Objective: To create a resource of induced pluripotent stem cells (iPSCs) as models for exploring mechanisms of pathogenesis in Lesch-Nyhan disease (LND).

Background: LND is an X-linked metabolic disorder with characteristic abnormalities that include dystonia, intellectual disability, and self-injurious behaviors. LND is caused by mutations in the HPRT1 gene, which encodes the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). The mechanisms responsible for neurological and behavioral abnormalities are unclear. Because there are no signs of degeneration in the LND brain, the neurobehavioral syndrome is thought to be due to neural dysfunction.

Methods: Fibroblasts from 3 healthy controls and 3 LND patients (with nonsense mutations c.151C>T or c.508C>T, or frame-shifting insertion 371insTT) were reprogrammed to iPSCs. Two independent lines were created for each case. Immunocytochemistry was performed on iPSCs to confirm expression of pluripotency markers. Karyotyping was performed. Pluripotency was verified by testing ability to differentiate to the three germ layers: ectoderm, mesoderm, and endoderm. Cells were then evaluated for gene expression and protein expression profiles.

Results: All 12 lines had immunostaining profiles consistent with pluripotency and possessed the ability to differentiate into all 3 germ layers. All lines had normal karyotypes. All lines expressed high levels of genes typical of pluripotent cells. RNA-Seq identified a total of 144 differentially expressed genes between LND and healthy controls at nominal p<0.001. After correcting for multiple comparisons, 16 genes remained significantly differentially expressed at FDR<0.1, including the HPRT1 gene. Gene Set Enrichment Analysis (GSEA) analysis revealed a number of biological pathways significantly altered. Studies addressing protein expression profiles and metabolic abnormalities are underway, as well as studies addressing ability to differentiate into neurons.

Conclusions: We have validated multiple iPSC lines from patients with LND and matched controls. The lines from patients show consistent abnormalities in gene expression profiles, and are being used now to evaluate dopamine neuron function.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/disease-modeling-of-lesch-nyhan-disease-using-induced-pluripotent-stem-cells/