Combining exome sequencing and functional studies to identify causal genes of ultra-rare neurodevelopmental disorders

Resumo

Neurodevelopmental disorders (NDDs) are a group of chronic diseases in which the development of the central nervous system is impaired, resulting in disability at the neuropsychiatric, motor and/or intellectual level. Some of these disorders are considered syndromic. For instance, intellectual disability (ID) may present comorbidity with other neurological conditions (such as seizures or behavioral problems), dysmorphic features and/or internal organ anomalies. The vast majority of syndromic NDDs have a genetic origin and are considered to be rare, affecting less than 1 in 2000 people. However, globally, these diseases represent a serious social and health issue. Even though the majority of them are monogenic, many of them remain with an unknown molecular basis. Next-generation sequencing technologies have played a critical role in the optimization of the diagnosis of NDDs during the last decades. In this thesis, we have used a combination of whole-exome sequencing (WES) and functional studies to establish the diagnosis of 9 cases tentatively diagnosed as Opitz C, a clinical entity that encompasses patients with very different molecular causes. We identified the genetic origin of the disorder in the 7 tested families, which happened to be different variants in different genes for each of them. We showed that WES is a powerful approach to identify the molecular basis of ultra-rare NDDs. A significantly higher diagnosis yield was reached compared with other studies, potentially explained by a deep analysis of the sequencing data using in silico predictors, followed by the performance of specific functional studies for each case. We identified four different variants putatively affecting splicing patterns of different genes (ASXL1, KAT6A, PIGT and FOXP1) and tested them directly using fibroblasts obtained from patients or indirectly using a mini-gene splicing assay. We assessed the effect of variants in DPH1 in protein function combining a biochemical technique with a protein structural model and we established a correlation between the results of the tests and the severity of the patients’ phenotype. We contributed to the delineation of a recently described syndrome caused by germline mutations in TRAF7 by gathering and describing a cohort of 45 patients. We also performed a transcriptomics analysis on fibroblasts from different patients carrying TRAF7 mutations, which showed alterations in the expression of different genes that might contribute to the phenotype. Aiming to characterize truncating mutations in MAGEL2, which are responsible for Schaaf-Yang syndrome (SYS), we performed different experiments that suggest a potential toxic effect of the produced truncated form of the protein, which lacks its most relevant functional domain. Finally, as a first step to establish a relevant in vitro model of SYS, we reprogrammed fibroblasts from different patients to induced pluripotent stem cells (iPSCs), which can be then differentiated to relevant neural cell types and brain organoids to further study the pathophysiological mechanisms underlying this disease.