In Silico Analysis and Predictions

Hello everyone and welcome to Science with Sandra!

For this edition I would like to highlight a recent publication. “GLUT-1DS resistant to ketogenic diet: from clinical feature to in silico analysis. An exemplificative case report with a literature review” by Professor Raffaele Falsaperla and his team. Professor Falsaperla is an Associate Professor of pediatrics and head of pediatric, emergency and neonatal intensive units in Catania, Italy. His team works on molecular dynamic simulations as individualized therapy in GLUT1 encephalopathy.

The publication describes how the ketogenic diet (KD) is the primary treatment for GLUT1 Deficiency, being most effective at controlling epileptic seizures. However, there are other symptoms that also impact patients and their families’ quality of life, such as movement disorders, for which the diet is not as effective apparently. In some patients, the diet fails to improve symptoms (resistant to KD); however, the reason why is not yet established, and there is little information about it. 

The goal of Professor Falsaperla and his team of pharmaceutical chemist and geneticist, was to propose a new clinical research approach based on precision medicine and molecular modeling. Their intention is to apply this approach to GLUT1 Deficiency patients who are resistant to the ketogenic dietary treatment. To do this, they conduct an in silico study (a simulation performed on a computer) of genetic and altered protein products to have a better understanding of the relationship between genotype and the symptoms experienced by the patient in relation with the diet.

For this publication, they analyzed the case of a GLUT1 Deficiency patient who was treated for seizures as an infant and was later diagnosed at age 8 with GLUT1 Deficiency when her treatment for seizures (phenobarbital) was discontinued. Once diagnosed, the patient started on the ketogenic dietary treatment (KDT) (3:1 ratio) and her seizures were controlled. Unfortunately, at age 10, she started experiencing episodes of dystonia lasting several hours and which disrupted her daily activities and resolved spontaneously. Several tests were performed including EEGs and brain MRI. However, doctors observed that her symptoms worsened when her ketone level exceeded 4 mmol/l. The doctors then decided to change her diet from a 3:1 to a 2.5:1 ratio and this resulted in ketones reduction and improvement of her symptoms with complete resolution.

One of the possible explanations the authors offer for the dystonia episodes occurrence at higher ketone levels is that these episodes could be caused by an excessive action of other mechanisms of the KDT mediated by ketone bodies. The authors describe how ketone bodies can inhibit directly or indirectly a receptor called AMPA receptor. This receptor is present in a wide range of neuron types and other cells in the brain and helps mediate fast excitatory synaptic transmission. According to the authors, AMPA receptors play a role in dopamine release in the striatum, a specific area of the brain. In addition, ketone bodies can also induce a decrease in dopamine release from the striatum. All of these mechanisms can contribute to the worsening of dystonic episodes during the highest level of ketosis due to an indirect action on the dopamine system with a reduction in the efficacy of dopamine action.

One of the interesting things in her case, is that when she first received genetic testing the results were negative for variants/mutations on the SLC2A1 gene (the gene that produces the GLUT1 protein). However, further testing detected a microdeletion on the SLC2A1 gene. The first test used was a Next Generation Sequencing test (NGS) which is a technology that is used for reading the DNA or RNA sequence that detects variant/mutation in the sequence. The second test used, and the one that detected her variant, is called Multiplex Ligation Probe Amplification (MLPA) which is a special molecular technique used to scan large deletions/duplications/multiplications on DNA sequences which are not detected by NGS or Sanger (another standard sequencing technique). 

Because the deletion had not yet been described in the literature, Prof. Falsaperla’s team decided to perform molecular modeling studies to elucidate the molecular mechanisms underlying this mutation. They conducted an in silico analysis of genomic and protein structure to have a better understanding of the effect of the deletion in the protein structure and function. The results of the analysis showed that the GLUT1 protein folds incorrectly, leading to impaired glucose transport. 

Finally, the authors discuss the importance of finding alternative and additional treatments for GLUT1 Deficiency patients, as well as exploring other treatments for patients who don’t respond to the diet or for whom the diet worsens their symptoms. In this regard, their goal with their in silico test is to carefully assess the type of mutation/variant present in the patient and to complement this information to patient registry data to evaluate the possible benefits and whether to start the KDT for treatment of recently diagnosed GLUT1 Deficiency patients.

We thank Professor Falsaperla and his team for the work they are doing to help our community and we thank him for reviewing this summary.

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