Roberto Dalto Fanganiello

Genetics of Craniosynostosis: Genes, Syndromes, Mutations and Genotype-Phenotype Correlations

Craniosynostosis is a very heterogeneous group of disorders, in the etiology of which genetics play an important role. Chromosomal alterations are important causative mechanisms of the syndromic forms of craniosynostosis accounting for at least 10% of the cases. Mutations in 7 genes are unequivocally associated with mendelian forms of syndromic craniosynostosis: FGFR1, FGFR2, FGFR3, TWIST1, EFNB1, MSX2 and RAB23. Mutations in 4 other genes, FBN1, POR, TGFBR1 and TGFBR2, are also associated with craniosynostosis, but not causing the major clinical feature of the phenotype or with an apparently low penetrance. The identification of these genes represented a great advance in the dissection of the genetics of craniosynostosis in the last 15 years, and today they explain the etiology of about 30% of the syndromic cases. The paucity in the identification of genes associated with this defect has partly been due to the rarity of familial cases. In contrast, very little is known about the molecular and cellular factors leading to nonsyndromic forms of craniosynostosis. Revealing the molecular pathology of craniosynostosis is also of great value for diagnosis, prognosis and genetic counseling. This chapter will review (1) the chromosomal regions associated with syndromic forms of the malformation, (2) the genes in which a large number of mutations have been reported by independent studies (FGFR1, FGFR2, FGFR3, TWIST1 and EFNB1) and (3) the molecular mechanisms and genotype-phenotype correlations of such mutations.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in nonimmunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Syndromes of the first and second pharyngeal arches: A review.

Our aim in this review is to discuss currently known mechanisms associated with three important syndromes of the first and second pharyngeal arches: Treacher Collins syndrome (TCS), Oculo‐auriculo‐vertebral syndrome (AOVS) and Auriculo‐Condylar syndrome (ACS) or question mark ear syndrome. TCS and ACS are autosomal dominant diseases, with nearly complete penetrance and wide spectrum of clinical variability. The phenotype of the latter has several overlapping features with OAVS, but OAVS may exist in both sporadic and autosomal dominant forms. Mutations in the TCOF1 gene are predicted to cause premature termination codons, leading to haploinsuficiency of the protein treacle and causing TCS. Low amount of treacle leads ultimately to a reduction in the number of cranial neural crest cells migrating to the first and second pharyngeal arches. Other than TCS, the genes associated with ACS and OAVS are still unknown. The first locus for ACS was mapped by our group to 1p21‐23 but there is genetic heretogeneity. Genetic heterogeneity is also present in OAVS. Based on the molecular analysis of balanced translocation in an OAVS patient, it has been suggested that abnormal expression of BAPX1 possibly due to epigenetic disregulation might be involved with the etiology of OAVS. Involvement of environmental events has also been linked to the causation of OAVS. Identification of factors leading to these disorders are important for a comprehensive delineation of the molecular pathways underlying the craniofacial development from the first and the second pharyngeal arches, for genetic counseling and to open alternative strategies for patient treatment.

Optimization of Parameters for a More Efficient Use of Adipose-Derived Stem Cells in Regenerative Medicine Therapies

Adipose tissue-derived stem cells (ASCs) association to fat in autologous lipotransfer is promising for a more effective soft tissue reconstruction, and optimization of protocols to isolate ASCs from lipoaspirate fat is much needed. We demonstrated that an increase in adipocyte differentiation is dependent on the number of ASCs. In a sample of 10 donors, we found a higher concentration of nucleated cells in the lower abdomen compared to flank (P = 0.015). In a sample of 6 donors we did not find differences in the cell yield obtained by manual or pump-assisted aspiration (P = 0.56). We suggest that the increase in the number of ASCs in the reinjected fat may enhance the efficiency of newly formed adipose tissue and that the anatomical region from which to harvest fat tissue needs to be considered to optimize the number of ASCs in the harvested tissue. Finally, pump-assisted aspiration can be used without any significant harm to the viability of cells.

Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells.

Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED) and MSC-like cells from iPSCs from SHED (iPS-SHED) and from human dermal fibroblasts (iPS-FIB). MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.

Novel Molecular Pathways Elicited by Mutant FGFR2 May Account for Brain Abnormalities in Apert Syndrome

Apert syndrome (AS), the most severe form craniosynostosis, is characterized by premature fusion of coronal sutures. Approximately 70% of AS patients carry S252W gain-of-function mutation in FGFR2. Besides the cranial phenotype, brain dysmorphologies are present and are not seen in other FGFR2-asociated craniosynostosis, such as Crouzon syndrome (CS). Here, we hypothesized that S252W mutation leads not only to overstimulation of FGFR2 downstream pathway, but likewise induces novel pathological signaling. First, we profiled global gene expression of wild-type and S252W periosteal fibroblasts stimulated with FGF2 to activate FGFR2. The great majority (92%) of the differentially expressed genes (DEGs) were divergent between each group of cell populations and they were regulated by different transcription factors. We than compared gene expression profiles between AS and CS cell populations and did not observe correlations. Therefore, we show for the first time that S252W mutation in FGFR2 causes a unique cell response to FGF2 stimulation. Since our gene expression results suggested that novel signaling elicited by mutant FGFR2 might be associated with central nervous system (CNS) development and maintenance, we next investigated if DEGs found in AS cells were also altered in the CNS of an AS mouse model. Strikingly, we validated Strc (stereocilin) in newborn Fgfr2S252W/+ mouse brain. Moreover, immunostaining experiments suggest a role for endothelial cells and cerebral vasculature in the establishment of characteristic CNS dysmorphologies in AS that has not been proposed by previous literature. Our approach thus led to the identification of new target genes directly or indirectly associated with FGFR2 which are contributing to the pathophysiology of AS.

Is bone transplantation the gold standard for repair of alveolar bone defects

New strategies to fulfill craniofacial bone defects have gained attention in recent years due to the morbidity of autologous bone graft harvesting. We aimed to evaluate the in vivo efficacy of bone tissue engineering strategy using mesenchymal stem cells associated with two matrices (bovine bone mineral and α-tricalcium phosphate), compared to an autologous bone transfer. A total of 28 adult, male, non-immunosuppressed Wistar rats underwent a critical-sized osseous defect of 5 mm diameter in the alveolar region. Animals were divided into five groups. Group 1 (n = 7) defects were repaired with autogenous bone grafts; Group 2 (n = 5) defects were repaired with bovine bone mineral free of cells; Group 3 (n = 5) defects were repaired with bovine bone mineral loaded with mesenchymal stem cells; Group 4 (n = 5) defects were repaired with α-tricalcium phosphate free of cells; and Group 5 (n = 6) defects were repaired with α-tricalcium phosphate loaded with mesenchymal stem cells. Groups 2–5 were compared to Group 1, the reference group. Healing response was evaluated by histomorphometry and computerized tomography. Histomorphometrically, Group 1 showed 60.27% ± 16.13% of bone in the defect. Groups 2 and 3 showed 23.02% ± 8.6% (p = 0.01) and 38.35% ± 19.59% (p = 0.06) of bone in the defect, respectively. Groups 4 and 5 showed 51.48% ± 11.7% (p = 0.30) and 61.80% ± 2.14% (p = 0.88) of bone in the defect, respectively. Animals whose bone defects were repaired with α-tricalcium phosphate and mesenchymal stem cells presented the highest bone volume filling the defects; both were not statistically different from autogenous bone.

FGFR2 mutation confers a less drastic gain of function in mesenchymal stem cells than in fibroblasts.

Gain-of-function mutations in FGFR2 cause Apert syndrome (AS), a disease characterized by craniosynostosis and limb bone defects both due to abnormalities in bone differentiation and remodeling. Although the periosteum is an important cell source for bone remodeling, its role in craniosynostosis remains poorly characterized. We hypothesized that periosteal mesenchymal stem cells (MSCs) and fibroblasts from AS patients have abnormal cell phenotypes that contribute to the recurrent fusion of the coronal sutures. MSCs and fibroblasts were obtained from the periostea of 3 AS patients (S252W) and 3 control individuals (WT). We evaluated the proliferation, migration, and osteogenic differentiation of these cells. Interestingly, S252W mutation had opposite effects on different cell types: S252W MSCs proliferated less than WT MSCs, while S252W fibroblasts proliferated more than WT fibroblasts. Under restrictive media conditions, only S252W fibroblasts showed enhanced migration. The presence of S252W mutation increased in vitro and in vivo osteogenic differentiation in both studied cell types, though the difference compared to WT cells was more pronounced in S252W fibroblasts. This osteogenic differentiation was reversed through inhibition of JNK. We demonstrated that S252W fibroblasts can induce osteogenic differentiation in periosteal MSCs but not in MSCs from another tissue. MSCs and fibroblasts responded differently to the pathogenic effects of the FGFR2S252W mutation. We propose that cells from the periosteum have a more important role in the premature fusion of cranial sutures than previously thought and that molecules in JNK pathway are strong candidates for the treatment of AS patients.

Cnbp ameliorates Treacher Collins Syndrome craniofacial anomalies through a pathway that involves redox-responsive genes.

Treacher Collins Syndrome (TCS) is a rare congenital disease (1:50 000 live births) characterized by craniofacial defects, including hypoplasia of facial bones, cleft palate and palpebral fissures. Over 90% of the cases are due to mutations in the TCOF1 gene, which codifies the nucleolar protein Treacle. Here we report a novel TCS-like zebrafish model displaying features that fully recapitulate the spectrum of craniofacial abnormalities observed in patients. As it was reported for a Tcof1+/− mouse model, Treacle depletion in zebrafish caused reduced rRNA transcription, stabilization of Tp53 and increased cell death in the cephalic region. An increase of ROS along with the overexpression of redox-responsive genes was detected; furthermore, treatment with antioxidants ameliorated the phenotypic defects of craniofacial anomalies in TCS-like larvae. On the other hand, Treacle depletion led to a lowering in the abundance of Cnbp, a protein required for proper craniofacial development. Tcof1 knockdown in transgenic zebrafish overexpressing cnbp resulted in barely affected craniofacial cartilage development, reinforcing the notion that Cnbp has a role in the pathogenesis of TCS. The cnbp overexpression rescued the TCS phenotype in a dose-dependent manner by a ROS-cytoprotective action that prevented the redox-responsive genes’ upregulation but did not normalize the synthesis of rRNAs. Finally, a positive correlation between the expression of CNBP and TCOF1 in mesenchymal cells from both control and TCS subjects was found. Based on this, we suggest CNBP as an additional target for new alternative therapeutic treatments to reduce craniofacial defects not only in TCS but also in other neurocristopathies.

Craniosynostosis and Chromosomal Alterations

A large number of patients with craniosynostosis and chromosomal rearrangements have been described in the last decades. Through a comparative analysis of these cases, we discuss in this chapter their