN in a near endogenous environment. These can be differentiated along a neurocortical lineage, to reflect the tissue affected in the Triple A Syndrome. We present detailed transcriptome analysis, which will inform further functional experiments, designed to provide insight into the pathogenesis of AAAS.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology