BSPED2018 Oral Communications Oral Communications 5 (9 abstracts)
Can novel stem cell models help unpick the pathogenesis of the Triple A syndrome?
Alexandra Rodrigues Da Costa 1 , Shamma Qarin 2 , Teisha Bradshaw 1 , David Watson 3 , Rathi Prasad 1 , Louise A Metherell 1 , Michael R Barnes 3 , William Skarnes 4 , J Paul Chapple 1 & Helen L Storr 1
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.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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