Tratamientos térmicos

3.3.1 A bstract

Mutations in the fibroblast growth factor receptor (FGFR) genes 1, 2, and 3 are causal in a num ber o f craniofacial dysostosis syndromes featuring craniosynostosis with basicranial and midfacial deformity. G reat clinical variablity is displayed in the pathologic phenotypes encountered. T o investigate the influence o f developmental genetics upon clinical diversity in these syndromes, the expression o f several genes implicated in their pathology was studied at sequential stages o f normal hum an embryo-foetal cranial base and facial ossification.

A t 8 weeks o f gestation, FGFR7, 2, and 3 are equally expressed throughout the pre - differentiated mesenchyme o f the cranium, the endochondral skull base, and midfacial mesenchyme. Both clinically significant isoforms o f FGFR2, Igllla/c and Igllla/b, are co - expressed in maxillary and basicranial ossification. By 10 - 13 weeks, FGFK1 and 2 are broadly expressed in epitheha, osteogenic and chondrogenic cell lineages. FG FRJ, however, is maximally expressed in dental epitheka and proliferating chondrocytes o f the skull base; but poorly expressed in the osteogenic tissues o f the midface. FGF2 and FGF4, b ut n ot

FGF7; and TGFJ31 and /l3, are expressed throughout both osteogenic and chondrogenic tissues in early hum an craniofacial skeletogenesis.

FG FR expression in the skull base proposes a pivotal role for syndromic growth dysplasia at this site, and the co — expression o f FG FR3 and STA Tl proteins suggests an inhibitory role for FG FR3 in basicranial chondrogenesis. Paucity o f FG FR3 expression in hum an midfacial development correlates with the relatively benign hum an m utant FG FR3 midfacial

phenotypes. T he regulation o f FG FR expression in hum an craniofacial skeletogenesis against background excess ligand and selected cofactors may therefore play a profound role in the pathologic craniofacial development o f children bearing F G F R mutations.

3.3.2 Introduction

T h a t the co — ordinated grow th o f the craniofacial skeleton w ith its so ft tissues and specialised organs proceeds so often un disrupted represents a trium ph o f m am m ahan developm ent. T he m echanism s controlling the interrelated developm ent o f the various tissues are com plex, and require delicate tem p or o — spatial co — ordination. P erhaps because o f this, the investigation o f specific aetiology for patterns o f craniofacial m alform ation has pro v ed frustrating. In the last decade, how ever, advances in m olecular genetics have identified a range o f single gene defects in the fibroblast grow th factor rec e p to r (FGFR)

gene family w hich are causal to a num ber o f craniofacial dysostosis syndrom es.

E ponym ously recognised for the clinicians w ho described them , the m o st well docum ented o f these are A pert syndrom e (Apert, 1906), Pfeiffer syndrom e (Pfeiffer, 1964), and C rouzon syndrom e (Crouzon, 1912). T hese and related syndrom es consist prim arily o f skeletal abnorm alities o f the craniofacial com plex and appendicular skeleton, thus exhibiting a joint role for F G F R signalling in lim b and craniofacial developm ent (Britto et al, 2000a). T he phenotypic range o f these and o ther related acrocephalosyndactyly syndrom es is w ide and overlapping. W hilst in the m ajority o f cases a clinical diagnosis can be m ade, o th er

phenotypes to n o t easily conform to eponym ous categories (PuUeyn et al, 1996). T h e craniofacial phenotype o f A p ert syndrom e reflects its restricted genotype. Pterional in drawing, a foreshortened skuU base, turribrachycephaly, severe m idfacial retru sio n w ith hypertelorism , and coronal sutural synostosis w ith a widely unossified m edian sagittal diastem a in place o f m etopic and sagittal sutures are strongly characteristic (K reiborg and Cohen, Jr., 1990;Kreiborg et al, 1993;Cohen and K reiborg, 1994). T h e n arrow phenotypic range reflects the finding that only two closely neighbouring lin k e r region’ m utations in the I g llla extraceUular subdom ain o f F G F R 2 cause 98% A p ert cases (O ldridge et al, 1999) as a result o f Hgand d ependent ‘gain o f function’ (A nderson et al, 1998c).

T he related syndrom es o f C rouzon, Pfeiffer, Saethre — C h otzen (Saethre, 1931;Chotzen, 1932), and Jackson Weiss (Jackson et al, 1976), how ever, show a m ore variable and

overlapping craniofacial phenotype. T h e skuU base synchondroses tend to early synostosis, and the m etopic and sagittal sutures fo rm and fuse w ith o u t an unossified m edian diastem a (K reiborg et al, 1993;CinaUi et al, 1995). Facial retrusion variably effects the supraorbital or midfacial skeleton, w ith a wide range o f severity. Phenotypic variabUity w ithin and betw een these syndrom es reflects their m utational base. A broad range o f m utations w ith effects upon the extraceUular and transm em brane dom ains o f the F G F R l and 2 proteins are causal, with greatest frequency in the I g lllc subdom ain o f the F G F R 2 -Ig IIIa /c iso fo rm (Burke et al, 1998). M any o f these m utations result in the creation o r rem oval o f an unpaired cysteine

residue, o r affect a neighbouring site, to cause conform ational change and confer ligand independent functional gain u p o n the recep to r (N eilson and Friesel, 1995;Neilson and Friesel, 1996;Galvin et al, 1996;Mangasarian et al, 1997;R obertson et al, 1998). In addition, m utations in F G F R 3 cause a range o f hum an phenotypes, w hich display variable cranial and skull base dysostosis w ith o r w ith o u t lim b anomaly (Rousseau et al, 1994;Shiang et al, 1994;Prinos et al, 1995;Meyers et al, 1995;Bellus et al, 1995a;Tavorm ina et al, 1995a;Bellus et al, 1995b;M oloney et al, 1997;M uenke et al, 1997;Reardon et al, 1997;Paznekas et al, 1998;Angle et al, 1998;Gripp et al, 1998b). In these syndrom es the midfacial appearance appears to be secondary to

basicranial deform ity; as displayed by the facial scoliosis w hich accom panies F G F R 3 - unicoronal synostosis and appears to ‘hinge’ up o n the affected cranial base (Persing et al, 1986).

M olecular factors influencing the genotype — phenotype relationship include the m echanism o f activation o f the m u tan t receptor, and the relative bio availability o f ligand, receptor, and cofactors in the m olecular cascades controlling craniofacial endochondral and

intram em branous ossification. F G F R transcript - m R N A expression studies have been previously rep o rted in chicken (Wilke et al, 1997) and in hum an foetal tissues (D elezoide et al, 1998;Chan and T h o ro g o o d , 1999). In this study, the expression o f F G F R 1, the Ig llla /c and

Ig llla / b isoform s o f FG FR2, and FG F R 3 is desribed in hum an basicranial and m id facial skeletogenesis at the level o f m R N A transcript and p rotein in a tem poral series o f hum an em bryo - foetal craniofacial tissues. T h e F G F R expression dom ains are contrasted w ith those o f the bone differentiation m arker, osteonectirr, and the ligand proteins F G F 2 , F G F 4 and FG F7. In addition, the co — factor cytokines T G F ^ l and TGF(33, w hich are im plicated in osseous differentiation, are detected in an attem pt to correlate genotype and phenotype in hum an F G F R - associated craniofacial dysostosis.

3.3.3 Materials

Methods

^Preparation of embryonic

early foetal material

H um an em bryo — foetal tissue aged 8 to 14 weeks (n=6) was provided by the H u m an Tissue R esource m aintained at the Institute o f Child H ealth and U niversity College H ospital, L ondon. Craniofacial tissue was collected and prepared as described above {S-2. /).

Studies of F G F R and allied gene expression

F G F R /, osteonectin, and the IG IIIa /c (BEK) and Ig llla /b (KGFR) isoform s o f F G F R 2 m R N A transcripts were detected by in situ hybridisation using isoform - specific riboprobes. P robe synthesis and in — situ hybridisation steps are described in S-2.2.

T he protein receptors F G F R l, F G F R 2, and FG FR 3; the ligand F G F s 2, 4, and 7; and the T G F P isoform s 1 and 3 proteins w ere detected by im m unohistochem istry using

comm ercially available specific antibodies (S-2.2.J). T h e anti — recep to r antibodies have epitopes corresponding to the carboxy — term inal sequences o f the recep to r protein, and therefore label b o th I g lllc and I g lllb isoform s in im m unohistochem istry studies.

3.3.4 Results