All ETDs from UAB

Advisory Committee Chair

Mary Macdougall

Advisory Committee Members

Amjad Javed

Dobrawa Napierala

Yi-Ping Li

Nathaniel Robin

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


Cleidocranial dysplasia (CCD, OMIM 119600) is a rare (1 in 1,000,000) human autosomal dominant skeletal disorder with unique craniofacial and dental features caused by mutations the gene coding for transcription factor RUNX2. RUNX2 is a key master regulator of osteoblast differentiation, chondrocyte maturation and tooth formation. CCD is commonly diagnosed based on bone defects with ~90% of cases presenting with supernumerary teeth or a “third dentition”. Mouse Runx2 null models (Runx2+/-), although having similar CCD skeletal defects, do not mimic the human dental phenotype of supernumerary teeth. Instead, Runx2-/- mouse have arrested tooth formation at the cap stage. Furthermore, based on the extended snout morphology, the mouse does not also serve as an ideal model for studying CCD craniofacial growth patterns. Among the reported CCD cases, ~70% are associated with RUNX2 mutations, with majority being missense mutations with remained due to copy number variations, chromosomal abnormalities or unknown molecular etiology. The significance of this work was to successfully delineate the genotypes and characterized the clinical phenotypes with emphasis on the craniofacial/dental complexes in three families with CCD (CCD-001, CCD-006 and CCD-011). In this study longitudinal medical and craniofacial/dental assessments were undertaken for furthering the knowledge of the genotype-phenotype correlation in dental tissues. With the identification, characterization and establishment of unique human CCD dental cell populations with identified RUNX2 mutations this work builds upon the existing knowledge for studying the critical tooth signaling pathways involved in CCD. The CCD Family 1 (CCD-001) were found to have RUNX2 (c.674G>A; R225Q) mutation which resulted in hypomorphic allele due to reduction in RUNX2 protein localization to the nucleus. Following immunocytochemistry and western blot analysis, the c.674G>A mutation was found to act as a dominant negative mutation. CCD-001 dental pulp cells demonstrated reduced TCF/LEF1 mRNA and protein. TCF/LEF1, a known binding partner of RUNX2 in nucleus revealed CCD-001 haploinsufficiency is a result of 1) reduced RUNX2 protein levels and 2) down-regulation of TCF/LEF1. The CCD-006 family demonstrated an AD inheritance pattern with no RUNX2 point mutation following DNA sequencing. Fluorescence in situ hybridization (FISH) analysis of all family members except paternal father revealed a chromosome 6 deletion. Chromosomal breakpoints were delineated with array comparative genomic hybridization revealing a 1 Mb deletion encompassing six genes SUPT3H, RUNX2, CLIC5, ENPP4, ENPP5 and RCAN2. The proband and other kindred karyotype were characterized 46, XY.ish del(6)(p12.3p12.3)(RP11-69J1-), and the mother’s karyotype was: 46, XX.ish del(6)(p12.3p12.3)(RP11-69J1-). Gene dosage analysis revealed decreased copy number of all six deleted genes in each affected family member. Quantitative RT-PCR analysis of bone and tooth cDNA revealed CLIC5 expression detectable only in bone. Longitudinal craniofacial analysis revealed mild maxillary anterior growth with 5-8mm increased incisor inclination and extrusion, growth, no noted vertical growth, increased posterior growth of mandible and increased molar extrusion over a five year period. Longitudinal analysis of craniofacial growth and facial morphology in CCD Family 11 (CCD-011) revealed decreased craniofacial bone and ankyloses in the permanent dentition. CCD-011 proband demonstrated significant transverse growth over the five year period, however, mild to no anterior-posterior or vertical growth was noted. These data demonstrated significant craniofacial growth attenuation with identified novel 397 kb microdeletion on chromosome 6p21 overlapping the RUNX2 locus. An altered extraction protocol for the supernumerary teeth was followed in this case due to decreased bone volume and heights. Craniofacial growth and morphologic analysis demonstrated atypical skull shape, persistent metopic suture and decreased mandibular size. The identification of altered critical tooth signaling pathways in human CCD dental cells was identified. Dental pulp cells isolated from the CCD-011 proband demonstrated a statistically significantly dyregulation profile as determined by mRNA next generation sequencing as compared to a sex- and age-matched control cells with comparable cellular passage number. Lentiviral infection of CCD-011 dental pulp cells revealed partial rescue of RUNX2 mRNA and protein. Post-infection of pLenti-RUNX2 demonstrated RUNX2 indirect/direct regulation of Insulin-like growth factor binding protein 2 (IGFBP2). Relative luciferase activity on IGFBP2 promoter revealed 24% down-regulation in HEK293T cells transiently transfected with pLenti-RUNX2-EGFP-Neo and pLenti-Empty-EGFP-Neo reporter plasmids. Immunohistochemistry of IGFBP2 in mouse 1, 3 and 5 day postnatal oral tissues revealed positive expression in oral epithelium, alveolar bone, surrounding muscle, odontoblasts, predentin, dental pulp and stellate reticulum. Furthermore, these studies provide novel insight into the genotype phenotype correlations associated with Cleidocranial Dysplasia.



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