Résumé
X-linked intellectual disability (XLID) refers to clinically and genetically heterogeneous disorders, accounting for 10–12% of all males with intellectual disability (ID). Previous studies have identified mutations in more than 100 genes located on the X chromosome. The first goals of this thesis were first to develop and to integrate the X chromosome exome sequencing (XES) in our diagnostic molecular laboratory for male patients with XLID, and thereafter to improve our knowledge in clinical and physiological insights concerning mutations in EIF2S3, a very rarely mutated-gene on the X chromosome.In the first instance, XES has been performed in a cohort of 93 familial cases with XLID, and then extended to a second cohort of 18 sporadic males with ID (without suspected X-linked inheritance). They were selected for XES because their mother had a skewed X chromosome inactivation suggesting being carrier of an XLID mutated-gene. This work allowed us to expand the diagnostic field in to 25% in the familial cases and up to 22% in the sporadic cases. In addition, it contributed to a better delineation of the clinical phenotype related to various X-linked genes (OPHN1, HUWE1, KDM5C). Finally, thanks to datasharing and diverse international collaborations, our results highlighted that females with a mutation on the X chromosome could also be symptomatic, not always protected by the X chromosome inactivation (XCI) pattern. In particular, we reported extremely skewed X chromosome inactivation (XCI) in favour of the normal allele in two different cohorts of symptomatic ID-females with HUWE1 mutation and with KDM5C mutation, for which we described the clinical features.
Furthermore, we focused on mutations in EIF2S3 encoding the γ subunit of eukaryotic translation initiation factor 2, eIF2, crucial for initiation of protein synthesis. On the one hand, we tended to delineate the phenotypic spectrum related to the gene. We suggest that severely affected patients with MEHMO syndrome (Mental deficiency with Epileptic seizures, Hypogonadism and Hypogenitalism, Microcephaly and Obesity) could be specifically related to a recurrent frameshift mutation (p.Ile465Serfs*4) in EIF2S3, while a few unique missense mutations cause incomplete phenotype of intellectual disability, microcephaly, growth retardation, epilepsy and inconstant glucose dysregulation. On the other hand, we performed functional studies and chose the in vivo zebrafish vertebrate model to unravel patholophysiological mechanisms leading to microcephaly and glucose dysregulation in EIF2S3-related disease. By using morpholino (MO) antisense oligonucleotides targeting translation of eif2s3 gene, we showed that knockdown of the zebrafish eif2s3 ortholog reproduced some features of the human phenotype such as a small head size with small eyes and decreased body length. The observed features seem to be specific as the rescue experiments with human or zebrafish mRNA partially restored the phenotype. Then we confirmed the pathogenicity of four missense mutations in EIF2S3 with additional rescue experiments in which co-injection eif2s3 MO with human EIF2S3 mRNA harbouring one of the mutations of interest failed to restore the small head size of zebrafish morphants. We further studied the biochemical and morphological consequences of eif2s3 deficiency in the zebrafish model using CRISPR/Cas9 genome editing to generate eif2s3 loss-of-function. Eif2s3 homozygous mutants displayed a statistically significant reduction of the head size. This was due, at least in part, to increased neuronal cell death in the early stage of nervous system development, as revealed by immunofluorescence and apoptosis studies. Next, as early-onset diabetes is a recurrent feature in severely affected patients with MEHMO syndrome, we provided additional pancreatic studies with our zebrafish mutant line and found high glucose levels. Immunofluorescence and whole-mount in situ hybridization analyses revealed both β cell and early pancreatic progenitor deficiency, not related to apoptosis or to abnormal proliferation. Finally, studies in cultured fibroblasts of patients with missense mutations showed the presence of the eIF2γ protein. However, in comparison with control fibroblasts, no increased apoptosis was observed neither under basal conditions nor using various apoptosis inducers.
In conclusion, whether apoptosis inconsistently found, our results in zebrafish provided new insights into disease mechanisms, suggesting apoptosis and early defects in cell differentiation. Further experiments, based among others on murine models and iPSCs studies, should be planned to better understand the consequences of EIF2S3 mutations, in particular their impacts in the neurogenesis and in the pancreatic function. We hope that this work, contributing to a better understanding of the physiological pathways involved in EIF2S3-related disease, will open the way to new therapeutic strategies in the future
| la date de réponse | 6 févr. 2020 |
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| langue originale | Anglais |
| L'institution diplômante |
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| Sponsors | Fonds de soutien Marguerite-Marie Delacroix |
| Superviseur | Isabelle Maystadt (Promoteur), Valérie Benoit (Copromoteur), Olivier De Backer (Président), Andre Goffinet (Jury), Julie Desir (Jury) & Hilde Van Esch (Jury) |