Molecular Medicine 8(10): 638–644, 2002 © 2002 North Shore-LIJ Research Institute Expression Profiles of Craniosynostosis-Derived Fibroblasts Francesco Carinci,1 Maria Bodo,2 Lara Tosi,3,4 Francesca Francioso,4 Rita Evangelisti,5 Furio Pezzetti,3,4 Luca Scapoli,3,5 Marcella Martinelli,5 Tiziano Baroni,2 Giordano Stabellini,6 Paolo Carinci,3,4 Catia Bellucci,2 Cinzia Lilli,2 and Stefano Volinia5,7 1Chair of Maxillofacial Surgery, University of Ferrara, Italy 2Institute of Histology and General Embryology, University of Perugia, Italy 3Centre of Molecular Genetics, CARISBO Foundation, Bologna, Italy 4Institute of Histology and General Embryology, University of Bologna, Italy 5Functional Genomics, Section of Histology, Department of Morphology and Embryology, University of Ferrara, Italy 6Department of Anatomy, University of Milan, Italy 7TIGEM, Telethon Institute of Genetics and Medicine, Napoli, Italy Accepted August 26, 2002 Abstract Background: Craniosynostosis syndromes, a group of respectively. The genetic profiles, as evaluated by DNA connective disorders characterized by abnormalities in microarrays, yielded different clusters of expressed se- vault osteogenesis and premature fusion of bone sutures, quence tag (ESTs) expression within the experiment. are associated with point mutations in FGF receptor fam- Expression profiles from craniosynostosis-derived fibrob- ily members. The cellular phenotype is characterized by lasts differ from those of wild-type fibroblasts (288 hu- abnormal extracellular matrix turnover. man ESTs, p Ͻ 0.01, pFDR ϭ 0.12). Furthermore, two Material and Methods: We used primary cultures of pe- ESTs clusters discriminate the Crouzon from Apert riosteal fibroblasts derived from two different craniosyn- fibroblasts. The differentially expressed genes cover a ostosis syndromes, the Apert and Crouzon syndromes. broad range of functional activities, including (1) bone The FGFR2 third immunoglobulin-like domain and its differentiation, (2) cell-cycle regulation, (3) apoptotic flanking linker regions were analyzed for mutation. DNA stimulation, and (4) signaling transduction, cytoskeleton, microarrays containing 19,200 cDNAs were used to study and vesicular transport. the gene expression profiles of Apert and Crouzon fibrob- Conclusions: The transcriptional program of craniosyn- lasts. The pathologic cells were compared to wild-type hu- ostosis fibroblasts differs from that of wild-type fibrob- man periosteal fibroblasts. lasts. Expression profiles of Crouzon and Apert fibroblasts Results: The P253R missense mutation and the G338R can also be distinguished by two EST expression clusters, mutation were observed in Apert and Crouzon fibroblasts, thus hinting at a different genetic background. Introduction Crouzon patients have brachycephaly, ocular prop- tosis, teleorbitism, and midfacial hypoplasia. The Mechanisms involving growth factors and different Apert clinical phenotype is more severe than the time-space expression of extracellular matrix (ECM) Crouzon, not only at the cranial level, but also in the components participate in skull osteogenesis. Cranial limbs where there is syndactyly of hands and feet. sutures contain osteogenic stem cells and periosteal Cranial base abnormalities are more severe and syn- fibroblasts that, during human embryo development, chondroses of cranial base are calcified at an earlier differentiate into osteoblasts that produce premineral- age, before vault sutures (1). In the Crouzon syn- ized osseous matrix. Abnormal ossification leads to pre- drome coronal and lambdoid sutures are the most mature fusion of cranial sutures and craniosynostosis affected (2). syndromes such as the Apert and Crouzon syndromes. ECM molecule composition plays a role in the The Apert syndrome is characterized by acro- morphogenetic processes that occur during skull brachycephalic vault, exorbitism, hypertelorism, development. Differences in ECM synthesis and and dystopy of the outer canthus; the nose is hooked secretion of glycosaminoglycans, collagens, proteo- and the mouth has the shape of an isosceles triangle. glycans, and fibronectin in fibroblasts and osteoblasts from Apert and Crouzon patients have been reported Correspondence and reprint requests should be addressed to: Maria Bodo, Sezione di Istologia ed Embriologia Sperimentale, (3–7). These changes in ECM were also affected by Via del Giochetto 06100 Perugia, Italy. Phone: (39)075-585.7432; exposure to growth factors such as transforming fax: (39)075-585.7434; e-mail: [email protected]. growth factor beta (TGF-␤) (8), interleukins (3,4,5,7), F. Carinci et al.: Craniosynostosis-Derived Fibroblasts 639 and the basic fibroblast growth factor (bFGF) (6,9). and eighth subculture. All fibroblasts were then incu- bFGF, the most abundant growth factor in the vault, bated with MEM with 0.5% fetal calf serum for 24 hr. regulates cell proliferation, migration, and differenti- ation; overall it has diverse roles in regulating bone Mutation Detection osteogenesis (10). As growth factors are secreted in Genomic DNA was extracted from Crouzon and an autocrine-paracrine manner and interact with Apert periosteal fibroblasts according to standard ECM, the ECM pattern may be considered another procedures (16). The FGFR2 third Ig-like domain signal of morphogenetic control. Indeed, it is a further and its flanking linker regions were analyzed for variable in phenotype determination. mutations. Primers designed from intronic sequences The molecular basis for craniosynostosis has flanking exon IIIa (forward 5Ј-accccagttgtgggtacctt-3Ј; been partially elucidated. Many human craniosyn- reverse 5Ј-tccccactcctcctttcttc3Ј) and exon IIIc (for- ostosis disorders have been linked to activating mu- ward 5Ј-aaaaggggccatttctgataa-3Ј; reverse 5Ј-ccacaat- tations in FGF receptors (FGFR) 1 and 2, but the cattcctgtgtcg-3) were used to amplify genomic DNA precise effects of FGF on the proliferation, matura- by PCR. Direct sequencing of the purified PCR prod- tion, and differentiation of the target osteoblastic uct was performed on both forward and reverse cells are still under investigation (11). Interestingly, strand on an ABI377 sequencer using the Big Die defects in the same gene induce craniosynostosis Terminator sequencing Kit. syndromes with a wide spectrum of clinical pheno- types and indeed several different point mutations DNA Microarrays have been mapped in FGFRs involved in cranial Expression profiling was performed essentially as development disorders. FGFRs are composed of a previously described (17). Briefly, total RNA was ex- single transmembrane domain, an extracellular do- tracted from normal and pathologic cells cultured as main composed of three immunoglobulin (Ig)-like above, using RNAzol. Ten micrograms of total RNA domains, and an intracellular domain containing were used for each sample. cDNA was synthesized by two tyrosine kinase subdomains. In the Crouzon using Superscript II (Invitrogen, Paisley, England, syndrome, several mutations in the third Ig-like UK) and amino-allyl dUTP (Sigma, St. Louis, MO, domain of the extracellular portion of FGFR2 have USA). Monoreactive Cy3 and Cy5 esters (Amersham been identified (12,13). In patients with Apert syn- Biosciences, Little Chalfont, England, UK) were used dromes, FGFR2 mutations produce missense substi- for indirect cDNA labeling. RNA from wild-type fi- tutions involving adjacent amino acids in the linker broblasts was labeled with Cy3 and used as common region between the second and third extracellular Ig reference against every other Cy5-labeled cDNAs. domain (14). Moreover, genes other than FGFR 1-4, Human 19.2K DNA microarrays, containing 19,200 ex- such as MSX2 and TWIST, can also cause craniosyn- pressed sequence tag (ESTs), corresponding to at least ostosis (15). 15,448 different Unigene clusters, were used (Ontario In this study we analyzed the expression profiles Cancer Institute, Ontario, Canada). One hundred mi- of periosteal fibroblasts from patients with cran- croliters of the sample and control cDNA in DIG Easy iosynostosis (Apert and Crouzon syndromes) and hybridization solution (Roche, Basel, Switzerland) compared them to those of wild-type periosteal were used at 37 ЊC overnight. Washing was performed fibroblasts. Furthermore, we aimed to identify FGFR- three times for 10 min with 1ϫ SSC, 0.1% SDS at regulated transcripts for investigating the molecular 42 ЊC, and three times for 5 min with 0.1ϫ SSC at room mechanism underlying abnormal FGFR signaling. temperature. Hybridized slides were scanned using GenePix 4000A (Axon Instruments, Foster City, CA, USA). Arabidopsis RNA was used as a reference for Materials and Methods RNA labeling. Cell Culture Images were analyzed by GENEPIX PRO 3.0 Human periosteal fibroblasts were obtained from (Axon Instruments) and data extracted as previously the galea-pericranium of cell parietal bones of two described (17). Four thousand four hundred and Crouzon patients (Crouzon Q and Crouzon K) and eight arrayed ESTs were present in at least 75% of one Apert patient (all aged 17–18 years) during cor- the samples. Spotted ESTs with expression levels rective surgery. Wild-type control fibroblasts origi- higher than reference have a positive log2 Cy5/Cy3 nated from explants of periosteum removed from ratio, whereas those with lower expression have a two age-matched subjects at the time of surgery for negative log2 Cy5/Cy3 ratio. repair of parietal bone fractures. Primary cultures To select the ESTs that distinguish between the were grown in Falcon flasks (Becton Dickinson, craniosynostosis