Thesis title: Molecular bases of GLUT1 deficiency syndrome: functional in vitro and in silico studies and a workflow implementation for variants' calling and annotation
GLUT1 deficiency syndrome (GLUT1 DS, OMIM #606777) is a rare genetic disorder characterized by insufficient glucose transport into the brain due to variants in the SLC2A1 gene, which encodes the GLUT1 protein, that facilitates glucose crossing the blood-brain barrier, essential for brain energy.
Classic GLUT1 DS phenotype is characterized by seizures, movement disorders, and cognitive/behavioral disturbances, however the expanding phenotypic spectrum includes various other symptoms, such as paroxysmal non-epileptic manifestations, and mild phenotypes.
Over 300 variants in SLC2A1 have been identified, ranging from large deletions to single nucleotide variants (SNVs) and insertions/deletions (INDELs), with significant phenotypic variability and with no clear genotype-phenotype correlations. Moreover, in about 20% of patients with clinical signs of the syndrome, no SLC2A1 mutation has been identified. The molecular pathogenetic mechanism is caused by haploinsufficiency, mainly due to loss-of-function variants, however it has been suggested, and only preliminary demonstrated, that missense variants may affect GLUT1 function in multiple ways.
In this work of thesis, we investigated the functional effects of selected SLC2A1 missense variants with different and complementary approaches, both in vitro and in silico, and we implemented an integrated analysis workflow to potentially increase the diagnostic yield of GLUT1 DS genetic tests. Glucose uptake assay detected a reduction of glucose transport in some mutants and, for three of them, immunofluorescence analysis showed an alteration of GLUT1 subcellular localization and trafficking, with evident accumulations in late endosomes/lysosomes. These results were concordant with in silico analysis by Molecular Dynamics simulations, showing an important steric hindrance hampering the correct glucose pathway.
We also implemented an integrative workflow for Next Generation Sequencing (NGS) data analysis for the SLC2A1 gene, including variant call, biological annotation, and filtering criteria to support functional and clinical interpretation of potentially pathogenic variants, especially those that are more difficult to interpret, namely missense variants, which are often classified as variants of uncertain significance (VUS). This workflow also considers variants that are usually filtered out in standard analysis (i.e. intronic, synonymous, UTRs) or not called by the standard pipelines (as Copy Number Variants and somatic variants), that can still prove to be pathogenic.
The detailed knowledge of different pathogenic mechanisms caused by GLUT1 mutations, the increase of diagnostic yield of genetics tests, and the resulting improvement of genotype-phenotype correlations may have important implications in genetic counseling and patient management, as a timely diagnosis is crucial for therapy, since ketogenic diet is the standard of care treatment.