La pratique du mariage entre apparentés au sein de la population libanaise, favorisée par des raisons sociales, religieuses, géographiques et aussi politiques, a vu apparaître des sous-groupes de populations de taille plus ou moins réduite, parfois à la limite d’isolats génétiques. Ceci a engendré une augmentation de la prévalence des maladies autosomiques récessives fréquentes mais aussi et surtout rares. Parmi ces dernières, les chondrodysplasies ont retenu notre attention. Elles sont caractérisées par un retard statural dû à un défaut du processus d’ossification endochondale, qui est responsable de la croissance des os longs. Au cours de ces dernières décennies, plus de 230 gènes responsables d’environ 400 maladies osseuses constitutionnelles ont été identifiés. Cependant, les bases moléculaires d'une centaine de dysplasies osseuses restent, à ce jour, inconnues. L’identification de gènes codant pour des protéines de nature extrêmement variée a contribué à la compréhension du mécanisme complexe d’ossification endochondrale. Mon travail de thèse, réalisé en cotutelle entre l’équipe de recherche « Bases moléculaires et physiopathologiques des chondrodysplasies » de l’hôpital Necker enfants-malades, à Paris en France et l’Unité de Génétique Médicale (UGM) de l’Université Saint-Joseph au Liban, a consisté à identifier des gènes impliqués dans des dysplasies osseuses autosomiques récessives dans quatre familles libanaises consanguines. Dans ce cadre, différentes stratégies ont été adoptées. La première a été une stratégie d’intersection des variations détectées par le séquençage de l’exome de deux patients, atteints d’une forme sévère de dysplasie spondylodysplastique létale et issus de deux familles libanaises consanguines et non apparentées (Familles A et B). Nous avons identifié une mutation homozygote du gène MAGMAS (NM_016069, p.Asn76Asp) (Mitochondria-associated granulocyte macrophage CSF-signaling molecule) à l’origine de la maladie dans les deux familles A et B. MAGMAS est une protéine associée à la mitochondrie et impliquée dans la régulation de l’import actif des protéines vers la matrice mitochondriale. Par immunohistochimie, nous avons montré que MAGMAS est spécifiquement exprimée au niveau de l’os et de la zone hypertrophique du cartilage. MAGMAS, ayant une fonction cruciale pour la survie, est très conservé entre les espèces. Après avoir généré des souches de levures exprimant une copie normale ou mutée du gène humain MAGMAS, nous avons validé l’effet délétère de la mutation p.Asn76Asp, i) sur la croissance des levures, en montrant que les souches portant le gène humain muté présentent un caractère thermosensible, ii) sur la fonction d’import des protéines vers la matrice mitochondriale, qui est altérée dans les souches mutées et iii) sur la stabilité de la protéine. Nous avons également observé un effet de la mutation sur la morphologie des mitochondries et des peroxysomes des cellules de levures, suggérant une induction de l’autophagie dans les souches de levures portant la mutation p.Asn76Asp. L’identification de mutations de MAGMAS dans une dysplasie osseuse sévère, permet d’attribuer à cette protéine un rôle spécifique dans le processus complexe d’ossification endochondrale. La deuxième stratégie a été une combinaison, au sein d’une même famille, d’une stratégie de cartographie par homozygotie et du séquençage de l’exome d’un seul patient. Cette approche a été utilisée dans une famille consanguine avec 3 enfants atteints porteurs d’une dysplasie rhizomélique (Famille C). Nous avons identifié une mutation homozygote du gène NWD1 (NACHT and WD repeat domain containing 1) (NM_001007525, p.Cys1376Tyr) responsable de la maladie dans cette famille C. Ce gène code pour une protéine ayant des domaines WD répétés qui lui confèrent un rôle dans divers mécanismes comme la transduction de signal, la régulation de la transcription, le transport vésiculaire et le contrôle du cycle cellulaire. (...) / Social, religious, geographic and political reasons have favored the consanguineous marriage in the Lebanese population. This led to an increase in the prevalence of autosomal recessive disorders, especially the rare entities including chondrodysplasias. This group of diseases is due to an impairment of the endochondral ossification process. Causative mutations have now been identified in over 230 different genes in more than 400 unique skeletal phenotypes. However, the genetic basis of over 100 different entities remains to be determined. My PhD research project, held between the research group « Bases moléculaires et physiopathologiques des chondrodysplasies » of Necker enfants-malades hospital (INSERM U781, PARIS, France) and the Medical Genetics Unit of Saint-Joseph University (Lebanon), aims to identify genes involved in autosomal recessive skeletal dysplasias in four consanguineous Lebanese families. Different strategies were carried out: the first consists in overlapping data from whole exome sequencing of two patients affected by a new lethal type of spondylodysplastic dysplasia and issued from two consanguineous unrelated Lebanese families (Families A and B). Here, we report a homozygous missense mutation in the Mitochondria-associated granulocyte macrophage CSF-signaling gene (MAGMAS: NM_016069, p.Asn76Asp) in this severe skeletal dysplasia. MAGMAS, also referred to as PAM16, is a mitochondria-associated protein, involved in pre-proteins import into mitochondria and essential for cell growth and development. We demonstrate that MAGMAS is expressed in trabecular bone and cartilage at early developmental stages underlining its specific role in skeletogenesis. We also give strong evidence of the deleterious effect of the identified mutation on the stability of the protein, its in-vivo activity and the viability of yeast strains. We also show that the mutation is able to induce autophagy in yeast cells. Reporting deleterious MAGMAS mutation in a skeletal dysplasia supports a key and specific role for this mitochondrial protein in ossification. Additional studies would be of interest to further understand the specific role of magmas in ossification. The second strategy was to combine, in a consanguineous family, homozygosity mapping with whole exome sequencing of one of the patients. This strategy was undertaken in family C with 3 patients affected by a rhizomelic dysplasia. It allowed us to identify a homozygous missense mutation in the NWD1 gene (NACHT and WD repeat domain containing 1: NM_001007525, p.Cys1376Tyr) as responsible for the skeletal dysplasia in this family. NWD1 belongs to a large group of WD-repeat domain-containing proteins that are involved in different physiological mechanisms such as signal transduction, transcription regulation, vesicular transport and cell cycle control. (...)

Les ataxies héréditaires représentent un groupe hétérogène de maladies neurodégénératives caractérisées par des anomalies de la coordination des mouvements associées à des troubles de l’équilibre et de la marche. L’immense diversité fonctionnelle des protéines touchées dans les ataxies autosomiques récessives (AR) souligne que celles-ci ne peuvent pas être classées selon les voies physiopathologiques en cause. De ce constat résulte une classification émergente des AR en fonction de la raison expliquant la nature « modérée » de l’atteinte neurologique, à savoir : (i) les mutations avec perte de fonction partielle, (ii) la présence redondante de protéines de la même famille fonctionnelle, (iii) la présence redondante d’autres voies détoxifiantes. L’objectif de ce travail était d’identifier de nouveaux gènes responsables d’AR syndromiques grâce à une stratégie couplant la cartographie par homozygotie et l’analyse d’exomes de larges familles consanguines. L’analyse par génotypage de l’une de ces familles nous a permis d’identifier 2 régions homozygotes partagées par les 3 enfants atteints par le syndrome de Lichtenstein-Knorr (ataxie-surdité). Parmi les variants présents dans ces régions, j’ai identifié une mutation faux-sens dans le gène SLC9A1 codant pour l'échangeur Na+/H+1, NHE1. Cette mutation transforme la Glycine 305, un petit acide aminé très conservé et localisé dans le 8ème domaine transmembranaire, en Arginine, acide aminé chargé positivement. Bien que NHE1 soit une protéine exprimée de façon ubiquitaire, 2 modèles souris « knock-out » de ce gène ont montré sa fonction essentielle au niveau des noyaux profonds cérébelleux, vestibulaires et cochléaires où a été observé une dégénérescence spécifique suite à l’inactivation du gène Slc9a1. Nous avons ensuite apporté la preuve de l’effet délétère de la mutation p.Gly305Arg en montrant une réduction importante mais pas totale de l’activité de l’échangeur muté ainsi qu’une abolition de son expression à la surface cellulaire, démontrant ainsi que SLC9A1 est le gène impliqué dans le syndrome de Lichtenstein-Knorr (Guissart et al. Hum Mol Genet 2015). L’analyse de l’exome d’une famille consanguine multi-générationnelle présentant une ataxie spino-cérébelleuse, une cécité et une surdité et dont le locus avait été identifié par notre équipe en 2000 sur le chromosome 6p23-p21 (SCAR3 ; MIM #271250) m’a permis d’identifier la mutation faux-sens homozygote p.Gly306Arg dans le gène SLC52A2, pourtant située sur le chromosome 8qter et déjà décrite chez des patients atteints du syndrome de Brown-Vialetto-Van Laere type 2, indiquant que la liaison génétique initialement publiée pour cette famille était due au hazard. Le séquençage d’exome d'une autre famille avec 2 enfants atteints d’une AR progressive et d’une rétinite pigmentaire et présentant une région homozygote partagée en 6p23-p21 m’a permis d’identifier la mutation faux-sens p.Ala912Val dans le gène PEX6. L'analyse rétrospective des marqueurs du peroxysome a montré un taux d’acide phytanique sérique très modérément augmenté, alors que les fibroblastes m’ont permis de confirmer le caractère pathogène de la mutation p.Ala912Val par l'absence de marquage à la catalase, la présence de structures peroxysomales anormales et une nette augmentation des AGTLC, indiquant une perte de fonction partielle de la protéine PEX6. Par conséquent, en dépit d’une liaison génétique initiale erronée, l’entité SCAR3 est confirmée et est causée par certaines mutations du gène PEX6 (Guissart et al. Eur J Hum Genet 2016). En conclusion, la présence des mutations faux-sens hypomorphes dans ces familles et les données de la littérature démontrent le concept selon lequel de nombreuses AR sont causées par des mutations de type « perte de fonction partielle » touchant une grande variété de voies physiopathologiques, ceci en raison de l’extrême sensibilité des neurones cérébelleux, spino-cérébelleux et sensitifs profonds, à des désordres métaboliques même légers. / Inherited ataxias are a heterogeneous group of neurodegerative diseases that are characterized by incoordination of movement and unsteadiness. The huge functional diversity of affected proteins in autosomal recessive ataxia highlights that these disorders cannot be classified according to relevant physiopathological pathways. Rather, current knowledge shows that no specific physiopathological pathway explains directly the appearance of the symptoms. This gives rise to an emerging recessive ataxia classification based on the reason explaining the “moderate” nature of neurological involvement, namely: (i) partial loss of function mutation, (ii) the presence of redundant functional family member proteins, (iii) the presence of redundant detoxifying pathways. The purpose of this work was to identify new causative genes for syndromic recessive ataxia using a strategy combining homozygosity mapping and exome analysis in large consanguineous families. Genotyping analysis of one of those families has enabled us to identify 2 significant regions of homozygosity shared by the 3 siblings affected by Lichtenstein-Knorr syndrome (ataxia-deafness): one of 23.6 Mb on chromosome 1 and the other of 5.5 Mb on chromosome 7. Among the variants located in the shared homozygous regions, I rapidly identified a missense mutation located in the SLC9A1 gene encoding for NHE1, the Na+/H+ exchanger family member 1. This mutation replaces Glycine 305, a small neutral highly conserved amino-acid, located in the 8th transmembrane segment and conserved in all investigated metazoans by Arginine, a positively charged amino acid. Despite NHE1 is ubiquitously expressed, extensive analysis of 2 Slc9a1 knock-out mice models revealed its crucial role in 3 regions of the central nervous system: vestibular nuclei, cochlear nuclei, and most prominently deep cerebellar nuclei. We then demonstrated the deleterious effect of the p.Gly305Arg mutation showing a significant but not complete reduction of the proton pump activity of NHE1 as well as absence of expression at the cell surface, thus demonstrating that SLC9A1 is the causative gene in Lichtenstein-Knorr syndrome (Guissart et al. Hum Mol Genet 2015). Exome analysis of a multigenerational consanguineous family with spinocerebellar ataxia, blindness and deafness for which we identified linkage to chromosome 6p23-p21 in 2000 (SCAR3; MIM #271250) allowed me to identify in all patients the homozygous p.Gly306Arg missense mutation in SLC52A2, yet located on chromosome 8qter and previously found mutated in patients with Brown-Vialetto-Van Laere type 2, indicating that the genetic linkage published for the SCAR3 family was a false positive result. High recombination rate in the telomeric region and use of widely spaced microsatellite markers explain why correct linkage was initially missed. Exome sequencing of another family with 2 children affected with progressive ataxia and retinitis pigmentosa and with linkage to 6p23-p21 revealed the p.Ala912Val mutation in PEX6. Retrospective analysis of peroxisomal markers showed very moderate increase of serum phytanic acid levels, but fibroblasts allowed me to confirm the pathogenicity of the p.Ala912Val mutation by absent peroxisomal catalase immunostaining, presence of peroxisomal ghosts with abnormal structure and markedly increased very long-chain fatty acids, indicating a partial loss of function of the PEX6 protein. Therefore, despite initial false genetic linkage, the SCAR3 locus is confirmed and is caused by some PEX6 mutation (Guissart et al. Eur J Hum Genet 2016). In conclusion, the identification of these hypomorphic missense mutations in ataxia families as well as literature data lend credence to the concept that numerous recessive ataxias are caused by partial loss of function mutations in a large variety of pathophysiological pathways, as a consequence of an exquisite sensitivity of cerebellar, spinocerebellar and deep sensory neurons to even mild metabolic insults.

Glioblastome machen etwa 47% aller intrinsischen Tumore des zentralen Nervensystems aus. Sie sind durch ein aggressives und invasives Wachstumsverhalten gekennzeichnet. Als erster wegweisender Schritt zur Therapie der Glioblastome gilt die frühe und möglichst vollständige Resektion, gefolgt von einer simultanen Radiochemotherapie. Dennoch sind Tumorrezidive binnen weniger Monate die Regel und es bestehen trotz intensiver Forschung bis heute kaum alternative Behandlungsoptionen. Das Verständnis für die Pathogenese der Glioblastome erfuhr in den letzten Jahren tiefgreifende Änderungen. Nach Berücksichtigung der molekularen Marker in der WHO- Klassifikation, wurden Glioblastome in zwei molekulare Gruppen unterteilt: die IDH-Wildtyp (95% der Fälle) und die IDH-mutierten Glioblastome. IDH-Wildtyp Glioblastome treten bei Patienten mit einem medianen Alter von 64 Jahren auf und gehen mit einer ungünstigen Prognose (medianes Überleben 14,2 Monate) einher. IDH-mutierte Glioblastome kommen vor allem bei jüngeren Patienten mit einem medianen Alter von 45 Jahren vor und weisen eine vergleichsweise bessere Prognose mit einem medianen Überleben von 4-5 Jahren auf. Bei IDH-Wildtyp Glioblastomen wurden am häufigsten TERTp-, und PTEN-Mutationen sowie EGFR-Amplifikationen beschrieben. Dabei stellt die TERTp-Mutation die häufigste somatische Alteration im Genom der IDH-Wildtyp Glioblastome dar. Die oben genannten molekularen Marker bieten eine solide Grundlage für die molekulare Diagnose der IDH- Wildtyp Glioblastome. Dennoch bleibt die Frage unbeantwortet, warum IDH-Wildtyp Glioblastome vor allem bei älteren Patienten auftreten und jüngere Patienten eine bessere Prognose besitzen. Unter der Annahme, dass IDH-Wildtyp Glioblastome ein altersspezifisches molekulargenetisches Profil aufweisen, welches wohlmöglich die Prognose beeinflusst, wurden daher die Tumor- und korrespondierenden Blutproben von 55 Patienten mittels Whole Exome Sequencing untersucht. Nach Filterung der Rohdaten wurden verschiedene Mutationen und Copy Number Variations identifiziert. Zur Validierung der Methode wurde zum einen ein Literaturabgleich der detektierten Alterationen durchgeführt und zum anderen einzelne Kandidatengene mittels Sanger Sequenzierung manuell bestätigt. Insgesamt wurden 1841 Mutationen auf 1544 verschiedenen Genen detektiert. Obwohl viele der 1544 Mutationen ohne Relevanz für die Pathogenese waren, konnte eine enorme Vielzahl an verschiedenen Treibermutationen nachgewiesen werden. Die manuell sequenzierte TERTp-Mutation war mit 76,4% am häufigsten aufgetreten. Weitere Treibermutationen, beispielsweise EGFR, TP53, PTEN, PI3K-Gruppe, NF1 und PDGFRA zeigten mit der Literatur vergleichbare Prävalenzen und betonten die Validität der Methode. Die aufgetretenen Copy Number Variations belegten sowohl auf chromosomaler (Chromosom 7-Amplifikation und Chromosom 10-Deletion), als auch auf genspezifischer Ebene (EGFR-, CDK6-, MET-, PDGFRA-Amplifikation und CDKN2A/B-, und PTEN-Deletion) die molekulargenetischen Charakteristika des IDH-Wildtyp Glioblastoms. Über eine Clusterung dieser Alterationen und Gegenüberstellung mit klinischen Eigenschaften konnten die typischen, publizierten Glioblastomsignaturen (proneural, klassisch, mesenchymal) beschrieben und mit dem Erkrankungsalter in Verbindung gebracht werden. Besonders eindrücklich zeigten unsere Daten, übereinstimmend mit der Literatur, dass eine proneurale Signatur mit einem jungen Erkrankungsalter und günstiger Prognose assoziiert war. Unerwartet zeigte ein Patient mit pädiatrischer Signatur und hohem Erkrankungsalter dennoch ein überdurchschnittlich vorteilhaftes Überleben, verglichen mit seiner Altersgruppe. Unabhängig von molekulargenetischen Alterationen, war ein junges Erkrankungsalter alleinstehend mit einer günstigeren Prognose verknüpft. Für einzelne molekulargenetische Alterationen konnte kein Zusammenhang mit dem Erkrankungsalter oder dem (Progressionsfreien-) Überleben hergestellt werden. Ein alterspezifisches, prognosebeeinflussendes Mutationsmuster konnte demnach nicht identifiziert werden. Limitierend muss dabei die geringe Kohortengröße (n=55) angemerkt werden. Eine Vergrößerung der Studienpopulation war aufgrund der geringen Inzidenz von jungen Patienten mit IDH-Wildtyp Glioblastomen in diesem Studiendesign nicht möglich und könnte perspektivisch in einem multizentrischen Ansatz oder einer langen Akquirierungsphase verwirklicht werden. Die Fülle an identifizierten Treibermutationen verdeutlichte nichtsdestotrotz die große intratumorale Heterogenität des Glioblastoms. Zusätzlich ermöglichte die enorme diagnostische Tiefe der verwendeten Methode die Identifikation der bisher nicht im Zusammenhang mit dem IDH-Wildtyp Glioblastom beschriebenen TET1-Deletion. Obwohl die detaillierte Rolle der TET1-Deletion für das Glioblastom nicht verstanden ist, liefern unsere Daten einen vielversprechenden Hinweis, dass ein Funktionsverlust des TET1-Enzyms, in Kombination mit EGFR-Amplifikation oder Deletion von PTEN oder CDKN2A/B, eine Auswirkung auf die Pathogenese des IDH-Wildtyp Glioblastoms besitzt und die Prognose negativ beeinflusst. Zukünftige molekulargenetische Sequenzierungen sind indiziert, um die Rolle der TET1- Deletion zu bestätigen und darüber hinaus die Pathogenese des Glioblastoms auf molekulargenetischer Ebene noch besser zu verstehen und weitere individualisierte Therapieansätze abzuleiten.:Abkürzungsverzeichnis 5 1 Einleitung 8 1.1 Klinische Grundlagen des Glioblastoms 8 1.1.1 Epidemiologie/Ätiologie 8 1.1.2 Symptomatik und Diagnostik 9 1.1.3 Therapie 10 1.1.4 Prognose 12 1.2 Pathologie und Molekulargenetische Veränderungen des Glioblastoms 12 1.2.1 Treibergene 13 1.2.2 Subgruppen 17 1.3 Fragestellung 18 2 Materialien 19 3 Methoden 23 3.1 Patientenrekrutierung 23 3.1.1 Erweiterte Einschlusskriterien 25 3.1.2 Ausschlusskriterien 25 3.2 Kohortendesign 25 3.3 Klinische Daten 26 3.4 Materialgewinnung 27 3.4.1 Proben-Lagerung 27 3.4.2 DNA-Extraktion 27 3.5 Sanger Sequenzierung Prä-WES 29 3.5.1 Primer-Design 29 3.5.2 Polymerase Kettenreaktion 29 3.5.3 Elektrophorese 31 3.5.4 Aufreinigung PCR-Produkt 33 3.5.5 Sequenzierreaktion 35 3.6 WES 38 3.6.1 Datensatz 41 3.6.2 CNV-Analyse 42 3.7 Sanger Sequenzierung Validierung 42 3.8 Statistische Auswertung 45 4 Ergebnisse 47 4.1 Deskriptive klinisch-pathologische Beschreibung der Kohorte 47 4.1.1 Klinisch-pathologische Zusammenhänge 54 4.2 TERTp-Sequenzierung 58 4.3 Datensatz WES 58 4.3.1 Somatische Mutationen 59 4.3.2 CNV-Analyse 67 4.4 Altersbezogene molekulargenetische Unterschiede 74 4.5 Zusammenhang zwischen Genotyp und OS sowie PFS 77 4.5.1 Somatische Mutationen 77 4.5.2 CNV-Analyse 78 5 Diskussion 82 5.1 Klinische Charakteristika von Patienten mit IDH-Wildtyp Glioblastom 82 5.2 Der prognostische Stellenwert von Tumoreigenschaften 84 5.3 Molekulargenetisches Profil des IDH-Wildtyp Glioblastoms 85 5.3.1 Somatische Mutationen und Copy Number Variations 85 5.3.2 Chromosomale Aberrationen 94 5.4 10q21.3-Deletion 95 6 Zusammenfassung 97 6.1 Deutsch 97 6.2 Englisch 99 Anlagen 101 Darstellung zur Geschlechtsneutralität im geschriebenen Wort 101 Erklärung zur Eröffnung des Promotionsverfahren 102 Erklärung zur Einhaltung der gesetzlichen Vorgaben 104 Anhang 105 Literaturverzeichnis 113 Abbildungsverzeichnis 125 Tabellenverzeichnis 127 Danksagung 128 / Glioblastomas account for approximately 47% of all intrinsic central nervous tumors. They are characterized by an aggressive and invasive growth pattern. Early and, if possible, complete resection followed by simultaneous radiochemotherapy is crucial for treatment success. However, tumor recurrence within a few months are very frequent. Despite intensive research, there are still hardly any alternative treatment options. The understanding of the pathogenesis of glioblastomas underwent profound changes in recent years. After considering molecular markers in the WHO classification, glioblastomas have been divided into two molecular groups: IDH-wild-type (95% of cases) and IDH-mutated glioblastomas. IDH-wild-type glioblastomas occur in patients with a median age of 64 years and are associated with an unfavorable prognosis (median survival 14.2 months). On the other hand, IDH-mutated glioblastomas are mainly associated with young patients with a median age of 45 years and have a rather good prognosis with a median survival of 4-5 years. In IDH- wild-type glioblastomas, TERTp-, and PTEN-mutations as well as EGFR-amplifications have been described most frequently. Among them, TERTp-mutation represents the most frequent somatic alteration in the genome of IDH-wild-type glioblastomas. The above molecular markers provide a solid basis for molecular diagnosis of IDH-wild-type glioblastomas. To date, however, the reason why IDH-wild-type glioblastomas appear primarily in older patients with younger patients having a better prognosis remains unclear. Assuming that IDH-wild-type glioblastomas have an age-specific molecular signature with possible impact on the outcome, we analyzed tumor and corresponding blood samples from 55 patients by whole exome sequencing. After filtering the raw data, various mutations and copy number variations were identified. To validate the method, a literature review as well as Sanger Sequencing of selected candidate genes was performed. A total of 1841 mutations on 1544 different genes were detected. Although many mutations appeared to be background mutations with no relevance to pathogenesis, an enormous number of different driver mutations remained. The manually sequenced TERTp-mutation was the most abundant at 76.4%. Other driver mutations, for example EGFR, TP53, PTEN, PI3K group, NF1 and PDGFRA showed prevalence comparable to published data and emphasized the validity of the method. The copy number variations that occurred concurred previously described molecular genetic characteristics of IDH wild-type glioblastoma at both chromosomal (chromosome 7 amplification and chromosome 10 deletion) and gene-specific levels (EGFR-, CDK6-, MET-, PDGFRA-amplification and CDKN2A/B-, and PTEN-deletion). Via clustering of these alterations and juxtaposition with clinical features, the typical glioblastoma signatures (proneural, classic, mesenchymal) could be described and related to age of diagnosis. In line with the literature, our data showed that a proneural signature was associated with younger patients and favorable prognosis. Unexpectedly, a patient with a pediatric signature and high age of diagnosis, showed a survival above average compared with his age group. Regardless of molecular alterations, young age was an independent characteristic of favorable prognosis. For individual genetic alterations, no association with age of diagnosis or (progression-free) survival could be established. Thus, an age-specific mutational pattern could not be identified. The relatively small cohort size (n=55) must be noted as a limiting factor. An increase of the study population was not possible in this study design due to the low incidence of young patients with IDH wild-type glioblastoma and could be realized in a multicenter approach or a long acquisition phase in the future. Nevertheless, the abundance of identified driver mutations highlighted the large intratumoral heterogeneity of glioblastoma. In addition, the tremendous diagnostic depth of the method used enabled the identification of the TET1-deletion not previously described in the context of IDH wild-type glioblastoma. Although the detailed role of TET1-deletion in glioblastoma is not yet understood, our data provides promising evidence that loss of function of the TET1-enzyme in combination with EGFR-amplification or deletion of PTEN or CDKN2A/B has an impact on the pathogenesis of IDH wild-type glioblastoma and negatively affects prognosis. Future genetic sequencing is indicated to confirm the role of TET1-deletion and moreover to get further understanding of the genomic pathogenesis of glioblastoma with the aim to derive individualized therapeutic approaches.:Abkürzungsverzeichnis 5 1 Einleitung 8 1.1 Klinische Grundlagen des Glioblastoms 8 1.1.1 Epidemiologie/Ätiologie 8 1.1.2 Symptomatik und Diagnostik 9 1.1.3 Therapie 10 1.1.4 Prognose 12 1.2 Pathologie und Molekulargenetische Veränderungen des Glioblastoms 12 1.2.1 Treibergene 13 1.2.2 Subgruppen 17 1.3 Fragestellung 18 2 Materialien 19 3 Methoden 23 3.1 Patientenrekrutierung 23 3.1.1 Erweiterte Einschlusskriterien 25 3.1.2 Ausschlusskriterien 25 3.2 Kohortendesign 25 3.3 Klinische Daten 26 3.4 Materialgewinnung 27 3.4.1 Proben-Lagerung 27 3.4.2 DNA-Extraktion 27 3.5 Sanger Sequenzierung Prä-WES 29 3.5.1 Primer-Design 29 3.5.2 Polymerase Kettenreaktion 29 3.5.3 Elektrophorese 31 3.5.4 Aufreinigung PCR-Produkt 33 3.5.5 Sequenzierreaktion 35 3.6 WES 38 3.6.1 Datensatz 41 3.6.2 CNV-Analyse 42 3.7 Sanger Sequenzierung Validierung 42 3.8 Statistische Auswertung 45 4 Ergebnisse 47 4.1 Deskriptive klinisch-pathologische Beschreibung der Kohorte 47 4.1.1 Klinisch-pathologische Zusammenhänge 54 4.2 TERTp-Sequenzierung 58 4.3 Datensatz WES 58 4.3.1 Somatische Mutationen 59 4.3.2 CNV-Analyse 67 4.4 Altersbezogene molekulargenetische Unterschiede 74 4.5 Zusammenhang zwischen Genotyp und OS sowie PFS 77 4.5.1 Somatische Mutationen 77 4.5.2 CNV-Analyse 78 5 Diskussion 82 5.1 Klinische Charakteristika von Patienten mit IDH-Wildtyp Glioblastom 82 5.2 Der prognostische Stellenwert von Tumoreigenschaften 84 5.3 Molekulargenetisches Profil des IDH-Wildtyp Glioblastoms 85 5.3.1 Somatische Mutationen und Copy Number Variations 85 5.3.2 Chromosomale Aberrationen 94 5.4 10q21.3-Deletion 95 6 Zusammenfassung 97 6.1 Deutsch 97 6.2 Englisch 99 Anlagen 101 Darstellung zur Geschlechtsneutralität im geschriebenen Wort 101 Erklärung zur Eröffnung des Promotionsverfahren 102 Erklärung zur Einhaltung der gesetzlichen Vorgaben 104 Anhang 105 Literaturverzeichnis 113 Abbildungsverzeichnis 125 Tabellenverzeichnis 127 Danksagung 128

Die Dissertationsarbeit untersucht genetische Varianten in Zellkulturmodellen des metastatischen und kastrationsresistenten Prostatakarzinoms. Außerdem werden Mechanismen der Chemoresistenz, insbesondere der Resistenz gegen Cisplatin und Docetaxel in diesen Zelllinien untersucht. / This Dissertation evaluates genetic variants found in cell culture models of metastatic castration resistant prostate cancer. Furthermore, mechanisms of resistance against the chemotherapeutic drugs cisplatin and docetaxel are investigated in these cell lines.

Massively parallel sequencing has rapidly revolutionized DNA and RNA research. Sample preparations are steadfastly advancing, sequencing costs have plummeted and throughput is ever growing. This progress has resulted in exponential growth in data generation with a corresponding demand for bioinformatic solutions. This thesis addresses methodological aspects of this sequencing revolution and applies it to selected biological topics. Papers I and II are technical in nature and concern sample preparation and data anal- ysis of RNA sequencing data. Paper I is focused on RNA degradation and paper II on generating strand specific RNA-seq libraries. Paper III and IV deal with current biological issues. In paper III, whole exomes of cancer patients undergoing chemotherapy are sequenced and their genetic variants associ- ated to their toxicity induced adverse drug reactions. In paper IV a comprehensive view of the gene expression of the endometrium is assessed from two time points of the menstrual cycle. Together these papers show relevant aspects of contemporary sequencing technologies and how it can be applied to diverse biological topics. / <p>QC 20160329</p>

During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules. As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample. Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools. / <p>QC 20160127</p>

A obesidade, que atinge uma grande parcela da população mundial é resultado do acumulo excessivo de gordura corporal, podendo afetar gravemente a saúde. É considerada multifatorial, com maior susceptibilidade ao ganho de massa em indivíduo que possuírem maior predisposição genética. Além disso, também está associada a doenças como diabetes, hipertensão, neoplasias e disfunções endócrinas. No Brasil, de acordo com a Organização Mundial de Saúde, cerca de 51,7% da população encontra-se com sobrepeso ou obesa. A literatura mostra que polimorfismos de nucleotídeos único (SNPs) são marcadores frequentemente usados em estudos de associação de doenças complexas, e trabalhos sobre obesidade focados em populações tri-híbridas, tal como é a brasileira, ainda são escassos. Este trabalho buscou identificar um conjunto de SNPs relacionados com o aumento do Índice de Massa Corpórea (IMC) a partir do exoma de ameríndios brasileiros e verificar se o padrão seria o mesmo na coorte de Ribeirão Preto - SP. O projeto desenvolvido foi devidamente aprovado pelo projeto pelo Comitê de Ética (CAAE 74509817.4.0000.5440). Um conjunto de 17 SNPs foi selecionado após o sequenciamento do exoma ameríndio, realizado pelo Dr. João Guerreiro, cada SNP foi validado posteriormente mediante a realização da genotipagem por PCR em tempo real, confirmando as mutações encontradas nos ameríndios. Para a coorte de Ribeirão Preto, utilizamos DNA genômico de 180 indivíduos, sendo 89 normais e 91 obesos, coletados previamente no Sistema Único de Saúde (SUS) por Barbieri et al (2006). A ancestralidade tri-híbrida de 45 indivíduos desses indivíduos foi confirmada por um conjunto de 15 polimorfismos do tipo inserção/deleção (InDels) escolhidos do painel desenvolvido por Santos et al. (2010), demostrando predominância europeia (87%), seguida de ameríndia (9%) e africana (4%). Os 17 SNPs selecionados apresentaram somente mutações sinônimas, onde rs2277552 e rs3731900 tiveram predição de patogenicidade positiva. A frequência do alelo mutante na coorte de Ribeirão Preto variou em função do SNP e da população comparada do banco de dados do Ensembl. A exemplo, rs8133052 exibiu diferença em seus valores quando a população comparada com a coorte de Ribeirão Preto era a total, 50% e 37% respectivamente. A análise de componentes principais (PCA) não foi sensível o bastante para separar o grupo obeso do controle. O teste de associação optou-se pelos modelos dominante e recessivo para todos os SNPs, porém nenhuma correlação foi observada quando o fenótipo observado era a obesidade (IMC>30). Entretanto, rs231591 para o MD e rs1824152 para o MR mostraram valores significativos de proteção para a obesidade central (razão entre cintura e estatura), com odds ratio de 0,27 (p = 0.0013) e 0.37 (p = 0.0056), respectivamente. Uma análise mais refinada considerando a influência do sexo dentro da coorte foi realizada e, dependendo do fenótipo e do sexo observado quatro SNPs (rs1824152, rs3732378, rs231591 e rs744224) apresentaram valores significativos de proteção. Destes três (rs1824152, rs231591 e rs744224) ainda não haviam sido relacionados diretamente com seu caráter protetivo para a obesidade. / Obesity affects a big portion of global population and it results from excessive accumulation of body fat, what can seriously affect health. It is known the obesity is multifactorial, and it will be stronger the greater is the individual genetic predisposition. Besides that, it is associated with many diseases like diabetes, hypertension, neoplasms, and endocrine dysfunctions. In Brazil, according to the World Health Organization, about 51.7% of the population is classified as being overweight or obese. Moreover, the literature shows that molecular markers called Single Nucleotide Polymorphisms (SNPs) are widely used in association studies of complex diseases, like obesity, and there are still few genetic researches whose focus is the obesity in mixed populations such as Brazil\'s. Therefore, after we got the acceptance for this work by the Ethics Committee (CAAE 74509817.4.0000.5440), we looked for a set of SNPs, based the obese Brazilian\'s indigenous exome, related to the increase of the Body Mass Index (BMI) and aimed to verify if that pattern is the same in a cohort of Ribeirão Preto (SP, Brazil). A set of 17 SNPs was selected after the indigenous exome was sequenced by Dr. João Guerreiro and each SNP was posteriorly validated by Real-Time PCR and verified by genotyping of the indigenous samples. For the cohort of Ribeirão Preto (SP), we used genomic DNA obtained from 180 individuals (89 normal-weight and 91 obese) from Sistema Único de Saúde (SUS) of Ribeirão Preto, previously collected by Barbieri (2006). We then confirmed the trihybrid ancestry of 45 individuals from the cohort using a set of 15 Insertion Deletion Polymorphisms (InDels) selected from the panel developed by Santos et al. (2010), what showed to be predominantly European, followed by African and Amerindian, with frequencies of 87%, 9% and 4%, respectively. The 17 SNPs showed synonymous mutations, from which rs2277552 and rs3731900 presented pathogenicity prediction. The cohort mutant allele frequency compared to Ensembl database varied in function of the SNP and the population. For example, rs8133052 varied when total population and the Ribeirão Preto cohort were compared, with 37% and 50%, respectively. The Principal Component Analysis (PCA) was not sensitive enough to separate obese from non-obese. The association test used the dominant and the recessive models for all SNPs, and no correlation was observed for the obese phenotype (IBM>30), independently of the model. However, rs231591 and rs1824152, for dominant and recessive models, in this order, showed values significantly associated to the protection against central obesity (ratio between waist and height), with odds ratio of 0,27 (p = 0,0013) and 0,37 (p = 0,0056), respectively. A more refined analysis of the associated test was realized, considering the influence of sex in the observed phenotype within the cohort. Four SNPs (rs1824152, rs3732378, rs231591 and rs744224) showed significant protection values, depending on the sex and the model adopted. From theses, three SNPs (rs1824152, rs231591 e rs744224) had never been directly associated to protection against obesity.

O desenvolvimento embrionário dos membros é um processo complexo e dinâmico. É controlado por diversos genes e diversos mecanismos morfogenéticos, muitos dos quais ainda não estão completamente elucidados. O objetivo deste estudo é identificar as alterações genéticas responsáveis por defeitos de membros em quatro famílias brasileiras. A família 1 apresenta três indivíduos com um espectro extremamente variável de displasia ectodérmica, ectrodactilia e fenda labiopalatina, características da síndrome EEC. O gene TP63 foi analisado por sequenciamento de Sanger e foi descoberta uma variante nunca descrita anteriormente, c.1037C&gt;A (p.Ala346Gly), em heterozigose, segregando com o fenótipo. Mutações no gene TP63 já foram associadas com casos da síndrome EEC. Concluímos que a variante no gene TP63 é a responsável pelo quadro clínico de EEC na família. A família 2 incluiu cinco indivíduos afetados por polegar trifalângico. O fenótipo de um dos indivíduos é mais grave e se caracteriza por, além do polegar trifalângico, pela presença de braço direito grosseiramente curto e deformado com dedos hipoplásicos, pé direito malformado com metatarsos hipoplásicos e orelha direita pequena e protuberante. Estudos de mapeamento gênico com arrays de SNPs revelaram Lod scores sugestivos de ligação com 8 regiões cromossômicas distintas. O sequenciamento massivo em paralelo do exoma com a amostra de um dos afetados, seguida da seleção das variantes presentes nas regiões com Lod scores sugestivos, revelou a presença da variante c.410dupG (p.Gly138Argfs&lowast;43) no gene SALL4, a qual nunca havia sido descrita. Mutações no SALL4 que alteram a matriz de leitura já foram associadas com casos da síndrome de Okihiro, a qual é caracterizada por defeitos radiais de membros e anomalia oftalmológica de Duane. A segregação da variante entre os indivíduos afetados foi confirmada por sequenciamento de Sanger. Concluimos que esta variante é responsável pelo quadro clínico da família, a qual apresenta a síndrome de Okihiro, porém sem a anomalia de Duane. A análise de todos os casos descritos com variantes no gene SALL4 sugere que há uma correlação entre defeitos nos membros inferiores e o tamanho reduzido da proteína truncada SALL4. A família 3 apresenta três indivíduos afetados por sindactilia completa com polidactilia pré-axial nas mãos, classificada como sindactilia do tipo IV. O sequenciamento massivo em paralelo do exoma com as amostras de três indivíduos afetados e dois normais não revelou a presença de qualquer alteração genética candidata a explicar o quadro clínico. O estudo do array-CGH revelou uma duplicação de aproximadamente 97 kb no gene LMBR1 abrangendo a região de enhancer (ZRS) do gene SHH. Duplicações no ZRS já foram identificadas em casos de sindactilia do tipo IV. Concluímos que a duplicação no ZRS é responsável pelo quadro. A revisão da literatura sobre os casos com duplicação dessa região sugere uma correlação entre duplicações com tamanho ao redor de 97 kb e o fenótipo clássico de sindactilia do tipo IV. A família 4 apresenta seis indivíduos afetados por uma síndrome relativamente nova descrita por nossa equipe em 2008, a síndrome de Santos. Essa síndrome é caracterizada por aplasia/hipoplasia fibular e anoniquia/hipoplasia ungueal dentre outros defeitos de membros. O estudo de ligação com array de SNP e marcadores de microssatélites, com posterior cálculo de Lod score, indicou duas regiões no cromossomo 3 como candidatas a conter a alteração genética responsável pelo fenótipo. Análise dos genes presentes nessas regiões indicou o gene WNT7A como o principal candidato. Alterações neste gene já foram associadas com a síndrome de Fuhrmann e a AARRS (Al-Awadi&frasl;Raas-Rothschild syndrome), de herança recessiva, as quais apresentam fenótipos correlatos aos presentes na síndrome de Santos. O sequenciamento de Sanger deste gene revelou a presença da variante c.934G&gt;A (p.Gly312Ser), não descrita, em homozigose em cinco dos seis indivíduos afetados da família. O sexto indivíduo afetado é heterozigoto e apresenta fenótipo muito menos grave. Não apresenta qualquer defeito fibular e ungueal, característicos da síndrome. Esse é o primeiro relato de um caso de defeitos de membros como resultado da presença de uma variante em heterozigose no gene WNT7A. O sequenciamento massivo em paralelo do exoma foi realizado com as amostras deste indivíduo e de um dos indivíduos afetados. Contudo, nenhuma outra variante se mostrou candidata a explicar o quadro clínico. Concluímos que a síndrome de Santos é o resultado desta mutação no gene WNT7A. Estudos funcionais in situ baseados na atividade da Luciferase estão em andamento para comprovar o efeito deletério desta variante / Limb development is a complex and dynamic process driven by many different genes and morphogenetic mechanisms. Many of them have not been properly clarified yet. The aim of this study is to identify genetic alterations responsible for limb defects in four Brazilian families. Family 1 includes three individuals affected by an extremely variable phenotype of ectrodactyly, ectodermal dysplasia and cleft lip/palate, and other clinical signs of EEC syndrome. The TP63 gene, associated with this condition, was analyzed through Sanger sequencing and a novel variant, c.1037C&gt;A (p.Ala346Gly), was found; the variant segregated in heterozygous state with the EEC phenotype. Mutations in TP63 gene are already known to be associated with EEC syndrome. Due to the extremely variable phenotype presented by the individuals in the family, the possibility of the mutation c.1037C&gt;G being related to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM cannot be ruled out. Family 2 presents five individuals affected by preaxial polydactyly (triphalangeal thumbs). The phenotype of the proband is more severe, being characterized by triphalangeal thumb, with the following manifestations: grossly shortened and deformed forearm, markedly hypoplastic and appendicular thumb and malformed right foot with hypoplastic metatarsals. Linkage studies by SNP-array pointed to suggestive Lod score values in 8 distinct chromosomal regions. Whole exome sequencing with the sample from one affected individual revealed a novel frameshift variant, c.410dupG (p.Gly138Argfs&lowast;43) in the SALL4 gene. Frameshift mutations in the SALL4 were already associated to Okihiro syndrome, which is characterized by radial limb defects and Duane anomaly. The segregation of the variant among the affected individuals was confirmed by Sanger sequencing. We concluded that this variant is the cause of the clinical signs in the family, which was classified as presenting Okihiro syndrome without Duane anomaly. The review of all clinical cases reported with SALL4 variants indicates a possible correlation between the foot malformation and the reduced size of the SALL4 protein. Family 3 has affected individuals with complete hand syndactyly associated to pre-axial polydactyly (syndactyly type IV). Whole exome sequencing was performed with the samples collected from the three affected individuals and two non-affected ones and no pathogenic variant was detected in genes related to limb development segregating with the phenotype. Through array-CGH, nevertheless, a duplication of about 97 kb including the enhancer (ZRS) of the SHH gene was detected. Duplications in ZRS region were already reported in Syndactyly type IV. We concluded that the 97-kb duplication is the cause of the syndactyly type IV in the family. A review of reported cases (including ours) suggested a possible correlation between the duplication with 97 kb size and the classic phenotype of syndactyly type IV. Family 4 has six individuals affected by a new syndrome (Santos syndrome) described by members of our group in 2008, a condition characterized by fibular agenesis/hipoplasia and ungual hypoplasia&frasl;anonychia, among other limb defects. Linkage study by SNP-array and microsatellite markers was performed and Lod score calculation pointed to two candidate regions on chromosome 3. Search for candidate genes revealed the WNT7A as the best candidate. Mutations in WNT7A gene, in homozygous state, are known to cause two other limb defect syndromes, Fuhrmann syndrome and AARRS (Al-Awadi/Raas-Rothschild syndrome), both presenting similar phenotypes to Santos syndrome. Sanger sequencing showed a novel variant, in homozygous state, c.934G&gt;A (p.Gly312Ser) in the WNT7A gene in five out of six affected individuals. One affected individual, much less severely affected than his relatives, carries the mutation in heterozygous state. He does not present fibular or ungual malformations, characteristic signals of the syndrome; instead, he is the carrier of a complex polydactyly in his left hand. This is the first report about a heterozygous variant in WNT7A gene resulting in limb defect. Whole exome sequencing was performed with the samples from two affected individuals. However, no other variant in candidate gene to explain the limb defects was detected. We concluded that Santos syndrome is caused by a mutation in the WNT7A gene. Functional assays, based on Luciferase activity, are in execution to test the deleterious effect of the c.934G&gt;A variant in the WNT7A gene

Deficiência intelectual (DI), associada ou não a outras alterações congênitas, é a razão mais frequente de procura por aconselhamento genético pelas famílias. Até alguns anos atrás, a realização de cariótipo, triagem para doenças metabólicas e fra(x) elucidavam apenas &sim;40% dos casos de pacientes com DI idiopática. Com o surgimento de arrays genômicos, as causas moleculares por trás de outros &sim;20% dos quadros de DI foram elucidadas; porém, mesmo com esse avanço, muitos pacientes ainda permanecem sem causa molecular clara que justifique o fenótipo. O sequenciamento do exoma (WES) é hoje um dos recursos disponíveis para o diagnóstico e possível elucidação das causas genéticas por trás da deficiência intelectual idiopática, abrindo caminho também à identificação de novos genes. O presente trabalho realizou o sequenciamento de exoma de 8 probandos que tinham em comum a deficiência intelectual esporádica, acompanhada ou não de outros sinais clínicos, e de seus genitores não afetados (trios). Esses pacientes foram previamente triados para a síndrome do X frágil, e submetidos a exame de array CGH para investigação de perdas e ganhos de segmentos cromossômicos, ambos com resultados negativos. O objetivo desse estudo foi detectar alterações e possivelmente novos genes associados com a DI, usando pipelines de padrões de herança mendeliano. Treze alterações em 9 genes foram detectadas por sequenciamento de exoma e confirmadas por sequenciamento Sanger: 8 mutações bialélicas em genes recessivos (TBC1D24, ADAMTSL2, NALCN, VPS13B), uma ligada ao X (MID1), e 4 alterações de novo (RYR2, GABBR2, CDK13, DDX3X); 5 dessas alterações ainda não haviam sido descritas nos bancos de dados consultados, caracterizando mutações novas. Dos 8 trios, em 5 identificamos alterações moleculares provavelmente responsáveis pelos quadros apresentados; dois desses casos foram em genes recessivos (mutações homozigotas ou em heterozigose composta) e potencialmente teriam sido detectados mesmo se apenas os probandos houvessem sido sequenciados. Para as alterações em heterozigose, porém, a avaliação dos genitores e constatação de status de novo da mutação foram importantes para avaliar o impacto da variante. Esse trabalho resultou em uma taxa de diagnóstico de 62,5%; mesmo considerando o pequeno tamanho da amostra, esse valor está bem acima dos 15-30% relatados na literatura quando essa metodologia é utilizada para o estudo de casos esporádicos de DI. Em dois casos, mutações foram identificadas em genes que só foram descritos como mutados recentemente e que ainda não são considerados genes de deficiência intelectual no OMIM: o gene CDK13 foi descrito como mutado em pacientes de uma única coorte com malformação cardíaca congênita (sindrômica ou não), porém sua contribuição para coortes de DI ainda não foi investigada. O gene GABBR2, identificado mutado em heterozigose em um dos nossos pacientes, já havia sido considerado um candidato potencial para DI, mas apenas 2 trabalhos detectaram mutações nesse gene entre pacientes com DI e epilepsia. Os resultados aqui apresentados substanciam o papel desses genes como implicados na DI sindrômica de herança autossômica dominante, e devem contribuir para serem considerados genes OMIM de deficiência intelectual / Intellectual disability (ID), associated or not with other congenital abnormalities, is the most frequent reason for families to seek genetic counseling. Until some years ago, karyotyping, metabolic disease and FRAXA screening elucidated only &sim;40% of patients with idiopathic ID. Importantly, with the introduction of genomic arrays, the molecular cause behind a further &sim;20% of ID cases was determined; however, despite this improvement, many patients are still not provided with a clear molecular explanation and cause for their phenotype. Nowadays, whole exome sequencing (WES) is one of the methods available for diagnosis and a further means of possible elucidation of the genetic causes of idiopathic intellectual disability; in many cases this method also allows identification of genes that have not been previously related to ID. In the present project, we sequenced the exome (WES) of 8 sporadic patients that all had ID, with or without other clinical signs, and their unaffected parents (trios); these patients had been previously screened for fragile X syndrome and for losses and gains of chromosomal segments by array CGH, both with negative results. The objective of this study was to detect mutations and possibly new genes associated with ID, using pipelines for Mendelian inheritance patterns. Thirteen mutations in 9candidate genes were detected by exome sequencing and confirmed by Sanger sequencing, among them 8 biallelic mutations in autossomal recessive genes (TBC1D24, ADAMTSL2, NALCN, VPS13B), one mutation in an X-linked gene (MID1), and 4 de novo alterations (RYR2, GABBR2, CDK13, DDX3X); 5 of these mutations had not been described in the databases consulted characterizing new variants. Of the 8 trios, we obtained a probable diagnosis of the molecular alteration responsible for the presented phenotypes in 5. Two of these cases were in recessive genes (homozygous mutations or compound heterozygous), and the mutations would probably have been detected even if only the probands had been sequenced. However, for the heterozygous mutations, the assessment of the parents and the confirmation of the de novo status of the mutation was important to evaluate the impact of the variant. This work resulted in a diagnosis rate of 62.5%; even considering the small sample size, this value is well above the average of 15-30% reported in the literature when the methodology used for the study of ID sporadic cases is considered. In two cases, mutations were detected in genes only recently described as mutated and which are not considered yet as OMIM ID genes. The CDK13 gene had already been described as mutated in a single cohort of patients with syndromic congenital heart defects, but its contribution to ID cohorts has not been established. The GABBR2 gene, where a heterozygous mutation was identified in the patient, had already been considered a potential candidate for ID; there are only 2 studies that detected mutations in this gene among patients with ID and epilepsy. This contribution may pave the way to establishing GABBR2 and CDK13 as causations of ID and acceptance by OMIM

La dysplasie fibromusculaire artérielle (DFM) est un groupe de pathologies vasculaires non inflammatoires, et non athéromateuses de la paroi artérielle. Elle est caractérisée par la sténose, l'occlusion, l’anévrisme ou la dissection des artères de petit et moyen calibres, en particulier les artères rénales et le tronc supra-aortique. La DFM est un facteur de risque de l’hypertension et de l’accident vasculaire cérébral. Elle touche essentiellement les femmes (80% des cas) de moins de 50 ans. La prévalence en population générale est inconnue et les estimations varient de 0.4% pour les formes cliniques à 4% dans une cohorte de donneurs de reins. Une agrégation familiale a été démontrée et une composante génétique suggérée. L'objectif de mon travail de thèse était de caractériser les bases génétiques la DFM. Dans la première partie, nous avons analysé des variants génétiques rares générés par séquençage d'exomes chez 16 cas apparentés de DFM issus de 7 fratries. Aucun gène majeur n’était muté pour l’ensemble des fratries ou pour au moins 3 fratries sur 7. Cependant, nous avons pu mettre en évidence puis validé un enrichissement en variants rares à fort potentiel fonctionnel de quatre gènes candidats pour la DFM (MYLK, OBSCN, DYNC2H1, RNF213) en combinant l’approche de séquençage d’exomes et l’étude d’association gène entier de 62767 variants rares (MAF < 5%) générés par génotypage avec la puce Exome-chip chez 249 cas non apparentés de DFM et 689 témoins. Cependant, l’implication de ces gènes dans la DFM doit être confirmée dans d’autres familles, et par des études de validations fonctionnelles. Dans la seconde partie, nous avons étudié l'association avec la DFM de 25606 variants fréquents (MAF ≥ 5%) de l’Exome-chip. Les résultats majeurs obtenus ont été répliqués dans une première étude (402 cas de DFM et 2537 témoins) puis dans 3 autres études incluant 512 cas de DFM et 669 témoins. La méta-analyse de l’ensemble a permis d’associer à la DFM le polymorphisme rs9349379-A situé dans l’intron du gène PHACTR1 (OR=1,39 [1,39-1,54] ; P=7,36 ×10-10). Ce variant est aussi un facteur de risque pour la maladie coronaire, la migraine et la dissection de l’artère cervicale. Des études complémentaires conduites chez 2458 volontaires non malades ont permis de montrer que l’allèle à risque pour la DFM, rs9349379-A est associé avec une augmentation de l’épaisseur intima média (P=1,97×10-4) et du rapport de la paroi sur la lumière artérielle (P=0,002), deux paramètres décrits comme augmentés chez les cas de DFM dans des études antérieures. Ensuite, PHACTR1 a été détecté par immunohistochimie dans l’endothélium et les cellules musculaires lisses de carotides dysplasiques et non dysplasiques avec une expression augmentée de PHACTR1 pour les porteurs de l’allèle à risque de DFM dans des cultures primaires de fibroblastes humains (N=86, P=0,003). Enfin, l’invalidation de Phactr1 chez le poisson zèbre conduit à une dilatation des vaisseaux indiquant un défaut du développement vasculaire. Ce travail confirme le caractère multifactoriel et hétérogène de la DFM et ouvre de nouvelles perspectives pour évaluer l’ensemble de la variabilité génomique des patients de DFM par des approches massives de génétique épidémiologique. / Fibromuscular dysplasia (FMD) is a group of nonatherosclerotic and noninflammatory vascular diseases leading to stenosis, aneurysm, dissection and/or occlusion of medium-sized arteries, in particular the renal and extracranial cervical arteries. Clinical manifestations of FMD are hypertension, dizziness, pulsatile tinnitus, transient ischemic attack or stroke, according to the involved arterial beds. FMD occurs predominantly (80% of cases) in females under 50 years with a variable prevalence estimation from 0.4% for asymptomatic clinical relevant forms to 4% in potential renal donors. The pathogenesis of FMD is unknown and a genetic origin is suspected given its demonstrated familial aggregation. The aim of my thesis work was to characterize genetic basis of FMD. In the first part of this thesis, we analyzed whole exome sequencing data in 16 related FMD cases from seven families. No gene harbors variants that were shared by all affected members in at least three out seven families. Using combined strategy of whole exome sequencing and gene based association study of 62,767 rare variants (MAF < 5%) generated by Exome‐chip arrays in 249 unrelated FMD cases and 689 controls, we have identified and validated an enrichment of rare and putatively functional variants in four candidates genes (MYLK, OBSCN, DYNC2H1 and RNF213). This results need to be validated in other FMD families and by functional analysis. In the second part, we analyzed 25,606 common variants (MAF ≥ 5%) generated by Exome‐chip array. Top loci were replicated in first replication study (402 cases and 2,537 controls) and in 3 others studies (512 cases and 669 controls). Meta-analysis of all including 1,154 unrelated FMD cases and 3,895 controls allowed identification of association between FMD and rs9349379-A (OR=1.39 [1.39-1.54]; P=7.4×10‐10). rs9349379 is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from 2,458 healthy volunteers indicated higher intima media thickness (P = 1.97×10‐4) and wall to lumen ratio (P = 0.002) in rs9349379‐A carriers, suggesting indices of carotid hypertrophy as previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379‐A carriers (N=86, P=0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impairment of vascular development. This work confirms the multifactorial and heterogeneous genetic architecture of the FMD and opens new opportunities to evaluate all of genomic variability of FMD patients with massive genetic epidemiology approaches.