Abstracts

Rationale: SLC35A2-congenital disorder of glycosylation (SLC35A2-CDG, also known as congenital disorder of glycosylation type llm), is an X-linked disorder associated with infantile seizures, hypsarrhythmia, hypotonia, and brain malformation (Quelhas et al., 2021). Also, somatic variants in SLC35A2 have been linked to malformations of cortical development (MCD) and drug-resistant epilepsy (Sim et al, 2018 and Bonduelle et al, 2021). SLC35A2 is a UDP-galactose translocator that aids in glycan synthesis by providing galactose to the Golgi and ER (Barbara et el., 2014). SLC35A2 loss of function has been clearly associated with disease, but pathogenesis remains understudied. An animal model would be useful to study the mechanism of disease.

Methods: We generated a floxed mouse carrying loxP sites around exon 3 of Slc35a2. First, to model the SLC35A2-CDG patient genotype, we crossed floxed mice with E2a-Cre mice to generate germline knockout of Slc35a2. Secondly, we modeled focal loss of Slc35a2, as observed in MCD patients, by crossing floxed mice with an Emx1-Cre line to generate forebrain-specific knockout of Slc35a2. To characterize the molecular phenotype of each model, we utilized PCR, immunofluorescence, and western blotting analyses. Also, we measured body and brain weight, gross brain anatomy, as well as muscle reflexes by the righting test. Survival plots were generated for each model to measure the severity of the effect of the deletion of Slc35a2.

Results: We were able to successfully generate heterozygous female Slc35a2 knockout mice with E2a-Cre and Emx1-Cre expression, respectively. Interestingly, hemizygous germline knockout male mice are not observed, and all hemizygous forebrain knockout male mice did not survive past 6 weeks (80% of them died within 4 weeks after birth, n=10). Hence, we focused on heterozygous females for additional study. In germline knockout mice, we confirmed the reduced amount of SLC35A2 in the heterozygous brain (about 50% compared to wild-type brain). In addition, body and brain weight at P21 were significantly reduced compared to wild type (40% and 25% reduction). Brain lateral ventricles were significantly enlarged, and the thickness of the cortex was reduced compared to wild type. In the forebrain-specific knockout model, intensity of SLC35A2 immunofluorescence signal was significantly reduced in heterozygous females. Reduced body weight in this line was not observed, but righting reflex was diminished. Currently, experiments are ongoing to fully examine the behavioral, neuroanatomical, and seizure phenotypes of these mice in order to understand their relevance as disease models._x000D_
Conclusions: In our study, we generated a novel conditional knockout mouse model of Slc35a2. We were able to confirm the reduction of Slc35a2 in our model and preliminary results suggest that these mice show some phenotypes relevant to human disease. Our new murine model is expected to contribute to understanding disease pathogenesis, which will open up new avenues for disease treatment plans.

Funding: This work is funded by Internal Startup funds for Tracy Bedrosian (Nationwide Children’s Hospital, Columbus, Ohio).