The aim of the proposed project is to identify and analyze mutations responsible for pyruvate dehydrogenase complex (PDC) deficiency, an inherited disorder of energy metabolism which causes lactic acidosis and neurologic disability. The project will be done in the context of the Physician Scientist Award which is a grant designed to allow clinicians to develop independent research skills and experience in a basic science. In Phase I of the award which is a grant designed to allow clinicians to develop independent research skills and experience in a basic science. In Phase I of the award, the candidate will attend courses in biochemistry and molecular biology as well as participate in conferences and seminars pertaining to different areas of metabolism. During this phase, the candidate will begin a research project involving the study of a series of patients with deficits in the first component of PDC, pyruvate dehydrogenase (E1) - the most common form of PDC deficiency - whose defects have been previously characterized at the protein and RNA levels. Mutant cDNA for E1 will be isolated from these subjects by the technique of reverse transcription using specific oligonucleotide primers complementary to sequences found in E1 cDNA followed by a polymerase chain reaction to amplify the newly synthesized cDNA. Mutant E1 cDNa will then be sequenced and compared to wild type E1. Subsequently, identified mutations will be analyzed as to their functional significance in causing the heterogeneous clinical and biochemical manifestations of E1 deficiency. Knowing the location of mutations responsible for E1 will allow for the development of improved techniques for the diagnosis of E1 deficiency, and determination of the specific mode of inheritance for this disorder. In phase II, knowledge and skills acquired during phase I will be applied to the further investigation of the structure and function of PDC and the pathology of PDC deficiency. Such studies will be facilitated by the development of in vitro systems of expression for mutant E1 genes. The long term goal of the project is for the candidate to develop into a competent physician scientist with the resources and capability to independently investigate disorders of energy metabolism.
