imprinting disease modelling by iPSCs and CRISPR/Cas9

Chromosomal rearrangement occurs in 1.8% of pregnancies (Forabosco A et al., 2009) and often lead to pregnancy interruption or early death of the newborn. The Kagami-Ogata syndrome is caused by rearrangements of a region on the maternal chromosome 14 that contains a cluster of genes regulated by imprinting mechanism. The Kagami-Ogata syndrome is often characterized by a severe phenotype that can, in some cases, cause the death of the patient in early ages. Even though the genes involved in the rearranged region are known, the molecular mechanisms related with the Kagami-Ogata syndrome have not been identified yet and the pathogenic role of these genes still needs to be investigated. This study focuses on a family in which the Kagami-Ogata syndrome occurred in all the progeny with different degrees in the phenotype severity. We used samples from this family to generate iPSCs aiming to demonstrate an effect of this case’s chromosomal rearrangement and, in particular, that the disease modelling using iPSCs is applicable for chr14 imprinting disorders. We also aimed to identify the gene(s) mainly responsible for the Kagami-Ogata syndrome’s clinical features and to reveal the phenotype variability in comparison with patUPD14 mouse model prediction. In order to verify the role of MEG3, that has been predicted as the most important gene in Kagami-Ogata syndrome and patUPD14 pathogenesis, we modified our patient-derived iPS cell lines to rescue the expression of intact MEG3. Because some phenotypes can be correlated with malfunctioning of nervous system and because MEG3, one of the main genes investigated in this study, is mainly expressed in the brain, we further differentiated our patient-derived iPSCs into the neuronal lineage to study the transcriptional changing related with Kagami-Ogata syndrome.