NOVEL DISEASE GENES DISCOVERY AND INNOVATIVE APPROACHES FOR INVESTIGATING NEURODEVELOPMENTAL DISORDERS

Neurodevelopmental disorders (NDDs) encompass a wide range of conditions, including Autism Spectrum Disorders (ASD), intellectual disability (ID), language delays, and epilepsy. These disorders exhibit significant genetic heterogeneity, involving all modes of inheritance. In the last years, the development of high-throughput technologies, like next-generation sequencing, has given the possibility to unravel the genetic mechanisms behind NDDs, and helped patients and their families to shorten the diagnostic odyssey giving a name to the disease they are facing. In 2015, the Laboratory of Medical Genetics and Rare Diseases at the University of Turin launched the NeuroWES project, a multicenter initiative in collaboration with the Autism Sequencing Consortium. This project aims to unravel the genetic basis of neurodevelopmental disorders (NDDs) using trio-exome sequencing. Overall, 417 families have been recruited from various hospitals across Italy and analyzed. Over 30% of these families received a report identifying a causative variant classified as pathogenic or likely pathogenic (ACMG classes 5 and 4). Additionally, less than 7% of cases harbored variants of uncertain significance (ACMG class 3). The project has also contributed to the identification of approximately seventeen novel genes, either through studies conducted in-house or in collaboration with international laboratories. Among these, DDX53 and ZMYM3 were identified in the NeuroWES cohort and studied in collaboration with other groups, culminating in their confirmed association with neurodevelopmental disorders. Several other candidate genes are under investigation. For instance, UBE2I, encoding UBC9 — a critical protein in the SUMOylation pathway — has shown promise in preliminary functional analyses involving few patients, though further experiments are needed to solidify its role in NDDs. Similarly, UPF1 is being intensively studied in collaboration with an Australian group that has collected data from over twenty patients with variants in this gene. Their preliminary findings strongly support UPF1 as a novel NDD gene. The NeuroWES cohort has also uncovered several unique and complex cases. For example: | 7 • Case 375: A dual genetic diagnosis was identified, with de novo pathogenic variants in TLK2 and DYNC1H1, both associated with overlapping phenotypic features. • Case 461: A missense variant in NFIB, affecting the last base of an exon was shown to result in partial exon skipping. This was demonstrated using cDNA analysis from the patient. • Case 402: An initial negative case was solved by re-analyzing the exome data in the light of previously unknown clinical information of the parents. In fact, a splicing variant in HDAC8 inherited from the mother, and a nonsense variant in CAPRIN1 inherited from the father were found, highlighting the critical importance of comprehensive clinical information and the value of reanalyzing exome data with improved bioinformatic pipelines. The NeuroWES Project has proven invaluable for the families involved, thanks to the numerous diagnoses made, continuous reanalysis of cases, and advancements in NDDs genetics. It has underscored the potential of exome sequencing as a routine tool for uncovering the genetic causes of NDDs. Finally, we show how we can contribute to a deeper understanding of known NDDs. For example, the Duplication 15q syndrome was further studied to explore the mechanisms underlying the disorder. An in vitro system was developed to analyze the electrophysiological signatures of neuronal networks derived from Dup15q patients’ cells. This platform will also be utilized to test potential treatment strategies for overcoming epilepsy resistance in patient-derived cell lines.