Torres Rosa J, Valentines-Casas Gerard, Cano-Estrada Claudia, Ontiveros Neus, López José M
Department of Biochemistry, Hospital La Paz Institute for Health Research (IdiPaz), 28046 Madrid, Spain.
Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, 28006 Madrid, Spain.
Cells. 2025 Jul 18;14(14):1105. doi: 10.3390/cells14141105.
Lesch-Nyhan disease (LND) is associated with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity due to mutations in the HPRT1 gene. Although the physiopathology of LND-related neurological manifestations remains unknown, a defective neuronal developmental process is the most widely accepted hypothesis. We generated an HPRT-deficient line from the pluripotent human embryonic cell line NT2/D1 by CRISPR-Cas9 and induced its differentiation along neuroectodermal lineages by retinoic acid treatment. As levels of folic acid in the culture media may affect results in LND models, we employed physiological levels of folate. The effect of HPRT deficiency on neural development-related gene expression was evaluated using two methodological approaches: a directed qPCR array of genes related to neuronal differentiation, and global gene expression by RNAseq. HPRT-deficient pluripotent cells presented altered expression of genes related to pluripotency in human embryonic stem cells, such as and , along with genes of the homeobox gene family. HPRT-deficient pluripotent cells were able to differentiate along neuro-ectodermal lineages but presented consistent dysregulation of several genes from the homeobox gene family, including and . GO enrichment analysis of up- and downregulated genes in HPRT-deficient cells showed that the most significant biological processes affected are related to development and nervous system development.
莱施-奈恩病（LND）与次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HPRT）活性完全缺乏有关，这是由于HPRT1基因突变所致。尽管LND相关神经学表现的生理病理学仍不清楚，但神经元发育过程缺陷是最被广泛接受的假说。我们通过CRISPR-Cas9从多能人类胚胎细胞系NT2/D1中生成了一个HPRT缺陷细胞系，并通过视黄酸处理诱导其沿神经外胚层谱系分化。由于培养基中的叶酸水平可能会影响LND模型的结果，我们采用了生理水平的叶酸。我们使用两种方法评估了HPRT缺陷对神经发育相关基因表达的影响：一种是针对神经元分化相关基因的定向定量PCR阵列，另一种是通过RNA测序进行全局基因表达分析。HPRT缺陷的多能细胞呈现出人类胚胎干细胞中与多能性相关基因（如 和 ）以及同源框基因家族基因表达的改变。HPRT缺陷的多能细胞能够沿神经外胚层谱系分化，但呈现出同源框基因家族中几个基因（包括 和 ）的持续失调。对HPRT缺陷细胞中上调和下调基因的基因本体（GO）富集分析表明，受影响最显著的生物学过程与发育和神经系统发育有关。
