Both gain- and loss-of-function mutations have recently implicated HCFC1 in neurodevelopmental disorders. Here, we extend our previous HCFC1 over-expression studies by employing short hairpin RNA to reduce the expression of Hcfc1 in embryonic neural cells. We show that in contrast to over-expression, loss of Hcfc1 favoured proliferation of neural progenitor cells at the expense of differentiation and promoted axonal growth of post-mitotic neurons. To further support the involvement of HCFC1 in neurological disorders, we report two novel HCFC1 missense variants found in individuals with intellectual disability (ID). One of these variants, together with three previously reported HCFC1 missense variants of unknown pathogenicity, were functionally assessed using multiple cell-based assays. We show that three out of the four variants tested result in a partial loss of HCFC1 function. While over-expression of the wild-type HCFC1 caused reduction in HEK293T cell proliferation and axonal growth of neurons, these effects were alleviated upon over-expression of three of the four HCFC1 variants tested. One of these partial loss-of-function variants disrupted a nuclear localization sequence and the resulting protein displayed reduced ability to localize to the cell nucleus. The other two variants displayed negative effects on the expression of the HCFC1 target gene MMACHC, which is responsible for the metabolism of cobalamin, suggesting that these individuals may also be susceptible to cobalamin deficiency. Together, our work identifies plausible cellular consequences of missense HCFC1 variants and identifies likely and relevant disease mechanisms that converge on embryonic stages of brain development.

Department of Biology and Medical Genetics Charles University 2nd Faculty of Medicine and University Hospital Motol Prague Czech Republic

Department of Human Molecular Genetics Max Planck Institute for Molecular Genetics Ihnestrasse 73 D 14195 Berlin Germany and

Department of Medical Genetics University Hospital Ostrava tr 17 listopadu 1790 708 52 Ostrava Czech Republic

INSERM UMR 1163 Laboratory of Molecular and Pathophysiological Bases of Cognitive Disorders Paris Descartes Sorbonne Paris Cité University Imagine Institute Necker Enfants Malades Hospital 75015 Paris France

School of Molecular and Biomedical Sciences University of Adelaide Adelaide 5000 Australia

School of Paediatrics and Reproductive Health

School of Paediatrics and Reproductive Health Robinson Research Institute and

School of Paediatrics and Reproductive Health Robinson Research Institute and School of Molecular and Biomedical Sciences University of Adelaide Adelaide 5000 Australia

Unit of Medical Genetics Department of Medical and Surgical Sciences DIMEC St Orsola Malpighi Hospital University of Bologna Bologna 40138 Italy

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