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MicroRNAs regulate posttranscriptional expression of target genes leading to inhibition or degradation of mRNA therefore inhibiting synthesis of protein coding genes. We reported that miRNA genes associated with response to ion-channel/transporter functions are differentially expressed in masseter of subjects with facial asymmetry. We have selected one species, miRNA 328, to test whether its expression associates with malocclusion types, asymmetry and TMD among orthognathic surgery patients. RNA was isolated from muscle of patients undergoing mandibular sagittal-split surgical procedures for non-syndromic skeletal discrepancies. Subjects were diagnosed with one of the following types of malocclusion, with or without TMD and facial asymmetry: Class-II or Class-III with vertical normal, open or deep bite. A two-step process was used for quantification of miRNAs. RNA was reverse transcribed and assayed using specific TaqManTM primers and probe for microRNA 328. Standard curve experiments were used for assay analysis. MicroRNA 328 expression values were low, ranging from 1-80pg throughout. Quantifiable differences were detected in comparisons between parameter groups. Statistical significant associations (P<0.04) were found between female Class III and male Class III malocclusion subjects. In addition, interesting differences were identified among TMD-related myalgia groups with p<0.20, and between male Class II and male Class III malocclusion subjects. Power analyses were done to determine the sample sizes needed for each group to attain significance. MicroRNA 328 is differentially expressed in masseter of subjects with Class-II/Class-III malocclusion and in TMD-related myalgia. Larger sample sizes are needed to verify whether microRNA 328 expression acts in both disorders. By power analysis, to reach p<0.05 with a power =0.8, 60 samples are needed in both TMD and Non-TMD groups. Differential expression of microRNA 328 seen in Class III females and males suggests a potential influence on muscle target genes, craniofacial development and pain. / Oral Biology

Alpha-actinins are myofibril anchor proteins, which influence contractile properties of skeletal muscle. ACTN2 is expressed in slow type I and fast type II fibers whereas ACTN3 is expressed only in fast fibers. ACTN3 homozygosity for the 577X stop codon (i.e. changing 577RR to 577XX - the R577X polymorphism) results in the absence of alpha-actinin-3 in about 18% of Europeans, diminished fast contractile ability, enhanced endurance performance, and reduced bone mass or bone mineral density. We have examined ACTN3 expression and genetic variation in masseter muscle of orthognathic surgery patients to determine genotype associations with malocclusion. To determine the associations between genotypes and malocclusions, clinical information, masseter muscle biopsies, and saliva samples were obtained from 60 subjects. Genotyping for ACTN3 SNPs, RT-PCR quantitation of muscle gene message, and muscle morphometric fiber type properties were compared to determine statistical differences between genotype and phenotype. We found muscle mRNA expression level was significantly different for ACTN3 SNP genotypes (p<0.01). The frequency of ACTN3 genotypes was significantly different for sagittal and vertical classifications of malocclusion with the clearest association being elevated 577XX genotype in skeletal Class II malocclusion (p = 0.003). This genotype also resulted in significantly smaller diameter of fast type II fibers in masseter muscle (p=0.002). In conclusion, ACTN3 577XX is overrepresented in skeletal Class II malocclusion, suggesting a biologic influence during bone growth. ACTN3 577XX is underrepresented in deep bite malocclusion, suggesting muscle differences contribute to variations in vertical facial dimensions. / Oral Biology

<p>Chiari Type I Malformation (CMI) is a developmental disorder characterized by displacement of the cerebellar tonsils below the base of the skull, resulting in significant neurologic morbidity. While there are multiple proposed mechanisms for tonsillar herniation, "classical" CMI is thought to occur due to a compromised posterior cranial fossa (PF). As CMI patients display a high degree of clinical variability, it is hypothesized that this heterogeneous disorder has a complex etiology influenced by multiple genetic and environmental factors. Despite the fact that multiple lines of evidence support a genetic contribution to disease, no genes have been identified to date. Thus, the primary goal of this dissertation is to begin to dissect the genetic etiology of this important disorder and gain a better understanding of what factors contribute to the observed disease heterogeneity.</p><p>In order to address these goals, two studies and three distinct analytic approaches were carried out. In the first study, 367 individuals from 66 nonsyndromic, CMI multiplex families provided the basis for a whole genome linkage screen to identify genomic regions likely to harbor CMI susceptibility genes. Results from the linkage screen using the complete collection of families yielded limited evidence for linkage, likely due to genetic heterogeneity. Thus, two separate analytic approaches were applied to the data to reduce phenotypic and hopefully genetic heterogeneity, thereby increasing power to identify disease genes. In the first approach, families were stratified based on the presence or absence of connective tissue disorder (CTD) related conditions as hereditary CTDs are commonly associated with CMI and the presumed mechanism for tonsillar herniation differs between CMI patients with CTDs and "classical" CMI patients. Stratified analyses resulted in increased evidence for linkage to multiple genomic regions. Of particular interest were two regions located on chromosomes 8 and 12, both of which harbor growth differentiation factors, GDF6 and GDF3, which have been implicated in Klippel-Feil syndrome (KFS). In the second approach, a comprehensive evaluation of the genetic contribution to the PF was performed, followed by ordered subset analysis (OSA) using heritable, disease-relevant PF traits to identify increased evidence for linkage within subsets of families that were similar with respect to cranial base morphological traits. Much of the PF was found to be heritable and results from OSA identified multiple genomic regions showing increased evidence for linkage, including regions on chromosomes 1 and 22 which implicated several strong biological candidates for disease. </p><p>In the second study, 44 pediatric, surgical CMI patients were ascertained in order to establish disease subtypes using whole genome expression profiles generated from patient blood and dura mater samples and radiological data consisting of PF morphometrics. Sparse k-means clustering as well as a modified version were used to cluster patients using the biological and radiological data both separately and collectively. The most significant patient classes were identified from the pure biological clustering analyses. Further characterization of these classes implicated strong biological candidates involved in endochondral ossification from the dura analysis and a blood gene expression profile exhibiting a global down-regulation in protein synthesis and related pathways that may be associated with comorbid conditions. </p><p>Collectively, these studies established several strong biological disease candidates, as well as emphasized the need to better understand and account for disease heterogeneity, re-evaluate the current diagnostic criteria for CMI, and continue to investigate the use of endophenotypes, such as cranial base morphometrics, when conducting genetic studies.</p> / Dissertation

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

Poliovirus is a small, structurally simple virus that can serve as a powerful model system for the elucidation of the basic processes involved in the genetic control of macromolecular synthesis. The physical and biochemical characterization of temperature-sensitive and drug-resistant mutants of this virus can provide insight into the normal sequence of events during the replication and assembly of the wild-type (wt) virus. The specific interference of the infecting virus with the major pathways of macromolecular synthesis in the host cell offers a possible inroad to the exploration of the relevant control systems. This research is divided into two major segments: (1) a temperature-sensitive mutant of poliovirus type 1, tsB9, and a guanidine-resistant mutant, gʳH, were characterized physiologically; (2) a physiological screening procedure that quickly and efficiently reveals the phenotype of a large number of poliovirus mutants in a short period of time was developed and validated. Two guanidine-resistant and twelve temperature-sensitive mutants of poliovirus type 1, Mahoney, were generated and compared with wt, defective-interfering particles, gʳH, and tsB9 using the screening procedure herein developed. The temperature-sensitive mutant, tsB9, appears to be a structural protein mutant bearing a related defect in ribonucleic acid synthesis at the restrictive temperature. The mutant gʳH was found to differ from the wt virus only in the guanidine resistance of its growth and ribonucleic acid synthesis, although a detailed electrophoretic analysis of its proteins was not done. Among the newly isolated mutants, strains were found with defects in each of the major viral functions except one. No mutant was found to be defective in the ability to inhibit host-cell protein synthesis. The screening procedure developed met all of the criteria set for it mentioned above. It has the potential of being adapted to many virus-cell systems for the rapid determination of mutant phenotype.