A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.

Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas-mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR family members because of duplications. Here, we report a novel plasmid-based miR inhibitor system (PMIS) that expresses a new RNA molecule, which inhibits miR family members in cells and mice. The PMIS engineered RNA optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. The PMIS shows no off-target effects or toxicity and is highly specific for miRs sharing identical seed sequences. Transgenic mice expressing both PMIS-miR-17-18 and PMIS-miR-19-92 show similar phenotypes of miR-17-92-knockout mice. Interestingly, mice only expressing PMIS-miR-17-18 have developmental defects distinct from mice only expressing PMIS-miR-19-92 demonstrating usefulness of the PMIS system to dissect different functions of miRs within clusters. Different PMIS miR inhibitors can be linked together to knock down multiple miRs expressed from different chromosomes. Inhibition of the miR-17-92, miR-106a-363 and miR-106b-25 clusters reveals new mechanisms and developmental defects for these miRs. We report a new tool to dissect the role of miRs in development without genome editing, inhibit miR function in cells and as a potential new therapeutic reagent.

The ontogeny of the chin: an analysis of allometric and biomechanical scaling.

The presence of a prominent chin in modern humans has been viewed by some researchers as an architectural adaptation to buttress the anterior corpus from bending stresses during mastication. In contrast, ontogenetic studies of mandibular symphyseal form suggest that a prominent chin results from the complex spatial interaction between the symphysis and surrounding soft tissue and skeletal anatomy during development. While variation in chin prominence is clearly influenced by differential growth and spatial constraints, it is unclear to what degree these developmental dynamics influence the mechanical properties of the symphysis. That is, do ontogenetic changes in symphyseal shape result in increased symphyseal bending resistance? We examined ontogenetic changes in the mechanical properties and shape of the symphysis using subjects from a longitudinal cephalometric growth study with ages ranging from 3 to 20+ years. We first examined whether ontogenetic changes in symphyseal shape were correlated with symphyseal vertical bending and wishboning resistance using multivariate regression. Secondly, we examined ontogenetic scaling of bending resistance relative to bending moment arm lengths. An ontogenetic increase in chin prominence was associated with decreased vertical bending resistance, while wishboning resistance was uncorrelated with ontogenetic development of the chin. Relative to bending moment arm lengths, vertical bending resistance scaled with significant negative allometry whereas wishboning resistance scaled isometrically. These results suggest a complex interaction between symphyseal ontogeny and bending resistance, and indicate that ontogenetic increases in chin projection do not provide greater bending resistance to the mandibular symphysis.

Candidate Gene Analyses of Skeletal Variation in Malocclusion.

This study evaluated associations between craniofacial candidate genes and skeletal variation in patients with malocclusion. Lateral cephalometric radiographs of 269 untreated adults with skeletal classes I, II, and III malocclusion were digitized with 14 landmarks. Two-dimensional coordinates were analyzed using Procrustes fit and principal component (PC) analysis to generate continuous malocclusion phenotypes. Skeletal class classifications (I, II, or III) were used as a categorical phenotype. Individuals were genotyped for 198 single-nucleotide polymorphisms (SNPs) in 71 craniofacial genes and loci. Phenotype-genotype associations were tested via multivariate linear regression for continuous phenotypes and multinomial logistic regression for skeletal malocclusion class. PC analysis resulted in 4 principal components (PCs) explaining 69% of the total skeletal facial variation. PC1 explained 32.7% of the variation and depicted vertical discrepancies ranging from skeletal deep to open bites. PC1 was associated with a SNP near PAX5 (P = 0.01). PC2 explained 21.7% and captured horizontal maxillomandibular discrepancies. PC2 was associated with SNPs upstream of SNAI3 (P = 0.0002) and MYO1H (P = 0.006). PC3 explained 8.2% and captured variation in ramus height, body length, and anterior cranial base orientation. PC3 was associated with TWIST1 (P = 0.000076). Finally, PC4 explained 6.6% and detected variation in condylar inclination as well as symphysis projection. PC4 was associated with PAX7 (P = 0.007). Furthermore, skeletal class II risk increased relative to class I with the minor alleles of SNPs in FGFR2 (odds ratio [OR] = 2.1, P = 0.004) and declined with SNPs in EDN1 (OR = 0.5, P = 0.007). Conversely, skeletal class III risk increased versus class I with SNPs in FGFR2 (OR 2.2, P = 0.005) and COL1A1 (OR = 2.1, P = 0.008) and declined with SNPs in TBX5 (OR = 0.5, P = 0.014). PAX5, SNAI3, MYO1H, TWIST1, and PAX7 are associated with craniofacial skeletal variation among patients with malocclusion, while FGFR2, EDN1, TBX5, and COL1A1 are associated with type of skeletal malocclusion.