A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects

Human Molecular Genetics · 15 authors, 26 centres

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This study generated the first patient mutation knock-in mouse model of ATR-X syndrome carrying the most common causative mutation, R246C (R245C in mice). The mutant mice recapitulate key features of the human disorder, including craniofacial defects, microcephaly, reduced body size, and impaired neurological function, providing a powerful tool for understanding disease mechanisms and testing therapies.

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**Background:** ATR-X syndrome is a rare X-linked disorder caused by hypomorphic mutations in the ATRX gene, characterized by intellectual disability, alpha-thalassemia, facial dysmorphism, microcephaly, short stature, and musculoskeletal defects. Complete deletion of Atrx in mice causes early embryonic lethality, so previous models relied on conditional knockouts. This study aimed to create a more patient-relevant model by introducing the most common patient mutation, R246C (R245C in mice), into the mouse genome.

**Methods:** The authors used CRISPR/Cas9-mediated editing in C57BL/6J zygotes to introduce the c.733C>T (p.R245C) mutation and a silent PAM-abolishing mutation (c.729C>T; p.I243I) into exon 9 of the mouse Atrx gene. Hemizygous male mice (Atrx^R245C/y) were generated and characterized. Protein levels were assessed by western blot in cortex at P0.5 and P60, and in spleen at P60. mRNA levels were measured by qPCR. Protein stability was evaluated using cycloheximide treatment in cultured cortical neurons. Heterochromatin binding was assessed by immunofluorescence for ATRX at pericentromeric foci. Morphological analyses included body weight, body length (X-ray), brain weight, craniofacial measurements (cranial index, zygomatic index), and muscle function tests (grip strength, gait analysis, rotarod, chronic treadmill exercise). Neurological function was evaluated using SHIRPA, open field, light/dark box, elevated zero maze, Barnes maze, social interaction test, Y-maze, and fear conditioning. Brain morphology was analyzed by MRI (75 μm isotropic voxels) and histological staining. Dendritic complexity was assessed by Sholl analysis of cultured hippocampal neurons.

**Key Results:** ATRX^R245C protein levels were ~60% of wild-type at P0.5 but declined to ~10% by 9 weeks of age in cortex, and to ~15% in spleen at P60. mRNA levels were only mildly reduced (~80% of controls). Cycloheximide treatment showed reduced stability of the mutant protein. Heterochromatin binding was impaired. Atrx^R245C/y mice had reduced body weight from weaning to 1 year, shorter body length (14 weeks: p=0.03; 52 weeks: p=0.007), reduced brain weight at 9 weeks (p=0.0002), and a shortened snout phenotype in ~58% of mutants. Cranial length was reduced at 14 weeks (p=0.047) and zygomatic length was reduced at both 14 and 40 weeks (p=0.004 for both). No muscle defects were detected. Neurologically, mutants showed reduced spontaneous activity (p=0.04), impaired spatial memory in the Barnes maze (days 1, 3, 4: p=0.004, 0.014, 0.0096), loss of social novelty preference (wild-type p=0.0004; mutant p>0.05), and impaired contextual fear memory (p=0.025). MRI revealed reduced total cerebellar volume (−6.0%, p<0.0001) and thinner corpus callosum in rostral (−12.3%, p=0.023) and medial (−28.0%, p=0.002) regions. Cultured hippocampal neurons showed reduced total dendritic length (p=0.043) and decreased dendritic branching (p=0.015).

**Clinical Implications:** This mouse model recapitulates several key features of ATR-X syndrome, including short stature, microcephaly, facial dysmorphism, and intellectual disability. The absence of alpha-thalassemia and genital abnormalities is explained by species-specific differences in the alpha-globin locus and the mutation type. The model provides a valuable tool for studying the molecular mechanisms of ATR-X syndrome, particularly the role of the ADD domain, and for testing potential therapeutic strategies. The finding that mutant protein levels decline postnatally suggests that protein instability may contribute more to pathogenicity than previously thought.

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other

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genetics

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ORIGINAL PAPER

PMC10360390

A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects

Human Molecular Genetics

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ABSTRACT

Abstract ATRX is a chromatin remodelling ATPase that is involved in transcriptional regulation, DNA damage repair and heterochromatin maintenance. It has been widely studied for its role in ALT-positive cancers, but its role in neurological function remains elusive. Hypomorphic mutations in the X-linked ATRX gene cause a rare form of intellectual disability combined with alpha-thalassemia called ATR-X syndrome in hemizygous males. Clinical features also include facial dysmorphism, microcephaly, short stature, musculoskeletal defects and genital abnormalities. As complete deletion of ATRX in mice results in early embryonic lethality, the field has largely relied on conditional knockout models to assess the role of ATRX in multiple tissues. Given that null alleles are not found in patients, a more patient-relevant model was needed. Here, we have produced and characterized the first patient mutation knock-in model of ATR-X syndrome, carrying the most common causative mutation, R246C. This is one of a cluster of missense mutations located in the chromatin-binding domain and disrupts its function. The knock-in mice recapitulate several aspects of the patient disorder, including craniofacial defects, microcephaly, reduced body size and impaired neurological function. They provide a powerful model for understanding the molecular mechanisms underlying ATR-X syndrome and testing potential therapeutic strategies.

CITATION EXPORT

Rebekah Tillotson, Keqin Yan, Julie Ruston et al.. A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects. Human Molecular Genetics. (2023). https://doi.org/10.1093/hmg/ddad075

AUTHORS

15 · 26 CENTRES

Rebekah Tillotson, Keqin Yan, Julie Ruston, Taylor DeYoung, Alex Córdova, Valérie Turcotte-Cardin, Yohan Yee, Christine Taylor, Shagana Visuvanathan, Christian Babbs, Evgueni A Ivakine, John G Sled, Brian J Nieman, David J Picketts, Monica J Justice

1 Genetics and Genome Biology Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
2 MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital/Headley Way, Oxford OX3 9DS, UK
3 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
4 Genetics and Genome Biology Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
5 Mouse Imaging Centre, The Hospital for Sick Children, Toronto, ON M5T 3H7, Canada
6 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
7 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
8 Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
9 Mouse Imaging Centre, The Hospital for Sick Children, Toronto, ON M5T 3H7, Canada
10 Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
11 Genetics and Genome Biology Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
12 Genetics and Genome Biology Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
13 MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital/Headley Way, Oxford OX3 9DS, UK
14 Genetics and Genome Biology Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
15 Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada
16 Mouse Imaging Centre, The Hospital for Sick Children, Toronto, ON M5T 3H7, Canada
17 Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
18 Translational Medicine Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
19 Mouse Imaging Centre, The Hospital for Sick Children, Toronto, ON M5T 3H7, Canada
20 Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
21 Translational Medicine Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
22 Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada
23 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
24 Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
25 Genetics and Genome Biology Program, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, ON M5G 0A4, Canada
26 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A1, Canada