Objectifs: Malgré leur grande hétérogénéité histologique et moléculaire, la prise en charge clinique des carcinomes ovariens de haut-grade (COHG) reste peu variable. Le pronostic sombre de cette pathologie implique un réel besoin des nouvelles thérapies. Au-delà des marqueurs pronostiques histologiques classiques et des enquêtes oncogénétiques, l’objectif de cette étude a consisté à rechercher des cibles moléculaires pharmacologiquement recrutables afin de pouvoir proposer aux patientes un accès à la thérapie innovante et personnalisée. Méthodes: Cette étude a été réalisée chez 53 patientes (pts) (âge moyen 58,9 ans, intervalle 25-87) de COHG histologiquement prouvés dont 45 pts de sous-type séreux. 19 pts ont fait l’objet d’une consultation et d’un test oncogénétique sur la base d’antécédents familiaux / personnel de cancer de sein/ovaire. chez. L’expression de P53 et de PTEN a été évaluée sur des tissus fixés au formol et inclus en paraffine par immunohistochimie. Les mutations somatiques de KRAS, NRAS, BRAF, PIK3CA et MET ont été recherchées par PCR-HRM (Polymerase Chain Reaction High Resolution Melting) puis vérifiées par NGS (Next Generation Sequencing) sur des extraits d'ADN préparés à partir d'échantillons de tumeurs congelés, prélevés au moment du diagnostic. Résultats: Des mutations germinales de BRCA1 / 2 ont été identifiées chez 7 pts, toutes atteintes des carcinomes séreux. Une mutation du gène KRAS (exon 2), 2 mutations du gène NRAS (exon 3), 6 mutations du gène PIK3CA (exon 5, 10 et 21) et 5 mutations du gène MET (exon 14 et 18) ont été identifiées chez les 53 tumeurs par NGS, dont deux mutations du gène NRAS et 2 mutations du gène PIK3CA détectées précédemment par PCR-HRM. Aucun profil mutationnel multiple n’a été retrouvé. La surexpression de P53 et la perte d’expression de PTEN ont été constatées chez 32 sur 53 (60%) et 19 sur 46 (41%) des tumeurs. L’analyse statistique n’a été réalisée que chez le sous-groupe de pts atteintes des carcinomes séreux à cause de l’effectif de l’étude. Avec un suivi médian de 38 mois (intervalle de 6-93), 35 pts ont eu une rechute de la maladie et 25 pts sont décédées. La survie sans progression à 2 ans est 28%, et la survie globale à 5 ans est 37%. La surexpression de P53 a été trouvée associée à une meilleure chimiosensibilité, une meilleure survie sans progression et une meilleure survie globale. Conclusion: Pour des COHG, au-delà des altérations de P53 et PTEN, des anomalies génétiques somatiques concernant les voies de signalisation PI3K et MAPK ne sont pas rares et peuvent être détectées par NGS. L’identification de ces anomalies somatiques pourrait offrir une possibilité des thérapies ciblées innovantes pour les patientes sur la base d’éléments diagnostics moléculaires. / Objectives: Despite the great histological and molecular heterogeneity, the clinical management of high-grade ovarian carcinoma remains univo-cal. As a major subgroup of ovarian carcinoma, high-grade ovarian carci-nomas (HGOC) need novel therapy. Additionally to conventional histolog-ical prognostic markers and oncogenetic investigations, molecular diag-nostic was performed using PCR-HRM (Polymerase Chain Reaction High Resolution Melting) and NGS (Next Generation Sequencing) to identify "druggable" targets that could provide access to innovative personalized therapy. Methods: This study was performed in 53 patients (pts) (mean age 58.9 years, range 25-87) with histologically proven HGOC of which 45 pts with serous carcinoma. BRCA1/2 germline mutations had been screened in 19 pts with familial/personal history of breast/ovarian cancer justifying on-cogenetic investigations. P53 and PTEN expression was assessed on for-malin fixed paraffin-embedded tissues using immunohistochemistry. So-matic mutations of KRAS, NRAS, BRAF, PIK3CA and MET were screened using PCR-HRM and then confirmed using NGS on DNA extracts from frozen tumor specimens taken at diagnosis. Results: Seven pts had BRCA1 / 2 germline mutations, all had serous carcinomas. One mutation of KRAS (exon 2), 2 mutations of NRAS (exon 3), 6 mutations of PIK3CA (exon 5, 10 and 21) and 5 mutations of MET (exon 14 and 18) were identified using NGS, of which 2 mutations of NRAS and 2 mutations de PIK3CA detected previously by PCR-HRM, no multiple mutation was detected. P53 overexpression and PTEN loss of expression was detected respectively in 32 of 53 (60%) and 19 of 46 (41%) of all the tumors. Because of the efffective of the study, statistical analyses were restricted to pts with serous carcinoma. With a median follow-up of 38 months (range 6-93), 35 pts had disease progression and 25 pts died during the follow-up. The 2-year progression-free survival (PFS) rate was 28% and 5-year overall survival (OS) rate was 37%. Overexpression of mutant P53 was found to be associated with chemosensitivity and longer PFS and OS. Conclusion: In HGOC, beside P53 and PTEN alterations, somatic genetic abnormalities of PI3K and MAPK signaling pathways can be detected us-ing NGS and provide molecular rationale for targeted therapies, potential-ly offering new therapeutic opportunities to the patients.

8 Summary The thesis "The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms" is focused on processing the massively parallel sequencing (MPS) data from a gene panel, whole-exome sequencing (WES) and whole-genome sequencing (WGS). The aim of the study was to develop a suitable pipeline to evaluate at least 250 MPS gene panel data, 150 WES data and 20 WGS data in order to improve molecular genetic testing of rare neurogenetic disorders. Associated data management and database implementation is also described. Targeted gene panel sequencing A custom-designed gene panel consisting of ge- nes previously associated with the disease was used. In the Epileptic Encephalopathy (EE) panel, two prerequisites need to be met for inclusion into the panel: the gene has to have been published in at least two independent publications OR at least in one publication but in multiple independent families. In the case of the EE panel, 112 genes were included. The targeted gene panel sequencing was then performed on 257 patients with EE. Pathogenic or likely pathogenic (according to ACMG criteria) variants have been found in 28% of patients (72 out of 257). Further analysis of the pathogenic or likely pathogenic variants was performed (76 in total); the variants were grouped by...

Deafblindness is a combined impairment of vision and hearing with an incidence of about 1: 8000 children and 1: 5500 adults. The most common genetic causes are the Stickler (STL) and Usher (USH) syndromes. The main goal of this work is to provide an up-to-date overview of STL and USH in the Czech and Slovak Republic (CR and SR), to determine the correlations between the genotype and phenotype in our population and the associated diagnostic criteria. Using sequencing and MLPA we examined 45 patients from 28 families for suspected STL. We found potentially causal variants of STL genes in 39 patients from 22 families. Fifteen different COL2A1 variants (8 being novel) were found in 28 patients from 18 families and 4 novel COL11A1 variants were found in 11 patients from 4 families. We identified the cause of the disease in 79 % of the families. The USH study involved 30 patients from 27 families. The most frequent cause was USH2A pathogenic variants, i.e. 19 variants in 14 families, 9 being novel. Less common were pathogenic variants in MYO7A (6 variants in 3 families, 5 being novel), USH1C and CDH23 (3 variants, 2 being novel, in 2 families both) genes. In 2 families, compound heterozygosity was found for variants in two different USH genes. The deafblindness etiology was clarified for 24 patients from...