Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid
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...
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2025-07-01
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| author | Rosa J. Torres Gerard Valentines-Casas Claudia Cano-Estrada Neus Ontiveros José M. López |
| author_facet | Rosa J. Torres Gerard Valentines-Casas Claudia Cano-Estrada Neus Ontiveros José M. López |
| author_sort | Rosa J. Torres |
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| description | 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 <i>DPPA3</i> and <i>CFAP95</i>, 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 <i>EN1</i> and <i>LMX1A</i>. 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. |
| format | Article |
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| institution | DOAJ |
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| publishDate | 2025-07-01 |
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| spelling | doaj-art-a64d71230eec4cd39915e71d61149be32025-08-20T03:08:00ZengMDPI AGCells2073-44092025-07-011414110510.3390/cells14141105Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic AcidRosa J. Torres0Gerard Valentines-Casas1Claudia Cano-Estrada2Neus Ontiveros3José M. López4Department of Biochemistry, Hospital La Paz Institute for Health Research (IdiPaz), 28046 Madrid, SpainInstitut de Neurociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, SpainInstitut de Neurociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, SpainInstitut de Neurociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, SpainInstitut de Neurociències, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, SpainLesch–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 <i>DPPA3</i> and <i>CFAP95</i>, 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 <i>EN1</i> and <i>LMX1A</i>. 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.https://www.mdpi.com/2073-4409/14/14/1105Lesch–NyhanHPRTnervous system developmentRNAseqpurine |
| spellingShingle | Rosa J. Torres Gerard Valentines-Casas Claudia Cano-Estrada Neus Ontiveros José M. López Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid Cells Lesch–Nyhan HPRT nervous system development RNAseq purine |
| title | Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid |
| title_full | Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid |
| title_fullStr | Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid |
| title_full_unstemmed | Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid |
| title_short | Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid |
| title_sort | gene expression analysis of hprt deficient cells maintained with physiological levels of folic acid |
| topic | Lesch–Nyhan HPRT nervous system development RNAseq purine |
| url | https://www.mdpi.com/2073-4409/14/14/1105 |
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