Hypomorphic GINS3 variants alter DNA replication and cause Meier-Gorlin syndrome
Mary E. McQuaid, Kashif Ahmed, Stephanie Tran, Justine Rousseau, Ranad Shaheen, Kristin D. Kernohan, Kyoko E. Yuki, Prerna Grover, Dominique Lévesque, Ema S. Dreseris, Sameen Ahmed, Lucie Dupuis, Jennifer Stimec, Mary Shago, Zuhair N. Al-Hassnan, Roch Tremblay, Philipp G. Maass, Michael D. Wilson, Eyal Grunebaum, Kym M. Boycott, François-Michel Boisvert, Sateesh Maddirevula, Eissa A. Faqeih, Fahad Almanjomi, Zaheer Ullah Khan, Fowzan S. Alkuraya, Philippe M. Campeau, Peter Kannu, Eric I. Campos, Hugo Wurtele
Mary E. McQuaid, Kashif Ahmed, Stephanie Tran, Justine Rousseau, Ranad Shaheen, Kristin D. Kernohan, Kyoko E. Yuki, Prerna Grover, Dominique Lévesque, Ema S. Dreseris, Sameen Ahmed, Lucie Dupuis, Jennifer Stimec, Mary Shago, Zuhair N. Al-Hassnan, Roch Tremblay, Philipp G. Maass, Michael D. Wilson, Eyal Grunebaum, Kym M. Boycott, François-Michel Boisvert, Sateesh Maddirevula, Eissa A. Faqeih, Fahad Almanjomi, Zaheer Ullah Khan, Fowzan S. Alkuraya, Philippe M. Campeau, Peter Kannu, Eric I. Campos, Hugo Wurtele
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Research Article Cell biology

Hypomorphic GINS3 variants alter DNA replication and cause Meier-Gorlin syndrome

Abstract

The eukaryotic CDC45/MCM2-7/GINS (CMG) helicase unwinds the DNA double helix during DNA replication. The GINS subcomplex is required for helicase activity and is, therefore, essential for DNA replication and cell viability. Here, we report the identification of 7 individuals from 5 unrelated families presenting with a Meier-Gorlin syndrome–like (MGS-like) phenotype associated with hypomorphic variants of GINS3, a gene not previously associated with this syndrome. We found that MGS-associated GINS3 variants affecting aspartic acid 24 (D24) compromised cell proliferation and caused accumulation of cells in S phase. These variants shortened the protein half-life, altered key protein interactions at the replisome, and negatively influenced DNA replication fork progression. Yeast expressing MGS-associated variants of PSF3 (the yeast GINS3 ortholog) also displayed impaired growth, S phase progression defects, and decreased Psf3 protein stability. We further showed that mouse embryos homozygous for a D24 variant presented intrauterine growth retardation and did not survive to birth, and that fibroblasts derived from these embryos displayed accelerated cellular senescence. Taken together, our findings implicate GINS3 in the pathogenesis of MGS and support the notion that hypomorphic variants identified in this gene impaired cell and organismal growth by compromising DNA replication.

Authors

Mary E. McQuaid, Kashif Ahmed, Stephanie Tran, Justine Rousseau, Ranad Shaheen, Kristin D. Kernohan, Kyoko E. Yuki, Prerna Grover, Dominique Lévesque, Ema S. Dreseris, Sameen Ahmed, Lucie Dupuis, Jennifer Stimec, Mary Shago, Zuhair N. Al-Hassnan, Roch Tremblay, Philipp G. Maass, Michael D. Wilson, Eyal Grunebaum, Kym M. Boycott, François-Michel Boisvert, Sateesh Maddirevula, Eissa A. Faqeih, Fahad Almanjomi, Zaheer Ullah Khan, Fowzan S. Alkuraya, Philippe M. Campeau, Peter Kannu, Eric I. Campos, Hugo Wurtele

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Figure 8

Mouse embryonic fibroblasts homozygous for Gins3D24N allele show increased senescence and decreased DNA replication.

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Mouse embryonic fibroblasts homozygous for Gins3D24N allele show increas...
(A) Senescence-associated β-galactosidase staining was assessed by bright-field microscopy in 3 separate experiments. Each point in the graph represents a separate MEF cell line. Results were combined by taking the mean of cell lines assayed in multiple experiments. A minimum of 500 cells were analyzed per experiment, and statistical analyses were performed using 2-tailed t tests. Original magnification, 100×. (B) Expression of senescence-associated genes, as measured by qPCR, normalized to Gins3WT/Gins3WT cell line. Each point represents an independently isolated cell line. The experiment was performed twice independently, and statistical analyses were performed using 2-tailed t tests. (C and D) Cells were fixed and immunostained with antibodies against the indicated protein. Foci number (C) and intensity (D) were assessed for a minimum of 50 cells from each cell line as described in Methods, and statistical analyses were performed using 2-tailed t tests. (E) Asynchronous cultures of primary MEFs were pulsed with 10 μM EdU for 2 hours prior to fixation. EdU content was assessed by flow cytometry (left). EdU levels in EdU+ cells are shown in scatter plot (right); bar represents median, and statistical analyses were performed using 2-tailed t tests. Experiment was performed in duplicate; a representative replicate is shown. (F) Cell cycle profile of cells from EdU experiment shown in E. P values were adjusted for multiple comparisons where appropriate (see Methods). For details on experimental procedures, see Methods. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.