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Molecular genetic analysis of UGDH gene in developmental and epileptic encephalopathy (DEE) (Bachelor thesis)

Παπαδοπούλου, Μαριάνθη/ Papadopoulou, Marianthi

The UDP Glucose 6-Dehydrogenase (UGDH) protein is the only human enzyme that catalyses the conversion of UDP-α-D-glucose to UDP-α-D-glucuronic acid. UDP-α-D-glucuronic acid is a component of the complex glycosaminoglycans and is therefore an intermediate sugar in carbohydrate metabolism. This role is one of the reasons why its expression is particularly required during embryonic development, for the proper development of the brain and neurons. Mutations in the gene that encodes for the UGDH enzyme have been associated with various types of diseases and pathological conditions, including some cancer types and congenital heart diseases. In 2020, one published article showed this gene to be responsible for some rare cases of developmental and epileptic encephalopathy. This thesis refers to a case report of the investigation of UGDH gene in an infant diagnosed with developmental and epileptic encephalopathy, as the genes usually responsible for the disease were not mutated. By the amplification of the coding gene fragments using PCR and following Sanger sequencing, compared to a control sample of a non-affected individual, two variants in heterozygosity were found. These could indicate that the cause of the disease is the existence of compound heterozygosity. In addition, an in silico analysis of these variants was performed with various bioinformatics tools to predict the damage they may cause and to categorize them as novel, pathogenic or likely pathogenic, based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. At last, using Reverse Transcription PCR (RT-PCR), we performed a semi-quantitative analysis of UGDH gene expression on the infant’s cDNA compared to a non-affected control cDNA, using GAPDH and B2M as housekeeping genes, in order to determine whether the pathogenic (likely null) allele is expressed at the mRNA level or is likely lost due to the mechanism of nonsense-mediated decay.