Identification and functional characterization of new disease genes and variants implicated in pediatric encephalopathies: from exome sequencing to in vivo investigation

Rare diseases are a heterogeneous group of clinical conditions that affect pediatric patients in about two-thirds of the cases and, most of them, have a genetic cause. These are chronic conditions, often complex to treat. The diagnosis is very challenging for many of these patients and, in about half of the pediatric cases presenting syndromes associated with cognitive impairment, identifying the cause of the disease remains complex. The Ospedale Pediatrico Bambino Gesù (OPBG) and above all the Unit of “Molecular genetics and functional genomics”, directed by Dr. Marco Tartaglia, carries out projects dedicated to find the genes implicated in rare diseases in orphan patients and explore the underlying pathogenetic mechanisms. In my PhD thesis, I have contributed to discover the molecular cause of forms of complex encephalopathies participating to whole-exome sequencing (WES) efforts that allowed to identify new disease genes of previously unknown pathologies. For one case, I investigated the pathogenesis using zebrafish as an in vivo model under expert supervision on animal experimentation. Specifically, the goal of my thesis was: 1. Initial identification, through the use of WES, of new genes responsible, when mutated, for rare neurodevelopment disorders (different forms of pediatric, isolated and syndromic encephalopathies); 2. The functional validation of a panel of mutations affecting one of these genes, through the use of small freshwater fish, Danio rerio (zebrafish), as an ideal model system for studying pathophysiology in embryonic development programs. During the first part of my PhD activity, I became confident with the WES technology starting from the DNA preparation library (from patients’ peripheral blood DNA samples) to sequencing reactions and row data analysis (derived from bioinformatics core of our research group). In this context, I focused my PhD work on the study of news forms of encephalopathies caused by mutations in genes encoding channels or subunits of ion channel (KCNK4; Bauer., et al., 2018) and encephalopathies caused by mutations in genes encoding proteins involved in the control of cytoskeletal dynamics and intracellular trafficking (CLTC; Manti., et al., 2018; and ARF3; manuscript in preparation). The identification of new disease-causing genes or genetic variants allow me to begin with the in vivo functional validation. Indeed, I focused the last period of my PhD work on the functional study of the variants identified in the ARF3 gene modeled in zebrafish. First, I performed studies of arf3 sequence to define the conservation among orthologs genes in human and in zebrafish. Second, I actively participated in the analysis of the phenotype of ARF3 transiently expressed mutants generated by my colleagues. Given the clinical features of patients (short stature with skeletal abnormalities and microcephaly), I investigated morphological characteristics of embryonic development leading to body axis formation and the possible impact on cephalic development. I deepened my analyses by looking at markers of early axis establishment (convergence and extension movements during early embryogenesis) via whole-mount in situ hybridization of specific mRNAs (krox20 and myoD) used as markers to reveal abnormalities in those processes. An enlargement of the medio-lateral (ML) axis as compared to a shortening of the anterior-posterior (AP) axis, which was evident in early mutant embryos after gastrulation as early as 15h of development. Fish had a shortened body axis and microcephaly. In summary, in my PhD I have contributed to find genetic diagnosis in some previously unsolved cases via next-generation sequencing and have actively participated in the in vivo validation of a new disease-gene causing a severe form of encephalopathy.