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Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes

European Journal of Human Genetics volume 31, pages 1228–1236 (2023)Cite this article

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Abstract

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

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Fig. 1: Pedigree and variant analysis in the three families with XCI-skewed female cases.
Fig. 2: Pedigrees and variants analysis in the three families with XCI skewed mothers of affected males.

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Data availability

All variants have been deposited into ClinVar: SCV002583290, SCV002583291, SCV002583292, SCV002583293, SCV002583294, SCV002583295, SCV002583296, SCV002583297, SCV002583298, SCV002583299, SCV002583300, SCV002583301.

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Acknowledgements

We are grateful to the patients and their families for their participation in this study. We acknowledge Samplix for the technical support.

Funding

This research received funding from the Italian Ministry for Education, University and Research (Ministero dell’Istruzione, dell’Università e della Ricerca - MIUR) PRIN2020 code 20203P8C3X to AB, “Associazione E.E. Rulfo per la genetica Medica” to AB, Fondazione Cassa di Risparmio di Torino to AB, and Fondazione Bambino Gesù (Vite Coraggiose) to MT. Sample collection was supported by the NIMH (U01MH111661 to JDB).

Author information

Authors and Affiliations

  1. Department of Medical Sciences, University of Turin, 10126, Turin, Italy

    Chiara Giovenino, Slavica Trajkova, Lisa Pavinato, Verdiana Pullano, Enza Ferrero, Silvia Carestiato, Antonina Rinninella, Anthony Battaglia, Flavia Palermo, Barbara Pasini & Alfredo Brusco

  2. Department of Public Health and Pediatrics, University of Turin, 10126, Turin, Italy

    Simona Cardaropoli, Diana Carli, Alessandro Mussa & Giovanni Battista Ferrero

  3. Department of Endocrinology and Genetics, University Clinic for Pediatric Diseases, Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, 1000, Skopje, Republic of North Macedonia

    Elena Sukarova-Angelovska

  4. Medical Genetics Unit, Città della Salute e della Scienza University Hospital, 10126, Turin, Italy

    Paola Salmin, Barbara Pasini & Alfredo Brusco

  5. Department of Biomedical and Biotechnological Sciences, Medical Genetics, University of Catania, 94124, Catania, Italy

    Antonina Rinninella

  6. Functional Genomics Lab, Department of Biotechnology, University of Verona, 37134, Verona, Italy

    Luca Bertoli, Antonio Fadda, Marzia Rossato & Massimo Delledonne

  7. Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy

    Paola Dimartino

  8. Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146, Rome, Italy

    Alessandro Bruselles & Marco Tartaglia

  9. Department of Biotechnology and Genetic Engineering, Philadelphia University, Amman, Jordan

    Tawfiq Froukh

  10. Medical Genetics Unit and Thalassemia Center, San Luigi University Hospital, University of Torino, Orbassano, TO, Italy

    Giorgia Mandrile

  11. Seaver Autism Center for Research and Treatment, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

    Silvia De Rubeis & Joseph D. Buxbaum

  12. Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

    Silvia De Rubeis & Joseph D. Buxbaum

  13. The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

    Silvia De Rubeis & Joseph D. Buxbaum

  14. Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

    Joseph D. Buxbaum

  15. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

    Joseph D. Buxbaum

  16. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

    Joseph D. Buxbaum

  17. U.O. Genetica Medica, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italia

    Tommaso Pippucci

Authors
  1. Chiara Giovenino
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  2. Slavica Trajkova
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  29. Alfredo Brusco
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Contributions

Conceptualization: CG, ST, AB; Data curation: CG, ST, AB, ES, FP, DC, AM, TF, GM, BP, GBF; Formal analysis: CG, ST, LP, SC, VP, SC, AR, AB, PD, AB, TP, MT; Investigation: CG, ST, LP, SC, VP, SC, LB, AF, PS, SDR, JB; Methodology: CG, PS, LB, AF, MR, MD, Visualization: Writing-original draft: CG, ST, AB; Writing-review & editing: EF, CG, ST, AB, ES, FP, DC, AM, TF, GM, BP, GBF, MR, MD.

Corresponding author

Correspondence to Alfredo Brusco.

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The authors declare no competing interests.

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All individuals and families from the different institutions agreed to participate in this study and signed appropriate consent forms. The Ethics Committee of Città della Salute e della Scienza University Hospital (n. 0060884) and University of Skopje (n. 03–6116/7) approved this study. Written informed consent for publication of clinical detail and patient photographs has been obtained from the parents or legal guardian.

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Giovenino, C., Trajkova, S., Pavinato, L. et al. Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes. Eur J Hum Genet 31, 1228–1236 (2023). https://doi.org/10.1038/s41431-023-01324-w

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