par Salik, Déborah 
Président du jury Casimir, Georges
Promoteur Smits, Guillaume
Co-Promoteur Richert, Bertrand
Publication Non publié, 2023-04-27
Président du jury Casimir, Georges
Promoteur Smits, Guillaume
Co-Promoteur Richert, Bertrand
Publication Non publié, 2023-04-27
Thèse de doctorat
| Résumé : | Although rare diseases seem exceptional on an individual level, they represent a significant global burden. Around 3 to 6% of the worldwide population suffer from a rare disease of which 80% are due to a genetic cause. Genodermatoses are a heterogeneous group of rare genetic skin disorders characterized by variable phenotypic expression and genetic heterogeneity making their diagnosis challenging. Owing to the complexity of these disorders, patients may remain undiagnosed for several years, during which the disease may worsen or even recur within the same family. Accurate diagnosis of genodermatoses is essential to offer genetic counseling and preconceptional guidance to families, to improve clinical management, and to develop innovative therapy. The aim of this PhD was to create a multi-omic strategy that included phenotyping, clinical exome and RNA-sequencing to enhance the genetic diagnosis of genodermatoses. Clinical implementation of next-generation sequencing (NGS) technologies such as whole-exome and clinical exome sequencing (WES/CES) has allowed the deciphering of numerous genodermatoses. Therefore, the first part of our work was to evaluate the diagnostic rate of CES in a cohort of children with genodermatosis suspicion. The yield of an in silico genodermatoses panel of 393 genes was compared to the analysis of the whole 4866 genes content of CES. It was performed in duo/trio/quartet on 56 children between 0 to 18 years with symptoms suggestive of genodermatosis, in a multistep variant analysis strategy. The diagnosis rate was 55.4%. Subsequent analysis of the entire CES yielded three supplementary diagnoses and two additional incidental findings. These results confirmed that sequencing is a powerful tool in the diagnosis of pediatric genodermatoses in clinical practice, making genodermatoses one of the most genetically diagnosed rare disorders using DNA-based NGS technologies. Moreover, we highlighted that our 393 genes panel was as efficient as the whole-CES analysis. Nevertheless, this technology leaves a large part of patients without a molecular diagnosis. The reason for persistent unsolved cases is that CES/WES does not allow the analysis of non-exonic sequences that impact gene expression and which may account for 9-30% of diagnoses. In addition, WES is not able to functionally validate non-synonymous, synonymous or silent nucleotide substitutions in exons or non-canonical splicing sequences affecting transcription. A transcriptome (RNA-sequencing or RNA-seq) allows for exploring the transcriptional effects of exonic, untranslated region (UTR) and non-coding mutations, by highlighting aberrant splicing and allele-specific expression. The addition of RNA-seq can increase the diagnostic rate by 7.5-36%. Hence, the second aim of this work was to elaborate a complete protocol for RNA-sequencing from primary fibroblast and keratinocyte cultures and evaluate its clinical contribution as a complementary tool to improve diagnostic rate in children with genodermatoses, not diagnosed by CES. We developed consistent and reproducible keratinocyte and fibroblast cultures and RNA-seq protocols. The streamlining of the bioinformatic analysis will be the key to implement transcriptome analysis in clinical routine.The skill and ability to establish accurate diagnoses provides us with the opportunity to better understand the underlying pathophysiological mechanisms of these diseases, therefore improving the prevention of associated comorbidities, along with access to specialized multidisciplinary care and expert teams. It also offers both the patients and their families essential psychological benefits. Moreover, these aptitudes allow for better informed genetic counseling. |
