Test design for Oculopharyngeal muscular dystrophy





Protein Binding Studies for Expanded Poly-A Repeats and Mutant PABP2 resulting from Oculopharyngeal Muscular Dystrophy

INTRODUCTION:
Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular genetic disorder. It has an autosomal dominant pattern of inheritance (Fried et al. 1975) in that the abnormal gene can be transmitted from only one parent. A child of an affected parent has a 50% chance of being affected. The disorder is found to be more prevalent among French-Canadians and is characterized by its late onset (approximately 50). Affected persons experience dropping eyelids (optosis), difficulty with swallowing (dysphagia), and some develop shoulder, hip or leg weaknesses (MDA publications 1998). Genetically, its mutation is quite unique. OPMD is caused by the expansion of a GCG (which codes for the amino acid alanine) 6 repeat (Brais et al. 1998), whereas most triplet repeat disorders are expansions of CAG (glutamine) repeats. Rare polymorphisms would be to have 7 consecutive GCG\'s, but the disease is mostly characterized by the mutation of having 8-15 consecutive GCG\'s. Other findings have shown that even the expansion of a 6 GCG repeat to 7 can also lead to OPMD (LaFontaine 1996). The severity of the disease depends on the number of extra alanines. Quite recently, scientists have found that the mutation occurs on chromosome 14 and is in the gene coding for a poly(A)-binding protein 2 gene (PABP2) (Brais et al. 1998). PABP2 was considered a good candidate for OPMD because it maps to the same location as the diseased gene, its mRNA is highly expressed in skeletal muscle, and the PAB2 protein is exclusively localized in the nucleus, where it acts as a factor in mRNA polyadenylation. The site of the additional GCG expansions in the PABP2 gene is at the polyalanine tract at the N terminus. From these findings, one may ask why this disease targets preferentially the skeletal muscle cells of the eyes and throat when the protein of the wild- type form of the mutant gene (PABP2) is expressed in all cells. In order to answer this probing question, binding studies involving the abnormal poly A stretches and the mutant PABP2 protein need to be performed. This will determine what other proteins (if any) are involved in this particular type of muscular dystrophy. In this project, I hypothesize that there are proteins from affected tissues which bind to the expanded poly A stretches as well as the mutant PABP2 protein. These proteins may also bind to them in varying amounts depending on the length of the expanded GCG repeats..
To find out if any proteins bind with extended repeats of the corresponding mutant PABP2 protein, affinity chromatography experiments can be done. This type of experiment will involve polystyrene beads, coated with synthetically made poly-A peptide representing the mutant protein domain, and are packed with various homogenated human muscle tissues. Human tissues will be used because the disease only seem to affect humans. The synthetic peptides will be of varying repeats of alanine, thus testing for the different severities of the disease. Other molecular studies investigating OPMD examined up to 13 repeated GCGs. Therefore, for the polystyrene beads experiment, repeats of 6 to 14 will be used. A test with 14 repeats will determine whether there is still a continual increase in severity after 13 repeats or whether the effects will be abolished past the 13 repeat mark.
An experiment to elucidate potential proteins that bind to the mutant PABP2 protein involved in OPMD is the yeast-two-hybrid system. The method uses the transcription of yeast reporter genes as a synthetic phenotype to detect protein-protein interactions. The approach takes advantage of the modular domain structure of eukaryotic transcription factors. Many transcription activators have at least two distinct functional domains, one that directs binding to specific DNA sequences and one that activates transcription. This modular structure is best illustrated by yeast experiments showing that the DNA-binding domains or activation domains can be exchanged from one transcription factor to the next and retain function. A crucial consequence of the modular nature of transcription activators is that the DNA-binding and activation domains do not need to be covalently attached to each other for activation to occur. Because of this, yeast transcription could be used to assay the interaction between two proteins if one of them