Development Of A Humanized Knockin Mouse Model For Rahman Syndrome

Diril M. K.

EU-CHROMNET European Network Conference on Chromatinopathies Congress, İzmir, Turkey, 25 - 27 November 2026, (Unpublished)

  • Publication Type: Conference Paper / Unpublished
  • City: İzmir
  • Country: Turkey
  • Dokuz Eylül University Affiliated: Yes

Abstract

Background/Aim

Rahman syndrome (RMNS) is a recently identified, rare genetic disorder characterized by overgrowth, intellectual disability, autism, and early aging. The disease is driven by de novo frameshift mutations in the HIST1H1E gene, which codes for the linker histone H1.4 (H1E). These mutations truncate the H1E C-terminal domain and introduce a novel 38-amino-acid acidic tail (RahCTD38) present in virtually all RMNS cases. This alters epigenetic charge dynamics, leading to aberrant chromatin organization. To investigate RMNS etiology in vivo, we aimed to develop a genetically humanized knockin (KI) mouse model.

Materials and Methods

The RMNS mouse model was generated utilizing the CRISPR-EZ technique. This highly efficient approach involves the direct electroporation of CRISPR-Cas9 ribonucleoprotein complexes, including specific guide RNAs and repair templates, into single-cell mouse embryos. This enabled the precise integration of the sequence encoding the human RahCTD38 tail directly into the endogenous mouse H1E locus. Modified embryos were subsequently transferred into pseudopregnant surrogates to generate founder knockin pups, which were bred to establish a stable colony.

Results We successfully generated the targeted humanized KI mice expressing the RMNS-associated H1E-RahCTD38 mutant protein. Precise on-target integration of the KI allele was genetically characterized and confirmed by Sanger sequencing. Basic phenotypic characterization to assess overall viability, fertility and development was carried out. Furthermore, presence of the mutant H1E protein across tissues was analyzed and validated by western blotting and immunohistochemistry.

Conclusion We established a novel humanized mouse model that accurately mirrors the genetic basis of RMNS, facilitating research on etiology and development of symptoms. This model will be further characterized by behavior tests, biochemical studies on mouse embryonic fibroblasts (MEF), comprehensive histological analysis to investigate the development of various RMNS-associated clinical phenotypes, and bioinformatics to investigate the underlying molecular mechanisms driving this rare disorder.

Keywords: Rahman Syndrome, Linker Histone H1E, Rare Genetic Disorders, Transgenic Mice, Epigenetics

Background/Aim

Rahman syndrome (RMNS) is a recently identified, rare genetic disorder characterized by overgrowth, intellectual disability, autism, and early aging. The disease is driven by de novo frameshift mutations in the HIST1H1E gene, which codes for the linker histone H1.4 (H1E). These mutations truncate the H1E C-terminal domain and introduce a novel 38-amino-acid acidic tail (RahCTD38) present in virtually all RMNS cases. This alters epigenetic charge dynamics, leading to aberrant chromatin organization. To investigate RMNS etiology in vivo, we aimed to develop a genetically humanized knockin (KI) mouse model.

Materials and Methods

The RMNS mouse model was generated utilizing the CRISPR-EZ technique. This highly efficient approach involves the direct electroporation of CRISPR-Cas9 ribonucleoprotein complexes, including specific guide RNAs and repair templates, into single-cell mouse embryos. This enabled the precise integration of the sequence encoding the human RahCTD38 tail directly into the endogenous mouse H1E locus. Modified embryos were subsequently transferred into pseudopregnant surrogates to generate founder knockin pups, which were bred to establish a stable colony.

Results We successfully generated the targeted humanized KI mice expressing the RMNS-associated H1E-RahCTD38 mutant protein. Precise on-target integration of the KI allele was genetically characterized and confirmed by Sanger sequencing. Basic phenotypic characterization to assess overall viability, fertility and development was carried out. Furthermore, presence of the mutant H1E protein across tissues was analyzed and validated by western blotting and immunohistochemistry.

Conclusion We established a novel humanized mouse model that accurately mirrors the genetic basis of RMNS, facilitating research on etiology and development of symptoms. This model will be further characterized by behavior tests, biochemical studies on mouse embryonic fibroblasts (MEF), comprehensive histological analysis to investigate the development of various RMNS-associated clinical phenotypes, and bioinformatics to investigate the underlying molecular mechanisms driving this rare disorder.

Keywords: Rahman Syndrome, Linker Histone H1E, Rare Genetic Disorders, Transgenic Mice, Epigenetics