UCLA

Objectives: The human craniofacial skeleton demonstrates considerable phenotypic variation. Half of all birth defects involve the skull and face, with the mandible being very commonly affected in a variety of syndromes. Heritability and genes responsible for craniofacial phenotypes are poorly understood and studied due to the complexity of the region and confounding environmental effects. The objective of this project is to assess and identify regions of significant variation in shape of the human mandible by utilizing reliable methods of morphometric analysis, particularly in 3D. The long-term goal of this project is to characterize genetic determinants of craniofacial phenotype. Three specific aims are proposed:

1) Define the parameters for each of the three skeletal growth patterns.

2) Analyze mandibular shapes of the skeletal patterns using morphometric methods to identify differences among the three groups.

3) Verify and establish methodology of salivary DNA collection and extraction for future down-stream applications.

Methods: Pre-treatment CBCTs of forty-five adult patients from the Orthodontic Clinic at the UCLA School of Dentistry were analyzed for measurement of craniofacial traits, 15 cases for each of the three skeletal growth patterns – brachyfacial, mesofacial, and dolichofacial. Inclusion criteria encompassed healthy adult non-growing patients at least 18 years old, male or female. Exclusion criteria comprised syndromic patients, alveolar/palatal cleft patients, individuals with hemimandibular or hemifacial asymmetry, history of orthodontic treatment involving orthopedic movement, and systemic disease that affects bone metabolism. All subjects had a high-resolution head CBCT scan obtained prior to orthodontic treatment as part of their diagnosis and treatment planning. The axial image slices were reconstructed and converted into DICOM format then exported into modified orthodontic analysis software for craniofacial analysis. Each hemimandible was segmented from the whole craniofacial structure, and three-dimensional anatomical landmarks were chosen to best describe its shape. Procrustes alignment of the landmark points was performed for both principal component analysis (PCA) and elliptical Fourier function analysis (EFFA) to identify where the variation in shape occurs between the averages of each of the three groups. Statistical significance was examined with ANOVA and Tukey’s HSD test. Extraction of high-quality genomic DNA of subjects was begun for future genome-wide association studies (GWAS) utilizing a non-invasive method of saliva collection and a DNA isolation kit. The purified DNA has been stored in -80?C for future experiments.

Results: Statistically significant area of variation between the three skeletal growth patterns was found with Procrustes and principal component analysis, and the results were further substantiated with elliptical Fourier function analysis. Additional trends were evident from the elliptical Fourier analysis. Target human gene was amplified and visualized on gels upon collection of saliva and DNA extraction following a troubleshot protocol.

Conclusions: Landmark-based analytical methods as the industry standard in morphometrics are useful in identifying shape differences. However, coupling with outline-based morphometrics is an even more powerful and informative tool in elucidating significant areas of variation in shape. Whole saliva can be effectively utilized for human DNA collection and isolation for future gene studies.