56a4e681e6e91808f57932b25645357d cells-14-01105.pdf f4bd9956d1269f65a2318f61c8324309b7b02318 cells-14-01105.pdf 2022385bdf0d4f7da6594f608e2422a5576a882060fd3ed90f9ea1970f0b809d cells-14-01105.pdf Title: Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid Subject: 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 DPPA3 and CFAP95, 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 EN1 and LMX1A. 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. Keywords: Lesch–Nyhan; HPRT; nervous system development; RNAseq; purine Author: Rosa J. Torres, Gerard Valentines-Casas, Claudia Cano-Estrada, Neus Ontiveros and José M. López Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Fri Jul 18 12:54:44 2025 CEST ModDate: Fri Jul 18 12:58:57 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 26 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1149279 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- QKZKVH+VnURWPalladioL Type 1 Custom yes yes yes 158 0 LMDPQP+URWPalladioL-Bold Type 1 Custom yes yes yes 159 0 IRBCVD+URWPalladioL-Roma Type 1 Custom yes yes yes 160 0 PWTABZ+URWPalladioL-Ital Type 1 Custom yes yes yes 161 0 COGLGI+CMSY10 Type 1 Builtin yes yes yes 212 0 CAMXTK+EURM10 Type 1 Builtin yes yes yes 247 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 279 0 MMNFAI+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 300 0 MMNFAH+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 301 0 MMNFAI+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 313 0 MMNEPG+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 314 0 MMNFAH+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 315 0 MMNFAI+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 338 0 MMNEPG+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 339 0 MMNFAH+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 340 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 398 0 MMNFAI+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 391 0 MMNEPG+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 392 0 MMNFAH+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 393 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-09 03:10:16 CEST RepresentationInformation: cells-14-01105.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-08 14:01:40 CEST Size: 1149279 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 724 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Generation of Pluripotent HPRT-Deficient Cells Item: Title: Cell Culture Medium and Differentiation of NT2/D1 Item: Title: 6-Thioguanine Toxicity Assay Item: Title: DNA Sequencing Item: Title: HPRT1 Expression Levels and HPRT Activity Item: Title: Hypoxanthine and Xanthine Determination Item: Title: Western Blotting Item: Title: Real-Time Quantitative PCR Array of Selected Genes Related to Neuronal Differentiation in HPRT-Deficient and Wild-Type NTD2/D1 Cells Item: Title: Differential Global Gene Expression by RNAseq in HPRT-Deficient and Wild-Type NTD2/D1 Cells Item: Title: Gene Ontology Analysis Item: Title: Results Children: Item: Title: Generation of Pluripotent HPRT-Deficient Cells Item: Title: Real-Time Quantitative PCR Array of Selected Genes Related to Neuronal Differentiation Children: Item: Title: Wild-Type Versus HPRT-Deficient Cells Item: Title: Differentiated Wild-Type Versus Differentiated HPRT-Deficient Cells Item: Title: Differential Global Gene Expression by RNAseq Children: Item: Title: Wild-Type Versus HPRT-Deficient Cells Item: Title: Differentiated Wild-Type Versus Differentiated HPRT-Deficient Cells Item: Title: Gene Ontology Enrichment Analysis Children: Item: Title: Wild-Type Versus HPRT-Deficient Cells Item: Title: Differentiated Wild-Type Versus Differentiated HPRT-Deficient Cells Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Gene Expression Analysis of HPRT-Deficient Cells Maintained with Physiological Levels of Folic Acid Author: Rosa J. Torres, Gerard Valentines-Casas, Claudia Cano-Estrada, Neus Ontiveros and José M. López Subject: 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 DPPA3 and CFAP95, 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 EN1 and LMX1A. 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. 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