Application of different methodologies for the detection of genetic variants in patients with neurodevelopmental disorders negative for single nucleotide variants

Neurodevelopmental disorders (NDDs) are a class of early-onset conditions that affect brain development and function, resulting in deficits in different areas such as cognition, communication and motor skills. These disorders affect approximately 15% of children and adolescents worldwide. A notable feature of NDDs is their frequent comorbidity with epilepsy, a neurological disorder characterized by recurrent seizures. It is estimated that 15-26% of people diagnosed with NDDs also present epilepsy. Both NDDs and epilepsy exhibit significant genetic and clinical heterogeneity, which poses a challenge in achieving accurate molecular diagnosis. Recent years have seen significant advances in the field of molecular diagnostics, particularly in the analysis of single nucleotide variations (SNVs) and copy number variations (CNVs). These genetic variations have been identified as key targets for improving diagnostic rates in NDDs and epilepsy, conditions that still have some of the highest rates of undiagnosed cases. The aim of this thesis work was to improve diagnostic outcomes by assessing the impact of genetic variations in NDDs and epilepsy disorders through a combined methodological approach. A group of 100 epilepsy patients, who tested negative for SNVs in ~ 1000 known causative genes, was selected for this study. Available whole-exome sequencing (WES) data was analysed using bioinformatic tools to identify CNVs. Five CNVs were detected and validated trough real-time PCR and comparative genomic hybridization (array-CGH). The findings confirmed the presence of CNVs in four patients, suggesting their potential genetic involvement. A parallel analysis was conducted on another cohort of 100 NDDs patients negative for SNVs and CNVs. Sanger sequencing was performed to identify the presence of variants in RNU4-2 small nuclear RNA, a key component of the major spliceosome, because recent studies have associated rare mutations in this snRNA with onset of NDDs. One patient was found to be positive for a recurrent de novo insertion, n.64-65insT, within an 18-base pair critical region of the RNU4-2 small nuclear RNA, confirming that RNU4-2 may be a causative gene for NDDs. In conclusion, by combining WES with Real Time PCR, CGH and Sanger sequencing, I was able to identify rare genetic variations that were previously undetected. These findings highlight the effectiveness of a combined methodological approach in improving the molecular diagnosis for NDDs and epilepsy.