Dissertations / Theses on the topic 'Skeletal Dysplasie'

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. (...)

Die Homöostase des Knochengewebes wird durch das balancierte Zusammenspiel von Ossifikation und Resorption gewährleistet. Eine in Relation zur Resorption zu starke Ossifikation führt zur Modellierungsstörung, Hyperostose und Sklerose. Knochenerkrankungen mit diesen Merkmalen werden als Sklerosierende Skelettdysplasien erfasst. Gegenstand der vorliegenden Arbeit sind fünf Skelettdysplasien aus dem Formenkreis der Sklerosierenden Skelettdysplasien: (1) Craniometaphysäre Dysplasie, autosomal dominante Form (MIM #123000); (2) Metaphysäre Dysplasie, Typ Braun-Tinschert (MIM *605946); (3) Caffey-Syndrom (MIM *114000); (4) McCune-Albright-Syndrom (MIM #174800); (5) Melorheostose (MIM 155950). Diese werden auf unterschiedlichen pathogenetischen Ebenen charakterisiert, die den Etappen des Weges entsprechen, der mit der Analyse des Phänotyps beginnt und zu einer Aufklärung des Basisdefektes führt. Die Arbeit gliedert sich ein in die Reihe von Bemühungen, zum molekularen Verständnis von Erkrankungen des Skelettsystems beizutragen.
Homeostasis of bone tissue is maintained by the balanced process of bone formation and resorption. Increased ossification in relation to resorption gives rise to conditions with modelling defects, hyperostosis and sclerosis. Skeletal diseases with these signs are classified as sclerosing bone dysplasias. The work presented here focuses on five skeletal dysplasias from the group of sclerosing bone dysplasias: (1) Craniometaphyseal dysplasia, autosomal dominant form (MIM #123000); (2) Metaphyseal dysplasia, Braun-Tinschert type (MIM *605946); (3) Caffey syndrome (MIM *114000); (4) McCune-Albright syndrome (MIM #174800); (5) Melorheostosis (MIM 155950). They were investigated at different pathogenetic levels that represent different steps on the path from phenotypic characterisation to clarification of the respective basic molecular defect. This work has contributed to our understanding of the molecular basis of skeletal diseases.

Dyggve Melchior Clausen (DMC) syndrome is an autosomal recessive skeletal dysplasia caused by mutations in the Dymeclin (DYM ) gene on chromosome 18q12-21. Affected individuals have multiple bony abnormalities and mental retardation.;The aim of this work was to elucidate the function of the DYM gene product (DYM) and determine the mechanisms by which mutation of the disease gene lead to cellular and clinical phenotype. Ten affected individuals from eight families were recruited to the study and five DYM mutations identified. These included two novel and complex genomic duplication/repetition events each predicted to result in a truncated transcript.;In-silico analyses of DYM suggest it encodes a transmembrane protein involved in protein sorting and targeting within the cell. The DYM transcript was shown by in-situ hybridization to be expressed at high levels in cartilage and brain, particularly in resting and hypertrophic chondrocytes. Sub-cellular localisation demonstrated the DYM gene product to be located within the endoplasmic reticulum.;Yeast two-hybrid analysis performed to detect DYM interacting proteins identified EGF-containing fibulin-like extracellular matrix protein (EFEMP1) and vacuolar protein sorting protein 25 (VPS25, human homologue known as EAP20). EFEMP1 is an extracellular matrix (ECM) protein and EAP20 a component of the endosomal sorting complex required for transport which acts in transport of transmembrane proteins for export and recycling.;Taken together, these findings indicate that DYM is an endoplasmic reticulum transmembrane protein required for cargo transport through the endosomal compartment. Abnormal chondrocyte differentiation and brain function occur in the absence of adequate functional DYM. Given that cartilage and brain both have substantial requirements for extracellular matrix, it is suggested that DYM contributes to the transport of components of the ECM.

Mutations in genes of importance for cartilage development may lead to skeletal malformations, chondroskeletal dysfunction and increased susceptibility to degenerative joint disease. Characterization of these mutations and identification of molecular pathways for the corresponding gene products have contributed to our understanding of mechanisms regulating skeletal patterning, endochondral ossification and joint formation. A five generation family segregating autosomal dominant osteochondritis dissecans (OCD) was identified. Affected family members presented with OCD in knees, hips and elbows, short stature, and early osteoarthritis. A genome wide scan and a multipoint linkage analysis identified aggrecan (ACAN) as a prime candidate gene. DNA sequence analysis of the ACAN-gene revealed heterozygosity for a missense mutation (c.6907G>A) in affected subjects, resulting in a p.V2303M substitution in the aggrecan G3 domain C-type lectin. This domain is important for the interaction with other proteins in the cartilage extracellular matrix. To determine the effect of the V2303M substitution on secretion and interaction, we performed binding studies with recombinant mutated and wild type G3 proteins. We found decreased affinity or complete loss of interaction between V2303M aggrecan and fibulin1, fibulin2 and tenascin-R. Analysis of articular cartilage from an affected family member confirmed that V2303M aggrecan is produced and present. In search for gene mutations associated with multiple epiphyseal dysplasia (MED) we considered the ACAN-gene a likely candidate. The ACAN-gene was analysed in 39 individuals with MED and screened negative for mutations in six previously known MED genes. Sequence analysis revealed a heterozygous missense mutation (c.1448G>T) in one adult male and compound heterozygous missense mutations (c.1366T>C and c.836G>A) in a five year old boy with healthy parents, each of them carrier for one of the mutations. A large family segregating autosomal dominant brachymesophalangia and OCD in finger joints was characterised. The clinical presentation in six affected family members was consistent with the diagnosis Brachydactyly type A1, in this family characterized by shortening of the middle phalanges, short ulnar styloid process, flattening of the metacarpal heads and mild osteoarthritis. The condition may be caused by mutations in the Indian hedgehog gene (IHH) or a yet unidentified gene on chromosome 5p13. Sequence analysis of the IHH-gene in affected individuals revealed a novel C to T transition (c.472C>T) leading to a p.158Arg>Cys substitution. Residue 158 in IHH is highly conserved throughout evolution and molecular structure modelling of IHH suggests that the R158C substitution leads to a conformational change at the site of interaction with the IHH-receptor. This supports that the substitution causes Brachydactyly type A1 in this family. In summary, we report on the clinical, radiological and molecular genetic characteristics of the three skeletal disorders OCD, MED and BDA1. Our results provide a novel molecular mechanism in the pathophysiology of familial osteochondritis dissecans confirming the importance of aggrecan C-type lectin for cartilage function. We also show that ACAN-gene mutations may be associated with MED extending the spectrum of skeletal dysplasias associated with the aggrecan gene. Finally, we report on a novel missense mutation in a conserved region of the IHH-gene associated with BDA1.

MCDS is an autosomal dominant skeletal dysplasia disorder caused by mutations in collagen X. In most cases, mutations in collagen X result in a misfolded protein which is retained within the ER of hypertrophic chondrocytes, causing increased ER stress. It has previously been demonstrated that increased ER stress causes hypertrophic chondrocytes to de-differentiate in an attempt to avoid the stress. The altered differentiation results in reduced cell hypertrophy and impaired vascular invasion accounting for reduced bone growth. The presence of increased ER stress in hypertrophic chondrocytes is sufficient to cause the MCDS pathology; therefore reducing ER stress may be beneficial in terms of improving the associated pathology. The autophagy enhancing drug carbamazepine (CBZ) has been shown to be capable of reducing ER stress in cells expressing the MCDS-causing p.N617K collagen X mutation. I show in this thesis that CBZ treatment reduced ER stress in HeLa cells transiently expressing a further 3 MCDS-causing collagen X mutations. I have also demonstrated that CBZ treatment induced the degradation of mutant collagen X proteins either through autophagy or proteasomal degradation depending on the nature of the mutation. The drug was tested in vivo using the p.N617K collagen X mouse model of MCDS. In MCDS mice, CBZ reduced the severity of the disease pathology based on histological analyses, restored hypertrophic chondrocyte differentiation toward normal, increased long bone growth rates and decreased the severity of the hip dysplasia. Gene expression analyses on RNA isolated from microdissected hypertrophic chondrocytes revealed that CBZ shifted the pattern of hypertrophic differentiation markers in MCDS mice toward the wild-type pattern, most likely through its stimulation of gene expression associated with intracellular proteolytic pathways. The results presented in this thesis have contributed to the identification of a potential treatment strategy for MCDS- the stimulation of intracellular proteolysis of mutant collagen X. CBZ is FDA approved for the use of epilepsy and bipolar disorder and has a strong safety record in humans. Therefore CBZ could be a potential treatment strategy for MCDS.

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.

Fibroblast Growth Factor Receptor 3 (FGFR3) appartiene alla famiglia dei recettori tirosin-chinasici e ha un ruolo importante come regolatore negativo del processo di ossificazione endocondrale. Mutazioni altamente attivanti di FGFR3 provocano gravi forme di displasie scheletriche. Durante i miei studi ho considerato se l’organizzazione del citoscheletro potesse essere influenzata della mutazione K650M, associata ad una grave acondroplasia con ritardo nello sviluppo e acantosis nigricans (SADDAN). FGFR3-SADDAN è un mutante altamente attivato che si trova accumulato in forma immatura glicosilata di 120kDa nel reticolo endoplasmico (ER). Da questo compartimento intracellulare è in grado di segnalare attraverso una via FRS2 e PLC indipendente e attivare le ERKs. Ci siamo chiesti se questo signalling anomalo dal reticolo endoplasmico potesse avere un ruolo importante nel determinare il fenotipo patologico del recettore. I dati che abbiamo ottenuto indicano che FGFR3-SADDAN recluta PLCPyk2 dal ER. Ciò si traduce in un aumento della fosforilazione di Pyk2 e paxillina, eventi associati alla disorganizzazione dell’actina citoscheletrica. Inoltre, durante il mio lavoro, abbiamo scoperto che abrogando l’interazione FGFR3/PLC, mediante la sostituzione Y754F in FGFR3-SADDAN, il recettore prosegue il suo pathway secretorio e raggiungere la membrana plasmatici, sulla quale è presente nella forma matura di 130kDa, pur rimanendo costitutivamente attivato. Crediamo che PLC giochi un ruolo chiave in quanto, legando FGFR3-SADDAN, impedisce che il recettore completi la sua biosintesi. Noi ipotizziamo che questo accada perchè si forma una “piattaforma” di segnalazione che coinvolge il recettore mutato e permette un signalling anomalo, forzando la permanenza di FGFR3-SADDAN nel reticolo endoplasmico. Questa via di segnalazione anomala dal ER determina inoltre la disorganizzazione delle strutture citoscheletriche suggerendo che questi eventi inducano displasie scheletriche mediate da FGFR3.
Fibroblast growth factor receptor 3 (FGFR3) belong to the tyrosine kinase receptor (RTK) family and plays a pivotal role in skeletal development being a negative regulator of bone growth as target disruption of the mouse FGFR3 gene causes a skeletal overgrowth. Many other mutations located in different domain of FGFR3 have been associated with skeletal diseases with graded severity, in particular gain-of-function mutation affecting the codon 650 within the critical kinase domain of FGFR3. The aim of our study was to investigate, in vitro, on the role by a mutant FGFR3 associated to the severe achondroplasia with developmental delay and achanthosis nigricans (SADDAN) on cytoskeletal organization. The SADDAN mutant revealed the unpaired trafficking of the immature mannose-rich 120kDa SADDAN receptor that remain localized in the ER, and transducers signal in its immature from leading to ERKs activation trough FRS2α and PLCγ-independent pathways. We have questioned whether the intracellular position of FGFR3 signalling has a critical role on the receptor-induced phenotype. Our findings indicate that PLCPyk2, paxillin interact with the immature FGFR3-SADDAN glycomers from the ER. These events are associated to an increased phosphorylation of paxillin/Pyk2 and the perturbed actin cytoskeltal organization. Preventing the PLC/FGFR3 interaction by the Y754F amino acid substitution in FGFR3 results in the failure of both Pyk2 recruitment and paxillin enhanced phosphorylation and restores the receptor full maturation on cell surface. We propose that PLC through its early engagement with the immature FGFR3-SADDAN confers a functional signalling activity to the receptor thus forcing its permanence in the ER. Altogether the data presented herein indicate that the interaction between PLC and the activated receptor in the ER are key events to determine the FGFR3-SADDAN-perturbed cytoskeletal organization and suggest that actin cytoskeleton is a target for the FGFR3-induced skeletal dysplasias.

O Sistema de Informação SDIS permite o armazenamento e a partilha de dados clínicos e pessoais de pacientes com displasias ósseas, assegurando a confidencialidade da informação. Permite, ainda, guardar informação acerca dos Grupos de Investigação e dos médicos que aí trabalham. O Sistema Esquelético é o sistema biológico cuja principal função é o suporte físico do organismo.As Displasias Ósseas são um grupo heterogéneo de doenças caracterizadas por alterações na forma, no tamanho e na constituição dos ossos e/ou cartilagens. Em 2011, foi feita uma tentativa de implementação desta aplicação, por alunos de Engenharia Informática da Universidade de Coimbra. Devido a um conjunto de falhas detectadas na mesma, esse trabalho apenas serviu de base ao desenvolvido neste novo projecto. A aplicação SDIS acrescenta várias funcionalidades às da primeira tentativa de implementação. De acordo com a arquitetura Cliente/Servidor, o Sistema de informação desenvolvido é composto por uma Base de Dados central, um Sistema gerenciador, uma interface de execução que corre num navegador Web, um Servidor Web e o Cliente da Aplicação.O Modelo em Cascata clássico conduziu a um desenvolvimento sistemático de Software, seguindo uma estratégia sequencial de conclusão de fases. Numa primeira fase a aplicação é utilizada nos três Centros de Investigação portugueses. Caso a sua utilização seja um sucesso, será alargada a outros grupos de investigação no estrangeiro. Palavras-chave "Partilha", "Confidencialidade", "Segurança", "Informação Clínica", "Dados Pessoais", “HTML”, "PHP", "MySQL", "Aplicação Web", "Software", "Displasia Óssea", "Médico", “Investigação

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.

Il recettore del fattore di crescita dei fibroblasti di tipo 3 (FGFR3) è un recettore transmembrana, avente attività tirosin-chinasica, esposto sulla superficie cellulare come monomero che dimerizza dopo legame specifico con i fattori di crescita dei fibroblasti (FGF). L’interazione FGF-FGFR3 porta ad autofosforilazione di specifici residui di tirosina (Tyr) presenti nel dominio tirosin-chinasico del recettore, evento cruciale per il reclutamento e l’attivazione di numerose proteine di segnalazione. FGFR3 ha un ruolo chiave nello sviluppo scheletrico come regolatore negativo dell'accrescimento delle ossa lunghe degli arti, inibendo la proliferazione e il differenziamento dei condrociti durante il processo di ossificazione endocondrale. In particolare, specifiche mutazioni germinali da “guadagno di funzione”, che rendono il recettore costitutivamente attivato, causano diverse displasie scheletriche. Nel presente studio, abbiamo esaminato le sostituzioni amminoacidiche K650M e K650E associate rispettivamente a due gravi displasie scheletriche, l’acondroplasia grave con ritardo dello sviluppo e acanthosis nigricans (SADDAN) e la displasia tanatofora di tipo II (TDII). In queste due sindromi, entrambe le mutazioni cadono nel dominio tirosin-chinasico di FGFR3 e portano all'attivazione costitutiva/ligando-indipendente del recettore. Studi precedenti hanno dimostrato che il recettore mutante, altamente fosforilato, non completa la sua biosintesi con conseguente accumulo della sua isoforma immatura di 120 kDa nel reticolo endoplasmatico, dal quale si attiva una trasduzione del segnale aberrante. Poiché diverse evidenze sperimentali indicano che le cellule trasfettate con il recettore mutante FGFR3-SADDAN mostrano una morfologia cellulare anomala, abbiamo ipotizzato che la trasduzione del segnale innescata da FGFR3-SADDAN potrebbe influenzare l'organizzazione dell’actina del citoscheletro. Quindi lo scopo del nostro studio è stato determinare, in vitro, il meccanismo molecolare attivato dal recettore FGFR3-SADDAN. Al fine di identificare i partner molecolari del mutante SADDAN coinvolti nelle alterazioni del citoscheletro, abbiamo analizzato la paxillina, una proteina citoscheletrica che svolge un ruolo critico nel controllo della riorganizzazione del citoscheletro e quindi dei cambiamenti della morfologia cellulare che sono necessari per la migrazione e la proliferazione cellulare. Abbiamo osservato che l'espressione del mutante SADDAN provoca una drastica alterazione dell'organizzazione della actina del citoscheletro, evento associato ad un aumento della fosforilazione della paxillina sulla tirosina 118 (Tyr118), un noto bersaglio delle chinasi FAK e c-Src. In aggiunta, mediante studi di immunofluorescenza, abbiamo osservato che il recettore SADDAN co-localizza parzialmente con la paxillina fosforilata all'interno della cellula. Abbiamo dimostrato, inoltre, che l'attività chinasica del recettore mutante SADDAN è richiesta per l’iperfosforilazione di paxillina. La presenza del mutante TDII, al contrario, non induce alterazioni della fosforilazione di paxillina, suggerendo dunque che le due varianti patologiche del recettore attivano un differente meccanismo molecolare. Abbiamo dimostrato, inoltre, che PLC-γ1, un effettore a valle della via di trasduzione del segnale attivata da FGFR3, svolge un ruolo nell’iperfosforilazione di paxillina. In modo particolare, i nostri dati mostrano che il recettore SADDAN promuove l’incremento della fosforilazione di paxillina mediante l'attivazione di c-Src e FAK. Complessivamente, i risultati del nostro studio contribuiscono a chiarire gli eventi molecolari che portano alla disorganizzazione dell’actina del citoscheletro causata dal mutante SADDAN.
Fibroblast growth factor receptor 3 (FGFR3) is a transmembrane tyrosine kinase receptor, exposed on cell surface as monomer that dimerizes upon specific binding with fibroblast growth factors (FGFs). The FGF-FGFR3 interaction results in tyrosine (Tyr) autophosphorylation within activation loop of FGFR3 tyrosine kinase domain, crucial event for the recruitment and activation of several signalling proteins. FGFR3 plays a key role in skeletal development as negative regulator of bone elongation by inhibiting the proliferation and differentiation of chondrocytes during the endochondral ossification. Accordingly, specific germline activating mutations in the FGFR3 gene cause several skeletal dysplasias. In the present study, we examined the Lys650Met (K650M) and Lys650Glu (K650E) amino acid substitutions associated with two severe skeletal disorders, respectively, severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN) and thanatophoric dysplasia type II (TDII). In these disorders, both substitutions are located in the FGFR3 kinase activation loop domain and lead to ligand-independent/constitutive activation of the receptor. Previous studies have shown that the highly phosphorylated mutant receptor does not complete its biosynthesis resulting in the accumulation of the 120-kDa immature isoform in the endoplasmic reticulum, from where an aberrant signalling is triggered. Since several experimental evidences indicated that cells transfected with SADDAN-FGFR3 construct show an abnormal cellular morphology, we hypothesized that the anomalous FGFR3 signalling could affect actin cytoskeleton organization. Accordingly, the aim of this study was to define, in vitro, the molecular mechanism triggered by SADDAN-FGFR3 signalling. To identify SADDAN-FGFR3 molecular partners involved in cytoskeleton alterations, we focused on paxillin, a focal adhesion-associated protein playing a crucial role in the control of cell morphology changes and cytoskeleton reorganization both required for cell migration and proliferation. We observed that the expression of the SADDAN-FGFR3 mutant causes drastic changes in actin cytoskeleton organization, event associated with an increase of paxillin phosphorylation at Tyr118, a well-known target of FAK and c-Src kinases. In addition, by immunofluorescence analysis we revealed that the SADDAN receptor partially colocalizes with phosphorylated paxillin. Moreover, we showed that paxillin hyper-phosphorylation requires the kinase activity of the SADDAN mutant receptor. Interestingly, we observed that paxillin is a specific target for SADDAN-FGFR3 since TDII-FGFR3 does not increase paxillin phosphorylation. Furthermore, we showed that PLC-γ1, a downstream effector of FGFR3 signalling, plays a role in paxillin hyper-phosphorylation. Interestingly, our results indicate that SADDAN-FGFR3 receptor enhances paxillin phosphorylation through c-Src and FAK activation. Overall our findings contribute to elucidate the molecular events leading to actin cytoskeleton disorganization by SADDAN-FGFR3 signalling pathway.

Introduction. Fetal short femur is defined by a femur length below the 5th percentile or -2 DS for the gestational age. The finding of a short femur represents a diagnostic dilemma for the various differential diagnosis. It may be associated with skeletal dysplasia, aneuploidies or genetic syndromes. In the isolated form, it may be an early sign of placental insufficiency and growth delay, or a normal variant in constitutionally small fetuses. Aims of the study: The aim of this study was: to examine postnatal outcome of pregnancies complicated by a short femur length; to compare outcomes in pregnancies with an early diagnosis of short FL (< 24 weeks of gestation) with pregnancies where this sign arises later in gestation (> 25 weeks of gestation); to analyse outcome differences in isolated and non-isolated form. A secondary aim of our research was a proposal of a diagnostic algorithm as a tool to guide clinicians in the management and counselling of pregnancy with isolated and not isolated short femur length. For this purpose, a revision of current literature data on the argument was carried out. Materials and Methods: A longitudinal prospective cohort study was conducted. All singleton pregnancies with a diagnosis of fetal femur < 5 centile were enrolled in the study. Patients were divided into two groups: patients with diagnosis of FL < 5th percentile at 14-24 weeks (group A) and at 25-40 weeks (group B). The differences in pregnancy complications and outcomes between the two groups were analysed. A comparison of the results of isolated and non-isolated forms was also carried out. For the secondary aim of our study we reviewed the literature and used meta-analytic technique to estimate accuracy of this marker in the prediction of Down Syndrome, IUGR and skeletal dysplasia. Correlation with poor perinatal outcome was also evaluated. Results: We enrolled 147 cases of short femur length in singleton pregnancies. In 61 (41,49%) cases short femur was associated to other fetal anomalies, in 86/147 fetuses (58,5%) was classified as isolated. Abnormal fetal karyotype (27,3% vs 3.7% p: 0.02) and skeletal dysplasia (19,7% vs 3.7% p: 0.002) were more frequent in group A. Cases of multiple abnormalities was diagnosed in 9 cases in group A and in 6 cases in group B with a difference not statistically significant (13.6% vs 7.4% p < 0.193). Diagnosis of isolated short femur was more common in group B (79% vs 33,4%, p: 0.000). In group B diagnosis of IUGR was made in 44.4% vs 19.7% of group A (p:0.002). The SGA prevalence had a difference statistically significant between the two groups (7.6% vs 24.7% p:0.007). The percentage of live birth was significant lower than group B (34.8% vs 97,6%). A comparison based on presence of an isolated short femur and not isolate finding (Group 1: Isolated - Group 2 not isolated) was also carried out. Abnormal fetal karyotype and (24,6% vs 7,0% p: 0.004), skeletal dysplasia (24,6% vs 1.2% p: 0.004) were more frequent in non-isolated group. Diagnosis of IUGR and SGA was more common in isolated group (47,7% vs 13,1%, p: 0.000, 25,6% vs 4,9% p 0.001) (table 4). The percentage of live birth was significant lower in not isolated group (45.9% vs 86% p 0.00). A higher incidence of neonatal complication, postnatal surgery and neonatal death were notice in not isolated group compared to isolated (57,69% vs17.45% p 0.019; 27,92% vs 4,2% p:0.003). Meta-analysis showed a higher incidence of short femur length in Down Syndrome fetuses (375/1326 28,2%) compared with euploid group (5809/188935, 3.07%) with an OR 5.12 (95% CI, 4.47-5.87). A higher incidence of IUGR/SGA was found in isolated short femur (455/3108, 14,6%) compared with the control group (11634/222362, 5.23%) with an OR of 4.12 (CI 95% 3.70-4.58). Conclusions. The diagnosis of short FL is often a challenge in obstetrics. The results of our study could help clinicians in counseling these patients in presence of this ultrasound findings. The diagnosis of a non-isolated short femur length before 24 weeks of gestation is associated to poor pregnancy outcome. When a short femur arises late in gestation and in isolated form, pregnancy outcome is better in term of chromosomal abnormalities but high rate of IUGR, SGA and neonatal complication is possible.