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Endocrine Abstracts (2018) 58 OC5.3 | DOI: 10.1530/endoabs.58.OC5.3

1Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London, London, UK; 2Wellcome Trust Sanger Institute, Cambridge, UK; 3Centre for Translational Bioinformatics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London, London, UK; 4The Jackson Laboratory for Genomic Medicine, Farmington, USA.


Triple A syndrome (AAAS) is a rare, incurable, homozygous disorder, characterised by tissue-specific degeneration resulting in adrenal failure and neurodisability. The AAAS gene encodes ALADIN, a nuclear pore complex (NPC) protein necessary for nuclear import of DNA protective molecules, important for redox homeostasis. ALADIN’s role is not fully characterised: its discovery at the centrosome and the endoplasmic reticulum suggests a role outside the NPC. The interrogation of ALADIN’s function is limited by suboptimal disease models not representative of the affected tissue type.

Aim: To generate cellular models of AAAS with isogenic controls and undertake characterisation.

Method: We developed induced pluripotent stem cell (iPSC) models of AAAS using CRISPR-Cas9 gene-editing: 1) Bi-allelic exon 2 deletion (AAAS-KO) and 2) 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 between AAAS-KO compared to WT by cell counting (P value 0.24). Immunofluorescence for Ki67 confirmed no significant changes in cellular proliferation (WT: 100% of cells exhibit Ki67, AAAS-KO cells: 88.46%, P-value 0.40). RNA sequencing was performed to identify transcriptomal differences between iPSC lines, comparing WT to AAAS-KO, AAAS-mutant and a heterozygous exon 2 deletion. This identified 8 genes with significantly altered transcription (q values <0.05, LogFC values >1.1 and <−1.1). Preliminary analysis suggests an impact of AAAS deficiency on genes involved in apoptosis and 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 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 a detailed transcriptome analysis, which will inform further functional experiments to clarify the pathogenesis of AAAS.

Volume 58

46th Meeting of the British Society for Paediatric Endocrinology and Diabetes

Birmingham, UK
07 Nov 2018 - 09 Nov 2018

British Society for Paediatric Endocrinology and Diabetes 

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