Identification of a novel synaptic protein, TMTC3, involved in periventricular nodular heterotopia with intellectual disability and epilepsy

Abstract

Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific expression of human TMTC3, suggesting a role for TMTC3 in seizure biology. In addition, we observed co-localization of TMTC3 in the rat brain with vesicular GABA transporter (VGAT), a presynaptic marker for inhibitory synapses. TMTC3 is localized at VGAT positive pre-synaptic terminals and boutons in the rat hypothalamus and piriform cortex, suggesting a role for TMTC3 in the regulation of GABAergic inhibitory synapses. TMTC3 did not co-localize with Vglut2, a presynaptic marker for excitatory neurons. Our data identified TMTC3 as a synaptic protein that is involved in PVNH with ID and epilepsy, in addition to its previously described association with cobblestone lissencephaly.

ER transmembrane protein TMTC3 contributes to O-mannosylation of E-cadherin, Cellular Adherence and Embryonic Gastrulation

“…Mutations in TMTC3 were identified in patients with Cobblestone lissencephaly and PVNH, common brain malformations caused by defects in neuronal migration (Jerber et al, 2016;Farhan et al, 2017). More specifically, three of the mutations (R488Efs, F562Lfs and G384E variants) identified in Cobblestone lissencephaly patients that possess a significant number of clinically observed neurological defects were found to be unstable in our protein degradation assay (Jerber et al, 2016) (Fig.…”

“…Point mutations in human TMTC2 and the knockout of Tmtc4 in mice result in hearing loss (Runge et al, 2016;Guillen Ahlers et al, 2018;Li et al, 2018). Ten mutations in TMTC3 are associated with neuronal cell migration diseases (Jerber et al, 2016;Farhan et al, 2017). Homozygous and compound heterozygous mutations resulting in single amino acid or frame shift changes in TMTC3 were identified in a cohort of families with recessive forms of Cobblestone lissencephaly, a severe brain malformation in which over migration of neurons and glial cells results in the formation of cortical dysplasia or brain development abnormalities (Jerber et al, 2016).…”

“…Homozygous and compound heterozygous mutations resulting in single amino acid or frame shift changes in TMTC3 were identified in a cohort of families with recessive forms of Cobblestone lissencephaly, a severe brain malformation in which over migration of neurons and glial cells results in the formation of cortical dysplasia or brain development abnormalities (Jerber et al, 2016). Another study identified heterozygous variants of TMTC3 in patients with periventricular nodular heterotopia (PVNH), a common brain malformation caused by the failure of neurons to migrate from the ventricular zone to the cortex (Farhan et al, 2017). While mutation or loss of the TMTC genes has been associated with a number of diseases, an understanding of how these mutations result in specific defects is unclear.…”

“…Additionally, the knockdown of Tmtc3 and Tmtc4 in Xenopus laevis resulted in an embryonic gastrulation delay phenotype and the delay was rescued by human TMTC3. There are eight disease variants of TMTC3 recently associated with Cobblestone lissencephaly and two associated with PVNH (Jerber et al, 2016;Farhan et al, 2017). While novel biochemical characterization of eight of these disease variants showed that they all localize to the ER, three of them are less stable than wild type (WT) and none of the missense mutations are able to rescue the delay in gastrulation caused by the knockdown of Tmtc3 in Xenopus laevis.…”

Recent Developments in Using <em>Drosophila</em> as a Model for Human Genetic Disease

“…Four affected siblings in an affected family were all found to be compound heterozygotes of two variants in TMTC3, one that produces a missense mutation (R71H) and another that produces a frameshift that truncates the normally 914amino-acid protein after amino acid 728 [21]. TMTC3 contains nine tetratricopeptide (TPR) repeats and the frameshift mutation results in the loss of the final three of these.…”