Re-arranged L-Myc Fusion (RLF) is a zinc finger DNA binding protein originally foundas fused with L-MYC in some cancers. De novo heterozygous mutations in the RLF gene have been recently found in a cohort of patients with an array of symptoms including intellectual disability (ID), recognizable facial dysmorphism, motor delay, behavioral alterations, and occasional congenital malformations. RLF protein has been involved with the regulation of critical elements of the epigenetic code which have been found altered in diseases phenotypically close to the condition of RLF patients. Details on RLF functions and its characterization in human settings are still missing. The aim of this project is the investigation of newly identified RLF mutations in patient deficits as leverage to find insights in pathophysiological mechanisms related to RLF. We generated human isogenic iPSC lines carrying different RLF mutations including as controls RLF deletion in one or both alleles (RLF KO).We are exploiting our iPSC models to evaluate the effect of RLF truncated proteins as well as complete or partial LoF on neural derivatives, through the differentiation of cortical neural progenitors (NPCs), post-mitotic neurons and cerebral organoids. We started to phenotypically analyze RLF WT, Het and KO iPSC cells, testing them for karyotype, pluripotency, multi-germ layer differentiation, and proliferation showing results indistinguishable from the parental line. Mutant NPCs (RLF Het and KO) didn't display defects in both key markers and proliferative rate. On the other hand, the differentiation in cortical neuronal cells was affected for the yield and showed a defect in the shape of the neurons. We next aim to expand phenotypical analysis to cell lines carrying patient-specific mutations and to 3D cortical spheroids. To better investigate the molecular role of RLF and its disease-inducing variants in neurological context, we performed preliminary genomic studies starting from RLF WT and KO iPSC lines, including transcriptomic analysis (RNA-seq), chromatin accessibility evaluation (ATAC-seq) and the profiling of DNA methylation (MeDIP-seq). We found a slightly higher prevalence for gene downregulation more than upregulation in RLF KO cells, that the mutant chromatin is less open than in control, and a highly methylated genome. Further analysis, including a proper integration between the datasets, and the expansion of genomic studies to the other cell lines and also to both 2D and 3D neural derivatives, will help to clarify the impact of the RLF absence and mutant forms on the chromatin at genome-wide level. In conclusion, through our iPSC-based in vitro modelling, we foresee to identify those loci on the genome that are (i) sensitive to RFL LoF and/or mutations, (ii) important for transcription in neural cells, and (iii) at the basis of the related phenotypes. These data may provide hints on the role of RLF for the pathophysiology of the associated neurological diseases.

Studio delle alterazioni molecolari indotte da mutazioni nel gene RLF in casi di disabilità intellettiva. Mutazioni in un gene chiamato RLF sono state individuate in pazienti con con disabilità intellettiva e alterazioni comportamentali. Le funzioni di gene e proteina associata sono poco note quindi le opzioni terapeutiche rimangono limitate. In questo progetto, abbiamo generato modelli sperimentali coerenti (di origine umana, con le mutazioni trovate nei pazienti, con cellule neurali) per lo studio del ruolo fisiologico di RLF e delle sue aberrazioni alla base della patologia umana. In questo momento stiamo ottenendo dati di sequenziamento del genoma e delle caratteristiche epigenetiche che ci stanno permettendo di indentificare meglio la funzione fisiologica di RLF e l'impatto delle sue mutazioni in particolare in modelli sperimentali che ricapitolano cellule neurali umane. A medio termine ci aspettiamo di generare quella conoscenza in grado poi di favorire lo sviluppo di terapie clinicamente rilevanti per i pazienti RLF.

Mental Retardation facial dymorphism, Kabuki-like

Ritardo mentale con dimorfismo facciale, simil-Kabuki