All ETDs from UAB

Advisory Committee Chair

Peter Jezewski

Advisory Committee Members

Heidi Erlandsen

Amjad Javed

Stephen Watts

Champion Cs Deivanayagam

Document Type


Date of Award


Degree Name by School

Master of Science in Dentistry (MScD) School of Dentistry


Orofacial clefting (OFC), also called cleft lip and/or palate, is the most common congenital birth anomaly that involve the craniofacial region in humans. The etiology of Non-Syndromic OFC (NS-OFC) is complex and multifactorial; a slew of genetic mutations, standing alone or in combinations, along with several environmental factors, play a major role in the disease process. Numerous linkage and association studies in humans, as well as expression studies and knockout mouse models, have implicated Muscle segment homeobox gene 1 (MSX1), which encodes a homeodomain/DNA binding domain (HD) containing transcription factor as a strong candidate contributing to NS-OFC and Tooth Agenesis (TA-congenital lack of teeth), both are part of a clinical spectrum of associated phenotypes. Interestingly, MSX1 mutations may account for as much as 2% of all facial clefting defects in humans. Multiple missense mutations in MSX1 among various populations have been reported. Of the proposed pathogenic mutations, the P147Q and R151S missense coding mutations were reported as private mutations and were found within a highly conserved set of amino acids upstream of the homeodomain in human MSX1. These two variants were hypothesized to affect the potential functions of the MSX1 protein, such as its binding interactions with other transcription factors and with DNA. Phosphorylation of proteins is known to be a regulator of binding interactions due to changes in protein conformation upon phosphorylation. Phosphorylation of MSX1 might facilitate binding to other proteins or DNA, but binding interactions have not been functionally characterized. Also, MSX2 is known to be phosphorylated, but MSX1 has not been previously reported to be phosphorylated. Thus our objective was to determine how these two rare coding variants affect MSX1 binding interactions through functional studies. Two truncated versions of the MSX1 protein, namely MH2-5 (E132-P242) and MH2-4 (E132-K233) were generated, as well as the mutant forms carrying the Proline to Glutamine substitution at amino acid 147 (P147Q) or Arginine to Serine substitution at amino acid 151 (R151S). Isothermal Titration Calorimetry (ITC) and enzyme kinase phosphorylation experiments followed by mass spectrometry detection of phosphorylated peptides, were utilized to evaluate the differences in the binding affinity and the degree/extent of phosphorylation respectively, between the wild type (WT) and mutant proteins. Furthermore, bioinformatic analyses identified a putative strong MSX1 DNA binding site within a highly conserved tissue specific enhancer (that included the growing tips of the maxillary prominences). The transgenic constructs necessary for generating future transgenic zebrafish lines were cloned. These will be useful for in vivo studies of the interactions of MSX1 to the enhancer. The results obtained and discussed herein suggest an interesting array of MSX1 phosphorylation observed in the mutants, ranging from creation of a novel phosphorylation site (R151S), to significant obliteration of phosphorylation at plausible sites (P147Q). ITC data revealed that the mutant P147Q showed increased binding affinity to DNA when compared to WT. Thus, these two missense mutations were shown to affect the degree of phosphorylation of the MSX1 protein and also observed to affect the binding of MSX1 to a consensus DNA sequence. Further investigations might help us understand how these mutations affect binding interaction between the MSX1-TALE protein (Three Amino acid Loop Extension) dimeric complex to DNA. Generation of msxe transient transgenic zebrafish lines was the first step towards developing a complete spatial and temporal in vivo assay of various DNA and protein sequence variants identified in patients with cleft and/or dental phenotypes. We believe it should offer insight into the fundamental control mechanisms of facial development.

Included in

Dentistry Commons



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