Skeletal dysplasias and dysostoses are a genotypically and phenotypically diverse group of disorders that affect the growth, development and maintenance of cartilage and bone. General disorders of bone affecting bones and cartilage throughout the body have been referred to as skeletal dysplasias, whereas defects that selectively affect certain bones or bone groups are called skeletal dysostoses. Despite this distinction, modern molecular techniques are showing that this division is somewhat superficial, given the similarity in their underlying causes. Although the rate of disease gene discovery has grown substantially since the advent of next-generation sequencing technologies, most of the disorders have unknown molecular defects. Skeletal dysostoses are rarely observed, occurring at such low incidence levels that no comprehensive study has ascertained their frequency. The effects range from mild growth inhibition to complete absence of entire bone groups. The axial skeleton is most often involved in skeletal dysostoses with common symptoms including poorly formed cranial bones, mandible, ribs and vertebrae. Several important signaling pathways control the migration and formation of mesodermal cells, which eventually differentiate into many elements of the vertebral column. The importance of these pathways, namely the T-box transcription factors, Wnt, Notch, and Smad pathways are integrally involved in the very early stages of vertebral development. Currently, the most cost-effective method of pathogenic gene discovery for rare genetic diseases is exome sequencing. Utilizing this technology, as well as SNP arrays for identity-by-descent loci mapping, two independent skeletal dysostosis cases with similar phenotypes were studied to determine pathogenic candidate genes. Next-generation sequencing and identity-by-descent analysis revealed a possible candidate gene, PM20D2, in one proband. The gene includes peptidase dimerization, peptidase M20/M25/M40, and N-myristolylation domains based on predicted functional analysis. It is implicated in various metabolic activities, having hydrolase, protein binding, and metallopeptidase molecular functions. Further investigation into this gene, as well as further studies of these probands is needed to understand the role, if any, the defect plays in the disease.

Mutations in the COL10A1 gene cause metaphyseal chondrodysplasia type Schmid (MCDS) by triggering ER stress and unfolded protein response (UPR). MCDS is characterised by a mild short-limb dwarfism accompanied by expansion of the cartilage growth plate hypertrophic zone (HZ) and altered differentiation of hypertrophic chondrocytes (HCs). ATF6 is one of the UPR mediators, which exists in two isoforms, ATF6α and ATF6β. Activation and up-regulation of ATF6α was a prominent biochemical sign of ER stress in a mouse model of MCDS, COL10a1 p.N617K. Although ATF6β is induced and activated in response to ER stress in a similar fashion to ATF6α, the role and significance of ATF6β in the pathology of many ER stress-associated diseases including MCDS is unknown. Here we utilized a combination of in vitro and in vivo approaches to define the precise role of each isoform of ATF6 in MCDS.To investigate the functions of ATF6α and ATF6β in vitro, we developed a MCDS cell model system (expressing either the wild type collagen X or one of the following MCDS-causing mutant forms of the protein: p.N617K, G618V, Y598D, and NC1del10) in which the expression of either ATF6α or ATF6β was efficiently silenced using siRNAs. ATF6α knockdown in HeLa cells expressing different MCDS-causing mutations suppressed the increased expression of UPR-associated genes such as BiP leading to an elevated ER stress, based on increased XBP1 splicing and/or ATF4 protein. In contrast, ATF6β knockdown did not significantly affect the mutant collagen X-induced increased expression of UPR-associated genes. Furthermore, the ER stress levels were significantly reduced in the ATF6β knockdown MCDS mutant cells based on the lower levels of XBP1 splicing and/or ATF4 protein detected. We then crossed the ATF6α/β knockout mice models with COL10a1 p.N617K mouse model of MCDS to investigate the function of ATF6α and ATF6β in vivo. Ablation of ATF6α in MCDS mice further- reduced the endochondral bone growth rate, further expanded the growth plate hypertrophic zone, and disrupted differentiation of HCs. Therefore, ATF6α appeared to play a chondroprotective role in MCDS as its deficiency caused an increase in the severity of the disease. Of particular note, the level of ER stress was further increased in the absence of ATF6α in MCDS, based on enhanced activities of PERK and IRE1 signalling pathways in compensation for the ATF6α loss. Paradoxically, ablation of ATF6β in MCDS mice reduced the intracellular retention of collagen X protein, and alleviated the ER stress as judged by the attenuated activities of PERK and IRE1 signalling pathways. The reduced ER stress resulting from deficiency for ATF6β in MCDS mice restored the expression of collagen X mRNA towards normal and improved the differentiation of HCs, causing a mark decrease in the expansion of HZ. The results presented within this thesis greatly increased our understanding of the function of ATF6α and ATF6β and their interplay in the pathogenesis of MCDS. We demonstrated an indispensable beneficiary role for ATF6α but a detrimental role for its closely related isoform, ATF6β, in pathology of MCDS. We also showed that the role of ATF6β should not be ignored. These findings may be used to develop a potential therapeutic strategy for MCDS through targeting and enhancing ATF6α-dependent and/or attenuating/blocking of ATF6β-dependent signalling pathways.

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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. (...)