A Dominant Negative Variant in MAST4 Causes a Developmental and Epileptic Encephalopathy
Abstract number :
2.462
Submission category :
12. Genetics / 12A. Human Studies
Year :
2023
Submission ID :
1349
Source :
www.aesnet.org
Presentation date :
12/3/2023 12:00:00 AM
Published date :
Authors :
Presenting Author: Kimberly Aldinger, PhD – Seattle Children's Research Institute
Moira Cornell, BS – Pharmacology – University of Washington; Alexandra Vilceanu, MD – Ludwig-Maximilians-Universität-München; Dan Doherty, MD PhD – Pediatrics – University of Washington; David Keays, PhD – Ludwig-Maximilians-Universität-München; Smita Yadav, PhD – Pharmacology – University of Washington; Scott Houghtaling, PhD – Center for Developmental Biology and Regenerative Medicine – Seattle Children's Research Institute
Rationale: De novo variants in MAST4, encoding a microtubule-associated serine-threonine kinase, are implicated in developmental and epileptic encephalopathy (DEE) etiology. We identified a patient with DEE, intellectual disability, axial and oral motor hypotonia, spasticity, and postnatal microcephaly and a de novo heterozygous splice site variant in MAST4 (642+6T >G). We aimed to investigate the molecular functions through which MAST4 variants could impact cellular functions.
Methods: We isolated RNA from cultured fibroblasts, transcribed into cDNA, and performed RTPCR. We PCR amplified full-length (FL) MAST4 from human fetal brain cDNA and cloned it into expression plasmids. We introduced seven MAST4 variants into FL MAST4 coding sequence. Domain dissection mutants were subcloned from sfGFP-MAST4. We performed an in vitro transcription and translation reaction using porcine tubulin extracts with biotinylated lysine labeled human MAST4. Reaction products were assayed for ability to bind to microtubules in the absence or presence of microtubule-associated proteins (MAPs). HEK cells were transfected with GFP-tagged WT or variant MAST4 and visualized under a confocal microscope. We used AlplaFold2.0 to predict MAST4 3D structure and impact of DEE variants.
Results: The proband variant in MAST4 is in the intron after exon 3. RTPCR revealed an amplicon shorter than the reference transcript. Sequencing confirmed skipping exon 3, an altered reading frame, and a nonsense codon in exon 6 (ΔEx3). We found MAST4 associates with Taxol-stabilized microtubules with enhanced affinity in the presence of MAPs. ΔEx3 abolishes MAST4 microtubule binding, whereas 6 other DEE variants do not alter MAST4 translation efficiency or its microtubule binding. We expressed GFP-tagged MAST4 constructs with DEE-associated variants in HEK cells. WT and 6/7 MAST4 variants were primarily cytoplasmic while ΔEx3 localized to the nucleus. To identify the mechanism by which MAST4 could achieve nuclear localization, we generated 6 GFP-tagged deletion constructs and expressed them in HEK cells. A truncated construct representing the ΔEx3 mutation (1-172) was nuclear while a construct containing the DUF domain (1-555) was cytoplasmic. There were two other potential masked NLS in MAST4, one in the kinase domain (556-905), and another in the proximal C-terminal region (1248-1848). AlphaFold2.0 prediction shows that all missense mutations lie in unstructured regions of MAST4 except for H1172R which lies in the PDZ domain. Alphafold2.0 analysis of the ΔEx3 MAST4 variant that localizes to the nucleus shows that the C-terminal tail coded by this variant that is absent from WT MAST4 folds in an alpha helix that might lead to protein stability and a dominant negative effect.
Conclusions: We show that MAST4 is a microtubule binding protein that localizes to the cytoplasm. 642+6T >G leads to a truncated protein with a dominant-negative nuclear localization defect of MAST4 due to the premature stop that exposes an NLS not otherwise active in the full length protein. Further investigation is required to decipher how dominant-negative MAST4 impacts brain development and function.
Funding: ITHS Catalyst Award; AES Junior Investigator Award; R03TR004179
Genetics